%PDF-1.3 1 0 obj << /Type /Catalog /Outlines 2 0 R /Pages 3 0 R >> endobj 2 0 obj << /Type /Outlines /Count 0 >> endobj 3 0 obj << /Type /Pages /Kids [6 0 R 170 0 R 258 0 R 260 0 R 287 0 R 303 0 R 312 0 R 363 0 R 401 0 R 405 0 R 408 0 R 410 0 R 412 0 R ] /Count 13 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R /F5 172 0 R /F6 173 0 R >> /XObject << /I1 12 0 R /I2 13 0 R /I3 270 0 R /I4 291 0 R /I5 294 0 R /I6 307 0 R /I7 316 0 R /I8 403 0 R /I9 404 0 R /I10 407 0 R >> >> /MediaBox [0.000 0.000 612.000 792.000] >> endobj 4 0 obj [/PDF /Text /ImageC ] endobj 5 0 obj << /Creator (DOMPDF) /CreationDate (D:20180721144024+00'00') /ModDate (D:20180721144024+00'00') /Title (Phylogenetic discordance of human and canine carcinoembryonic antigen \(CEA, CEACAM\) families, but striking identity of the CEA receptors will impact comparative oncology studies. 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Thalhammer)] TJ ET 0.271 0.267 0.267 rg BT 195.861 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 201.282 601.446 Td /F1 9.8 Tf [(Robert Kammerer)] TJ ET 0.271 0.267 0.267 rg BT 278.755 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 284.176 601.446 Td /F1 9.8 Tf [(Erika Jensen-Jarolim)] TJ ET 0.271 0.267 0.267 rg BT 26.250 589.542 Td /F1 9.8 Tf [(Weichselbaumer M, Willmann M, Reifinger M, Singer J, Bajna E, Sobanov Y, Mechtcherikova D, Selzer E, Thalhammer JG, )] TJ ET BT 26.250 577.637 Td /F1 9.8 Tf [(Kammerer R, Jensen-Jarolim E. Phylogenetic discordance of human and canine carcinoembryonic antigen \(CEA, CEACAM\) )] TJ ET BT 26.250 565.732 Td /F1 9.8 Tf [(families, but striking identity of the CEA receptors will impact comparative oncology studies.. PLOS Currents Tree of Life. 2011 )] TJ ET BT 26.250 553.827 Td /F1 9.8 Tf [(Nov 11 . Edition 1. doi: 10.1371/currents.RRN1223.)] TJ ET q 15.000 26.071 577.500 525.375 re W n 0.271 0.267 0.267 rg BT 26.250 524.725 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 504.771 Td /F1 9.8 Tf [(Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following )] TJ ET BT 26.250 492.866 Td /F1 9.8 Tf [(this line, carcinoembryonic antigen \(CEA, CEACAM5\) could be a therapeutic target not only for human but also for canine )] TJ ET BT 26.250 480.961 Td /F1 9.8 Tf [(\(Canis lupus familiaris; dog\) patients. CEACAM5 interacts with CEA-receptor \(CEAR\) in the cytoplasm of human cancer cells. )] TJ ET BT 26.250 469.056 Td /F1 9.8 Tf [(Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and )] TJ ET BT 26.250 457.152 Td /F1 9.8 Tf [(canine cancer.)] TJ ET BT 26.250 445.247 Td /F1 9.8 Tf [(Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted )] TJ ET BT 569.744 445.247 Td /F1 9.8 Tf [(to )] TJ ET BT 26.250 433.342 Td /F1 9.8 Tf [(23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 )] TJ ET BT 26.250 421.437 Td /F1 9.8 Tf [(kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma )] TJ ET BT 26.250 409.533 Td /F1 9.8 Tf [(cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be )] TJ ET BT 26.250 397.628 Td /F1 9.8 Tf [(most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to )] TJ ET BT 26.250 385.723 Td /F1 9.8 Tf [(canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in )] TJ ET BT 26.250 373.818 Td /F1 9.8 Tf [(contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on )] TJ ET BT 26.250 361.914 Td /F1 9.8 Tf [(canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly )] TJ ET BT 26.250 350.009 Td /F1 9.8 Tf [(suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a )] TJ ET BT 26.250 338.104 Td /F1 9.8 Tf [(99% amino acid similarity of human and canine CEA receptor \(CEAR\) within the phylogenetic tree. CEAR could be detected in )] TJ ET BT 26.250 326.199 Td /F1 9.8 Tf [(four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by )] TJ ET BT 26.250 314.295 Td /F1 9.8 Tf [(immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act )] TJ ET BT 26.250 302.390 Td /F1 9.8 Tf [(as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the )] TJ ET BT 26.250 290.485 Td /F1 9.8 Tf [(complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a )] TJ ET BT 26.250 278.580 Td /F1 9.8 Tf [(comprehensive interspecies target for novel cancer therapeutics.)] TJ ET BT 26.250 241.978 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 222.024 Td /F1 9.8 Tf [(Comparative oncology trials represent a novel preclinical infrastructure for speeding up the development of anticancer )] TJ ET BT 26.250 210.119 Td /F1 9.8 Tf [(therapeutics )] TJ ET 0.267 0.267 0.267 rg BT 82.069 210.119 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 106.463 210.119 Td /F1 9.8 Tf [(. In contrast to known preclinical animal models, comparative studies take advantage of spontaneously )] TJ ET BT 26.250 198.214 Td /F1 9.8 Tf [(occurring cancers, for instance in dogs \(Canis lupus familiaris\) )] TJ ET 0.267 0.267 0.267 rg BT 297.163 198.214 Td /F1 9.8 Tf [([3])] TJ ET 0.271 0.267 0.267 rg BT 308.005 198.214 Td /F1 9.8 Tf [(. Following specific guidelines )] TJ ET 0.267 0.267 0.267 rg BT 439.133 198.214 Td /F1 9.8 Tf [([4])] TJ ET 0.271 0.267 0.267 rg BT 449.975 198.214 Td /F1 9.8 Tf [(, these studies also assess )] TJ ET BT 26.250 186.309 Td /F1 9.8 Tf [(the response of the veterinary patients to novel drugs in terms of clinical benefit)] TJ ET 0.267 0.267 0.267 rg BT 368.202 186.309 Td /F1 9.8 Tf [( [5] [6] [7])] TJ ET 0.271 0.267 0.267 rg BT 408.860 186.309 Td /F1 9.8 Tf [(. Mammary neoplasms are the most )] TJ ET BT 26.250 174.405 Td /F1 9.8 Tf [(common tumors in female dogs )] TJ ET 0.267 0.267 0.267 rg BT 164.963 174.405 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 175.805 174.405 Td /F1 9.8 Tf [(, with incidence depending on the population studied and varying from 205 per 100,000 )] TJ ET 0.267 0.267 0.267 rg BT 554.105 174.405 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 564.947 174.405 Td /F1 9.8 Tf [( to )] TJ ET BT 26.250 162.500 Td /F1 9.8 Tf [(111 per 10,000 female dogs between 3 and 10 years of age )] TJ ET 0.267 0.267 0.267 rg BT 286.955 162.500 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 303.218 162.500 Td /F1 9.8 Tf [(. 30 to 50% of all canine mammary tumors are malignant and )] TJ ET BT 26.250 150.595 Td /F1 9.8 Tf [(50 to 75% of these recur or metastasize within 1 to 2 years )] TJ ET 0.267 0.267 0.267 rg BT 282.577 150.595 Td /F1 9.8 Tf [([11] [12])] TJ ET 0.271 0.267 0.267 rg BT 317.814 150.595 Td /F1 9.8 Tf [(. For these reasons more effective therapy strategies are )] TJ ET BT 26.250 138.690 Td /F1 9.8 Tf [(urgently needed for companion animals, at the same time their disease mirrors the settings of human oncology patients.)] TJ ET BT 26.250 119.286 Td /F1 9.8 Tf [(Especially immunological targeting of cancer for diagnosis and therapy is much more advanced in humans )] TJ ET 0.267 0.267 0.267 rg BT 487.396 119.286 Td /F1 9.8 Tf [([13] [14])] TJ ET 0.271 0.267 0.267 rg BT 522.632 119.286 Td /F1 9.8 Tf [(. Therefore, )] TJ ET BT 26.250 107.381 Td /F1 9.8 Tf [(we suggest that careful, comparative evaluation of most important tumor targets should be undertaken to increase the basic )] TJ ET BT 26.250 95.476 Td /F1 9.8 Tf [(knowledge for comparative studies.)] TJ ET BT 26.250 76.071 Td /F1 9.8 Tf [(An important example for a potential immunological cancer target is carcinoembryonic antigen \(CEACAM5\), which represents a )] TJ ET BT 26.250 64.167 Td /F1 9.8 Tf [(classical tumor marker and is routinely exploited for diagnosis in man. In its soluble form CEACAM5 binds to the )] TJ ET BT 26.250 52.262 Td /F1 9.8 Tf [(carcinoembryonic antigen receptor \(CEAR\), a member of the heterogeneous nuclear ribonucleoproteins \(hnRNP\) family, )] TJ ET BT 26.250 40.357 Td /F1 9.8 Tf [(offering the likely potential to deliver prometastatic signals to neoplastic cells )] TJ ET 0.267 0.267 0.267 rg BT 357.887 40.357 Td /F1 9.8 Tf [([15] [16])] TJ ET 0.271 0.267 0.267 rg BT 393.123 40.357 Td /F1 9.8 Tf [(. In many types of human cancers )] TJ ET Q q 15.000 634.458 577.500 103.542 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Phylogenetic discordance of human and canine )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(carcinoembryonic antigen \(CEA, CEACAM\) families, but )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(striking identity of the CEA receptors will impact comparative )] TJ ET BT 15.000 643.198 Td /F2 21.0 Tf [(oncology studies.)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 625.192 Td /F3 9.8 Tf [(November 11, 2011)] TJ ET BT 96.510 625.192 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 101.385 625.192 Td /F3 9.8 Tf [(Tree of Life)] TJ ET BT 26.250 613.351 Td /F1 9.8 Tf [(Marlene Weichselbaumer)] TJ ET 0.271 0.267 0.267 rg BT 136.776 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 142.197 613.351 Td /F1 9.8 Tf [(Michael Willmann)] TJ ET 0.271 0.267 0.267 rg BT 218.579 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 224.000 613.351 Td /F1 9.8 Tf [(Martin Reifinger)] TJ ET 0.271 0.267 0.267 rg BT 292.805 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 298.226 613.351 Td /F1 9.8 Tf [(Josef Singer)] TJ ET 0.271 0.267 0.267 rg BT 352.417 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 357.838 613.351 Td /F1 9.8 Tf [(Erika Bajna)] TJ ET 0.271 0.267 0.267 rg BT 407.689 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 413.110 613.351 Td /F1 9.8 Tf [(Yuriy Sobanov)] TJ ET 0.271 0.267 0.267 rg BT 476.515 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 481.936 613.351 Td /F1 9.8 Tf [(Diana Mechtcherikova)] TJ ET 0.271 0.267 0.267 rg BT 26.250 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 31.671 601.446 Td /F1 9.8 Tf [(Edgar Selzer)] TJ ET 0.271 0.267 0.267 rg BT 88.026 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 93.447 601.446 Td /F1 9.8 Tf [(Johann G. Thalhammer)] TJ ET 0.271 0.267 0.267 rg BT 195.861 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 201.282 601.446 Td /F1 9.8 Tf [(Robert Kammerer)] TJ ET 0.271 0.267 0.267 rg BT 278.755 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 284.176 601.446 Td /F1 9.8 Tf [(Erika Jensen-Jarolim)] TJ ET 0.271 0.267 0.267 rg BT 26.250 589.542 Td /F1 9.8 Tf [(Weichselbaumer M, Willmann M, Reifinger M, Singer J, Bajna E, Sobanov Y, Mechtcherikova D, Selzer E, Thalhammer JG, )] TJ ET BT 26.250 577.637 Td /F1 9.8 Tf [(Kammerer R, Jensen-Jarolim E. Phylogenetic discordance of human and canine carcinoembryonic antigen \(CEA, CEACAM\) )] TJ ET BT 26.250 565.732 Td /F1 9.8 Tf [(families, but striking identity of the CEA receptors will impact comparative oncology studies.. PLOS Currents Tree of Life. 2011 )] TJ ET BT 26.250 553.827 Td /F1 9.8 Tf [(Nov 11 . Edition 1. doi: 10.1371/currents.RRN1223.)] TJ ET q 15.000 26.071 577.500 525.375 re W n 0.271 0.267 0.267 rg BT 26.250 524.725 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 504.771 Td /F1 9.8 Tf [(Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following )] TJ ET BT 26.250 492.866 Td /F1 9.8 Tf [(this line, carcinoembryonic antigen \(CEA, CEACAM5\) could be a therapeutic target not only for human but also for canine )] TJ ET BT 26.250 480.961 Td /F1 9.8 Tf [(\(Canis lupus familiaris; dog\) patients. CEACAM5 interacts with CEA-receptor \(CEAR\) in the cytoplasm of human cancer cells. )] TJ ET BT 26.250 469.056 Td /F1 9.8 Tf [(Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and )] TJ ET BT 26.250 457.152 Td /F1 9.8 Tf [(canine cancer.)] TJ ET BT 26.250 445.247 Td /F1 9.8 Tf [(Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted )] TJ ET BT 569.744 445.247 Td /F1 9.8 Tf [(to )] TJ ET BT 26.250 433.342 Td /F1 9.8 Tf [(23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 )] TJ ET BT 26.250 421.437 Td /F1 9.8 Tf [(kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma )] TJ ET BT 26.250 409.533 Td /F1 9.8 Tf [(cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be )] TJ ET BT 26.250 397.628 Td /F1 9.8 Tf [(most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to )] TJ ET BT 26.250 385.723 Td /F1 9.8 Tf [(canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in )] TJ ET BT 26.250 373.818 Td /F1 9.8 Tf [(contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on )] TJ ET BT 26.250 361.914 Td /F1 9.8 Tf [(canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly )] TJ ET BT 26.250 350.009 Td /F1 9.8 Tf [(suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a )] TJ ET BT 26.250 338.104 Td /F1 9.8 Tf [(99% amino acid similarity of human and canine CEA receptor \(CEAR\) within the phylogenetic tree. CEAR could be detected in )] TJ ET BT 26.250 326.199 Td /F1 9.8 Tf [(four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by )] TJ ET BT 26.250 314.295 Td /F1 9.8 Tf [(immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act )] TJ ET BT 26.250 302.390 Td /F1 9.8 Tf [(as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the )] TJ ET BT 26.250 290.485 Td /F1 9.8 Tf [(complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a )] TJ ET BT 26.250 278.580 Td /F1 9.8 Tf [(comprehensive interspecies target for novel cancer therapeutics.)] TJ ET BT 26.250 241.978 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 222.024 Td /F1 9.8 Tf [(Comparative oncology trials represent a novel preclinical infrastructure for speeding up the development of anticancer )] TJ ET BT 26.250 210.119 Td /F1 9.8 Tf [(therapeutics )] TJ ET 0.267 0.267 0.267 rg BT 82.069 210.119 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 106.463 210.119 Td /F1 9.8 Tf [(. In contrast to known preclinical animal models, comparative studies take advantage of spontaneously )] TJ ET BT 26.250 198.214 Td /F1 9.8 Tf [(occurring cancers, for instance in dogs \(Canis lupus familiaris\) )] TJ ET 0.267 0.267 0.267 rg BT 297.163 198.214 Td /F1 9.8 Tf [([3])] TJ ET 0.271 0.267 0.267 rg BT 308.005 198.214 Td /F1 9.8 Tf [(. Following specific guidelines )] TJ ET 0.267 0.267 0.267 rg BT 439.133 198.214 Td /F1 9.8 Tf [([4])] TJ ET 0.271 0.267 0.267 rg BT 449.975 198.214 Td /F1 9.8 Tf [(, these studies also assess )] TJ ET BT 26.250 186.309 Td /F1 9.8 Tf [(the response of the veterinary patients to novel drugs in terms of clinical benefit)] TJ ET 0.267 0.267 0.267 rg BT 368.202 186.309 Td /F1 9.8 Tf [( [5] [6] [7])] TJ ET 0.271 0.267 0.267 rg BT 408.860 186.309 Td /F1 9.8 Tf [(. Mammary neoplasms are the most )] TJ ET BT 26.250 174.405 Td /F1 9.8 Tf [(common tumors in female dogs )] TJ ET 0.267 0.267 0.267 rg BT 164.963 174.405 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 175.805 174.405 Td /F1 9.8 Tf [(, with incidence depending on the population studied and varying from 205 per 100,000 )] TJ ET 0.267 0.267 0.267 rg BT 554.105 174.405 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 564.947 174.405 Td /F1 9.8 Tf [( to )] TJ ET BT 26.250 162.500 Td /F1 9.8 Tf [(111 per 10,000 female dogs between 3 and 10 years of age )] TJ ET 0.267 0.267 0.267 rg BT 286.955 162.500 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 303.218 162.500 Td /F1 9.8 Tf [(. 30 to 50% of all canine mammary tumors are malignant and )] TJ ET BT 26.250 150.595 Td /F1 9.8 Tf [(50 to 75% of these recur or metastasize within 1 to 2 years )] TJ ET 0.267 0.267 0.267 rg BT 282.577 150.595 Td /F1 9.8 Tf [([11] [12])] TJ ET 0.271 0.267 0.267 rg BT 317.814 150.595 Td /F1 9.8 Tf [(. For these reasons more effective therapy strategies are )] TJ ET BT 26.250 138.690 Td /F1 9.8 Tf [(urgently needed for companion animals, at the same time their disease mirrors the settings of human oncology patients.)] TJ ET BT 26.250 119.286 Td /F1 9.8 Tf [(Especially immunological targeting of cancer for diagnosis and therapy is much more advanced in humans )] TJ ET 0.267 0.267 0.267 rg BT 487.396 119.286 Td /F1 9.8 Tf [([13] [14])] TJ ET 0.271 0.267 0.267 rg BT 522.632 119.286 Td /F1 9.8 Tf [(. Therefore, )] TJ ET BT 26.250 107.381 Td /F1 9.8 Tf [(we suggest that careful, comparative evaluation of most important tumor targets should be undertaken to increase the basic )] TJ ET BT 26.250 95.476 Td /F1 9.8 Tf [(knowledge for comparative studies.)] TJ ET BT 26.250 76.071 Td /F1 9.8 Tf [(An important example for a potential immunological cancer target is carcinoembryonic antigen \(CEACAM5\), which represents a )] TJ ET BT 26.250 64.167 Td /F1 9.8 Tf [(classical tumor marker and is routinely exploited for diagnosis in man. In its soluble form CEACAM5 binds to the )] TJ ET BT 26.250 52.262 Td /F1 9.8 Tf [(carcinoembryonic antigen receptor \(CEAR\), a member of the heterogeneous nuclear ribonucleoproteins \(hnRNP\) family, )] TJ ET BT 26.250 40.357 Td /F1 9.8 Tf [(offering the likely potential to deliver prometastatic signals to neoplastic cells )] TJ ET 0.267 0.267 0.267 rg BT 357.887 40.357 Td /F1 9.8 Tf [([15] [16])] TJ ET 0.271 0.267 0.267 rg BT 393.123 40.357 Td /F1 9.8 Tf [(. In many types of human cancers )] TJ ET Q q 15.000 634.458 577.500 103.542 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Phylogenetic discordance of human and canine )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(carcinoembryonic antigen \(CEA, CEACAM\) families, but )] TJ ET BT 15.000 668.146 Td /F2 21.0 Tf [(striking identity of the CEA receptors will impact comparative )] TJ ET BT 15.000 643.198 Td /F2 21.0 Tf [(oncology studies.)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 625.192 Td /F3 9.8 Tf [(November 11, 2011)] TJ ET BT 96.510 625.192 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 101.385 625.192 Td /F3 9.8 Tf [(Tree of Life)] TJ ET BT 26.250 613.351 Td /F1 9.8 Tf [(Marlene Weichselbaumer)] TJ ET 0.271 0.267 0.267 rg BT 136.776 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 142.197 613.351 Td /F1 9.8 Tf [(Michael Willmann)] TJ ET 0.271 0.267 0.267 rg BT 218.579 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 224.000 613.351 Td /F1 9.8 Tf [(Martin Reifinger)] TJ ET 0.271 0.267 0.267 rg BT 292.805 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 298.226 613.351 Td /F1 9.8 Tf [(Josef Singer)] TJ ET 0.271 0.267 0.267 rg BT 352.417 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 357.838 613.351 Td /F1 9.8 Tf [(Erika Bajna)] TJ ET 0.271 0.267 0.267 rg BT 407.689 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 413.110 613.351 Td /F1 9.8 Tf [(Yuriy Sobanov)] TJ ET 0.271 0.267 0.267 rg BT 476.515 613.351 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 481.936 613.351 Td /F1 9.8 Tf [(Diana Mechtcherikova)] TJ ET 0.271 0.267 0.267 rg BT 26.250 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 31.671 601.446 Td /F1 9.8 Tf [(Edgar Selzer)] TJ ET 0.271 0.267 0.267 rg BT 88.026 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 93.447 601.446 Td /F1 9.8 Tf [(Johann G. Thalhammer)] TJ ET 0.271 0.267 0.267 rg BT 195.861 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 201.282 601.446 Td /F1 9.8 Tf [(Robert Kammerer)] TJ ET 0.271 0.267 0.267 rg BT 278.755 601.446 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 284.176 601.446 Td /F1 9.8 Tf [(Erika Jensen-Jarolim)] TJ ET 0.271 0.267 0.267 rg BT 26.250 589.542 Td /F1 9.8 Tf [(Weichselbaumer M, Willmann M, Reifinger M, Singer J, Bajna E, Sobanov Y, Mechtcherikova D, Selzer E, Thalhammer JG, )] TJ ET BT 26.250 577.637 Td /F1 9.8 Tf [(Kammerer R, Jensen-Jarolim E. Phylogenetic discordance of human and canine carcinoembryonic antigen \(CEA, CEACAM\) )] TJ ET BT 26.250 565.732 Td /F1 9.8 Tf [(families, but striking identity of the CEA receptors will impact comparative oncology studies.. PLOS Currents Tree of Life. 2011 )] TJ ET BT 26.250 553.827 Td /F1 9.8 Tf [(Nov 11 . Edition 1. doi: 10.1371/currents.RRN1223.)] TJ ET q 15.000 26.071 577.500 525.375 re W n 0.271 0.267 0.267 rg BT 26.250 524.725 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 504.771 Td /F1 9.8 Tf [(Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following )] TJ ET BT 26.250 492.866 Td /F1 9.8 Tf [(this line, carcinoembryonic antigen \(CEA, CEACAM5\) could be a therapeutic target not only for human but also for canine )] TJ ET BT 26.250 480.961 Td /F1 9.8 Tf [(\(Canis lupus familiaris; dog\) patients. CEACAM5 interacts with CEA-receptor \(CEAR\) in the cytoplasm of human cancer cells. )] TJ ET BT 26.250 469.056 Td /F1 9.8 Tf [(Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and )] TJ ET BT 26.250 457.152 Td /F1 9.8 Tf [(canine cancer.)] TJ ET BT 26.250 445.247 Td /F1 9.8 Tf [(Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted )] TJ ET BT 569.744 445.247 Td /F1 9.8 Tf [(to )] TJ ET BT 26.250 433.342 Td /F1 9.8 Tf [(23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 )] TJ ET BT 26.250 421.437 Td /F1 9.8 Tf [(kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma )] TJ ET BT 26.250 409.533 Td /F1 9.8 Tf [(cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be )] TJ ET BT 26.250 397.628 Td /F1 9.8 Tf [(most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to )] TJ ET BT 26.250 385.723 Td /F1 9.8 Tf [(canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in )] TJ ET BT 26.250 373.818 Td /F1 9.8 Tf [(contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on )] TJ ET BT 26.250 361.914 Td /F1 9.8 Tf [(canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly )] TJ ET BT 26.250 350.009 Td /F1 9.8 Tf [(suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a )] TJ ET BT 26.250 338.104 Td /F1 9.8 Tf [(99% amino acid similarity of human and canine CEA receptor \(CEAR\) within the phylogenetic tree. CEAR could be detected in )] TJ ET BT 26.250 326.199 Td /F1 9.8 Tf [(four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by )] TJ ET BT 26.250 314.295 Td /F1 9.8 Tf [(immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act )] TJ ET BT 26.250 302.390 Td /F1 9.8 Tf [(as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the )] TJ ET BT 26.250 290.485 Td /F1 9.8 Tf [(complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a )] TJ ET BT 26.250 278.580 Td /F1 9.8 Tf [(comprehensive interspecies target for novel cancer therapeutics.)] TJ ET BT 26.250 241.978 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 222.024 Td /F1 9.8 Tf [(Comparative oncology trials represent a novel preclinical infrastructure for speeding up the development of anticancer )] TJ ET BT 26.250 210.119 Td /F1 9.8 Tf [(therapeutics )] TJ ET 0.267 0.267 0.267 rg BT 82.069 210.119 Td /F1 9.8 Tf [([1] [2])] TJ ET 0.271 0.267 0.267 rg BT 106.463 210.119 Td /F1 9.8 Tf [(. In contrast to known preclinical animal models, comparative studies take advantage of spontaneously )] TJ ET BT 26.250 198.214 Td /F1 9.8 Tf [(occurring cancers, for instance in dogs \(Canis lupus familiaris\) )] TJ ET 0.267 0.267 0.267 rg BT 297.163 198.214 Td /F1 9.8 Tf [([3])] TJ ET 0.271 0.267 0.267 rg BT 308.005 198.214 Td /F1 9.8 Tf [(. Following specific guidelines )] TJ ET 0.267 0.267 0.267 rg BT 439.133 198.214 Td /F1 9.8 Tf [([4])] TJ ET 0.271 0.267 0.267 rg BT 449.975 198.214 Td /F1 9.8 Tf [(, these studies also assess )] TJ ET BT 26.250 186.309 Td /F1 9.8 Tf [(the response of the veterinary patients to novel drugs in terms of clinical benefit)] TJ ET 0.267 0.267 0.267 rg BT 368.202 186.309 Td /F1 9.8 Tf [( [5] [6] [7])] TJ ET 0.271 0.267 0.267 rg BT 408.860 186.309 Td /F1 9.8 Tf [(. Mammary neoplasms are the most )] TJ ET BT 26.250 174.405 Td /F1 9.8 Tf [(common tumors in female dogs )] TJ ET 0.267 0.267 0.267 rg BT 164.963 174.405 Td /F1 9.8 Tf [([8])] TJ ET 0.271 0.267 0.267 rg BT 175.805 174.405 Td /F1 9.8 Tf [(, with incidence depending on the population studied and varying from 205 per 100,000 )] TJ ET 0.267 0.267 0.267 rg BT 554.105 174.405 Td /F1 9.8 Tf [([9])] TJ ET 0.271 0.267 0.267 rg BT 564.947 174.405 Td /F1 9.8 Tf [( to )] TJ ET BT 26.250 162.500 Td /F1 9.8 Tf [(111 per 10,000 female dogs between 3 and 10 years of age )] TJ ET 0.267 0.267 0.267 rg BT 286.955 162.500 Td /F1 9.8 Tf [([10])] TJ ET 0.271 0.267 0.267 rg BT 303.218 162.500 Td /F1 9.8 Tf [(. 30 to 50% of all canine mammary tumors are malignant and )] TJ ET BT 26.250 150.595 Td /F1 9.8 Tf [(50 to 75% of these recur or metastasize within 1 to 2 years )] TJ ET 0.267 0.267 0.267 rg BT 282.577 150.595 Td /F1 9.8 Tf [([11] [12])] TJ ET 0.271 0.267 0.267 rg BT 317.814 150.595 Td /F1 9.8 Tf [(. For these reasons more effective therapy strategies are )] TJ ET BT 26.250 138.690 Td /F1 9.8 Tf [(urgently needed for companion animals, at the same time their disease mirrors the settings of human oncology patients.)] TJ ET BT 26.250 119.286 Td /F1 9.8 Tf [(Especially immunological targeting of cancer for diagnosis and therapy is much more advanced in humans )] TJ ET 0.267 0.267 0.267 rg BT 487.396 119.286 Td /F1 9.8 Tf [([13] [14])] TJ ET 0.271 0.267 0.267 rg BT 522.632 119.286 Td /F1 9.8 Tf [(. Therefore, )] TJ ET BT 26.250 107.381 Td /F1 9.8 Tf [(we suggest that careful, comparative evaluation of most important tumor targets should be undertaken to increase the basic )] TJ ET BT 26.250 95.476 Td /F1 9.8 Tf [(knowledge for comparative studies.)] TJ ET BT 26.250 76.071 Td /F1 9.8 Tf [(An important example for a potential immunological cancer target is carcinoembryonic antigen \(CEACAM5\), which represents a )] TJ ET BT 26.250 64.167 Td /F1 9.8 Tf [(classical tumor marker and is routinely exploited for diagnosis in man. In its soluble form CEACAM5 binds to the )] TJ ET BT 26.250 52.262 Td /F1 9.8 Tf [(carcinoembryonic antigen receptor \(CEAR\), a member of the heterogeneous nuclear ribonucleoproteins \(hnRNP\) family, )] TJ ET BT 26.250 40.357 Td /F1 9.8 Tf [(offering the likely potential to deliver prometastatic signals to neoplastic cells )] TJ ET 0.267 0.267 0.267 rg BT 357.887 40.357 Td /F1 9.8 Tf [([15] [16])] TJ ET 0.271 0.267 0.267 rg BT 393.123 40.357 Td /F1 9.8 Tf [(. In many types of human cancers )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 12 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 3579>> stream xy8~JT`QAp lW\P @*0 eV+s.O ! 3͌F"  A;A7;A7;A7f}=>>ڶ4ͭ$ q3lڶm&˲[IALf}XX}VeAQ;Syx=_<&1q!c좢ʲTʹbY8᩺۶; k4tv_eYF>"I9Y1!t T&N|4M`QoxZ.51rstKPD9cLy^QN0 Zd.a_,9ޣ\HQp Pqwal|V^eACt_bLb Izs\FeQr:%c1fcl>(exZQUUWQUU4p<-<K wЈX8[Ԛ)OSD?+wn Xj9=g{UUb(Ht݂zYrjZ wuxmreFNA/8Nǣ</eY #7oX9vlC2UUM]1^׵`[ y۶abT+Ư kܼHeЖN}j+7 nQGQtp08ҘÀl61q&W >4 |TUUUUQhaV+u Aه몎)2ɷA LXo7tMDQEjJIJl\}۲\}D@߯z>_T7P۶u]׶mZMhhܔ ,ֲ,ې1e1gydY4̥&#nXc^AOC';TX=cse߃~l#K7˜窪dТEwu>ۭ.D#T$+>6Fy(;dP1'bP+a<2˭'J[Dь 8$ wF~VܕxK8P^+D? 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208 0 R 210 0 R 212 0 R 214 0 R 216 0 R 218 0 R 220 0 R 222 0 R 224 0 R 226 0 R 228 0 R 230 0 R 232 0 R 234 0 R 236 0 R 238 0 R 240 0 R 242 0 R 244 0 R 246 0 R 248 0 R 250 0 R 252 0 R 254 0 R 256 0 R ] /Contents 171 0 R >> endobj 171 0 obj << /Length 30067 >> stream 0.271 0.267 0.267 rg q 15.000 41.396 577.500 735.604 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(CEACAM5 is specifically expressed as a cell surface glycoprotein exhibiting several functions like regulating intercellular )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(adhesion, differentiation and anoikis, cell polarization and tissue architecture )] TJ ET 0.267 0.267 0.267 rg BT 357.906 755.571 Td /F1 9.8 Tf [([17])] TJ ET 0.271 0.267 0.267 rg BT 374.169 755.571 Td /F1 9.8 Tf [(. In humans the level of overexpression )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(determines tumor cell migration, invasion and formation of distant metastases and may therefore be a measure of malignancy )] TJ ET 0.267 0.267 0.267 rg BT 26.250 731.762 Td /F1 9.8 Tf [([18])] TJ ET 0.271 0.267 0.267 rg BT 42.513 731.762 Td /F1 9.8 Tf [(. High levels of CEACAM5 expression are found in human patients in 80 90% of all colorectal cancers, in up to 50% of all )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(breast and lung cancers and in about 15% of gastrointestinal and pancreatic carcinomas as well as in many other epithelial )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(cancers )] TJ ET 0.267 0.267 0.267 rg BT 63.095 707.952 Td /F1 9.8 Tf [([19])] TJ ET 0.271 0.267 0.267 rg BT 79.358 707.952 Td /F1 9.8 Tf [(. It has, therefore, been recognized as a clinically relevant target for passive and active immunotherapy. CEA has )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(been the focus of multiple preclinical and clinical human studies using antibodies or fragments for targeted therapy )] TJ ET 0.267 0.267 0.267 rg BT 521.560 696.048 Td /F1 9.8 Tf [([20] [21] [22] )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [([23] [24] [25])] TJ ET 0.271 0.267 0.267 rg BT 80.460 684.143 Td /F1 9.8 Tf [( or vaccines for active stimulation of B- and T-cell immunity )] TJ ET 0.267 0.267 0.267 rg BT 337.285 684.143 Td /F1 9.8 Tf [([26] [27] [28] [29] [30])] TJ ET 0.271 0.267 0.267 rg BT 429.442 684.143 Td /F1 9.8 Tf [(, including one of our own studies )] TJ ET 0.267 0.267 0.267 rg BT 26.250 672.238 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 42.513 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(Preclinical vaccination studies based on human CEACAM5 have been pursued in mice and also in dogs )] TJ ET 0.267 0.267 0.267 rg BT 477.119 652.833 Td /F1 9.8 Tf [([29] [32] [33])] TJ ET 0.271 0.267 0.267 rg BT 531.329 652.833 Td /F1 9.8 Tf [(. These )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(efforts were based on the fact that carcinoembryonic antigen had been previously proposed as tumor marker in several canine )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(immunohistochemical studies using anti-human CEACAM5 antibodies )] TJ ET 0.267 0.267 0.267 rg BT 330.772 629.024 Td /F1 9.8 Tf [([34] [35] [36] )] TJ ET 0.271 0.267 0.267 rg BT 387.692 629.024 Td /F1 9.8 Tf [(such as Col-1 )] TJ ET 0.267 0.267 0.267 rg BT 450.005 629.024 Td /F1 9.8 Tf [([37])] TJ ET 0.271 0.267 0.267 rg BT 466.267 629.024 Td /F1 9.8 Tf [(. Monoclonal antibody )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(Col-1 is originally known for its excellent diagnostic properties for the detection of premalignant and malignant lesions and )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(metastases in humans )] TJ ET 0.267 0.267 0.267 rg BT 126.499 605.214 Td /F1 9.8 Tf [([38] [39] [40] [41])] TJ ET 0.271 0.267 0.267 rg BT 199.683 605.214 Td /F1 9.8 Tf [(. This antibody also served as the model for anti-idiotypic antibody production to elicit )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(specific immune responses )] TJ ET 0.267 0.267 0.267 rg BT 146.536 593.310 Td /F1 9.8 Tf [([42] )] TJ ET 0.271 0.267 0.267 rg BT 165.509 593.310 Td /F1 9.8 Tf [(and it has recently been humanized for safer clinical application as radio-immunoconjugate in )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(human patients )] TJ ET 0.267 0.267 0.267 rg BT 95.621 581.405 Td /F1 9.8 Tf [([43])] TJ ET 0.271 0.267 0.267 rg BT 111.884 581.405 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(Thus CEACAM5 or Col-1 based diagnostic and therapeutic strategies in canine patients are further developed without precise )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(knowledge of the target molecule. Human CEACAM5 belongs to the CEACAM1-related molecules within the CEA gene family. )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Although no unambiguous orthologous gene to human CEACAM5 was found in the dog genome )] TJ ET 0.267 0.267 0.267 rg BT 444.106 538.191 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 460.369 538.191 Td /F1 9.8 Tf [( our phylogenetic analysis )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(provides evidence that molecules related to the canine CEACAM1-family show the highest similarity to human CEACAM5 \(Fig. )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(1\). However, the lack of the sequence of canine CEACAM5 in the database is not )] TJ ET BT 380.136 514.381 Td /F5 9.8 Tf [(a priori)] TJ ET BT 409.932 514.381 Td /F1 9.8 Tf [( proof that the gene is absent.)] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(Therefore, it is to date still unclear whether CEACAMs are relevant targets for comparative studies. Based on the current )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(knowledge of the phylogenetic associations, we aimed here to verify the antigenic relationship of human and canine CEACAMs )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(as well as of CEAR.)] TJ ET BT 26.250 434.564 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 414.610 Td /F6 9.8 Tf [(Patients material and Immunohistochemistry)] TJ ET BT 26.250 395.205 Td /F1 9.8 Tf [(The study was conducted with tissue samples from 30 canine mammary tumor patients. Tissue specimens of canine mammary )] TJ ET BT 26.250 383.301 Td /F1 9.8 Tf [(glands were obtained either during diagnostic biopsy or during mastectomy at the University of Veterinary Medicine, Vienna. )] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(Formalin-fixed, paraffin embedded sections \(2 m\) of mammary carcinoma tissue were dewaxed and rehydrated. Heat induced )] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(epitope retrieval was performed in the Lab Vision PT Module \(Thermo Fisher Scientific, Waltham, MA, USA\) by incubating )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(slides in ethylenediaminetetraacetic acid \(EDTA\) buffer at pH 8.0. Further steps were performed in the Lab Vision Autostainer )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). Endogenous peroxidase activity was inhibited using Hydrogen Peroxidase Block und Ultra V Block )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). For immunostaining Col-1, a monoclonal mouse anti-human CEACAM5 IgG2a antibody \(Invitrogen, )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(San Diego, CA, USA\) was diluted 1:50 in Ultra Plus Ab Diluent \(Thermo Fisher Scientific\) and incubated at RT for 30 min. )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(Afterwards slides were incubated with Primary Antibody Enhancer \(Thermo Fisher Scientific\) for 15 min followed by HRP )] TJ ET BT 26.250 288.063 Td /F1 9.8 Tf [(\(horseradish peroxidase\) Polymer \(Thermo Fisher Scientific\) for 20 min at RT. Specific immunoperoxidase staining activity was )] TJ ET BT 26.250 276.158 Td /F1 9.8 Tf [(detected using DAB substrate \(Thermo Fisher Scientific\). Slides were counterstained with hematoxylin and mounted in Neo )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(Mount \(Merck, Darmstadt, Germany\). To determine CEAR expression, tissue tumor samples of 10 of the above mentioned )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(mammary cancer patients were selected based on positive Col-1 staining and treated as mentioned above. Non-specific binding )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(was blocked with 5% FCS. Slides were incubated with mouse anti-hnRNP antibody \(recognizing isoforms M1 M4\) from )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(Invitrogen, 1:100 in 5% FCS, for 60 min at RT and afterwards with HRP Polymer \(Dako, Carpinteria, CA, USA\) for 10 min at RT. )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(Further steps were performed as described above and stainings evaluated by microscopy \(Tissuegnostics, Vienna, Austria\) with )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(a 20X/0.5 or a 40X/1.3 oil objective lens.)] TJ ET BT 26.250 185.325 Td /F6 9.8 Tf [(Cell lines)] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(Canine mammary carcinoma cell lines CF33 and CF41 and human colon adenocarcinoma cell line HT29 are available from the )] TJ ET BT 26.250 154.015 Td /F1 9.8 Tf [(American Type Culture Collection \(ATCC, Rockville, MD, USA; Cat. No.: CRL-6227 for CF33 and CRL-6232 for CF41, Cat. No.: )] TJ ET BT 26.250 142.110 Td /F1 9.8 Tf [(HTB-38 for HT29\). All cell lines were cultivated according to the distributors protocol in DMEM medium supplemented with 10% )] TJ ET BT 26.250 130.206 Td /F1 9.8 Tf [(heat inactivated fetal calve serum \(FCS\), 2 mM l-glutamine, penicillin \(5,000 U/mL\) and streptomycin \(100 g/mL\). The canine )] TJ ET BT 26.250 118.301 Td /F1 9.8 Tf [(mammary carcinoma cell lines Sh1b and P114 were a kind gift of Dr. Gerard Rutteman \(Department of Clinic Science and )] TJ ET BT 26.250 106.396 Td /F1 9.8 Tf [(Companion Animals, University of Utrecht, The Netherlands\) and were maintained in DMEM/F12 supplemented with 10% FCS, )] TJ ET BT 26.250 94.491 Td /F1 9.8 Tf [(2 mM l-glutamine and 10 g/mL gentamicin sulfate. Canine oral melanoma cell line TLM-1 was kindly provided by Dr. Jaime F. )] TJ ET BT 26.250 82.587 Td /F1 9.8 Tf [(Modiano \(University of Minnesota, Veterinary Clinical Sciences Department, St. Paul, MN, USA\). TLM-1 cells were cultured in )] TJ ET BT 26.250 70.682 Td /F1 9.8 Tf [(DMEM containing 10% FCS. Cell lines were kept at 37 C in a humidified atmosphere of 5% CO2.)] TJ ET BT 26.250 51.277 Td /F6 9.8 Tf [(Western Blotting)] TJ ET Q q 15.000 41.396 577.500 735.604 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(CEACAM5 is specifically expressed as a cell surface glycoprotein exhibiting several functions like regulating intercellular )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(adhesion, differentiation and anoikis, cell polarization and tissue architecture )] TJ ET 0.267 0.267 0.267 rg BT 357.906 755.571 Td /F1 9.8 Tf [([17])] TJ ET 0.271 0.267 0.267 rg BT 374.169 755.571 Td /F1 9.8 Tf [(. In humans the level of overexpression )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(determines tumor cell migration, invasion and formation of distant metastases and may therefore be a measure of malignancy )] TJ ET 0.267 0.267 0.267 rg BT 26.250 731.762 Td /F1 9.8 Tf [([18])] TJ ET 0.271 0.267 0.267 rg BT 42.513 731.762 Td /F1 9.8 Tf [(. High levels of CEACAM5 expression are found in human patients in 80 90% of all colorectal cancers, in up to 50% of all )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(breast and lung cancers and in about 15% of gastrointestinal and pancreatic carcinomas as well as in many other epithelial )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(cancers )] TJ ET 0.267 0.267 0.267 rg BT 63.095 707.952 Td /F1 9.8 Tf [([19])] TJ ET 0.271 0.267 0.267 rg BT 79.358 707.952 Td /F1 9.8 Tf [(. It has, therefore, been recognized as a clinically relevant target for passive and active immunotherapy. CEA has )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(been the focus of multiple preclinical and clinical human studies using antibodies or fragments for targeted therapy )] TJ ET 0.267 0.267 0.267 rg BT 521.560 696.048 Td /F1 9.8 Tf [([20] [21] [22] )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [([23] [24] [25])] TJ ET 0.271 0.267 0.267 rg BT 80.460 684.143 Td /F1 9.8 Tf [( or vaccines for active stimulation of B- and T-cell immunity )] TJ ET 0.267 0.267 0.267 rg BT 337.285 684.143 Td /F1 9.8 Tf [([26] [27] [28] [29] [30])] TJ ET 0.271 0.267 0.267 rg BT 429.442 684.143 Td /F1 9.8 Tf [(, including one of our own studies )] TJ ET 0.267 0.267 0.267 rg BT 26.250 672.238 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 42.513 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(Preclinical vaccination studies based on human CEACAM5 have been pursued in mice and also in dogs )] TJ ET 0.267 0.267 0.267 rg BT 477.119 652.833 Td /F1 9.8 Tf [([29] [32] [33])] TJ ET 0.271 0.267 0.267 rg BT 531.329 652.833 Td /F1 9.8 Tf [(. These )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(efforts were based on the fact that carcinoembryonic antigen had been previously proposed as tumor marker in several canine )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(immunohistochemical studies using anti-human CEACAM5 antibodies )] TJ ET 0.267 0.267 0.267 rg BT 330.772 629.024 Td /F1 9.8 Tf [([34] [35] [36] )] TJ ET 0.271 0.267 0.267 rg BT 387.692 629.024 Td /F1 9.8 Tf [(such as Col-1 )] TJ ET 0.267 0.267 0.267 rg BT 450.005 629.024 Td /F1 9.8 Tf [([37])] TJ ET 0.271 0.267 0.267 rg BT 466.267 629.024 Td /F1 9.8 Tf [(. Monoclonal antibody )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(Col-1 is originally known for its excellent diagnostic properties for the detection of premalignant and malignant lesions and )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(metastases in humans )] TJ ET 0.267 0.267 0.267 rg BT 126.499 605.214 Td /F1 9.8 Tf [([38] [39] [40] [41])] TJ ET 0.271 0.267 0.267 rg BT 199.683 605.214 Td /F1 9.8 Tf [(. This antibody also served as the model for anti-idiotypic antibody production to elicit )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(specific immune responses )] TJ ET 0.267 0.267 0.267 rg BT 146.536 593.310 Td /F1 9.8 Tf [([42] )] TJ ET 0.271 0.267 0.267 rg BT 165.509 593.310 Td /F1 9.8 Tf [(and it has recently been humanized for safer clinical application as radio-immunoconjugate in )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(human patients )] TJ ET 0.267 0.267 0.267 rg BT 95.621 581.405 Td /F1 9.8 Tf [([43])] TJ ET 0.271 0.267 0.267 rg BT 111.884 581.405 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(Thus CEACAM5 or Col-1 based diagnostic and therapeutic strategies in canine patients are further developed without precise )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(knowledge of the target molecule. Human CEACAM5 belongs to the CEACAM1-related molecules within the CEA gene family. )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Although no unambiguous orthologous gene to human CEACAM5 was found in the dog genome )] TJ ET 0.267 0.267 0.267 rg BT 444.106 538.191 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 460.369 538.191 Td /F1 9.8 Tf [( our phylogenetic analysis )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(provides evidence that molecules related to the canine CEACAM1-family show the highest similarity to human CEACAM5 \(Fig. )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(1\). However, the lack of the sequence of canine CEACAM5 in the database is not )] TJ ET BT 380.136 514.381 Td /F5 9.8 Tf [(a priori)] TJ ET BT 409.932 514.381 Td /F1 9.8 Tf [( proof that the gene is absent.)] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(Therefore, it is to date still unclear whether CEACAMs are relevant targets for comparative studies. Based on the current )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(knowledge of the phylogenetic associations, we aimed here to verify the antigenic relationship of human and canine CEACAMs )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(as well as of CEAR.)] TJ ET BT 26.250 434.564 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 414.610 Td /F6 9.8 Tf [(Patients material and Immunohistochemistry)] TJ ET BT 26.250 395.205 Td /F1 9.8 Tf [(The study was conducted with tissue samples from 30 canine mammary tumor patients. Tissue specimens of canine mammary )] TJ ET BT 26.250 383.301 Td /F1 9.8 Tf [(glands were obtained either during diagnostic biopsy or during mastectomy at the University of Veterinary Medicine, Vienna. )] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(Formalin-fixed, paraffin embedded sections \(2 m\) of mammary carcinoma tissue were dewaxed and rehydrated. Heat induced )] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(epitope retrieval was performed in the Lab Vision PT Module \(Thermo Fisher Scientific, Waltham, MA, USA\) by incubating )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(slides in ethylenediaminetetraacetic acid \(EDTA\) buffer at pH 8.0. Further steps were performed in the Lab Vision Autostainer )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). Endogenous peroxidase activity was inhibited using Hydrogen Peroxidase Block und Ultra V Block )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). For immunostaining Col-1, a monoclonal mouse anti-human CEACAM5 IgG2a antibody \(Invitrogen, )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(San Diego, CA, USA\) was diluted 1:50 in Ultra Plus Ab Diluent \(Thermo Fisher Scientific\) and incubated at RT for 30 min. )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(Afterwards slides were incubated with Primary Antibody Enhancer \(Thermo Fisher Scientific\) for 15 min followed by HRP )] TJ ET BT 26.250 288.063 Td /F1 9.8 Tf [(\(horseradish peroxidase\) Polymer \(Thermo Fisher Scientific\) for 20 min at RT. Specific immunoperoxidase staining activity was )] TJ ET BT 26.250 276.158 Td /F1 9.8 Tf [(detected using DAB substrate \(Thermo Fisher Scientific\). Slides were counterstained with hematoxylin and mounted in Neo )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(Mount \(Merck, Darmstadt, Germany\). To determine CEAR expression, tissue tumor samples of 10 of the above mentioned )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(mammary cancer patients were selected based on positive Col-1 staining and treated as mentioned above. Non-specific binding )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(was blocked with 5% FCS. Slides were incubated with mouse anti-hnRNP antibody \(recognizing isoforms M1 M4\) from )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(Invitrogen, 1:100 in 5% FCS, for 60 min at RT and afterwards with HRP Polymer \(Dako, Carpinteria, CA, USA\) for 10 min at RT. )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(Further steps were performed as described above and stainings evaluated by microscopy \(Tissuegnostics, Vienna, Austria\) with )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(a 20X/0.5 or a 40X/1.3 oil objective lens.)] TJ ET BT 26.250 185.325 Td /F6 9.8 Tf [(Cell lines)] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(Canine mammary carcinoma cell lines CF33 and CF41 and human colon adenocarcinoma cell line HT29 are available from the )] TJ ET BT 26.250 154.015 Td /F1 9.8 Tf [(American Type Culture Collection \(ATCC, Rockville, MD, USA; Cat. No.: CRL-6227 for CF33 and CRL-6232 for CF41, Cat. No.: )] TJ ET BT 26.250 142.110 Td /F1 9.8 Tf [(HTB-38 for HT29\). All cell lines were cultivated according to the distributors protocol in DMEM medium supplemented with 10% )] TJ ET BT 26.250 130.206 Td /F1 9.8 Tf [(heat inactivated fetal calve serum \(FCS\), 2 mM l-glutamine, penicillin \(5,000 U/mL\) and streptomycin \(100 g/mL\). The canine )] TJ ET BT 26.250 118.301 Td /F1 9.8 Tf [(mammary carcinoma cell lines Sh1b and P114 were a kind gift of Dr. Gerard Rutteman \(Department of Clinic Science and )] TJ ET BT 26.250 106.396 Td /F1 9.8 Tf [(Companion Animals, University of Utrecht, The Netherlands\) and were maintained in DMEM/F12 supplemented with 10% FCS, )] TJ ET BT 26.250 94.491 Td /F1 9.8 Tf [(2 mM l-glutamine and 10 g/mL gentamicin sulfate. Canine oral melanoma cell line TLM-1 was kindly provided by Dr. Jaime F. )] TJ ET BT 26.250 82.587 Td /F1 9.8 Tf [(Modiano \(University of Minnesota, Veterinary Clinical Sciences Department, St. Paul, MN, USA\). TLM-1 cells were cultured in )] TJ ET BT 26.250 70.682 Td /F1 9.8 Tf [(DMEM containing 10% FCS. Cell lines were kept at 37 C in a humidified atmosphere of 5% CO2.)] TJ ET BT 26.250 51.277 Td /F6 9.8 Tf [(Western Blotting)] TJ ET Q q 15.000 41.396 577.500 735.604 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(CEACAM5 is specifically expressed as a cell surface glycoprotein exhibiting several functions like regulating intercellular )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(adhesion, differentiation and anoikis, cell polarization and tissue architecture )] TJ ET 0.267 0.267 0.267 rg BT 357.906 755.571 Td /F1 9.8 Tf [([17])] TJ ET 0.271 0.267 0.267 rg BT 374.169 755.571 Td /F1 9.8 Tf [(. In humans the level of overexpression )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(determines tumor cell migration, invasion and formation of distant metastases and may therefore be a measure of malignancy )] TJ ET 0.267 0.267 0.267 rg BT 26.250 731.762 Td /F1 9.8 Tf [([18])] TJ ET 0.271 0.267 0.267 rg BT 42.513 731.762 Td /F1 9.8 Tf [(. High levels of CEACAM5 expression are found in human patients in 80 90% of all colorectal cancers, in up to 50% of all )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(breast and lung cancers and in about 15% of gastrointestinal and pancreatic carcinomas as well as in many other epithelial )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(cancers )] TJ ET 0.267 0.267 0.267 rg BT 63.095 707.952 Td /F1 9.8 Tf [([19])] TJ ET 0.271 0.267 0.267 rg BT 79.358 707.952 Td /F1 9.8 Tf [(. It has, therefore, been recognized as a clinically relevant target for passive and active immunotherapy. CEA has )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(been the focus of multiple preclinical and clinical human studies using antibodies or fragments for targeted therapy )] TJ ET 0.267 0.267 0.267 rg BT 521.560 696.048 Td /F1 9.8 Tf [([20] [21] [22] )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [([23] [24] [25])] TJ ET 0.271 0.267 0.267 rg BT 80.460 684.143 Td /F1 9.8 Tf [( or vaccines for active stimulation of B- and T-cell immunity )] TJ ET 0.267 0.267 0.267 rg BT 337.285 684.143 Td /F1 9.8 Tf [([26] [27] [28] [29] [30])] TJ ET 0.271 0.267 0.267 rg BT 429.442 684.143 Td /F1 9.8 Tf [(, including one of our own studies )] TJ ET 0.267 0.267 0.267 rg BT 26.250 672.238 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 42.513 672.238 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(Preclinical vaccination studies based on human CEACAM5 have been pursued in mice and also in dogs )] TJ ET 0.267 0.267 0.267 rg BT 477.119 652.833 Td /F1 9.8 Tf [([29] [32] [33])] TJ ET 0.271 0.267 0.267 rg BT 531.329 652.833 Td /F1 9.8 Tf [(. These )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(efforts were based on the fact that carcinoembryonic antigen had been previously proposed as tumor marker in several canine )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(immunohistochemical studies using anti-human CEACAM5 antibodies )] TJ ET 0.267 0.267 0.267 rg BT 330.772 629.024 Td /F1 9.8 Tf [([34] [35] [36] )] TJ ET 0.271 0.267 0.267 rg BT 387.692 629.024 Td /F1 9.8 Tf [(such as Col-1 )] TJ ET 0.267 0.267 0.267 rg BT 450.005 629.024 Td /F1 9.8 Tf [([37])] TJ ET 0.271 0.267 0.267 rg BT 466.267 629.024 Td /F1 9.8 Tf [(. Monoclonal antibody )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(Col-1 is originally known for its excellent diagnostic properties for the detection of premalignant and malignant lesions and )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(metastases in humans )] TJ ET 0.267 0.267 0.267 rg BT 126.499 605.214 Td /F1 9.8 Tf [([38] [39] [40] [41])] TJ ET 0.271 0.267 0.267 rg BT 199.683 605.214 Td /F1 9.8 Tf [(. This antibody also served as the model for anti-idiotypic antibody production to elicit )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(specific immune responses )] TJ ET 0.267 0.267 0.267 rg BT 146.536 593.310 Td /F1 9.8 Tf [([42] )] TJ ET 0.271 0.267 0.267 rg BT 165.509 593.310 Td /F1 9.8 Tf [(and it has recently been humanized for safer clinical application as radio-immunoconjugate in )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(human patients )] TJ ET 0.267 0.267 0.267 rg BT 95.621 581.405 Td /F1 9.8 Tf [([43])] TJ ET 0.271 0.267 0.267 rg BT 111.884 581.405 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 562.000 Td /F1 9.8 Tf [(Thus CEACAM5 or Col-1 based diagnostic and therapeutic strategies in canine patients are further developed without precise )] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(knowledge of the target molecule. Human CEACAM5 belongs to the CEACAM1-related molecules within the CEA gene family. )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Although no unambiguous orthologous gene to human CEACAM5 was found in the dog genome )] TJ ET 0.267 0.267 0.267 rg BT 444.106 538.191 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 460.369 538.191 Td /F1 9.8 Tf [( our phylogenetic analysis )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(provides evidence that molecules related to the canine CEACAM1-family show the highest similarity to human CEACAM5 \(Fig. )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(1\). However, the lack of the sequence of canine CEACAM5 in the database is not )] TJ ET BT 380.136 514.381 Td /F5 9.8 Tf [(a priori)] TJ ET BT 409.932 514.381 Td /F1 9.8 Tf [( proof that the gene is absent.)] TJ ET BT 26.250 494.976 Td /F1 9.8 Tf [(Therefore, it is to date still unclear whether CEACAMs are relevant targets for comparative studies. Based on the current )] TJ ET BT 26.250 483.072 Td /F1 9.8 Tf [(knowledge of the phylogenetic associations, we aimed here to verify the antigenic relationship of human and canine CEACAMs )] TJ ET BT 26.250 471.167 Td /F1 9.8 Tf [(as well as of CEAR.)] TJ ET BT 26.250 434.564 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 414.610 Td /F6 9.8 Tf [(Patients material and Immunohistochemistry)] TJ ET BT 26.250 395.205 Td /F1 9.8 Tf [(The study was conducted with tissue samples from 30 canine mammary tumor patients. Tissue specimens of canine mammary )] TJ ET BT 26.250 383.301 Td /F1 9.8 Tf [(glands were obtained either during diagnostic biopsy or during mastectomy at the University of Veterinary Medicine, Vienna. )] TJ ET BT 26.250 371.396 Td /F1 9.8 Tf [(Formalin-fixed, paraffin embedded sections \(2 m\) of mammary carcinoma tissue were dewaxed and rehydrated. Heat induced )] TJ ET BT 26.250 359.491 Td /F1 9.8 Tf [(epitope retrieval was performed in the Lab Vision PT Module \(Thermo Fisher Scientific, Waltham, MA, USA\) by incubating )] TJ ET BT 26.250 347.586 Td /F1 9.8 Tf [(slides in ethylenediaminetetraacetic acid \(EDTA\) buffer at pH 8.0. Further steps were performed in the Lab Vision Autostainer )] TJ ET BT 26.250 335.682 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). Endogenous peroxidase activity was inhibited using Hydrogen Peroxidase Block und Ultra V Block )] TJ ET BT 26.250 323.777 Td /F1 9.8 Tf [(\(Thermo Fisher Scientific\). For immunostaining Col-1, a monoclonal mouse anti-human CEACAM5 IgG2a antibody \(Invitrogen, )] TJ ET BT 26.250 311.872 Td /F1 9.8 Tf [(San Diego, CA, USA\) was diluted 1:50 in Ultra Plus Ab Diluent \(Thermo Fisher Scientific\) and incubated at RT for 30 min. )] TJ ET BT 26.250 299.967 Td /F1 9.8 Tf [(Afterwards slides were incubated with Primary Antibody Enhancer \(Thermo Fisher Scientific\) for 15 min followed by HRP )] TJ ET BT 26.250 288.063 Td /F1 9.8 Tf [(\(horseradish peroxidase\) Polymer \(Thermo Fisher Scientific\) for 20 min at RT. Specific immunoperoxidase staining activity was )] TJ ET BT 26.250 276.158 Td /F1 9.8 Tf [(detected using DAB substrate \(Thermo Fisher Scientific\). Slides were counterstained with hematoxylin and mounted in Neo )] TJ ET BT 26.250 264.253 Td /F1 9.8 Tf [(Mount \(Merck, Darmstadt, Germany\). To determine CEAR expression, tissue tumor samples of 10 of the above mentioned )] TJ ET BT 26.250 252.348 Td /F1 9.8 Tf [(mammary cancer patients were selected based on positive Col-1 staining and treated as mentioned above. Non-specific binding )] TJ ET BT 26.250 240.444 Td /F1 9.8 Tf [(was blocked with 5% FCS. Slides were incubated with mouse anti-hnRNP antibody \(recognizing isoforms M1 M4\) from )] TJ ET BT 26.250 228.539 Td /F1 9.8 Tf [(Invitrogen, 1:100 in 5% FCS, for 60 min at RT and afterwards with HRP Polymer \(Dako, Carpinteria, CA, USA\) for 10 min at RT. )] TJ ET BT 26.250 216.634 Td /F1 9.8 Tf [(Further steps were performed as described above and stainings evaluated by microscopy \(Tissuegnostics, Vienna, Austria\) with )] TJ ET BT 26.250 204.729 Td /F1 9.8 Tf [(a 20X/0.5 or a 40X/1.3 oil objective lens.)] TJ ET BT 26.250 185.325 Td /F6 9.8 Tf [(Cell lines)] TJ ET BT 26.250 165.920 Td /F1 9.8 Tf [(Canine mammary carcinoma cell lines CF33 and CF41 and human colon adenocarcinoma cell line HT29 are available from the )] TJ ET BT 26.250 154.015 Td /F1 9.8 Tf [(American Type Culture Collection \(ATCC, Rockville, MD, USA; Cat. No.: CRL-6227 for CF33 and CRL-6232 for CF41, Cat. No.: )] TJ ET BT 26.250 142.110 Td /F1 9.8 Tf [(HTB-38 for HT29\). All cell lines were cultivated according to the distributors protocol in DMEM medium supplemented with 10% )] TJ ET BT 26.250 130.206 Td /F1 9.8 Tf [(heat inactivated fetal calve serum \(FCS\), 2 mM l-glutamine, penicillin \(5,000 U/mL\) and streptomycin \(100 g/mL\). The canine )] TJ ET BT 26.250 118.301 Td /F1 9.8 Tf [(mammary carcinoma cell lines Sh1b and P114 were a kind gift of Dr. Gerard Rutteman \(Department of Clinic Science and )] TJ ET BT 26.250 106.396 Td /F1 9.8 Tf [(Companion Animals, University of Utrecht, The Netherlands\) and were maintained in DMEM/F12 supplemented with 10% FCS, )] TJ ET BT 26.250 94.491 Td /F1 9.8 Tf [(2 mM l-glutamine and 10 g/mL gentamicin sulfate. Canine oral melanoma cell line TLM-1 was kindly provided by Dr. Jaime F. )] TJ ET BT 26.250 82.587 Td /F1 9.8 Tf [(Modiano \(University of Minnesota, Veterinary Clinical Sciences Department, St. Paul, MN, USA\). TLM-1 cells were cultured in )] TJ ET BT 26.250 70.682 Td /F1 9.8 Tf [(DMEM containing 10% FCS. 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analysis. Briefly, frozen tissue )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(samples were homogenized in 1 ml lysis buffer \(10 mM Tris-HCL pH 7.4, 1% SDS\). Cells were cultured as described above, )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(washed with ice cold PBS and lysed in 500 l ice cold lysis buffer \(10 mM Tris-HCL pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(ethylene glycol tetraacetic acid \(EGTA\), 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate\). Lysis buffers contained )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(protease inhibitor cocktail \(P8340; Sigma-Aldrich, St. Louis, MO, USA\). The lysates were boiled for 5 min and centrifuged at )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(1000 g. Equal amounts of protein were separated by 10% SDS-PAGE and blotted to polyvinylidene fluoride \(PVDF\) )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(membranes. To assess equal loading of lysates, we performed reversible Ponceau staining of the membrane using Ponceau S )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(solution \(Sigma-Aldrich\). 5% dried milk powder in TBS containing 0.1% Tween was used for blocking nonspecific binding sites. )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(Membranes were incubated with Col-1 antibody diluted 1:250 overnight at 4 C. Anti-mouse IgG2a antibody \(SouthernBiotech, )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(Birmingham, AL, USA\) was used as isotype control. HRP conjugated secondary anti-mouse antibody \(Abcam, Cambridge, )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(UK\) was applied for 1 h at room temperature. For detection of CEA receptor expression in canine and human cell lines, Western )] TJ ET BT 26.250 624.619 Td /F1 9.8 Tf [(blot analysis was performed as described above. Membranes were incubated overnight using either rabbit anti-human CEA )] TJ ET BT 26.250 612.714 Td /F1 9.8 Tf [(receptor \(hnRNP M4 long isoform 1\) antibody \(USB, Swampscott, MA, USA\) or mouse anti-hnRNP \(M1-M4\), each diluted )] TJ ET BT 26.250 600.810 Td /F1 9.8 Tf [(1:1000. Next, appropriate HRP conjugated secondary anti-rabbit \(Cell Signaling, Danvers, MA, USA\) or anti-mouse antibody )] TJ ET BT 26.250 588.905 Td /F1 9.8 Tf [(was applied 1:2000 over a period of 1 h and bound antibodies were detected with the SuperSignal CL-HRP Substrate system )] TJ ET BT 26.250 577.000 Td /F1 9.8 Tf [(\(Pierce Biotechnology, Rockford, IL, USA\).)] TJ ET BT 26.250 557.595 Td /F6 9.8 Tf [(Transient transfection of TLM-1 cells with plasmids encoding CEACAM variants)] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Canine TLM-1 cells were cultured in DMEM medium containing 10% calf serum at 37C in 5% CO2. For transfection, cells were )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(seeded overnight in 6-well plates to reach 50% confluency the next day. Cells were transfected with 4 g of plasmid encoding )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(canine CEACAM1, canCEACAM23, canCEACAM24, canCEACAM25, canCEACAM28 and canCEACAM30 using the CaPO4 )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(method according the manufacturers protocol \(Stratagene, Agilent Technologies, Santa Clara, CA, USA\). To estimate efficiency )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(of transfection, cells were routinely transfected with an empty vector pEGFP-c2 \(Clontech Laboratories, Mountain View, CA, )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(USA\) encoding green fluorescent protein and analyzed with an inverted microscope \(Axioobserver Z1, Zeiss, Oberkochen, )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(Germany\) equipped with a high resolution microscopy camera \(AxioCam MRc, Zeiss\) and a filter set for fluorescein )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(isothiocyanate /enhanced green fluorescent protein \(FITC/EGFP\) fluorescence. Wells with transfection efficiencies of ? 50% )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(were collected for Western Blot analysis.)] TJ ET BT 26.250 423.548 Td /F6 9.8 Tf [(Flow Cytometry)] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(Cells were trypsinized, counted and resuspended in fluorescence-activated cell sorting \(FACS\) buffer \(1 x PBS supplemented )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(with 2 % heat-inactivated goat serum\). For intracellular staining 2 x 105 cells were fixed with formaldehyde for 20 min at RT in )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(the dark, washed once with FACS buffer and afterwards permeabilized with prechilled methanol for 30 min on ice. Next, treated )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(cells were washed once with FACS buffer and incubated with 5 ?g Col-1 antibody or isotype control antibody in FACS buffer for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(1 h at 4 C. For surface expression analysis, fixing and permeabilization steps were omitted. Stained cells were again washed )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(with FACS buffer, followed by incubation of FITC conjugated anti-mouse IgG \(Jackson ImmunoResearch Lab, West Grove, )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(PA, USA\) for 30 min at 4 C. After washing the pellet was resuspended in 300 ?l FACS buffer and 10,000 events were counted )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(using a FACSCalibur flow cytometer \(Becton Dickinson, San Jose, CA, USA\). Data analysis was performed via CellQuest )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(software.)] TJ ET BT 26.250 289.500 Td /F6 9.8 Tf [(Cell Viability Assay)] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(Tumor cell proliferation was analyzed by a modified tetrazolium dye assay \(EZ4U Cell Proliferation Assay; Biomedica Group, )] TJ ET BT 26.250 258.191 Td /F1 9.8 Tf [(Vienna, Austria\) based on the ability of living cells to reduce tetrazolium salts into formazan derivates. Cells were seeded in 96-)] TJ ET BT 26.250 246.286 Td /F1 9.8 Tf [(well plates at 2 x 104 cells per well and allowed to adhere overnight under standard culture conditions. 1 g Col-1 or isotype )] TJ ET BT 26.250 234.381 Td /F1 9.8 Tf [(control antibody per 200 l medium was applied over a period of 24 h and 48 h. Control wells were given media alone or treated )] TJ ET BT 26.250 222.477 Td /F1 9.8 Tf [(with 0.9% Triton X-100 for 10 min prior to addition of the modified tetrazolium dye. The quantity of reduced formazan is directly )] TJ ET BT 26.250 210.572 Td /F1 9.8 Tf [(proportional to living cells. Therefore absorbance was measured after 1 h incubation at 450 nm with 620 nm as a reference with )] TJ ET BT 26.250 198.667 Td /F1 9.8 Tf [(a 96-well plate reader. All experiments were done in triplicates.)] TJ ET BT 26.250 179.262 Td /F6 9.8 Tf [(Signaling Pathway Assay)] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(Phosphorylation assays were performed in CF33 and HT29 cells after 24 h serum starvation in DMEM medium containing 0.1% )] TJ ET BT 26.250 147.953 Td /F1 9.8 Tf [(FCS. After culturing for 24 h, cells were then treated with either fresh medium containing 0.1 % serum alone \(control\) or with 2 )] TJ ET BT 26.250 136.048 Td /F1 9.8 Tf [(g/mL Col-1 antibody in the same medium for the time periods indicated. Next, cell monolayers were washed with ice-cold PBS, )] TJ ET BT 26.250 124.143 Td /F1 9.8 Tf [(lysed and subjected to Western blot analysis as described above. The phosphorylation patterns were determined by probing the )] TJ ET BT 26.250 112.239 Td /F1 9.8 Tf [(blots with anti-AKT, anti-phospho AKT \(targeting serin 473\), anti-phospho AKT \(targeting threonine 308\), anti-phospho p44/42 )] TJ ET BT 26.250 100.334 Td /F1 9.8 Tf [(MAPK \(all from Cell Signaling\) and anti-MAPK antibody \(Santa Cruz Biotechnology, Santa Cruz, CA, USA\) all diluted 1:1000, )] TJ ET BT 26.250 88.429 Td /F1 9.8 Tf [(followed by HRP conjugated anti-rabbit antibody before detection using the ECL method \(Amersham, Arlington Heights, IL, )] TJ ET BT 26.250 76.524 Td /F1 9.8 Tf [(USA\). Densitometric analyses were carried out using ImageJ software.)] TJ ET BT 26.250 57.120 Td /F6 9.8 Tf [(Sequence and phylogenetic analyses)] TJ ET BT 26.250 37.715 Td /F1 9.8 Tf [(Phylogenetic analyses based on nucleotide and amino acid sequence alignments were conducted in Molecular evolutionary )] TJ ET Q q 15.000 23.429 577.500 753.571 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Expression of the canine Col-1 target in canine mammary carcinoma tissue samples and cell lysates was assessed by sodium )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(dodecylsulfate \(SDS\) gel electrophoresis using reducing conditions followed by Western blot analysis. Briefly, frozen tissue )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(samples were homogenized in 1 ml lysis buffer \(10 mM Tris-HCL pH 7.4, 1% SDS\). Cells were cultured as described above, )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(washed with ice cold PBS and lysed in 500 l ice cold lysis buffer \(10 mM Tris-HCL pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(ethylene glycol tetraacetic acid \(EGTA\), 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate\). Lysis buffers contained )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(protease inhibitor cocktail \(P8340; Sigma-Aldrich, St. Louis, MO, USA\). The lysates were boiled for 5 min and centrifuged at )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(1000 g. Equal amounts of protein were separated by 10% SDS-PAGE and blotted to polyvinylidene fluoride \(PVDF\) )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(membranes. To assess equal loading of lysates, we performed reversible Ponceau staining of the membrane using Ponceau S )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(solution \(Sigma-Aldrich\). 5% dried milk powder in TBS containing 0.1% Tween was used for blocking nonspecific binding sites. )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(Membranes were incubated with Col-1 antibody diluted 1:250 overnight at 4 C. Anti-mouse IgG2a antibody \(SouthernBiotech, )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(Birmingham, AL, USA\) was used as isotype control. HRP conjugated secondary anti-mouse antibody \(Abcam, Cambridge, )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(UK\) was applied for 1 h at room temperature. For detection of CEA receptor expression in canine and human cell lines, Western )] TJ ET BT 26.250 624.619 Td /F1 9.8 Tf [(blot analysis was performed as described above. Membranes were incubated overnight using either rabbit anti-human CEA )] TJ ET BT 26.250 612.714 Td /F1 9.8 Tf [(receptor \(hnRNP M4 long isoform 1\) antibody \(USB, Swampscott, MA, USA\) or mouse anti-hnRNP \(M1-M4\), each diluted )] TJ ET BT 26.250 600.810 Td /F1 9.8 Tf [(1:1000. Next, appropriate HRP conjugated secondary anti-rabbit \(Cell Signaling, Danvers, MA, USA\) or anti-mouse antibody )] TJ ET BT 26.250 588.905 Td /F1 9.8 Tf [(was applied 1:2000 over a period of 1 h and bound antibodies were detected with the SuperSignal CL-HRP Substrate system )] TJ ET BT 26.250 577.000 Td /F1 9.8 Tf [(\(Pierce Biotechnology, Rockford, IL, USA\).)] TJ ET BT 26.250 557.595 Td /F6 9.8 Tf [(Transient transfection of TLM-1 cells with plasmids encoding CEACAM variants)] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Canine TLM-1 cells were cultured in DMEM medium containing 10% calf serum at 37C in 5% CO2. For transfection, cells were )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(seeded overnight in 6-well plates to reach 50% confluency the next day. Cells were transfected with 4 g of plasmid encoding )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(canine CEACAM1, canCEACAM23, canCEACAM24, canCEACAM25, canCEACAM28 and canCEACAM30 using the CaPO4 )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(method according the manufacturers protocol \(Stratagene, Agilent Technologies, Santa Clara, CA, USA\). To estimate efficiency )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(of transfection, cells were routinely transfected with an empty vector pEGFP-c2 \(Clontech Laboratories, Mountain View, CA, )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(USA\) encoding green fluorescent protein and analyzed with an inverted microscope \(Axioobserver Z1, Zeiss, Oberkochen, )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(Germany\) equipped with a high resolution microscopy camera \(AxioCam MRc, Zeiss\) and a filter set for fluorescein )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(isothiocyanate /enhanced green fluorescent protein \(FITC/EGFP\) fluorescence. Wells with transfection efficiencies of ? 50% )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(were collected for Western Blot analysis.)] TJ ET BT 26.250 423.548 Td /F6 9.8 Tf [(Flow Cytometry)] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(Cells were trypsinized, counted and resuspended in fluorescence-activated cell sorting \(FACS\) buffer \(1 x PBS supplemented )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(with 2 % heat-inactivated goat serum\). For intracellular staining 2 x 105 cells were fixed with formaldehyde for 20 min at RT in )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(the dark, washed once with FACS buffer and afterwards permeabilized with prechilled methanol for 30 min on ice. Next, treated )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(cells were washed once with FACS buffer and incubated with 5 ?g Col-1 antibody or isotype control antibody in FACS buffer for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(1 h at 4 C. For surface expression analysis, fixing and permeabilization steps were omitted. Stained cells were again washed )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(with FACS buffer, followed by incubation of FITC conjugated anti-mouse IgG \(Jackson ImmunoResearch Lab, West Grove, )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(PA, USA\) for 30 min at 4 C. After washing the pellet was resuspended in 300 ?l FACS buffer and 10,000 events were counted )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(using a FACSCalibur flow cytometer \(Becton Dickinson, San Jose, CA, USA\). Data analysis was performed via CellQuest )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(software.)] TJ ET BT 26.250 289.500 Td /F6 9.8 Tf [(Cell Viability Assay)] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(Tumor cell proliferation was analyzed by a modified tetrazolium dye assay \(EZ4U Cell Proliferation Assay; Biomedica Group, )] TJ ET BT 26.250 258.191 Td /F1 9.8 Tf [(Vienna, Austria\) based on the ability of living cells to reduce tetrazolium salts into formazan derivates. Cells were seeded in 96-)] TJ ET BT 26.250 246.286 Td /F1 9.8 Tf [(well plates at 2 x 104 cells per well and allowed to adhere overnight under standard culture conditions. 1 g Col-1 or isotype )] TJ ET BT 26.250 234.381 Td /F1 9.8 Tf [(control antibody per 200 l medium was applied over a period of 24 h and 48 h. Control wells were given media alone or treated )] TJ ET BT 26.250 222.477 Td /F1 9.8 Tf [(with 0.9% Triton X-100 for 10 min prior to addition of the modified tetrazolium dye. The quantity of reduced formazan is directly )] TJ ET BT 26.250 210.572 Td /F1 9.8 Tf [(proportional to living cells. Therefore absorbance was measured after 1 h incubation at 450 nm with 620 nm as a reference with )] TJ ET BT 26.250 198.667 Td /F1 9.8 Tf [(a 96-well plate reader. All experiments were done in triplicates.)] TJ ET BT 26.250 179.262 Td /F6 9.8 Tf [(Signaling Pathway Assay)] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(Phosphorylation assays were performed in CF33 and HT29 cells after 24 h serum starvation in DMEM medium containing 0.1% )] TJ ET BT 26.250 147.953 Td /F1 9.8 Tf [(FCS. After culturing for 24 h, cells were then treated with either fresh medium containing 0.1 % serum alone \(control\) or with 2 )] TJ ET BT 26.250 136.048 Td /F1 9.8 Tf [(g/mL Col-1 antibody in the same medium for the time periods indicated. Next, cell monolayers were washed with ice-cold PBS, )] TJ ET BT 26.250 124.143 Td /F1 9.8 Tf [(lysed and subjected to Western blot analysis as described above. The phosphorylation patterns were determined by probing the )] TJ ET BT 26.250 112.239 Td /F1 9.8 Tf [(blots with anti-AKT, anti-phospho AKT \(targeting serin 473\), anti-phospho AKT \(targeting threonine 308\), anti-phospho p44/42 )] TJ ET BT 26.250 100.334 Td /F1 9.8 Tf [(MAPK \(all from Cell Signaling\) and anti-MAPK antibody \(Santa Cruz Biotechnology, Santa Cruz, CA, USA\) all diluted 1:1000, )] TJ ET BT 26.250 88.429 Td /F1 9.8 Tf [(followed by HRP conjugated anti-rabbit antibody before detection using the ECL method \(Amersham, Arlington Heights, IL, )] TJ ET BT 26.250 76.524 Td /F1 9.8 Tf [(USA\). Densitometric analyses were carried out using ImageJ software.)] TJ ET BT 26.250 57.120 Td /F6 9.8 Tf [(Sequence and phylogenetic analyses)] TJ ET BT 26.250 37.715 Td /F1 9.8 Tf [(Phylogenetic analyses based on nucleotide and amino acid sequence alignments were conducted in Molecular evolutionary )] TJ ET Q q 15.000 23.429 577.500 753.571 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Expression of the canine Col-1 target in canine mammary carcinoma tissue samples and cell lysates was assessed by sodium )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(dodecylsulfate \(SDS\) gel electrophoresis using reducing conditions followed by Western blot analysis. Briefly, frozen tissue )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(samples were homogenized in 1 ml lysis buffer \(10 mM Tris-HCL pH 7.4, 1% SDS\). Cells were cultured as described above, )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(washed with ice cold PBS and lysed in 500 l ice cold lysis buffer \(10 mM Tris-HCL pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(ethylene glycol tetraacetic acid \(EGTA\), 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate\). Lysis buffers contained )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(protease inhibitor cocktail \(P8340; Sigma-Aldrich, St. Louis, MO, USA\). The lysates were boiled for 5 min and centrifuged at )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(1000 g. Equal amounts of protein were separated by 10% SDS-PAGE and blotted to polyvinylidene fluoride \(PVDF\) )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(membranes. To assess equal loading of lysates, we performed reversible Ponceau staining of the membrane using Ponceau S )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(solution \(Sigma-Aldrich\). 5% dried milk powder in TBS containing 0.1% Tween was used for blocking nonspecific binding sites. )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(Membranes were incubated with Col-1 antibody diluted 1:250 overnight at 4 C. Anti-mouse IgG2a antibody \(SouthernBiotech, )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(Birmingham, AL, USA\) was used as isotype control. HRP conjugated secondary anti-mouse antibody \(Abcam, Cambridge, )] TJ ET BT 26.250 636.524 Td /F1 9.8 Tf [(UK\) was applied for 1 h at room temperature. For detection of CEA receptor expression in canine and human cell lines, Western )] TJ ET BT 26.250 624.619 Td /F1 9.8 Tf [(blot analysis was performed as described above. Membranes were incubated overnight using either rabbit anti-human CEA )] TJ ET BT 26.250 612.714 Td /F1 9.8 Tf [(receptor \(hnRNP M4 long isoform 1\) antibody \(USB, Swampscott, MA, USA\) or mouse anti-hnRNP \(M1-M4\), each diluted )] TJ ET BT 26.250 600.810 Td /F1 9.8 Tf [(1:1000. Next, appropriate HRP conjugated secondary anti-rabbit \(Cell Signaling, Danvers, MA, USA\) or anti-mouse antibody )] TJ ET BT 26.250 588.905 Td /F1 9.8 Tf [(was applied 1:2000 over a period of 1 h and bound antibodies were detected with the SuperSignal CL-HRP Substrate system )] TJ ET BT 26.250 577.000 Td /F1 9.8 Tf [(\(Pierce Biotechnology, Rockford, IL, USA\).)] TJ ET BT 26.250 557.595 Td /F6 9.8 Tf [(Transient transfection of TLM-1 cells with plasmids encoding CEACAM variants)] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(Canine TLM-1 cells were cultured in DMEM medium containing 10% calf serum at 37C in 5% CO2. For transfection, cells were )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(seeded overnight in 6-well plates to reach 50% confluency the next day. Cells were transfected with 4 g of plasmid encoding )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(canine CEACAM1, canCEACAM23, canCEACAM24, canCEACAM25, canCEACAM28 and canCEACAM30 using the CaPO4 )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(method according the manufacturers protocol \(Stratagene, Agilent Technologies, Santa Clara, CA, USA\). To estimate efficiency )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(of transfection, cells were routinely transfected with an empty vector pEGFP-c2 \(Clontech Laboratories, Mountain View, CA, )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(USA\) encoding green fluorescent protein and analyzed with an inverted microscope \(Axioobserver Z1, Zeiss, Oberkochen, )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(Germany\) equipped with a high resolution microscopy camera \(AxioCam MRc, Zeiss\) and a filter set for fluorescein )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(isothiocyanate /enhanced green fluorescent protein \(FITC/EGFP\) fluorescence. Wells with transfection efficiencies of ? 50% )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(were collected for Western Blot analysis.)] TJ ET BT 26.250 423.548 Td /F6 9.8 Tf [(Flow Cytometry)] TJ ET BT 26.250 404.143 Td /F1 9.8 Tf [(Cells were trypsinized, counted and resuspended in fluorescence-activated cell sorting \(FACS\) buffer \(1 x PBS supplemented )] TJ ET BT 26.250 392.238 Td /F1 9.8 Tf [(with 2 % heat-inactivated goat serum\). For intracellular staining 2 x 105 cells were fixed with formaldehyde for 20 min at RT in )] TJ ET BT 26.250 380.334 Td /F1 9.8 Tf [(the dark, washed once with FACS buffer and afterwards permeabilized with prechilled methanol for 30 min on ice. Next, treated )] TJ ET BT 26.250 368.429 Td /F1 9.8 Tf [(cells were washed once with FACS buffer and incubated with 5 ?g Col-1 antibody or isotype control antibody in FACS buffer for )] TJ ET BT 26.250 356.524 Td /F1 9.8 Tf [(1 h at 4 C. For surface expression analysis, fixing and permeabilization steps were omitted. Stained cells were again washed )] TJ ET BT 26.250 344.619 Td /F1 9.8 Tf [(with FACS buffer, followed by incubation of FITC conjugated anti-mouse IgG \(Jackson ImmunoResearch Lab, West Grove, )] TJ ET BT 26.250 332.715 Td /F1 9.8 Tf [(PA, USA\) for 30 min at 4 C. After washing the pellet was resuspended in 300 ?l FACS buffer and 10,000 events were counted )] TJ ET BT 26.250 320.810 Td /F1 9.8 Tf [(using a FACSCalibur flow cytometer \(Becton Dickinson, San Jose, CA, USA\). Data analysis was performed via CellQuest )] TJ ET BT 26.250 308.905 Td /F1 9.8 Tf [(software.)] TJ ET BT 26.250 289.500 Td /F6 9.8 Tf [(Cell Viability Assay)] TJ ET BT 26.250 270.096 Td /F1 9.8 Tf [(Tumor cell proliferation was analyzed by a modified tetrazolium dye assay \(EZ4U Cell Proliferation Assay; Biomedica Group, )] TJ ET BT 26.250 258.191 Td /F1 9.8 Tf [(Vienna, Austria\) based on the ability of living cells to reduce tetrazolium salts into formazan derivates. Cells were seeded in 96-)] TJ ET BT 26.250 246.286 Td /F1 9.8 Tf [(well plates at 2 x 104 cells per well and allowed to adhere overnight under standard culture conditions. 1 g Col-1 or isotype )] TJ ET BT 26.250 234.381 Td /F1 9.8 Tf [(control antibody per 200 l medium was applied over a period of 24 h and 48 h. Control wells were given media alone or treated )] TJ ET BT 26.250 222.477 Td /F1 9.8 Tf [(with 0.9% Triton X-100 for 10 min prior to addition of the modified tetrazolium dye. The quantity of reduced formazan is directly )] TJ ET BT 26.250 210.572 Td /F1 9.8 Tf [(proportional to living cells. Therefore absorbance was measured after 1 h incubation at 450 nm with 620 nm as a reference with )] TJ ET BT 26.250 198.667 Td /F1 9.8 Tf [(a 96-well plate reader. All experiments were done in triplicates.)] TJ ET BT 26.250 179.262 Td /F6 9.8 Tf [(Signaling Pathway Assay)] TJ ET BT 26.250 159.858 Td /F1 9.8 Tf [(Phosphorylation assays were performed in CF33 and HT29 cells after 24 h serum starvation in DMEM medium containing 0.1% )] TJ ET BT 26.250 147.953 Td /F1 9.8 Tf [(FCS. After culturing for 24 h, cells were then treated with either fresh medium containing 0.1 % serum alone \(control\) or with 2 )] TJ ET BT 26.250 136.048 Td /F1 9.8 Tf [(g/mL Col-1 antibody in the same medium for the time periods indicated. Next, cell monolayers were washed with ice-cold PBS, )] TJ ET BT 26.250 124.143 Td /F1 9.8 Tf [(lysed and subjected to Western blot analysis as described above. The phosphorylation patterns were determined by probing the )] TJ ET BT 26.250 112.239 Td /F1 9.8 Tf [(blots with anti-AKT, anti-phospho AKT \(targeting serin 473\), anti-phospho AKT \(targeting threonine 308\), anti-phospho p44/42 )] TJ ET BT 26.250 100.334 Td /F1 9.8 Tf [(MAPK \(all from Cell Signaling\) and anti-MAPK antibody \(Santa Cruz Biotechnology, Santa Cruz, CA, USA\) all diluted 1:1000, )] TJ ET BT 26.250 88.429 Td /F1 9.8 Tf [(followed by HRP conjugated anti-rabbit antibody before detection using the ECL method \(Amersham, Arlington Heights, IL, )] TJ ET BT 26.250 76.524 Td /F1 9.8 Tf [(USA\). Densitometric analyses were carried out using ImageJ software.)] TJ ET BT 26.250 57.120 Td /F6 9.8 Tf [(Sequence and phylogenetic analyses)] TJ ET BT 26.250 37.715 Td /F1 9.8 Tf [(Phylogenetic analyses based on nucleotide and amino acid sequence alignments were conducted in Molecular evolutionary )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 260 0 obj << /Type /Page /Parent 3 0 R /Annots [ 262 0 R 264 0 R 266 0 R 268 0 R 271 0 R 273 0 R 275 0 R 277 0 R 279 0 R 281 0 R 283 0 R 285 0 R ] /Contents 261 0 R >> endobj 261 0 obj << /Length 19536 >> stream 0.271 0.267 0.267 rg q 15.000 38.579 577.500 738.421 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(genetics analysis \(MEGA5\) )] TJ ET 0.267 0.267 0.267 rg BT 149.782 767.476 Td /F1 9.8 Tf [([45] [46])] TJ ET 0.271 0.267 0.267 rg BT 185.019 767.476 Td /F1 9.8 Tf [(. The Maximum Likelihood method based on the JTT matrix-based model \(protein )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 79.358 755.571 Td /F1 9.8 Tf [([47])] TJ ET 0.271 0.267 0.267 rg BT 95.621 755.571 Td /F1 9.8 Tf [( or based on the Tamura-Nei model \(DNA sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 331.874 755.571 Td /F1 9.8 Tf [([48])] TJ ET 0.271 0.267 0.267 rg BT 348.137 755.571 Td /F1 9.8 Tf [(, with bootstrap testing \(500 replicates\) was applied. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(The multiple amino acid sequence alignment shown in Figure 1 and in Supplementary Figure 1 were performed with ClustalW at )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(NPS@ \(Network Protein Sequence @nalysis;http://npsa-pbil.ibcp.fr/\) and verified using MEGA5.)] TJ ET BT 26.250 712.357 Td /F6 9.8 Tf [(Similarity of CEACAMs and CEAR between Humans and Dogs)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Human and canine CEACAMs and CEAR proteins from ncbi \(http://www.ncbi.nlm.nih.gov/protein\) were aligned using BLAST )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(\(Basic Local Alignment Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi\). Sequences stored under the accession number )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(NP_001091026 \(canine CEACAM1\), NP_001091021 \(canine CEACAM23\), NP_001091023 \(canine CEACAM24\), ABL76056 )] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(\(canine CEACAM25\), ABL76057 \(canine CEACAM28\), NP_001091022 \(canine CEACAM30\), NP_005959.2 \(human CEAR\), )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(XP_854270.1 \(canine CEAR\), NP_001178152.1 \(bovine CEAR\), NP_001103381.1 \(Rat CEAR\), NP_084080.1 \(Mouse CEAR\), )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(NP_001026103.1 \(Chicken CEAR\), AAI34826.1 \(Xenopus CEAR\), NP_001133640.1 \(Salmon CEAR\) and XP_683303.4 )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(\(Zebrafish CEAR\) were used for homology alignment searches.)] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(Phylogenetic Trees and Data have been submitted to TreeBASE under study number 12028 \( http://treebase.org/treebase-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(web/search/study/summary.html?id=12028 \))] TJ ET BT 26.250 553.612 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 533.658 Td /F6 9.8 Tf [(Phylogenetic comparison of human and canine CEACAMs)] TJ ET BT 26.250 514.253 Td /F1 9.8 Tf [(Our BLAST analysis across the whole sequences revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 26.250 502.348 Td /F1 9.8 Tf [(similarities between 55% and 72% between human CEACAM5 and canine CEACAM1-related molecules \(Fig. 1a\). Phylogenetic )] TJ ET BT 26.250 490.443 Td /F1 9.8 Tf [(analysis comparing N domain exon nucleotide sequences of human and canine CEACAM1-related CEACAMs and CEACAM19, )] TJ ET BT 26.250 478.539 Td /F1 9.8 Tf [(respectively, indicated that CEACAM1-related CEACAMs between the different species are more closely related than )] TJ ET BT 26.250 466.634 Td /F1 9.8 Tf [(CEACAM1-related and other CEACAM types within the same species. The results were corroborated by three independent )] TJ ET BT 26.250 454.729 Td /F1 9.8 Tf [(methods, Maximum likelihood \(ML\), Maximum parsimony \(MP\) using MEGA5 and Neighbor-joining \(NJ\) using ClustalW \(data )] TJ ET BT 26.250 442.824 Td /F1 9.8 Tf [(not shown\) \(Fig. 1b\). Amino acid sequences of mature CEACAM1-related N domains without leader peptide were aligned using )] TJ ET BT 26.250 430.920 Td /F1 9.8 Tf [(ClustalW. Especially the N domain of human CEACAM5 contains multiple regions that are highly similar to regions of canine )] TJ ET BT 26.250 419.015 Td /F1 9.8 Tf [(CEACAM1-related CEACAM N-domains \(Fig. 1c\).)] TJ ET 0.965 0.965 0.965 rg 26.250 108.698 555.000 300.436 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 409.134 m 581.250 409.134 l 581.250 408.384 l 26.250 408.384 l f 26.250 108.698 m 581.250 108.698 l 581.250 109.448 l 26.250 109.448 l f q 112.500 0 0 112.500 35.250 286.884 cm /I3 Do Q q 35.250 119.948 537.000 160.936 re W n 0.271 0.267 0.267 rg BT 35.250 271.360 Td /F4 9.8 Tf [(Fig. 1: Comparison of human CEA \(CEACAM5\) with canine CEACAMs. \(a\) BLAST analysis across the whole )] TJ ET BT 35.250 259.455 Td /F4 9.8 Tf [(sequences of human)] TJ ET BT 35.250 240.085 Td /F1 9.8 Tf [(CEACAM5 and canine CEACAM1-related molecules revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 35.250 226.349 Td /F1 9.8 Tf [(similarities between 55% and 72%. \(b\) A phylogenetic tree was constructed using MEGA5 from N domain exon nucleotide )] TJ ET BT 35.250 212.613 Td /F1 9.8 Tf [(sequences of selected human and canine CEACAM1-related CEACAMs and CEACAM19, respectively. It shows that )] TJ ET BT 35.250 198.877 Td /F1 9.8 Tf [(CEACAM1-related CEACAMs between the different species are more closely related than CEACAM1-related and other )] TJ ET BT 35.250 185.140 Td /F1 9.8 Tf [(CEACAM types within the same species. \(c\) Especially the N domain of human CEACAM5 contains multiple regions that )] TJ ET BT 35.250 171.404 Td /F1 9.8 Tf [(are highly similar to regions of canine CEACAM1-related CEACAM N domains. Amino acid sequences of mature )] TJ ET BT 35.250 157.668 Td /F1 9.8 Tf [(CEACAM1-related N domains without leader peptide were aligned using ClustalW. The following color code was used: red, )] TJ ET BT 35.250 143.932 Td /F1 9.8 Tf [(identical amino acids; green, strongly similar amino acids; blue, weakly similar amino acids; black, non-conserved amino )] TJ ET BT 35.250 130.195 Td /F1 9.8 Tf [(acids.)] TJ ET Q BT 26.250 91.674 Td /F6 9.8 Tf [(Immunoreactivity of Col-1 in canine mammary cancer cells)] TJ ET BT 26.250 72.270 Td /F1 9.8 Tf [(In 23 out of 30 investigated canine mammary cancer patients Col-1 reactive tumor cells could be detected via )] TJ ET BT 26.250 60.365 Td /F1 9.8 Tf [(immunohistochemical analysis, revealing individually varying staining intensities. Figure 2a shows typical examples of positive )] TJ ET BT 26.250 48.460 Td /F1 9.8 Tf [(immunoreactivity which was loca lized predominantly in the cytoplasm of tumor cells.)] TJ ET Q q 15.000 38.579 577.500 738.421 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(genetics analysis \(MEGA5\) )] TJ ET 0.267 0.267 0.267 rg BT 149.782 767.476 Td /F1 9.8 Tf [([45] [46])] TJ ET 0.271 0.267 0.267 rg BT 185.019 767.476 Td /F1 9.8 Tf [(. The Maximum Likelihood method based on the JTT matrix-based model \(protein )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 79.358 755.571 Td /F1 9.8 Tf [([47])] TJ ET 0.271 0.267 0.267 rg BT 95.621 755.571 Td /F1 9.8 Tf [( or based on the Tamura-Nei model \(DNA sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 331.874 755.571 Td /F1 9.8 Tf [([48])] TJ ET 0.271 0.267 0.267 rg BT 348.137 755.571 Td /F1 9.8 Tf [(, with bootstrap testing \(500 replicates\) was applied. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(The multiple amino acid sequence alignment shown in Figure 1 and in Supplementary Figure 1 were performed with ClustalW at )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(NPS@ \(Network Protein Sequence @nalysis;http://npsa-pbil.ibcp.fr/\) and verified using MEGA5.)] TJ ET BT 26.250 712.357 Td /F6 9.8 Tf [(Similarity of CEACAMs and CEAR between Humans and Dogs)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Human and canine CEACAMs and CEAR proteins from ncbi \(http://www.ncbi.nlm.nih.gov/protein\) were aligned using BLAST )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(\(Basic Local Alignment Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi\). Sequences stored under the accession number )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(NP_001091026 \(canine CEACAM1\), NP_001091021 \(canine CEACAM23\), NP_001091023 \(canine CEACAM24\), ABL76056 )] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(\(canine CEACAM25\), ABL76057 \(canine CEACAM28\), NP_001091022 \(canine CEACAM30\), NP_005959.2 \(human CEAR\), )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(XP_854270.1 \(canine CEAR\), NP_001178152.1 \(bovine CEAR\), NP_001103381.1 \(Rat CEAR\), NP_084080.1 \(Mouse CEAR\), )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(NP_001026103.1 \(Chicken CEAR\), AAI34826.1 \(Xenopus CEAR\), NP_001133640.1 \(Salmon CEAR\) and XP_683303.4 )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(\(Zebrafish CEAR\) were used for homology alignment searches.)] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(Phylogenetic Trees and Data have been submitted to TreeBASE under study number 12028 \( http://treebase.org/treebase-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(web/search/study/summary.html?id=12028 \))] TJ ET BT 26.250 553.612 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 533.658 Td /F6 9.8 Tf [(Phylogenetic comparison of human and canine CEACAMs)] TJ ET BT 26.250 514.253 Td /F1 9.8 Tf [(Our BLAST analysis across the whole sequences revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 26.250 502.348 Td /F1 9.8 Tf [(similarities between 55% and 72% between human CEACAM5 and canine CEACAM1-related molecules \(Fig. 1a\). Phylogenetic )] TJ ET BT 26.250 490.443 Td /F1 9.8 Tf [(analysis comparing N domain exon nucleotide sequences of human and canine CEACAM1-related CEACAMs and CEACAM19, )] TJ ET BT 26.250 478.539 Td /F1 9.8 Tf [(respectively, indicated that CEACAM1-related CEACAMs between the different species are more closely related than )] TJ ET BT 26.250 466.634 Td /F1 9.8 Tf [(CEACAM1-related and other CEACAM types within the same species. The results were corroborated by three independent )] TJ ET BT 26.250 454.729 Td /F1 9.8 Tf [(methods, Maximum likelihood \(ML\), Maximum parsimony \(MP\) using MEGA5 and Neighbor-joining \(NJ\) using ClustalW \(data )] TJ ET BT 26.250 442.824 Td /F1 9.8 Tf [(not shown\) \(Fig. 1b\). Amino acid sequences of mature CEACAM1-related N domains without leader peptide were aligned using )] TJ ET BT 26.250 430.920 Td /F1 9.8 Tf [(ClustalW. Especially the N domain of human CEACAM5 contains multiple regions that are highly similar to regions of canine )] TJ ET BT 26.250 419.015 Td /F1 9.8 Tf [(CEACAM1-related CEACAM N-domains \(Fig. 1c\).)] TJ ET 0.965 0.965 0.965 rg 26.250 108.698 555.000 300.436 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 409.134 m 581.250 409.134 l 581.250 408.384 l 26.250 408.384 l f 26.250 108.698 m 581.250 108.698 l 581.250 109.448 l 26.250 109.448 l f q 112.500 0 0 112.500 35.250 286.884 cm /I3 Do Q q 35.250 119.948 537.000 160.936 re W n 0.271 0.267 0.267 rg BT 35.250 271.360 Td /F4 9.8 Tf [(Fig. 1: Comparison of human CEA \(CEACAM5\) with canine CEACAMs. \(a\) BLAST analysis across the whole )] TJ ET BT 35.250 259.455 Td /F4 9.8 Tf [(sequences of human)] TJ ET BT 35.250 240.085 Td /F1 9.8 Tf [(CEACAM5 and canine CEACAM1-related molecules revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 35.250 226.349 Td /F1 9.8 Tf [(similarities between 55% and 72%. \(b\) A phylogenetic tree was constructed using MEGA5 from N domain exon nucleotide )] TJ ET BT 35.250 212.613 Td /F1 9.8 Tf [(sequences of selected human and canine CEACAM1-related CEACAMs and CEACAM19, respectively. It shows that )] TJ ET BT 35.250 198.877 Td /F1 9.8 Tf [(CEACAM1-related CEACAMs between the different species are more closely related than CEACAM1-related and other )] TJ ET BT 35.250 185.140 Td /F1 9.8 Tf [(CEACAM types within the same species. \(c\) Especially the N domain of human CEACAM5 contains multiple regions that )] TJ ET BT 35.250 171.404 Td /F1 9.8 Tf [(are highly similar to regions of canine CEACAM1-related CEACAM N domains. Amino acid sequences of mature )] TJ ET BT 35.250 157.668 Td /F1 9.8 Tf [(CEACAM1-related N domains without leader peptide were aligned using ClustalW. The following color code was used: red, )] TJ ET BT 35.250 143.932 Td /F1 9.8 Tf [(identical amino acids; green, strongly similar amino acids; blue, weakly similar amino acids; black, non-conserved amino )] TJ ET BT 35.250 130.195 Td /F1 9.8 Tf [(acids.)] TJ ET Q BT 26.250 91.674 Td /F6 9.8 Tf [(Immunoreactivity of Col-1 in canine mammary cancer cells)] TJ ET BT 26.250 72.270 Td /F1 9.8 Tf [(In 23 out of 30 investigated canine mammary cancer patients Col-1 reactive tumor cells could be detected via )] TJ ET BT 26.250 60.365 Td /F1 9.8 Tf [(immunohistochemical analysis, revealing individually varying staining intensities. Figure 2a shows typical examples of positive )] TJ ET BT 26.250 48.460 Td /F1 9.8 Tf [(immunoreactivity which was loca lized predominantly in the cytoplasm of tumor cells.)] TJ ET Q q 15.000 38.579 577.500 738.421 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(genetics analysis \(MEGA5\) )] TJ ET 0.267 0.267 0.267 rg BT 149.782 767.476 Td /F1 9.8 Tf [([45] [46])] TJ ET 0.271 0.267 0.267 rg BT 185.019 767.476 Td /F1 9.8 Tf [(. The Maximum Likelihood method based on the JTT matrix-based model \(protein )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 79.358 755.571 Td /F1 9.8 Tf [([47])] TJ ET 0.271 0.267 0.267 rg BT 95.621 755.571 Td /F1 9.8 Tf [( or based on the Tamura-Nei model \(DNA sequences\) )] TJ ET 0.267 0.267 0.267 rg BT 331.874 755.571 Td /F1 9.8 Tf [([48])] TJ ET 0.271 0.267 0.267 rg BT 348.137 755.571 Td /F1 9.8 Tf [(, with bootstrap testing \(500 replicates\) was applied. )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(The multiple amino acid sequence alignment shown in Figure 1 and in Supplementary Figure 1 were performed with ClustalW at )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(NPS@ \(Network Protein Sequence @nalysis;http://npsa-pbil.ibcp.fr/\) and verified using MEGA5.)] TJ ET BT 26.250 712.357 Td /F6 9.8 Tf [(Similarity of CEACAMs and CEAR between Humans and Dogs)] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(Human and canine CEACAMs and CEAR proteins from ncbi \(http://www.ncbi.nlm.nih.gov/protein\) were aligned using BLAST )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(\(Basic Local Alignment Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi\). Sequences stored under the accession number )] TJ ET BT 26.250 669.143 Td /F1 9.8 Tf [(NP_001091026 \(canine CEACAM1\), NP_001091021 \(canine CEACAM23\), NP_001091023 \(canine CEACAM24\), ABL76056 )] TJ ET BT 26.250 657.238 Td /F1 9.8 Tf [(\(canine CEACAM25\), ABL76057 \(canine CEACAM28\), NP_001091022 \(canine CEACAM30\), NP_005959.2 \(human CEAR\), )] TJ ET BT 26.250 645.333 Td /F1 9.8 Tf [(XP_854270.1 \(canine CEAR\), NP_001178152.1 \(bovine CEAR\), NP_001103381.1 \(Rat CEAR\), NP_084080.1 \(Mouse CEAR\), )] TJ ET BT 26.250 633.429 Td /F1 9.8 Tf [(NP_001026103.1 \(Chicken CEAR\), AAI34826.1 \(Xenopus CEAR\), NP_001133640.1 \(Salmon CEAR\) and XP_683303.4 )] TJ ET BT 26.250 621.524 Td /F1 9.8 Tf [(\(Zebrafish CEAR\) were used for homology alignment searches.)] TJ ET BT 26.250 602.119 Td /F1 9.8 Tf [(Phylogenetic Trees and Data have been submitted to TreeBASE under study number 12028 \( http://treebase.org/treebase-)] TJ ET BT 26.250 590.214 Td /F1 9.8 Tf [(web/search/study/summary.html?id=12028 \))] TJ ET BT 26.250 553.612 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 533.658 Td /F6 9.8 Tf [(Phylogenetic comparison of human and canine CEACAMs)] TJ ET BT 26.250 514.253 Td /F1 9.8 Tf [(Our BLAST analysis across the whole sequences revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 26.250 502.348 Td /F1 9.8 Tf [(similarities between 55% and 72% between human CEACAM5 and canine CEACAM1-related molecules \(Fig. 1a\). Phylogenetic )] TJ ET BT 26.250 490.443 Td /F1 9.8 Tf [(analysis comparing N domain exon nucleotide sequences of human and canine CEACAM1-related CEACAMs and CEACAM19, )] TJ ET BT 26.250 478.539 Td /F1 9.8 Tf [(respectively, indicated that CEACAM1-related CEACAMs between the different species are more closely related than )] TJ ET BT 26.250 466.634 Td /F1 9.8 Tf [(CEACAM1-related and other CEACAM types within the same species. The results were corroborated by three independent )] TJ ET BT 26.250 454.729 Td /F1 9.8 Tf [(methods, Maximum likelihood \(ML\), Maximum parsimony \(MP\) using MEGA5 and Neighbor-joining \(NJ\) using ClustalW \(data )] TJ ET BT 26.250 442.824 Td /F1 9.8 Tf [(not shown\) \(Fig. 1b\). Amino acid sequences of mature CEACAM1-related N domains without leader peptide were aligned using )] TJ ET BT 26.250 430.920 Td /F1 9.8 Tf [(ClustalW. Especially the N domain of human CEACAM5 contains multiple regions that are highly similar to regions of canine )] TJ ET BT 26.250 419.015 Td /F1 9.8 Tf [(CEACAM1-related CEACAM N-domains \(Fig. 1c\).)] TJ ET 0.965 0.965 0.965 rg 26.250 108.698 555.000 300.436 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 409.134 m 581.250 409.134 l 581.250 408.384 l 26.250 408.384 l f 26.250 108.698 m 581.250 108.698 l 581.250 109.448 l 26.250 109.448 l f q 112.500 0 0 112.500 35.250 286.884 cm /I3 Do Q q 35.250 119.948 537.000 160.936 re W n 0.271 0.267 0.267 rg BT 35.250 271.360 Td /F4 9.8 Tf [(Fig. 1: Comparison of human CEA \(CEACAM5\) with canine CEACAMs. \(a\) BLAST analysis across the whole )] TJ ET BT 35.250 259.455 Td /F4 9.8 Tf [(sequences of human)] TJ ET BT 35.250 240.085 Td /F1 9.8 Tf [(CEACAM5 and canine CEACAM1-related molecules revealed amino acid sequence identities ranging from 42 59% and )] TJ ET BT 35.250 226.349 Td /F1 9.8 Tf [(similarities between 55% and 72%. \(b\) A phylogenetic tree was constructed using MEGA5 from N domain exon nucleotide )] TJ ET BT 35.250 212.613 Td /F1 9.8 Tf [(sequences of selected human and canine CEACAM1-related CEACAMs and CEACAM19, respectively. It shows that )] TJ ET BT 35.250 198.877 Td /F1 9.8 Tf [(CEACAM1-related CEACAMs between the different species are more closely related than CEACAM1-related and other )] TJ ET BT 35.250 185.140 Td /F1 9.8 Tf [(CEACAM types within the same species. \(c\) Especially the N domain of human CEACAM5 contains multiple regions that )] TJ ET BT 35.250 171.404 Td /F1 9.8 Tf [(are highly similar to regions of canine CEACAM1-related CEACAM N domains. Amino acid sequences of mature )] TJ ET BT 35.250 157.668 Td /F1 9.8 Tf [(CEACAM1-related N domains without leader peptide were aligned using ClustalW. The following color code was used: red, )] TJ ET BT 35.250 143.932 Td /F1 9.8 Tf [(identical amino acids; green, strongly similar amino acids; blue, weakly similar amino acids; black, non-conserved amino )] TJ ET BT 35.250 130.195 Td /F1 9.8 Tf [(acids.)] TJ ET Q BT 26.250 91.674 Td /F6 9.8 Tf [(Immunoreactivity of Col-1 in canine mammary cancer cells)] TJ ET BT 26.250 72.270 Td /F1 9.8 Tf [(In 23 out of 30 investigated canine mammary cancer patients Col-1 reactive tumor cells could be detected via )] TJ ET BT 26.250 60.365 Td /F1 9.8 Tf [(immunohistochemical analysis, revealing individually varying staining intensities. Figure 2a shows typical examples of positive )] TJ ET BT 26.250 48.460 Td /F1 9.8 Tf [(immunoreactivity which was loca lized predominantly in the cytoplasm of tumor cells.)] TJ ET Q q 112.500 0 0 112.500 35.250 286.884 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 262 0 obj << /Type /Annot /Subtype /Link /A 263 0 R /Border [0 0 0] /H /I /Rect [ 149.7825 766.5743 185.0190 776.4950 ] >> endobj 263 0 obj << /Type /Action >> endobj 264 0 obj << /Type /Annot /Subtype /Link /A 265 0 R /Border [0 0 0] /H /I /Rect [ 79.3582 754.6696 95.6213 764.5902 ] >> endobj 265 0 obj << /Type /Action >> endobj 266 0 obj << /Type /Annot /Subtype /Link /A 267 0 R /Border [0 0 0] /H /I /Rect [ 331.8735 754.6696 348.1365 764.5902 ] >> endobj 267 0 obj << /Type /Action >> endobj 268 0 obj << /Type /Annot /Subtype /Link /A 269 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 286.8840 147.7500 399.3840 ] >> endobj 269 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/treeoflife/files/2011/11/fig.1weichselbaumer.tif) >> endobj 270 0 obj << /Type /XObject /Subtype /Image /Width 150 /Height 150 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 8027>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   " }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q!KތN#Ҍgޗ2=(ȣ5OlwF脣2}v_Ӫ߫O1^)m&oqm /!v}]V[&UlWd Æe<7ӫ߫O1\4'xP5_k ;YVF3 yC]&̶Я#{FG"B^I[E|SᲺ=Kgc[FRQ]R d'zoŮ{%fQӭو$ew[;~8G/iէᨽ7r&VBdm, XcswlĞVD|EFmv(?Uf*#=;;g=h,ifhB #F}KZ-XZS{RiRM;w8ʻO^jʷջSG/+noРEۀW~N/DEmLE \ WrMG7-_{0(TcsH?7$7OxCO6:NY"0_.XaGa/GmOXf=>j{AY#]G@ҍI\0˵G 3OA]C^ޑ%qT1s @9^62?6QODIP\ݴvzSz2 T88eq?J۷VŗS N y?}j?chc~/!gԴs#+ pTkZxJ_bvg~ܗA gi8s#f^7_2޵_Z)8Z'8}h@ F('gj7p\ŧL"o=浧WWcveU8>ƊjQ]j @!_#Ey}z;[28oL$،*%Fuϊu2۶4wEӁN $)ڶ:aRIIyw;噘TpNmUt4Kc˕. 2c b  mD]CV[hA4~)ŔT~ƻ oM6yڕQgJ dP[:\[:nhʋpт3MsZq+=V#7,0<S@i16*hη,>way~]b5+GD713}ˮ\"-x~&Ϸe$#.#9$bX 3TOR[y+FHȸ=MWuGQV1\G#gq6z7 Z=ZTmʴLFUWkp9^^O::ʛAr/|3 6&ݗ$l d&:d :RѥJ>֮J'R&6MXtͬoH LtY/y;c9ԋb#2\D '#Un~)EսH̻ΖΊB .X ʄzu <]`нӗr?[mOe\F /" HaZŪG]Y)-"sFZ!!Կ_i(uOF㕡(??Q_-?h 1@ S-?3s~HiȇФQO}hU\(>jKin+ˋgUYbAp~e>W)=>KkYmu+v܎vlwr#Z^κ欩0/{O܆$av) bqxW6zmIaՂ 7*ȼO-A= Mq(v.]3( cA$zV}2]t #[X.]$HXv* Q&MYi[|OQ~j>i-?y8۹8 X𮁦aaZŝ%%MPnĚW$-ghpn߱ i X]YqBxGɸHѾfqrO_SYVwmu5]<2&\<17|}I[=ۖBnbqVN/u CP4]-U卶Gl!IH6\+{J/\=*yK#[3:QI ( QF(?? #:@5M%ն6$G2, Uw` tvڔ/Y<-!yc :{KˇZӵ_-%ao2_['EF*FAn@:Z'OrO~Ѣ\,)QV|z"[ 6SЈPK@O@ p? a W]ś\_2)J$=Z':gk-J7%WC@ޥj,mQ+,wYI aʏA#c\:~'p%ZZHd>XycG=Ic>/QRmJt ,*oC,V@>"X_=$(,deە/<=-œSolȕTrF~5'!z\VHڷ*>b9mI5bq9ƯCJ_HgU)`FlCm5;_ivA ֫1 DAaT (<H#bOcڭp0lr9Nxɑ|d,ls"a `GpA6?տkq4?nio[876e軎KɧJWX bJ>խ@ tY2 қR[F'4\uA[V$0Dă kTz:TE%/zJZ(J((hұG)c} BZy)#&Rv4ތV-Z(ӯ{x{$m;Q ƏƌV+c:hN!/V6pd_;Zhh]Sz݆tF{bD3_hhh+^:7miڅ[RTfC|}m~4~4bj?h [>Rw6:1R(ki;V~ Nkcw 46H@=v3n}D}i3QY\ H.rB%E.U둜0GqUu\h:MZ]_vE`>U?@hCN5m2[j'H!M&{24j4o=з0[&d3p(nV~ {IZ]Xs{*a'3"ר~n \ʌ CU-/w4h6 \BdݒNs _TSSԷjRJ^ZO);8| &\Iĸd1 blsN?dW$@|_`c @$qݑׇ\ ?Ǐ5yb}pL_{:!@?tqƲ~2j^ari8Gl>CN '@~Uxm[Dt >Hqw7Jɾt&jwQ!o rAɆN>uru-b}Ee@qx]JմoO5EM[@3{ *2Hm¸x^rUWHaQ\eaь찀sZ%~[Ej/tE^I5 p-JO}=yro2I)vrޙn])V&dȨ+aؑHڝ]iu? 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In cell lysates of CF33 and CF41 cells, but not in Sh1b and P114 cells, we observed a specific protein band at 60 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(kDa, whereas staining with isotype control antibody remained negative \(Fig. 2b\). The result was confirmed by blotting lysa tes )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(from spontaneous mammary carcinoma of canine patients that had proven positive for Col-1 reactivity by immunohistochemistry )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(\(Fig. 2c\). In 3 out of 4 samples from dog patients, Col-1 binding was revealed at 60 kDa and weaker at 120 kDa. In dog 4, like in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(human HT29 colon cancer cells \(Fig. 2b\), Col-1 binding was observed at approximately 180 kDa corresponding to the molecular )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(weight of human CEACAM5. Next we investigated whether Col-1 would recognize important members of the canine CEACAM )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(family. We selected canine TLM-1 melanoma cells as host for transfection and optimal species-specific expression of canine )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(CEACAM1, -23, -24, -25, -28 and -30, all belonging to the canine CEACAM1 family. Col-1 binding was only found in cells )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(transfected with human CEACAM5 but not in cells transfected with any of the canine CEACAMs \(Fig. 2d\). These data indicate )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(that Col-1 does not crossreact with the above-mentioned canine CEACAM isoforms.)] TJ ET 0.965 0.965 0.965 rg 26.250 322.544 555.000 316.004 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.548 m 581.250 638.548 l 581.250 637.798 l 26.250 637.798 l f 26.250 322.544 m 581.250 322.544 l 581.250 323.294 l 26.250 323.294 l f q 112.500 0 0 112.500 35.250 516.298 cm /I4 Do Q q 35.250 333.794 537.000 176.504 re W n 0.271 0.267 0.267 rg BT 35.250 500.774 Td /F4 9.8 Tf [(Fig. 2: Col-1 is immunoreactive in canine mammary cancer.)] TJ ET BT 35.250 481.404 Td /F1 9.8 Tf [(\(a\) Sections of canine tumor specimens were stained immunohistochemically with Col-1 antibody. The same region of the )] TJ ET BT 35.250 467.668 Td /F1 9.8 Tf [(tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a 40X/1.3 oil objective lens. \(b\) Western blot analysis )] TJ ET BT 35.250 453.931 Td /F1 9.8 Tf [(revealed a Col-1 immunoreactive signal in canine mammary carcinoma cells CF33 and CF41 at approximately 60 kDa. )] TJ ET BT 35.250 440.195 Td /F1 9.8 Tf [(P114 and Sh1b remained negative. Human colon cancer cells HT29 served as positive control showing a strong Col-1 )] TJ ET BT 35.250 426.459 Td /F1 9.8 Tf [(signal at 180 kDa. \(c\) Tumor tissue samples from four different canine mammary carcinoma patients also displayed a Col-1 )] TJ ET BT 35.250 412.723 Td /F1 9.8 Tf [(reactive protein band. Samples from dog 1 to 3 showed positive signals at 60 kDa and 120 kDa, respectively, whereas the )] TJ ET BT 35.250 398.986 Td /F1 9.8 Tf [(protein detected in the sample from dog 4 had an apparent molecular weight of approximately 180 kDa. \(d\) Total lysates )] TJ ET BT 35.250 385.250 Td /F1 9.8 Tf [(derived from TLM-1 cells transfected with canine CEACAM23, CEACAM24, CEACAM25, CEACAM28, CEACAM30, )] TJ ET BT 35.250 371.514 Td /F1 9.8 Tf [(CEACAM1 \(lane 1-6\) or human CEACAM5 cDNA \(lane 7\) were blotted. Col-1 did not bind to any of the tested canine )] TJ ET BT 35.250 357.778 Td /F1 9.8 Tf [(variants, but only to cells transfected with human CEACAM5 cDNA. Murine monoclonal antibody Col-1 \(i\) or IgG2a isotype )] TJ ET BT 35.250 344.041 Td /F1 9.8 Tf [(control antibody \(ii\) were detected by HRP conjugated anti-mouse antibody.)] TJ ET Q BT 26.250 305.520 Td /F6 9.8 Tf [(Intracellular binding in flow cytometry and no effects on growth by Col-1)] TJ ET BT 26.250 286.116 Td /F1 9.8 Tf [(To verify the expression of the epitope recognized by Col-1 in canine cancer cell lines we performed flow cytometry assays. In )] TJ ET BT 26.250 274.211 Td /F1 9.8 Tf [(contrast to human HT29 cells which showed strong membranous Col-1 binding, all tested canine cells remained negative \(Fig. )] TJ ET BT 26.250 262.306 Td /F1 9.8 Tf [(3a\). However, specific cytoplasmic Col-1 staining was revealed after permeabilizing CF33 and CF41, and weak in Sh1b and )] TJ ET BT 26.250 250.401 Td /F1 9.8 Tf [(P114 cells \(Fig. 3b\). Staining with the isotype control antibody remained negative in all intracellular and surface expression )] TJ ET BT 26.250 238.497 Td /F1 9.8 Tf [(assays. In agreement with the Western blotting experiments these results confirmed that Col-1 binds to a specific target in )] TJ ET BT 26.250 226.592 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines.)] TJ ET 0.965 0.965 0.965 rg 26.250 70.556 555.000 146.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 216.711 m 581.250 216.711 l 581.250 215.961 l 26.250 215.961 l f q 112.500 0 0 112.500 35.250 94.461 cm /I5 Do Q q 35.250 70.556 537.000 17.905 re W n Q Q q 15.000 70.556 577.500 706.444 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(The presence of a Col-1 binding signal in canine mammary cancer cells was confirmed by Western blot analysis using reducing )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(conditions. In cell lysates of CF33 and CF41 cells, but not in Sh1b and P114 cells, we observed a specific protein band at 60 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(kDa, whereas staining with isotype control antibody remained negative \(Fig. 2b\). The result was confirmed by blotting lysa tes )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(from spontaneous mammary carcinoma of canine patients that had proven positive for Col-1 reactivity by immunohistochemistry )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(\(Fig. 2c\). In 3 out of 4 samples from dog patients, Col-1 binding was revealed at 60 kDa and weaker at 120 kDa. In dog 4, like in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(human HT29 colon cancer cells \(Fig. 2b\), Col-1 binding was observed at approximately 180 kDa corresponding to the molecular )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(weight of human CEACAM5. Next we investigated whether Col-1 would recognize important members of the canine CEACAM )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(family. We selected canine TLM-1 melanoma cells as host for transfection and optimal species-specific expression of canine )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(CEACAM1, -23, -24, -25, -28 and -30, all belonging to the canine CEACAM1 family. Col-1 binding was only found in cells )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(transfected with human CEACAM5 but not in cells transfected with any of the canine CEACAMs \(Fig. 2d\). These data indicate )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(that Col-1 does not crossreact with the above-mentioned canine CEACAM isoforms.)] TJ ET 0.965 0.965 0.965 rg 26.250 322.544 555.000 316.004 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.548 m 581.250 638.548 l 581.250 637.798 l 26.250 637.798 l f 26.250 322.544 m 581.250 322.544 l 581.250 323.294 l 26.250 323.294 l f q 112.500 0 0 112.500 35.250 516.298 cm /I4 Do Q q 35.250 333.794 537.000 176.504 re W n 0.271 0.267 0.267 rg BT 35.250 500.774 Td /F4 9.8 Tf [(Fig. 2: Col-1 is immunoreactive in canine mammary cancer.)] TJ ET BT 35.250 481.404 Td /F1 9.8 Tf [(\(a\) Sections of canine tumor specimens were stained immunohistochemically with Col-1 antibody. The same region of the )] TJ ET BT 35.250 467.668 Td /F1 9.8 Tf [(tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a 40X/1.3 oil objective lens. \(b\) Western blot analysis )] TJ ET BT 35.250 453.931 Td /F1 9.8 Tf [(revealed a Col-1 immunoreactive signal in canine mammary carcinoma cells CF33 and CF41 at approximately 60 kDa. )] TJ ET BT 35.250 440.195 Td /F1 9.8 Tf [(P114 and Sh1b remained negative. Human colon cancer cells HT29 served as positive control showing a strong Col-1 )] TJ ET BT 35.250 426.459 Td /F1 9.8 Tf [(signal at 180 kDa. \(c\) Tumor tissue samples from four different canine mammary carcinoma patients also displayed a Col-1 )] TJ ET BT 35.250 412.723 Td /F1 9.8 Tf [(reactive protein band. Samples from dog 1 to 3 showed positive signals at 60 kDa and 120 kDa, respectively, whereas the )] TJ ET BT 35.250 398.986 Td /F1 9.8 Tf [(protein detected in the sample from dog 4 had an apparent molecular weight of approximately 180 kDa. \(d\) Total lysates )] TJ ET BT 35.250 385.250 Td /F1 9.8 Tf [(derived from TLM-1 cells transfected with canine CEACAM23, CEACAM24, CEACAM25, CEACAM28, CEACAM30, )] TJ ET BT 35.250 371.514 Td /F1 9.8 Tf [(CEACAM1 \(lane 1-6\) or human CEACAM5 cDNA \(lane 7\) were blotted. Col-1 did not bind to any of the tested canine )] TJ ET BT 35.250 357.778 Td /F1 9.8 Tf [(variants, but only to cells transfected with human CEACAM5 cDNA. Murine monoclonal antibody Col-1 \(i\) or IgG2a isotype )] TJ ET BT 35.250 344.041 Td /F1 9.8 Tf [(control antibody \(ii\) were detected by HRP conjugated anti-mouse antibody.)] TJ ET Q BT 26.250 305.520 Td /F6 9.8 Tf [(Intracellular binding in flow cytometry and no effects on growth by Col-1)] TJ ET BT 26.250 286.116 Td /F1 9.8 Tf [(To verify the expression of the epitope recognized by Col-1 in canine cancer cell lines we performed flow cytometry assays. In )] TJ ET BT 26.250 274.211 Td /F1 9.8 Tf [(contrast to human HT29 cells which showed strong membranous Col-1 binding, all tested canine cells remained negative \(Fig. )] TJ ET BT 26.250 262.306 Td /F1 9.8 Tf [(3a\). However, specific cytoplasmic Col-1 staining was revealed after permeabilizing CF33 and CF41, and weak in Sh1b and )] TJ ET BT 26.250 250.401 Td /F1 9.8 Tf [(P114 cells \(Fig. 3b\). Staining with the isotype control antibody remained negative in all intracellular and surface expression )] TJ ET BT 26.250 238.497 Td /F1 9.8 Tf [(assays. In agreement with the Western blotting experiments these results confirmed that Col-1 binds to a specific target in )] TJ ET BT 26.250 226.592 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines.)] TJ ET 0.965 0.965 0.965 rg 26.250 70.556 555.000 146.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 216.711 m 581.250 216.711 l 581.250 215.961 l 26.250 215.961 l f q 112.500 0 0 112.500 35.250 94.461 cm /I5 Do Q q 35.250 70.556 537.000 17.905 re W n Q Q q 15.000 70.556 577.500 706.444 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(The presence of a Col-1 binding signal in canine mammary cancer cells was confirmed by Western blot analysis using reducing )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(conditions. In cell lysates of CF33 and CF41 cells, but not in Sh1b and P114 cells, we observed a specific protein band at 60 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(kDa, whereas staining with isotype control antibody remained negative \(Fig. 2b\). The result was confirmed by blotting lysa tes )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(from spontaneous mammary carcinoma of canine patients that had proven positive for Col-1 reactivity by immunohistochemistry )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(\(Fig. 2c\). In 3 out of 4 samples from dog patients, Col-1 binding was revealed at 60 kDa and weaker at 120 kDa. In dog 4, like in )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(human HT29 colon cancer cells \(Fig. 2b\), Col-1 binding was observed at approximately 180 kDa corresponding to the molecular )] TJ ET BT 26.250 696.048 Td /F1 9.8 Tf [(weight of human CEACAM5. Next we investigated whether Col-1 would recognize important members of the canine CEACAM )] TJ ET BT 26.250 684.143 Td /F1 9.8 Tf [(family. We selected canine TLM-1 melanoma cells as host for transfection and optimal species-specific expression of canine )] TJ ET BT 26.250 672.238 Td /F1 9.8 Tf [(CEACAM1, -23, -24, -25, -28 and -30, all belonging to the canine CEACAM1 family. Col-1 binding was only found in cells )] TJ ET BT 26.250 660.333 Td /F1 9.8 Tf [(transfected with human CEACAM5 but not in cells transfected with any of the canine CEACAMs \(Fig. 2d\). These data indicate )] TJ ET BT 26.250 648.429 Td /F1 9.8 Tf [(that Col-1 does not crossreact with the above-mentioned canine CEACAM isoforms.)] TJ ET 0.965 0.965 0.965 rg 26.250 322.544 555.000 316.004 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.548 m 581.250 638.548 l 581.250 637.798 l 26.250 637.798 l f 26.250 322.544 m 581.250 322.544 l 581.250 323.294 l 26.250 323.294 l f q 112.500 0 0 112.500 35.250 516.298 cm /I4 Do Q q 35.250 333.794 537.000 176.504 re W n 0.271 0.267 0.267 rg BT 35.250 500.774 Td /F4 9.8 Tf [(Fig. 2: Col-1 is immunoreactive in canine mammary cancer.)] TJ ET BT 35.250 481.404 Td /F1 9.8 Tf [(\(a\) Sections of canine tumor specimens were stained immunohistochemically with Col-1 antibody. The same region of the )] TJ ET BT 35.250 467.668 Td /F1 9.8 Tf [(tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a 40X/1.3 oil objective lens. \(b\) Western blot analysis )] TJ ET BT 35.250 453.931 Td /F1 9.8 Tf [(revealed a Col-1 immunoreactive signal in canine mammary carcinoma cells CF33 and CF41 at approximately 60 kDa. )] TJ ET BT 35.250 440.195 Td /F1 9.8 Tf [(P114 and Sh1b remained negative. Human colon cancer cells HT29 served as positive control showing a strong Col-1 )] TJ ET BT 35.250 426.459 Td /F1 9.8 Tf [(signal at 180 kDa. \(c\) Tumor tissue samples from four different canine mammary carcinoma patients also displayed a Col-1 )] TJ ET BT 35.250 412.723 Td /F1 9.8 Tf [(reactive protein band. Samples from dog 1 to 3 showed positive signals at 60 kDa and 120 kDa, respectively, whereas the )] TJ ET BT 35.250 398.986 Td /F1 9.8 Tf [(protein detected in the sample from dog 4 had an apparent molecular weight of approximately 180 kDa. \(d\) Total lysates )] TJ ET BT 35.250 385.250 Td /F1 9.8 Tf [(derived from TLM-1 cells transfected with canine CEACAM23, CEACAM24, CEACAM25, CEACAM28, CEACAM30, )] TJ ET BT 35.250 371.514 Td /F1 9.8 Tf [(CEACAM1 \(lane 1-6\) or human CEACAM5 cDNA \(lane 7\) were blotted. Col-1 did not bind to any of the tested canine )] TJ ET BT 35.250 357.778 Td /F1 9.8 Tf [(variants, but only to cells transfected with human CEACAM5 cDNA. Murine monoclonal antibody Col-1 \(i\) or IgG2a isotype )] TJ ET BT 35.250 344.041 Td /F1 9.8 Tf [(control antibody \(ii\) were detected by HRP conjugated anti-mouse antibody.)] TJ ET Q BT 26.250 305.520 Td /F6 9.8 Tf [(Intracellular binding in flow cytometry and no effects on growth by Col-1)] TJ ET BT 26.250 286.116 Td /F1 9.8 Tf [(To verify the expression of the epitope recognized by Col-1 in canine cancer cell lines we performed flow cytometry assays. In )] TJ ET BT 26.250 274.211 Td /F1 9.8 Tf [(contrast to human HT29 cells which showed strong membranous Col-1 binding, all tested canine cells remained negative \(Fig. )] TJ ET BT 26.250 262.306 Td /F1 9.8 Tf [(3a\). However, specific cytoplasmic Col-1 staining was revealed after permeabilizing CF33 and CF41, and weak in Sh1b and )] TJ ET BT 26.250 250.401 Td /F1 9.8 Tf [(P114 cells \(Fig. 3b\). Staining with the isotype control antibody remained negative in all intracellular and surface expression )] TJ ET BT 26.250 238.497 Td /F1 9.8 Tf [(assays. In agreement with the Western blotting experiments these results confirmed that Col-1 binds to a specific target in )] TJ ET BT 26.250 226.592 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines.)] TJ ET 0.965 0.965 0.965 rg 26.250 70.556 555.000 146.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 216.711 m 581.250 216.711 l 581.250 215.961 l 26.250 215.961 l f q 112.500 0 0 112.500 35.250 94.461 cm /I5 Do Q q 35.250 70.556 537.000 17.905 re W n Q Q q 112.500 0 0 112.500 35.250 516.298 cm /I4 Do Q q 112.500 0 0 112.500 35.250 94.461 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 289 0 obj << /Type /Annot /Subtype /Link /A 290 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 516.2977 147.7500 628.7977 ] >> endobj 290 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/treeoflife/files/2011/11/fig.2weichselbaumer-comp.jpg) >> endobj 291 0 obj << /Type /XObject /Subtype /Image /Width 150 /Height 150 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 7184>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   " }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?R2( dP;ёI@ EdP 2(;&17Pz|r9-QFEQFEqEdPdQ@e?iޕs[ǨZ?<\m )_mÔ_ww 1M|ugLX1c\e\-!nR^}s^<ҌlR8H)S?\  ]d8c6pGni O Y!Y\0#F<Ө-!4@ yX9oe8^g/^0e$|,k*8LWĺMv->sc#+!\:ݴ4Yۺm" \ q\I#a❢ϲ#< ˸NᜯNi1M_㼵oZ} Y :5~%ӡ ]rllZs1L̸;Hn`<=tXwh4C+pGښ;F[+cJ,uSsas2pG.GgcIspRQ4F8cG!GlWӟD@bdU᱓]1{i,bX7ܤnS=:_7֑+jiWr" :-ԧ[*>'uDMCQ[pyU,w;pX5GA{bYyP[YT:&Ш]*C#ZޭsU?3UP[%Ho~^{ij47 9ʱ˒FnkoR!vb`* A9z&hZBPlR9.F !B{Ίjx^Εjzikq] ܪV ~qמk;J{Icsj$hdSá$a0k[Mq#j F͵}qw%,KEyx@:HS'FCI!]|g.tKAo i%8-@ Q@)~5⋉Q q'+ӵ+-L\7ͱR.LjN0Z~=zgt YW #}G^qˉlni/1]29_/8ԕ*n]YnĻdYFC  JɆɵM?LҴ-v$7,ez*I*"Vk[u]y?)Kmm#+Js=ʪѹe<$9SOuI Hfyb9;qZqzmΚ}L9Bnc]!eF8ep3V庎!pA ^:`ӫ(6Һ.pRVlo5#snBd6rO 瓜~ygᵯBpQ!|g{l0J߯;pʹ$w_c~Γ z|0$6[*szUUu1tg?m5` l͸15g+,vȪchX$Ev= z p̖-ğd;h+)偿 d2lA$p^uYyڜZ,|$Gq7mcW DA]Gfυ +9R d* [3|[ 3#6?qm7 XƠV`W4؍;Eb\ۊ+ſ kvX ɴ`{~x4P#$OG!QaT;eßԠ/$18n}y<,7 ;yqsn-Dj$)bha#Cm$v" jPlQ2W9}q{{ڂ"V,d IsڜQuE4QuC,dt 8<ҥPkn}.s,E{  #9(Ǧ[ 1!^9VRI6q𯶭"i%@'eLت~<3j&$ s7`tXT. 7QfXt!gv` dM;+c ŷr#v+숾-'I9ݞ֢;q/Lnݜ{t~Rۢ |TU|%y#*pTd5_A/ӠLHzϹ$Ƶׂ]; rN>k4k-l [ke%jIֻ(P;Njy-3MkiKi 䜱A}7>JMOծ 1RmV32vxë-VS,鏶1³,-9 AWП 573hX/YSGr%D+pH Aq\gxP~wB8F}(&>nͨ ~U+" =k0vÆFq|  cVi6QIns @/6֒9#T-+'σ2xW $A9J~iA=)ecd酼3rïٯ9e|^Bc4W?t;s̍?J("RQz1F(yjZ0]4;y}N]J9eTf 븣_ ~+47:\Okȵ S:ʑ)r]X,bC*z(sWO kzZ}mw{,qJI`G>R “](e kK6\^^"%%Iڹ31s6OLPqǽ.(5 {dx@OFF;"V!+lW%Kv [pwzXbHA]nf'ql.?\Q\|A7gQk{"5VD%A <{7$Tԫ1dl#_<ſkǥ,_cGH晟(ڨ#Sn$PsڕkN;@Y(9zh&hf};ÚժO%6IZ$#{g2{א"S.dKEɍHцxi)E{mV< )y$($wD'HWZSL I<˺wW^T}5ܞ4wGnO O Nm߉z&>0ٞUpF_X]iyHPF 3Qr;|x\ Nހ1n7 \ˎ5e#_ΐmjWy:bM#jyt  pܨ8gE@"+5xIayEeV*,4(EjfA[?rO$2@B*-gb2 @<_xLtF AbTvxrl`z2222:WXiyѬ,bX"5@ z)?>Q<¹V4xC~;cV?֗'>{z 1n2$(UITe|L|IRc=4fњlFFYH AIvzyX$o)csWed}5^'%i^ǪAY3w(t$ƪǙr$׉4QTQړh@ Lt\()1\PzQLRހ(j:4:s$# a,7JI1@uhzrGo}m%º%I(th|;U6Ґ\(PX2pxأI1@ <jƞݼgs\a (f^YZw2HwkosCwX‘4'ko񢻬Q@(P1(b((P1EbQEQ@(P( endstream endobj 292 0 obj << /Type /Annot /Subtype /Link /A 293 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 94.4610 147.7500 206.9610 ] >> endobj 293 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/treeoflife/files/2011/11/fig.3weichselbaumer-comp.tif) >> endobj 294 0 obj << /Type /XObject /Subtype /Image /Width 150 /Height 150 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 6475>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   " }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q(ҏƏƀ=(ҏƸGU`j욄7W2""% 1gvzWjf19WC$qmA f^bUk I YX $'a@~+-f8iY ~Q.I* waYUtV6̱CkdRjkylm{z[xUUyJ`PGYQYN'sBFCiϔ$s@v t4? 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None of the canine cancer cells \(CF33, CF41, Sh1b and )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(P114\) showed any extracellular Col-1 binding. \(b\) Permeabilization of canine cells revealed a strong intracellular signal in )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(CF33 and CF41, and weak Col-1 binding in Sh1b and P114. Cell line were stained using mAb Col-1 \(bold line\) or isotype )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(matched control antibody \(fine line\) followed by FITC-conjugated secondary antibody.)] TJ ET Q BT 26.250 635.472 Td /F1 9.8 Tf [(The potency of Col-1 to affect proliferation of canine and human tumor cells in vitro was evaluated. CF33 and HT29 were )] TJ ET BT 26.250 623.567 Td /F1 9.8 Tf [(selected due to the highest staining intensity in Western blot and flow cytometry analysis. When Col-1 was applied over a period )] TJ ET BT 26.250 611.662 Td /F1 9.8 Tf [(of 24 h and 48 h, neither in canine nor in human cells significant effects on cell proliferation could be achieved as compared to )] TJ ET BT 26.250 599.757 Td /F1 9.8 Tf [(the isotype control antibody \(data not shown\).)] TJ ET BT 26.250 580.353 Td /F6 9.8 Tf [(Differences in AKT phosphorylation between human and canine cells)] TJ ET BT 26.250 560.948 Td /F1 9.8 Tf [(Our results of the phosphorylation assay revealed that phospho-AKT levels in human HT29 cells were undetectable after serum )] TJ ET BT 26.250 549.043 Td /F1 9.8 Tf [(starvation, but upregulated after both 7.5 and 15 min of Col-1 treatment. In contrast, phospho-AKT levels in canine CF33 cells )] TJ ET BT 26.250 537.138 Td /F1 9.8 Tf [(remained elevated even after prolonged serum starvation and were subsequently downregulated in the presence of Col-1. Two )] TJ ET BT 26.250 525.234 Td /F1 9.8 Tf [(different antibodies against phospho-AKT were used, both showed similar results, indicating phosphorylation at multiple )] TJ ET BT 26.250 513.329 Td /F1 9.8 Tf [(locations. Total AKT levels were unaffected in both cell lines. Under the same conditions, total MAPK as well as MAPK )] TJ ET BT 26.250 501.424 Td /F1 9.8 Tf [(phosphorylation levels remained unchanged in HT29 as well as in CF33 cells \(Fig. 4\).)] TJ ET 0.965 0.965 0.965 rg 26.250 246.052 555.000 245.491 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 491.543 m 581.250 491.543 l 581.250 490.793 l 26.250 490.793 l f 26.250 246.052 m 581.250 246.052 l 581.250 246.802 l 26.250 246.802 l f q 112.500 0 0 112.500 35.250 369.293 cm /I6 Do Q q 35.250 257.302 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 353.769 Td /F4 9.8 Tf [(Fig. 4: Effects of Col-1 treatment on phosphorylation pattern of AKT and MAPK pathways in human versus canine )] TJ ET BT 35.250 341.865 Td /F4 9.8 Tf [(cancer cells.)] TJ ET BT 35.250 322.495 Td /F1 9.8 Tf [(\(a\) In human HT29 colon cancer cells Col-1 treatment after 7.5 min and 15 min induced AKT phosphorylation detected by )] TJ ET BT 35.250 308.758 Td /F1 9.8 Tf [(two different anti-phosphoAKT antibodies \(pAKT\(Ser473\), pAKT\(Thr308\)\). In contrast, a slight decrease of AKT )] TJ ET BT 35.250 295.022 Td /F1 9.8 Tf [(phosphorylation was observed in CF33 cells with both antibodies. Results of densitometric analysis using ImageJ software )] TJ ET BT 35.250 281.286 Td /F1 9.8 Tf [(are given below blots. The signal intensities at 48 h incubation were used as a reference. \(b\) Phosphorylation levels of both )] TJ ET BT 35.250 267.550 Td /F1 9.8 Tf [(MAPK \(ERK1 and ERK2\) isoforms were unaffected in both cell lines by Col-1 treatment.)] TJ ET Q BT 26.250 229.029 Td /F6 9.8 Tf [(High similarity of the human and canine carcinoembryonic antigen receptor)] TJ ET BT 26.250 209.624 Td /F1 9.8 Tf [(Soluble human CEACAM5 binds to a specific CEA receptor \(CEAR\), which is a member of the hnRNP family. By BLAST )] TJ ET BT 26.250 197.719 Td /F1 9.8 Tf [(analysis we found a homologue to human CEAR in the genome of dog, and exemplarily for other vertebrates, in bovine, rat, )] TJ ET BT 26.250 185.814 Td /F1 9.8 Tf [(mouse, chicken, xenopus, salmon and zebrafish. The amino acid identity of CEAR in mammalians was above 98%, and 99% )] TJ ET BT 26.250 173.910 Td /F1 9.8 Tf [(between human and dog, respectively \(Fig. 5a\). Further, homologies of CEARs among chicken, xenopus, salmon and zebrafish )] TJ ET BT 26.250 162.005 Td /F1 9.8 Tf [(ranged lower, from 50 to 73% amino acid identities. The whole sequence alignments are given in supplementary figure 1. Based )] TJ ET BT 26.250 150.100 Td /F1 9.8 Tf [(on these data a phylogenetic tree was constructed for CEAR with MEGA5 \(Fig. 5b\) and approved by ClustalW \(data not shown\), )] TJ ET BT 26.250 138.195 Td /F1 9.8 Tf [(which indicates that the interspecies relationship of this molecule closely follows the principle of evolution: Bovine and canine )] TJ ET BT 26.250 126.291 Td /F1 9.8 Tf [(CEAR are more closely related to human CEAR than any other depicted species \(Fig. 5b\). Immunoblots with anti-human CEAR )] TJ ET BT 26.250 114.386 Td /F1 9.8 Tf [(antibodies directed against two different isoforms identified not only human CEAR in HT29 cells but also canine CEAR in all )] TJ ET BT 26.250 102.481 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines tested \(Fig. 5c\). Next, tissue sections of 10 different Col-1 positive canine mammary )] TJ ET BT 26.250 90.576 Td /F1 9.8 Tf [(cancer patients were immunostained for CEAR. All 10 tissue samples showed positive immunoreactivity with anti-human )] TJ ET BT 26.250 78.672 Td /F1 9.8 Tf [(hnRNP \(M1-M4\) antibody, mainly in the nuclear region of the tumor cells. In Figure 5d, CEAR staining is shown in samples from )] TJ ET BT 26.250 66.767 Td /F1 9.8 Tf [(the same two patients depicted in Figure 2a.)] TJ ET Q q 15.000 56.886 577.500 720.114 re W n 0.965 0.965 0.965 rg 26.250 652.495 555.000 124.505 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 652.495 m 581.250 652.495 l 581.250 653.245 l 26.250 653.245 l f q 35.250 663.745 537.000 113.255 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Binding of Col-1 to canine or human mammary cancer cell lines determined by flow cytometry.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(a\) HT29 showed high surface expression levels as expected. None of the canine cancer cells \(CF33, CF41, Sh1b and )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(P114\) showed any extracellular Col-1 binding. \(b\) Permeabilization of canine cells revealed a strong intracellular signal in )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(CF33 and CF41, and weak Col-1 binding in Sh1b and P114. Cell line were stained using mAb Col-1 \(bold line\) or isotype )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(matched control antibody \(fine line\) followed by FITC-conjugated secondary antibody.)] TJ ET Q BT 26.250 635.472 Td /F1 9.8 Tf [(The potency of Col-1 to affect proliferation of canine and human tumor cells in vitro was evaluated. CF33 and HT29 were )] TJ ET BT 26.250 623.567 Td /F1 9.8 Tf [(selected due to the highest staining intensity in Western blot and flow cytometry analysis. When Col-1 was applied over a period )] TJ ET BT 26.250 611.662 Td /F1 9.8 Tf [(of 24 h and 48 h, neither in canine nor in human cells significant effects on cell proliferation could be achieved as compared to )] TJ ET BT 26.250 599.757 Td /F1 9.8 Tf [(the isotype control antibody \(data not shown\).)] TJ ET BT 26.250 580.353 Td /F6 9.8 Tf [(Differences in AKT phosphorylation between human and canine cells)] TJ ET BT 26.250 560.948 Td /F1 9.8 Tf [(Our results of the phosphorylation assay revealed that phospho-AKT levels in human HT29 cells were undetectable after serum )] TJ ET BT 26.250 549.043 Td /F1 9.8 Tf [(starvation, but upregulated after both 7.5 and 15 min of Col-1 treatment. In contrast, phospho-AKT levels in canine CF33 cells )] TJ ET BT 26.250 537.138 Td /F1 9.8 Tf [(remained elevated even after prolonged serum starvation and were subsequently downregulated in the presence of Col-1. Two )] TJ ET BT 26.250 525.234 Td /F1 9.8 Tf [(different antibodies against phospho-AKT were used, both showed similar results, indicating phosphorylation at multiple )] TJ ET BT 26.250 513.329 Td /F1 9.8 Tf [(locations. Total AKT levels were unaffected in both cell lines. Under the same conditions, total MAPK as well as MAPK )] TJ ET BT 26.250 501.424 Td /F1 9.8 Tf [(phosphorylation levels remained unchanged in HT29 as well as in CF33 cells \(Fig. 4\).)] TJ ET 0.965 0.965 0.965 rg 26.250 246.052 555.000 245.491 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 491.543 m 581.250 491.543 l 581.250 490.793 l 26.250 490.793 l f 26.250 246.052 m 581.250 246.052 l 581.250 246.802 l 26.250 246.802 l f q 112.500 0 0 112.500 35.250 369.293 cm /I6 Do Q q 35.250 257.302 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 353.769 Td /F4 9.8 Tf [(Fig. 4: Effects of Col-1 treatment on phosphorylation pattern of AKT and MAPK pathways in human versus canine )] TJ ET BT 35.250 341.865 Td /F4 9.8 Tf [(cancer cells.)] TJ ET BT 35.250 322.495 Td /F1 9.8 Tf [(\(a\) In human HT29 colon cancer cells Col-1 treatment after 7.5 min and 15 min induced AKT phosphorylation detected by )] TJ ET BT 35.250 308.758 Td /F1 9.8 Tf [(two different anti-phosphoAKT antibodies \(pAKT\(Ser473\), pAKT\(Thr308\)\). In contrast, a slight decrease of AKT )] TJ ET BT 35.250 295.022 Td /F1 9.8 Tf [(phosphorylation was observed in CF33 cells with both antibodies. Results of densitometric analysis using ImageJ software )] TJ ET BT 35.250 281.286 Td /F1 9.8 Tf [(are given below blots. The signal intensities at 48 h incubation were used as a reference. \(b\) Phosphorylation levels of both )] TJ ET BT 35.250 267.550 Td /F1 9.8 Tf [(MAPK \(ERK1 and ERK2\) isoforms were unaffected in both cell lines by Col-1 treatment.)] TJ ET Q BT 26.250 229.029 Td /F6 9.8 Tf [(High similarity of the human and canine carcinoembryonic antigen receptor)] TJ ET BT 26.250 209.624 Td /F1 9.8 Tf [(Soluble human CEACAM5 binds to a specific CEA receptor \(CEAR\), which is a member of the hnRNP family. By BLAST )] TJ ET BT 26.250 197.719 Td /F1 9.8 Tf [(analysis we found a homologue to human CEAR in the genome of dog, and exemplarily for other vertebrates, in bovine, rat, )] TJ ET BT 26.250 185.814 Td /F1 9.8 Tf [(mouse, chicken, xenopus, salmon and zebrafish. The amino acid identity of CEAR in mammalians was above 98%, and 99% )] TJ ET BT 26.250 173.910 Td /F1 9.8 Tf [(between human and dog, respectively \(Fig. 5a\). Further, homologies of CEARs among chicken, xenopus, salmon and zebrafish )] TJ ET BT 26.250 162.005 Td /F1 9.8 Tf [(ranged lower, from 50 to 73% amino acid identities. The whole sequence alignments are given in supplementary figure 1. Based )] TJ ET BT 26.250 150.100 Td /F1 9.8 Tf [(on these data a phylogenetic tree was constructed for CEAR with MEGA5 \(Fig. 5b\) and approved by ClustalW \(data not shown\), )] TJ ET BT 26.250 138.195 Td /F1 9.8 Tf [(which indicates that the interspecies relationship of this molecule closely follows the principle of evolution: Bovine and canine )] TJ ET BT 26.250 126.291 Td /F1 9.8 Tf [(CEAR are more closely related to human CEAR than any other depicted species \(Fig. 5b\). Immunoblots with anti-human CEAR )] TJ ET BT 26.250 114.386 Td /F1 9.8 Tf [(antibodies directed against two different isoforms identified not only human CEAR in HT29 cells but also canine CEAR in all )] TJ ET BT 26.250 102.481 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines tested \(Fig. 5c\). Next, tissue sections of 10 different Col-1 positive canine mammary )] TJ ET BT 26.250 90.576 Td /F1 9.8 Tf [(cancer patients were immunostained for CEAR. All 10 tissue samples showed positive immunoreactivity with anti-human )] TJ ET BT 26.250 78.672 Td /F1 9.8 Tf [(hnRNP \(M1-M4\) antibody, mainly in the nuclear region of the tumor cells. In Figure 5d, CEAR staining is shown in samples from )] TJ ET BT 26.250 66.767 Td /F1 9.8 Tf [(the same two patients depicted in Figure 2a.)] TJ ET Q q 15.000 56.886 577.500 720.114 re W n 0.965 0.965 0.965 rg 26.250 652.495 555.000 124.505 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 652.495 m 581.250 652.495 l 581.250 653.245 l 26.250 653.245 l f q 35.250 663.745 537.000 113.255 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Binding of Col-1 to canine or human mammary cancer cell lines determined by flow cytometry.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(\(a\) HT29 showed high surface expression levels as expected. None of the canine cancer cells \(CF33, CF41, Sh1b and )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(P114\) showed any extracellular Col-1 binding. \(b\) Permeabilization of canine cells revealed a strong intracellular signal in )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(CF33 and CF41, and weak Col-1 binding in Sh1b and P114. Cell line were stained using mAb Col-1 \(bold line\) or isotype )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(matched control antibody \(fine line\) followed by FITC-conjugated secondary antibody.)] TJ ET Q BT 26.250 635.472 Td /F1 9.8 Tf [(The potency of Col-1 to affect proliferation of canine and human tumor cells in vitro was evaluated. CF33 and HT29 were )] TJ ET BT 26.250 623.567 Td /F1 9.8 Tf [(selected due to the highest staining intensity in Western blot and flow cytometry analysis. When Col-1 was applied over a period )] TJ ET BT 26.250 611.662 Td /F1 9.8 Tf [(of 24 h and 48 h, neither in canine nor in human cells significant effects on cell proliferation could be achieved as compared to )] TJ ET BT 26.250 599.757 Td /F1 9.8 Tf [(the isotype control antibody \(data not shown\).)] TJ ET BT 26.250 580.353 Td /F6 9.8 Tf [(Differences in AKT phosphorylation between human and canine cells)] TJ ET BT 26.250 560.948 Td /F1 9.8 Tf [(Our results of the phosphorylation assay revealed that phospho-AKT levels in human HT29 cells were undetectable after serum )] TJ ET BT 26.250 549.043 Td /F1 9.8 Tf [(starvation, but upregulated after both 7.5 and 15 min of Col-1 treatment. In contrast, phospho-AKT levels in canine CF33 cells )] TJ ET BT 26.250 537.138 Td /F1 9.8 Tf [(remained elevated even after prolonged serum starvation and were subsequently downregulated in the presence of Col-1. Two )] TJ ET BT 26.250 525.234 Td /F1 9.8 Tf [(different antibodies against phospho-AKT were used, both showed similar results, indicating phosphorylation at multiple )] TJ ET BT 26.250 513.329 Td /F1 9.8 Tf [(locations. Total AKT levels were unaffected in both cell lines. Under the same conditions, total MAPK as well as MAPK )] TJ ET BT 26.250 501.424 Td /F1 9.8 Tf [(phosphorylation levels remained unchanged in HT29 as well as in CF33 cells \(Fig. 4\).)] TJ ET 0.965 0.965 0.965 rg 26.250 246.052 555.000 245.491 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 491.543 m 581.250 491.543 l 581.250 490.793 l 26.250 490.793 l f 26.250 246.052 m 581.250 246.052 l 581.250 246.802 l 26.250 246.802 l f q 112.500 0 0 112.500 35.250 369.293 cm /I6 Do Q q 35.250 257.302 537.000 105.991 re W n 0.271 0.267 0.267 rg BT 35.250 353.769 Td /F4 9.8 Tf [(Fig. 4: Effects of Col-1 treatment on phosphorylation pattern of AKT and MAPK pathways in human versus canine )] TJ ET BT 35.250 341.865 Td /F4 9.8 Tf [(cancer cells.)] TJ ET BT 35.250 322.495 Td /F1 9.8 Tf [(\(a\) In human HT29 colon cancer cells Col-1 treatment after 7.5 min and 15 min induced AKT phosphorylation detected by )] TJ ET BT 35.250 308.758 Td /F1 9.8 Tf [(two different anti-phosphoAKT antibodies \(pAKT\(Ser473\), pAKT\(Thr308\)\). In contrast, a slight decrease of AKT )] TJ ET BT 35.250 295.022 Td /F1 9.8 Tf [(phosphorylation was observed in CF33 cells with both antibodies. Results of densitometric analysis using ImageJ software )] TJ ET BT 35.250 281.286 Td /F1 9.8 Tf [(are given below blots. The signal intensities at 48 h incubation were used as a reference. \(b\) Phosphorylation levels of both )] TJ ET BT 35.250 267.550 Td /F1 9.8 Tf [(MAPK \(ERK1 and ERK2\) isoforms were unaffected in both cell lines by Col-1 treatment.)] TJ ET Q BT 26.250 229.029 Td /F6 9.8 Tf [(High similarity of the human and canine carcinoembryonic antigen receptor)] TJ ET BT 26.250 209.624 Td /F1 9.8 Tf [(Soluble human CEACAM5 binds to a specific CEA receptor \(CEAR\), which is a member of the hnRNP family. By BLAST )] TJ ET BT 26.250 197.719 Td /F1 9.8 Tf [(analysis we found a homologue to human CEAR in the genome of dog, and exemplarily for other vertebrates, in bovine, rat, )] TJ ET BT 26.250 185.814 Td /F1 9.8 Tf [(mouse, chicken, xenopus, salmon and zebrafish. The amino acid identity of CEAR in mammalians was above 98%, and 99% )] TJ ET BT 26.250 173.910 Td /F1 9.8 Tf [(between human and dog, respectively \(Fig. 5a\). Further, homologies of CEARs among chicken, xenopus, salmon and zebrafish )] TJ ET BT 26.250 162.005 Td /F1 9.8 Tf [(ranged lower, from 50 to 73% amino acid identities. The whole sequence alignments are given in supplementary figure 1. Based )] TJ ET BT 26.250 150.100 Td /F1 9.8 Tf [(on these data a phylogenetic tree was constructed for CEAR with MEGA5 \(Fig. 5b\) and approved by ClustalW \(data not shown\), )] TJ ET BT 26.250 138.195 Td /F1 9.8 Tf [(which indicates that the interspecies relationship of this molecule closely follows the principle of evolution: Bovine and canine )] TJ ET BT 26.250 126.291 Td /F1 9.8 Tf [(CEAR are more closely related to human CEAR than any other depicted species \(Fig. 5b\). Immunoblots with anti-human CEAR )] TJ ET BT 26.250 114.386 Td /F1 9.8 Tf [(antibodies directed against two different isoforms identified not only human CEAR in HT29 cells but also canine CEAR in all )] TJ ET BT 26.250 102.481 Td /F1 9.8 Tf [(canine mammary carcinoma cell lines tested \(Fig. 5c\). Next, tissue sections of 10 different Col-1 positive canine mammary )] TJ ET BT 26.250 90.576 Td /F1 9.8 Tf [(cancer patients were immunostained for CEAR. All 10 tissue samples showed positive immunoreactivity with anti-human )] TJ ET BT 26.250 78.672 Td /F1 9.8 Tf [(hnRNP \(M1-M4\) antibody, mainly in the nuclear region of the tumor cells. In Figure 5d, CEAR staining is shown in samples from )] TJ ET BT 26.250 66.767 Td /F1 9.8 Tf [(the same two patients depicted in Figure 2a.)] TJ ET Q q 112.500 0 0 112.500 35.250 369.293 cm /I6 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 305 0 obj << /Type /Annot /Subtype /Link /A 306 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 369.2932 147.7500 481.7932 ] >> endobj 306 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/treeoflife/files/2011/11/fig.4weichselbaumer-comp.tif) >> endobj 307 0 obj << /Type /XObject /Subtype /Image /Width 150 /Height 150 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 6114>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   " }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Qǵ~)h:tQxT{WkV9Y>UUq|w 뿘vڶ-|Ѝw{ Kj+&Ec+:,*<ƍzU1@;,{QjUҾ,>:y/`/&/ ڙvrd j? JZO—ԙcڊ(1Kj3Fh(ǵPj? J(qE%M˭3QǦ[]=QeF'rVǃ>?:Euơw^{ƒLp:WB?V-" 7*r>:/ s^յuIgjdS^]oyiz]~;O&!Ү:rFNXq'} |pGOC5x?/[My2[$hϙWh͞+?jo|U:_sGI5=a@bfUe`3NqR=q2xkUӼSc=̈́žfpc`?S@d'iշy5v#I-[&֗HQxVxᵄʊqm'aqq ISnV[,\#͆Wc,P=#<|sss}hhN; y fp@~!?gxJ]J6^6YfS's:cK'n VY>՟ZT*z ߑ}(ޟjZu}I,g\[jG;DpTs;WsM 8x+gZQJM"Re۴zjXu07% Z䚗JtZ]އ3[s^=,FWTST;PڌzֿY D!_i]H~;zYizn\i:cFlvGbH9㌐K&k, O>"ECj h˨ 3E0!|mbi g4in[L[I%C6fLo<[ۮc<yZ-Jj7V4<+OA䏭ym/ t߈W:,Sq?ej/&vf/$`y0|;Ωߘt{gY%w >9w$9}8Ycu[A6iM"TW82㎃im|l>1{ud#4mfC0Y8H>}Qܽȶ2#EopʰʰTi2_z׍.o=s]o1wF7 q@|Og-{i(Đ\[ #qO?skok&^ FvWdv,~kphO&s0nns9qt:O,ilfN{,mlAyS@H_<3ga Z@( x{طVPwI*AdbR2dADzu4V&d"67U+]OJ7m w73y|?եov`dzH, ,[1&7"-0Kb6g*(=YVVzw+YI8;3n2Nh8U+]EKh#E^ $p#N޴PZ6x[M:~ ~񶫱F+&\j:͔7RB$Q80w'תCY)YT\xBt{Kúޭa%Oq7ۂ(idV9\c@׾ͨOլN1]˴mTE.C+%{54xr13V-!ߎ&N>8m>CܣlpdkBKW{a_2k=æ!k? 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It shows that within )] TJ ET BT 35.250 592.384 Td /F1 9.8 Tf [(mammalians canine CEAR is most closely related to human CEAR; other species like bird, frog and fish range lower .\(c\) )] TJ ET BT 35.250 578.647 Td /F1 9.8 Tf [(Two different antibodies specific for human CEAR, rabbit anti-CEAR long antibody \(i\) as well as mouse anti-hnRNP )] TJ ET BT 35.250 564.911 Td /F1 9.8 Tf [(antibody \(ii\) identified a CEAR-specific signal in canine P114, Sh1b, CF41 and CF33 cell lysates at ~70 kDa similar as well )] TJ ET BT 35.250 551.175 Td /F1 9.8 Tf [(as in human HT29 cancer cell lysates serving as a positive control. \(d\) Immunostaining of primary lesions of canine )] TJ ET BT 35.250 537.439 Td /F1 9.8 Tf [(mammary cancer patients which were positive for Col-1 staining was mainly observed in the nuclear region of tumor cells )] TJ ET BT 35.250 523.702 Td /F1 9.8 Tf [(with anti-hnRNP antibody. The same region of the tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a )] TJ ET BT 35.250 509.966 Td /F1 9.8 Tf [(40X/1.3 oil objective lens.)] TJ ET Q BT 26.250 454.247 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 434.293 Td /F1 9.8 Tf [(Using Col-1 monoclonal anti-CEACAM5 antibody we have previously developed a mimotope-based anti-CEACAM5 vaccine for )] TJ ET BT 26.250 422.388 Td /F1 9.8 Tf [(the treatment of human carcinoma. Mimotope vaccination prevented the growth of transplanted Meth-A/CEA tumor cells in a )] TJ ET BT 26.250 410.484 Td /F1 9.8 Tf [(BALB/c mouse model )] TJ ET 0.267 0.267 0.267 rg BT 122.707 410.484 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 138.970 410.484 Td /F1 9.8 Tf [(. Inspired by the concept of comparative oncology we intended to test the applicability of the )] TJ ET BT 26.250 398.579 Td /F1 9.8 Tf [(mimotope vaccine in canine cancer patients. Therefore, we focused here on the canine target molecule of Col-1 being a )] TJ ET BT 26.250 386.674 Td /F1 9.8 Tf [(putative CEACAM homologue. We soon realized the complexity of this approach because the CEACAM protein family exhibits )] TJ ET BT 26.250 374.769 Td /F1 9.8 Tf [(an extreme diversity within different species in vertebrates )] TJ ET 0.267 0.267 0.267 rg BT 279.302 374.769 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 295.565 374.769 Td /F1 9.8 Tf [(. This is connected to the fact that they play a role in multiple )] TJ ET BT 26.250 362.865 Td /F1 9.8 Tf [(processes, including homotypic and heterotypic adhesion, cellular differentiation and as pathogen receptors. In most )] TJ ET BT 26.250 350.960 Td /F1 9.8 Tf [(mammalian orders orthologs to CEACAM1 exist )] TJ ET 0.267 0.267 0.267 rg BT 235.934 350.960 Td /F1 9.8 Tf [([49])] TJ ET 0.271 0.267 0.267 rg BT 252.197 350.960 Td /F1 9.8 Tf [(. The primordial CEACAM1 gene was subject to species-specific gene )] TJ ET BT 26.250 339.055 Td /F1 9.8 Tf [(amplification leading to groups of CEACAM1-related genes which vary considerably between mammalian species. In humans )] TJ ET BT 26.250 327.150 Td /F1 9.8 Tf [(CEACAM5 is a glycosylphosphatidylinositol \(GPI\)-anchored molecule of the CEACAM1-related CEACAMs which can be )] TJ ET BT 26.250 315.246 Td /F1 9.8 Tf [(cleaved by GPI-phospholipase D \(GPI-PLD\), canine CEACAM1-related CEACAMs are mostly ITAM- \(Immunoreceptor Tyrosine-)] TJ ET BT 26.250 303.341 Td /F1 9.8 Tf [(based Activation Motif-\) bearing transmembrane proteins encoded by at least five functional genes )] TJ ET 0.267 0.267 0.267 rg BT 453.281 303.341 Td /F1 9.8 Tf [([50] [51])] TJ ET 0.271 0.267 0.267 rg BT 488.517 303.341 Td /F1 9.8 Tf [(. Interestingly, in the )] TJ ET BT 26.250 291.436 Td /F1 9.8 Tf [(canine genome a CEACAM5 equivalent could not be found )] TJ ET 0.267 0.267 0.267 rg BT 283.679 291.436 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 299.942 291.436 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 272.031 Td /F1 9.8 Tf [(In accordance with previous immunohistochemistry studies )] TJ ET 0.267 0.267 0.267 rg BT 283.084 272.031 Td /F1 9.8 Tf [([35] [36] [37])] TJ ET 0.271 0.267 0.267 rg BT 337.294 272.031 Td /F1 9.8 Tf [( and being aware of the lack of a known canine )] TJ ET BT 26.250 260.127 Td /F1 9.8 Tf [(CEACAM5 ortholog, our immunohistochemical analysis showed specific Col-1 staining in 23 of 30 canine mammary cancer )] TJ ET BT 26.250 248.222 Td /F1 9.8 Tf [(patients. When we probed lysates of 4 primary mammary tumors we found that only one of the samples showed a specific Col-1 )] TJ ET BT 26.250 236.317 Td /F1 9.8 Tf [(signal at the expected molecular weight of 180 kDa of the human CEACAM5 molecule. The human 180 kDa CEACAM5 is built )] TJ ET BT 26.250 224.412 Td /F1 9.8 Tf [(up by 6 immunoglobulin domains and additionally decorated by glycans of the N-glycosylated complex which contribute to about )] TJ ET BT 26.250 212.508 Td /F1 9.8 Tf [(half of the molecules mass )] TJ ET 0.267 0.267 0.267 rg BT 147.628 212.508 Td /F1 9.8 Tf [([30] [52])] TJ ET 0.271 0.267 0.267 rg BT 182.864 212.508 Td /F1 9.8 Tf [(. As in 3 of the 4 tested canine mammary cancer samples faster migrating bands were )] TJ ET BT 26.250 200.603 Td /F1 9.8 Tf [(detected at ~60 and ~120 kDa by Col-1, suggestive that they could represent fragments of the canine 180 kDa CEACAM )] TJ ET BT 26.250 188.698 Td /F1 9.8 Tf [(molecule. Also in the canine mammary carcinoma cell lines CF33 and CF41 60 kDa molecules were specifically identified by )] TJ ET BT 26.250 176.793 Td /F1 9.8 Tf [(Col-1.)] TJ ET BT 26.250 157.389 Td /F1 9.8 Tf [(Our phylogenetic search indicated that in the canine genome CEACAM1-related CEACAMs would be most closely related to )] TJ ET BT 26.250 145.484 Td /F1 9.8 Tf [(human CEACAM5. However, a transfection experiment indicated that most widely expressed canine CEACAM1-related )] TJ ET BT 26.250 133.579 Td /F1 9.8 Tf [(molecules, namely CEACAM1, -23, -24, -25, -28 or -30, were not recognized by Col-1. Having used canine TLM-1 melanoma )] TJ ET BT 26.250 121.674 Td /F1 9.8 Tf [(cells for transfection we exclude that the negative binding was due to incorrect species-specific post-translational modification.)] TJ ET Q q 15.000 32.865 577.500 744.135 re W n 0.965 0.965 0.965 rg 26.250 488.469 555.000 288.531 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 488.469 m 581.250 488.469 l 581.250 489.219 l 26.250 489.219 l f q 112.500 0 0 112.500 35.250 654.750 cm /I7 Do Q q 35.250 499.719 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 639.226 Td /F4 9.8 Tf [(Fig. 5: CEA-receptor \(CEAR\) is phylogenetically highly conserved and expressed in canine mammary carcinoma.)] TJ ET BT 35.250 619.856 Td /F1 9.8 Tf [(\(a\) BLAST analysis revealed more than 98% homology in selected mammalians, and 99% amino acid sequence homology )] TJ ET BT 35.250 606.120 Td /F1 9.8 Tf [(between human and canine CEAR. \(b\) Using MEGA5 a phylogenetic tree was constructed. It shows that within )] TJ ET BT 35.250 592.384 Td /F1 9.8 Tf [(mammalians canine CEAR is most closely related to human CEAR; other species like bird, frog and fish range lower .\(c\) )] TJ ET BT 35.250 578.647 Td /F1 9.8 Tf [(Two different antibodies specific for human CEAR, rabbit anti-CEAR long antibody \(i\) as well as mouse anti-hnRNP )] TJ ET BT 35.250 564.911 Td /F1 9.8 Tf [(antibody \(ii\) identified a CEAR-specific signal in canine P114, Sh1b, CF41 and CF33 cell lysates at ~70 kDa similar as well )] TJ ET BT 35.250 551.175 Td /F1 9.8 Tf [(as in human HT29 cancer cell lysates serving as a positive control. \(d\) Immunostaining of primary lesions of canine )] TJ ET BT 35.250 537.439 Td /F1 9.8 Tf [(mammary cancer patients which were positive for Col-1 staining was mainly observed in the nuclear region of tumor cells )] TJ ET BT 35.250 523.702 Td /F1 9.8 Tf [(with anti-hnRNP antibody. The same region of the tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a )] TJ ET BT 35.250 509.966 Td /F1 9.8 Tf [(40X/1.3 oil objective lens.)] TJ ET Q BT 26.250 454.247 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 434.293 Td /F1 9.8 Tf [(Using Col-1 monoclonal anti-CEACAM5 antibody we have previously developed a mimotope-based anti-CEACAM5 vaccine for )] TJ ET BT 26.250 422.388 Td /F1 9.8 Tf [(the treatment of human carcinoma. Mimotope vaccination prevented the growth of transplanted Meth-A/CEA tumor cells in a )] TJ ET BT 26.250 410.484 Td /F1 9.8 Tf [(BALB/c mouse model )] TJ ET 0.267 0.267 0.267 rg BT 122.707 410.484 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 138.970 410.484 Td /F1 9.8 Tf [(. Inspired by the concept of comparative oncology we intended to test the applicability of the )] TJ ET BT 26.250 398.579 Td /F1 9.8 Tf [(mimotope vaccine in canine cancer patients. Therefore, we focused here on the canine target molecule of Col-1 being a )] TJ ET BT 26.250 386.674 Td /F1 9.8 Tf [(putative CEACAM homologue. We soon realized the complexity of this approach because the CEACAM protein family exhibits )] TJ ET BT 26.250 374.769 Td /F1 9.8 Tf [(an extreme diversity within different species in vertebrates )] TJ ET 0.267 0.267 0.267 rg BT 279.302 374.769 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 295.565 374.769 Td /F1 9.8 Tf [(. This is connected to the fact that they play a role in multiple )] TJ ET BT 26.250 362.865 Td /F1 9.8 Tf [(processes, including homotypic and heterotypic adhesion, cellular differentiation and as pathogen receptors. In most )] TJ ET BT 26.250 350.960 Td /F1 9.8 Tf [(mammalian orders orthologs to CEACAM1 exist )] TJ ET 0.267 0.267 0.267 rg BT 235.934 350.960 Td /F1 9.8 Tf [([49])] TJ ET 0.271 0.267 0.267 rg BT 252.197 350.960 Td /F1 9.8 Tf [(. The primordial CEACAM1 gene was subject to species-specific gene )] TJ ET BT 26.250 339.055 Td /F1 9.8 Tf [(amplification leading to groups of CEACAM1-related genes which vary considerably between mammalian species. In humans )] TJ ET BT 26.250 327.150 Td /F1 9.8 Tf [(CEACAM5 is a glycosylphosphatidylinositol \(GPI\)-anchored molecule of the CEACAM1-related CEACAMs which can be )] TJ ET BT 26.250 315.246 Td /F1 9.8 Tf [(cleaved by GPI-phospholipase D \(GPI-PLD\), canine CEACAM1-related CEACAMs are mostly ITAM- \(Immunoreceptor Tyrosine-)] TJ ET BT 26.250 303.341 Td /F1 9.8 Tf [(based Activation Motif-\) bearing transmembrane proteins encoded by at least five functional genes )] TJ ET 0.267 0.267 0.267 rg BT 453.281 303.341 Td /F1 9.8 Tf [([50] [51])] TJ ET 0.271 0.267 0.267 rg BT 488.517 303.341 Td /F1 9.8 Tf [(. Interestingly, in the )] TJ ET BT 26.250 291.436 Td /F1 9.8 Tf [(canine genome a CEACAM5 equivalent could not be found )] TJ ET 0.267 0.267 0.267 rg BT 283.679 291.436 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 299.942 291.436 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 272.031 Td /F1 9.8 Tf [(In accordance with previous immunohistochemistry studies )] TJ ET 0.267 0.267 0.267 rg BT 283.084 272.031 Td /F1 9.8 Tf [([35] [36] [37])] TJ ET 0.271 0.267 0.267 rg BT 337.294 272.031 Td /F1 9.8 Tf [( and being aware of the lack of a known canine )] TJ ET BT 26.250 260.127 Td /F1 9.8 Tf [(CEACAM5 ortholog, our immunohistochemical analysis showed specific Col-1 staining in 23 of 30 canine mammary cancer )] TJ ET BT 26.250 248.222 Td /F1 9.8 Tf [(patients. When we probed lysates of 4 primary mammary tumors we found that only one of the samples showed a specific Col-1 )] TJ ET BT 26.250 236.317 Td /F1 9.8 Tf [(signal at the expected molecular weight of 180 kDa of the human CEACAM5 molecule. The human 180 kDa CEACAM5 is built )] TJ ET BT 26.250 224.412 Td /F1 9.8 Tf [(up by 6 immunoglobulin domains and additionally decorated by glycans of the N-glycosylated complex which contribute to about )] TJ ET BT 26.250 212.508 Td /F1 9.8 Tf [(half of the molecules mass )] TJ ET 0.267 0.267 0.267 rg BT 147.628 212.508 Td /F1 9.8 Tf [([30] [52])] TJ ET 0.271 0.267 0.267 rg BT 182.864 212.508 Td /F1 9.8 Tf [(. As in 3 of the 4 tested canine mammary cancer samples faster migrating bands were )] TJ ET BT 26.250 200.603 Td /F1 9.8 Tf [(detected at ~60 and ~120 kDa by Col-1, suggestive that they could represent fragments of the canine 180 kDa CEACAM )] TJ ET BT 26.250 188.698 Td /F1 9.8 Tf [(molecule. Also in the canine mammary carcinoma cell lines CF33 and CF41 60 kDa molecules were specifically identified by )] TJ ET BT 26.250 176.793 Td /F1 9.8 Tf [(Col-1.)] TJ ET BT 26.250 157.389 Td /F1 9.8 Tf [(Our phylogenetic search indicated that in the canine genome CEACAM1-related CEACAMs would be most closely related to )] TJ ET BT 26.250 145.484 Td /F1 9.8 Tf [(human CEACAM5. However, a transfection experiment indicated that most widely expressed canine CEACAM1-related )] TJ ET BT 26.250 133.579 Td /F1 9.8 Tf [(molecules, namely CEACAM1, -23, -24, -25, -28 or -30, were not recognized by Col-1. Having used canine TLM-1 melanoma )] TJ ET BT 26.250 121.674 Td /F1 9.8 Tf [(cells for transfection we exclude that the negative binding was due to incorrect species-specific post-translational modification.)] TJ ET Q q 15.000 32.865 577.500 744.135 re W n 0.965 0.965 0.965 rg 26.250 488.469 555.000 288.531 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 488.469 m 581.250 488.469 l 581.250 489.219 l 26.250 489.219 l f q 112.500 0 0 112.500 35.250 654.750 cm /I7 Do Q q 35.250 499.719 537.000 149.031 re W n 0.271 0.267 0.267 rg BT 35.250 639.226 Td /F4 9.8 Tf [(Fig. 5: CEA-receptor \(CEAR\) is phylogenetically highly conserved and expressed in canine mammary carcinoma.)] TJ ET BT 35.250 619.856 Td /F1 9.8 Tf [(\(a\) BLAST analysis revealed more than 98% homology in selected mammalians, and 99% amino acid sequence homology )] TJ ET BT 35.250 606.120 Td /F1 9.8 Tf [(between human and canine CEAR. \(b\) Using MEGA5 a phylogenetic tree was constructed. It shows that within )] TJ ET BT 35.250 592.384 Td /F1 9.8 Tf [(mammalians canine CEAR is most closely related to human CEAR; other species like bird, frog and fish range lower .\(c\) )] TJ ET BT 35.250 578.647 Td /F1 9.8 Tf [(Two different antibodies specific for human CEAR, rabbit anti-CEAR long antibody \(i\) as well as mouse anti-hnRNP )] TJ ET BT 35.250 564.911 Td /F1 9.8 Tf [(antibody \(ii\) identified a CEAR-specific signal in canine P114, Sh1b, CF41 and CF33 cell lysates at ~70 kDa similar as well )] TJ ET BT 35.250 551.175 Td /F1 9.8 Tf [(as in human HT29 cancer cell lysates serving as a positive control. \(d\) Immunostaining of primary lesions of canine )] TJ ET BT 35.250 537.439 Td /F1 9.8 Tf [(mammary cancer patients which were positive for Col-1 staining was mainly observed in the nuclear region of tumor cells )] TJ ET BT 35.250 523.702 Td /F1 9.8 Tf [(with anti-hnRNP antibody. The same region of the tumor tissue sample was evaluated by microscopy with a 20X/0.5 and a )] TJ ET BT 35.250 509.966 Td /F1 9.8 Tf [(40X/1.3 oil objective lens.)] TJ ET Q BT 26.250 454.247 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 434.293 Td /F1 9.8 Tf [(Using Col-1 monoclonal anti-CEACAM5 antibody we have previously developed a mimotope-based anti-CEACAM5 vaccine for )] TJ ET BT 26.250 422.388 Td /F1 9.8 Tf [(the treatment of human carcinoma. Mimotope vaccination prevented the growth of transplanted Meth-A/CEA tumor cells in a )] TJ ET BT 26.250 410.484 Td /F1 9.8 Tf [(BALB/c mouse model )] TJ ET 0.267 0.267 0.267 rg BT 122.707 410.484 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 138.970 410.484 Td /F1 9.8 Tf [(. Inspired by the concept of comparative oncology we intended to test the applicability of the )] TJ ET BT 26.250 398.579 Td /F1 9.8 Tf [(mimotope vaccine in canine cancer patients. Therefore, we focused here on the canine target molecule of Col-1 being a )] TJ ET BT 26.250 386.674 Td /F1 9.8 Tf [(putative CEACAM homologue. We soon realized the complexity of this approach because the CEACAM protein family exhibits )] TJ ET BT 26.250 374.769 Td /F1 9.8 Tf [(an extreme diversity within different species in vertebrates )] TJ ET 0.267 0.267 0.267 rg BT 279.302 374.769 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 295.565 374.769 Td /F1 9.8 Tf [(. This is connected to the fact that they play a role in multiple )] TJ ET BT 26.250 362.865 Td /F1 9.8 Tf [(processes, including homotypic and heterotypic adhesion, cellular differentiation and as pathogen receptors. In most )] TJ ET BT 26.250 350.960 Td /F1 9.8 Tf [(mammalian orders orthologs to CEACAM1 exist )] TJ ET 0.267 0.267 0.267 rg BT 235.934 350.960 Td /F1 9.8 Tf [([49])] TJ ET 0.271 0.267 0.267 rg BT 252.197 350.960 Td /F1 9.8 Tf [(. The primordial CEACAM1 gene was subject to species-specific gene )] TJ ET BT 26.250 339.055 Td /F1 9.8 Tf [(amplification leading to groups of CEACAM1-related genes which vary considerably between mammalian species. In humans )] TJ ET BT 26.250 327.150 Td /F1 9.8 Tf [(CEACAM5 is a glycosylphosphatidylinositol \(GPI\)-anchored molecule of the CEACAM1-related CEACAMs which can be )] TJ ET BT 26.250 315.246 Td /F1 9.8 Tf [(cleaved by GPI-phospholipase D \(GPI-PLD\), canine CEACAM1-related CEACAMs are mostly ITAM- \(Immunoreceptor Tyrosine-)] TJ ET BT 26.250 303.341 Td /F1 9.8 Tf [(based Activation Motif-\) bearing transmembrane proteins encoded by at least five functional genes )] TJ ET 0.267 0.267 0.267 rg BT 453.281 303.341 Td /F1 9.8 Tf [([50] [51])] TJ ET 0.271 0.267 0.267 rg BT 488.517 303.341 Td /F1 9.8 Tf [(. Interestingly, in the )] TJ ET BT 26.250 291.436 Td /F1 9.8 Tf [(canine genome a CEACAM5 equivalent could not be found )] TJ ET 0.267 0.267 0.267 rg BT 283.679 291.436 Td /F1 9.8 Tf [([44])] TJ ET 0.271 0.267 0.267 rg BT 299.942 291.436 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 272.031 Td /F1 9.8 Tf [(In accordance with previous immunohistochemistry studies )] TJ ET 0.267 0.267 0.267 rg BT 283.084 272.031 Td /F1 9.8 Tf [([35] [36] [37])] TJ ET 0.271 0.267 0.267 rg BT 337.294 272.031 Td /F1 9.8 Tf [( and being aware of the lack of a known canine )] TJ ET BT 26.250 260.127 Td /F1 9.8 Tf [(CEACAM5 ortholog, our immunohistochemical analysis showed specific Col-1 staining in 23 of 30 canine mammary cancer )] TJ ET BT 26.250 248.222 Td /F1 9.8 Tf [(patients. When we probed lysates of 4 primary mammary tumors we found that only one of the samples showed a specific Col-1 )] TJ ET BT 26.250 236.317 Td /F1 9.8 Tf [(signal at the expected molecular weight of 180 kDa of the human CEACAM5 molecule. The human 180 kDa CEACAM5 is built )] TJ ET BT 26.250 224.412 Td /F1 9.8 Tf [(up by 6 immunoglobulin domains and additionally decorated by glycans of the N-glycosylated complex which contribute to about )] TJ ET BT 26.250 212.508 Td /F1 9.8 Tf [(half of the molecules mass )] TJ ET 0.267 0.267 0.267 rg BT 147.628 212.508 Td /F1 9.8 Tf [([30] [52])] TJ ET 0.271 0.267 0.267 rg BT 182.864 212.508 Td /F1 9.8 Tf [(. As in 3 of the 4 tested canine mammary cancer samples faster migrating bands were )] TJ ET BT 26.250 200.603 Td /F1 9.8 Tf [(detected at ~60 and ~120 kDa by Col-1, suggestive that they could represent fragments of the canine 180 kDa CEACAM )] TJ ET BT 26.250 188.698 Td /F1 9.8 Tf [(molecule. Also in the canine mammary carcinoma cell lines CF33 and CF41 60 kDa molecules were specifically identified by )] TJ ET BT 26.250 176.793 Td /F1 9.8 Tf [(Col-1.)] TJ ET BT 26.250 157.389 Td /F1 9.8 Tf [(Our phylogenetic search indicated that in the canine genome CEACAM1-related CEACAMs would be most closely related to )] TJ ET BT 26.250 145.484 Td /F1 9.8 Tf [(human CEACAM5. However, a transfection experiment indicated that most widely expressed canine CEACAM1-related )] TJ ET BT 26.250 133.579 Td /F1 9.8 Tf [(molecules, namely CEACAM1, -23, -24, -25, -28 or -30, were not recognized by Col-1. 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expression of the Col-1 target was further demonstrated by flow cytometry where, in contrast to human HT29 cells with )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(membranous and cytoplasmic expression, the antigen was detected only intracellularly in the canine mammary CF41 and CF33 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cells. This is in accordance with the present immunohistochemistry in canine mammary tumor samples, and previous reports on )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(cytoplasmic CEA staining in human head and neck )] TJ ET 0.267 0.267 0.267 rg BT 248.433 731.762 Td /F1 9.8 Tf [([40])] TJ ET 0.271 0.267 0.267 rg BT 264.696 731.762 Td /F1 9.8 Tf [( and colorectal cancer )] TJ ET 0.267 0.267 0.267 rg BT 362.781 731.762 Td /F1 9.8 Tf [([53])] TJ ET 0.271 0.267 0.267 rg BT 379.044 731.762 Td /F1 9.8 Tf [(. As it has been repeatedly demonstrated that )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(also intracellular targets can be approached by natural and engineered antibodies )] TJ ET 0.267 0.267 0.267 rg BT 381.228 719.857 Td /F1 9.8 Tf [([54] [55] [56])] TJ ET 0.271 0.267 0.267 rg BT 435.438 719.857 Td /F1 9.8 Tf [(, this fact would not contradict )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(the principle of antibody-mediated targeting.)] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(To investigate the potential functional relevance of Col-1 targeting in human or canine cancer we performed growth inhibition )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(and signal transduction assays. Col-1 treatment did neither induce growth inhibition in canine CF33 mammary cancer cells nor )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(in human HT29 colon cancer cells. In line with that we previously ascribed the cytotoxic effect of a Col-1 mimotope vaccine to )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(antibody-mediated tumoricidity )] TJ ET 0.267 0.267 0.267 rg BT 161.171 652.833 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 177.434 652.833 Td /F1 9.8 Tf [(. Our data from signal transduction assays indicate that Col-1 targeting of human and canine )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(cells leads to divergent effects on AKT phosphorylation, which was increased in human HT29 cells but slightly decreased in )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(canine CF33 cells. Therefore, thorough antibody validation prior to use across species is mandatory. The AKT signaling )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(pathway plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(and cell migration. Possibly, the biological effect of Col-1 targeting may also depend on the activation state of the AKT signaling )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(pathway in the different species. MAPK pathway, likewise being involved in multiple biological effects, such as regulation of )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(gene expression, mitosis, proliferation and cell survival, remained unaffected by Col-1 treatment in both human and canine )] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(cancer cells.)] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(A homolog of heterogeneous nuclear protein M4 \(hnRNP M4\) termed CEA-receptor \(CEAR\) has previously been shown on the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(membrane as well as in the cytoplasm of human colon cancer cells )] TJ ET 0.267 0.267 0.267 rg BT 317.775 538.191 Td /F1 9.8 Tf [([57])] TJ ET 0.271 0.267 0.267 rg BT 334.038 538.191 Td /F1 9.8 Tf [(. The fact that CEAR may co-localize with CEA is )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(suggestive for signaling function of this complex )] TJ ET 0.267 0.267 0.267 rg BT 235.427 526.286 Td /F1 9.8 Tf [([16])] TJ ET 0.271 0.267 0.267 rg BT 251.690 526.286 Td /F1 9.8 Tf [(. We report here for the first time that CEAR is much higher conserved )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(among mammalians than in other vertebrates, like bird, frog, and fish. With 99% amino acid identity, conservation of CEAR is )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(especially pronounced in the human and canine species. Further, CEAR is highly expressed in human as well as canine )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(mammary carcinoma. Using anti-human CEAR antibody, it could not only be detected in all tested canine cancer cell lines but )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(also in 10 out of 10 tissue samples from spontaneous canine mammary cancers. As mentioned above no CEACAM5 equivalent )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(with the potential to act as soluble form has been found in the dog genome so far. Our data show that anti-human CEACAM5 )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(antibody Col-1 specifically detects 180, 120 and 60 kDa molecules in canine cancer cells which are most likely non-related to )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(the canine CEACAM1-related proteins. Future studies will reveal whether these molecules may adapt ligand function to the )] TJ ET BT 26.250 431.048 Td /F1 9.8 Tf [(canine CEAR.)] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(The fact that the inter-species heterogeneity of CEACAM molecules in general was greater than expected was in striking )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(contrast to the high conservation of the CEA receptor molecule between humans and dogs. Our initial expectations that )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(spontaneous mammary carcinoma in dogs could serve as a model for human breast cancer with respect to CEACAM targeting )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(could thus, discordant to the intriguing comparative oncology concept, not be fulfilled. Though, the finding that CEAR is highly )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(homologous between humans and dogs, and specifically expressed in canine mammary cancer might open up new avenues for )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(anticancer developments based on the comparative oncology strategy.)] TJ ET BT 26.250 315.517 Td /F4 12.0 Tf [(Acknowledgments)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(The authors want to thank Philipp Starkl for helpful discussions. Erika Jensen-Jarolim is designated as corresponding author of )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(this manuscript.)] TJ ET BT 26.250 247.055 Td /F4 12.0 Tf [(Funding information)] TJ ET BT 26.250 227.101 Td /F1 9.8 Tf [(This study was supported by the RotePfote \(http://www.rotepfote.at\) grant #FA648A1301. MW is a recipient of a Von Fircks-)] TJ ET BT 26.250 215.196 Td /F1 9.8 Tf [(Scholarship of the University of Veterinary Medicine, Vienna. JS is supported by the CCHD program of the Austrian Science )] TJ ET BT 26.250 203.292 Td /F1 9.8 Tf [(Fund FWF \(#APW01205FW\).)] TJ ET BT 26.250 166.689 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 146.735 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET Q q 15.000 -315.396 577.500 1092.396 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Specific expression of the Col-1 target was further demonstrated by flow cytometry where, in contrast to human HT29 cells with )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(membranous and cytoplasmic expression, the antigen was detected only intracellularly in the canine mammary CF41 and CF33 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cells. This is in accordance with the present immunohistochemistry in canine mammary tumor samples, and previous reports on )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(cytoplasmic CEA staining in human head and neck )] TJ ET 0.267 0.267 0.267 rg BT 248.433 731.762 Td /F1 9.8 Tf [([40])] TJ ET 0.271 0.267 0.267 rg BT 264.696 731.762 Td /F1 9.8 Tf [( and colorectal cancer )] TJ ET 0.267 0.267 0.267 rg BT 362.781 731.762 Td /F1 9.8 Tf [([53])] TJ ET 0.271 0.267 0.267 rg BT 379.044 731.762 Td /F1 9.8 Tf [(. As it has been repeatedly demonstrated that )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(also intracellular targets can be approached by natural and engineered antibodies )] TJ ET 0.267 0.267 0.267 rg BT 381.228 719.857 Td /F1 9.8 Tf [([54] [55] [56])] TJ ET 0.271 0.267 0.267 rg BT 435.438 719.857 Td /F1 9.8 Tf [(, this fact would not contradict )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(the principle of antibody-mediated targeting.)] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(To investigate the potential functional relevance of Col-1 targeting in human or canine cancer we performed growth inhibition )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(and signal transduction assays. Col-1 treatment did neither induce growth inhibition in canine CF33 mammary cancer cells nor )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(in human HT29 colon cancer cells. In line with that we previously ascribed the cytotoxic effect of a Col-1 mimotope vaccine to )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(antibody-mediated tumoricidity )] TJ ET 0.267 0.267 0.267 rg BT 161.171 652.833 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 177.434 652.833 Td /F1 9.8 Tf [(. Our data from signal transduction assays indicate that Col-1 targeting of human and canine )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(cells leads to divergent effects on AKT phosphorylation, which was increased in human HT29 cells but slightly decreased in )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(canine CF33 cells. Therefore, thorough antibody validation prior to use across species is mandatory. The AKT signaling )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(pathway plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(and cell migration. Possibly, the biological effect of Col-1 targeting may also depend on the activation state of the AKT signaling )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(pathway in the different species. MAPK pathway, likewise being involved in multiple biological effects, such as regulation of )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(gene expression, mitosis, proliferation and cell survival, remained unaffected by Col-1 treatment in both human and canine )] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(cancer cells.)] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(A homolog of heterogeneous nuclear protein M4 \(hnRNP M4\) termed CEA-receptor \(CEAR\) has previously been shown on the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(membrane as well as in the cytoplasm of human colon cancer cells )] TJ ET 0.267 0.267 0.267 rg BT 317.775 538.191 Td /F1 9.8 Tf [([57])] TJ ET 0.271 0.267 0.267 rg BT 334.038 538.191 Td /F1 9.8 Tf [(. The fact that CEAR may co-localize with CEA is )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(suggestive for signaling function of this complex )] TJ ET 0.267 0.267 0.267 rg BT 235.427 526.286 Td /F1 9.8 Tf [([16])] TJ ET 0.271 0.267 0.267 rg BT 251.690 526.286 Td /F1 9.8 Tf [(. We report here for the first time that CEAR is much higher conserved )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(among mammalians than in other vertebrates, like bird, frog, and fish. With 99% amino acid identity, conservation of CEAR is )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(especially pronounced in the human and canine species. Further, CEAR is highly expressed in human as well as canine )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(mammary carcinoma. Using anti-human CEAR antibody, it could not only be detected in all tested canine cancer cell lines but )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(also in 10 out of 10 tissue samples from spontaneous canine mammary cancers. As mentioned above no CEACAM5 equivalent )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(with the potential to act as soluble form has been found in the dog genome so far. Our data show that anti-human CEACAM5 )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(antibody Col-1 specifically detects 180, 120 and 60 kDa molecules in canine cancer cells which are most likely non-related to )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(the canine CEACAM1-related proteins. Future studies will reveal whether these molecules may adapt ligand function to the )] TJ ET BT 26.250 431.048 Td /F1 9.8 Tf [(canine CEAR.)] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(The fact that the inter-species heterogeneity of CEACAM molecules in general was greater than expected was in striking )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(contrast to the high conservation of the CEA receptor molecule between humans and dogs. Our initial expectations that )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(spontaneous mammary carcinoma in dogs could serve as a model for human breast cancer with respect to CEACAM targeting )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(could thus, discordant to the intriguing comparative oncology concept, not be fulfilled. Though, the finding that CEAR is highly )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(homologous between humans and dogs, and specifically expressed in canine mammary cancer might open up new avenues for )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(anticancer developments based on the comparative oncology strategy.)] TJ ET BT 26.250 315.517 Td /F4 12.0 Tf [(Acknowledgments)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(The authors want to thank Philipp Starkl for helpful discussions. Erika Jensen-Jarolim is designated as corresponding author of )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(this manuscript.)] TJ ET BT 26.250 247.055 Td /F4 12.0 Tf [(Funding information)] TJ ET BT 26.250 227.101 Td /F1 9.8 Tf [(This study was supported by the RotePfote \(http://www.rotepfote.at\) grant #FA648A1301. MW is a recipient of a Von Fircks-)] TJ ET BT 26.250 215.196 Td /F1 9.8 Tf [(Scholarship of the University of Veterinary Medicine, Vienna. JS is supported by the CCHD program of the Austrian Science )] TJ ET BT 26.250 203.292 Td /F1 9.8 Tf [(Fund FWF \(#APW01205FW\).)] TJ ET BT 26.250 166.689 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 146.735 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET Q q 15.000 -315.396 577.500 1092.396 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Specific expression of the Col-1 target was further demonstrated by flow cytometry where, in contrast to human HT29 cells with )] TJ ET BT 26.250 755.571 Td /F1 9.8 Tf [(membranous and cytoplasmic expression, the antigen was detected only intracellularly in the canine mammary CF41 and CF33 )] TJ ET BT 26.250 743.667 Td /F1 9.8 Tf [(cells. This is in accordance with the present immunohistochemistry in canine mammary tumor samples, and previous reports on )] TJ ET BT 26.250 731.762 Td /F1 9.8 Tf [(cytoplasmic CEA staining in human head and neck )] TJ ET 0.267 0.267 0.267 rg BT 248.433 731.762 Td /F1 9.8 Tf [([40])] TJ ET 0.271 0.267 0.267 rg BT 264.696 731.762 Td /F1 9.8 Tf [( and colorectal cancer )] TJ ET 0.267 0.267 0.267 rg BT 362.781 731.762 Td /F1 9.8 Tf [([53])] TJ ET 0.271 0.267 0.267 rg BT 379.044 731.762 Td /F1 9.8 Tf [(. As it has been repeatedly demonstrated that )] TJ ET BT 26.250 719.857 Td /F1 9.8 Tf [(also intracellular targets can be approached by natural and engineered antibodies )] TJ ET 0.267 0.267 0.267 rg BT 381.228 719.857 Td /F1 9.8 Tf [([54] [55] [56])] TJ ET 0.271 0.267 0.267 rg BT 435.438 719.857 Td /F1 9.8 Tf [(, this fact would not contradict )] TJ ET BT 26.250 707.952 Td /F1 9.8 Tf [(the principle of antibody-mediated targeting.)] TJ ET BT 26.250 688.548 Td /F1 9.8 Tf [(To investigate the potential functional relevance of Col-1 targeting in human or canine cancer we performed growth inhibition )] TJ ET BT 26.250 676.643 Td /F1 9.8 Tf [(and signal transduction assays. Col-1 treatment did neither induce growth inhibition in canine CF33 mammary cancer cells nor )] TJ ET BT 26.250 664.738 Td /F1 9.8 Tf [(in human HT29 colon cancer cells. In line with that we previously ascribed the cytotoxic effect of a Col-1 mimotope vaccine to )] TJ ET BT 26.250 652.833 Td /F1 9.8 Tf [(antibody-mediated tumoricidity )] TJ ET 0.267 0.267 0.267 rg BT 161.171 652.833 Td /F1 9.8 Tf [([31])] TJ ET 0.271 0.267 0.267 rg BT 177.434 652.833 Td /F1 9.8 Tf [(. Our data from signal transduction assays indicate that Col-1 targeting of human and canine )] TJ ET BT 26.250 640.929 Td /F1 9.8 Tf [(cells leads to divergent effects on AKT phosphorylation, which was increased in human HT29 cells but slightly decreased in )] TJ ET BT 26.250 629.024 Td /F1 9.8 Tf [(canine CF33 cells. Therefore, thorough antibody validation prior to use across species is mandatory. The AKT signaling )] TJ ET BT 26.250 617.119 Td /F1 9.8 Tf [(pathway plays a key role in multiple cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription )] TJ ET BT 26.250 605.214 Td /F1 9.8 Tf [(and cell migration. Possibly, the biological effect of Col-1 targeting may also depend on the activation state of the AKT signaling )] TJ ET BT 26.250 593.310 Td /F1 9.8 Tf [(pathway in the different species. MAPK pathway, likewise being involved in multiple biological effects, such as regulation of )] TJ ET BT 26.250 581.405 Td /F1 9.8 Tf [(gene expression, mitosis, proliferation and cell survival, remained unaffected by Col-1 treatment in both human and canine )] TJ ET BT 26.250 569.500 Td /F1 9.8 Tf [(cancer cells.)] TJ ET BT 26.250 550.095 Td /F1 9.8 Tf [(A homolog of heterogeneous nuclear protein M4 \(hnRNP M4\) termed CEA-receptor \(CEAR\) has previously been shown on the )] TJ ET BT 26.250 538.191 Td /F1 9.8 Tf [(membrane as well as in the cytoplasm of human colon cancer cells )] TJ ET 0.267 0.267 0.267 rg BT 317.775 538.191 Td /F1 9.8 Tf [([57])] TJ ET 0.271 0.267 0.267 rg BT 334.038 538.191 Td /F1 9.8 Tf [(. The fact that CEAR may co-localize with CEA is )] TJ ET BT 26.250 526.286 Td /F1 9.8 Tf [(suggestive for signaling function of this complex )] TJ ET 0.267 0.267 0.267 rg BT 235.427 526.286 Td /F1 9.8 Tf [([16])] TJ ET 0.271 0.267 0.267 rg BT 251.690 526.286 Td /F1 9.8 Tf [(. We report here for the first time that CEAR is much higher conserved )] TJ ET BT 26.250 514.381 Td /F1 9.8 Tf [(among mammalians than in other vertebrates, like bird, frog, and fish. With 99% amino acid identity, conservation of CEAR is )] TJ ET BT 26.250 502.476 Td /F1 9.8 Tf [(especially pronounced in the human and canine species. Further, CEAR is highly expressed in human as well as canine )] TJ ET BT 26.250 490.572 Td /F1 9.8 Tf [(mammary carcinoma. Using anti-human CEAR antibody, it could not only be detected in all tested canine cancer cell lines but )] TJ ET BT 26.250 478.667 Td /F1 9.8 Tf [(also in 10 out of 10 tissue samples from spontaneous canine mammary cancers. As mentioned above no CEACAM5 equivalent )] TJ ET BT 26.250 466.762 Td /F1 9.8 Tf [(with the potential to act as soluble form has been found in the dog genome so far. Our data show that anti-human CEACAM5 )] TJ ET BT 26.250 454.857 Td /F1 9.8 Tf [(antibody Col-1 specifically detects 180, 120 and 60 kDa molecules in canine cancer cells which are most likely non-related to )] TJ ET BT 26.250 442.953 Td /F1 9.8 Tf [(the canine CEACAM1-related proteins. Future studies will reveal whether these molecules may adapt ligand function to the )] TJ ET BT 26.250 431.048 Td /F1 9.8 Tf [(canine CEAR.)] TJ ET BT 26.250 411.643 Td /F1 9.8 Tf [(The fact that the inter-species heterogeneity of CEACAM molecules in general was greater than expected was in striking )] TJ ET BT 26.250 399.738 Td /F1 9.8 Tf [(contrast to the high conservation of the CEA receptor molecule between humans and dogs. Our initial expectations that )] TJ ET BT 26.250 387.834 Td /F1 9.8 Tf [(spontaneous mammary carcinoma in dogs could serve as a model for human breast cancer with respect to CEACAM targeting )] TJ ET BT 26.250 375.929 Td /F1 9.8 Tf [(could thus, discordant to the intriguing comparative oncology concept, not be fulfilled. Though, the finding that CEAR is highly )] TJ ET BT 26.250 364.024 Td /F1 9.8 Tf [(homologous between humans and dogs, and specifically expressed in canine mammary cancer might open up new avenues for )] TJ ET BT 26.250 352.119 Td /F1 9.8 Tf [(anticancer developments based on the comparative oncology strategy.)] TJ ET BT 26.250 315.517 Td /F4 12.0 Tf [(Acknowledgments)] TJ ET BT 26.250 295.563 Td /F1 9.8 Tf [(The authors want to thank Philipp Starkl for helpful discussions. Erika Jensen-Jarolim is designated as corresponding author of )] TJ ET BT 26.250 283.658 Td /F1 9.8 Tf [(this manuscript.)] TJ ET BT 26.250 247.055 Td /F4 12.0 Tf [(Funding information)] TJ ET BT 26.250 227.101 Td /F1 9.8 Tf [(This study was supported by the RotePfote \(http://www.rotepfote.at\) grant #FA648A1301. MW is a recipient of a Von Fircks-)] TJ ET BT 26.250 215.196 Td /F1 9.8 Tf [(Scholarship of the University of Veterinary Medicine, Vienna. 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PLoS One, 2010. 5\(6\): p. e11013)] TJ ET BT 26.250 188.550 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 188.550 Td /F1 9.8 Tf [(Paoloni, M.C., et al., Launching a novel preclinical infrastructure: comparative oncology trials consortium directed therapeutic )] TJ ET BT 26.250 176.646 Td /F1 9.8 Tf [(targeting of TNFalpha to cancer vasculature. PLoS One, 2009. 4\(3\): p. e4972)] TJ ET BT 26.250 157.241 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 157.241 Td /F1 9.8 Tf [(Gordon, I., et al., The Comparative Oncology Trials Consortium: using spontaneously occurring cancers in dogs to inform the )] TJ ET BT 26.250 145.336 Td /F1 9.8 Tf [(cancer drug development pathway. PLoS Med, 2009. 6\(10\): p. e1000161)] TJ ET BT 26.250 125.931 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 125.931 Td /F1 9.8 Tf [(Khanna, C., et al., Guiding the optimal translation of new cancer treatments from canine to human cancer patients. Clin )] TJ ET BT 26.250 114.027 Td /F1 9.8 Tf [(Cancer Res, 2009. 15\(18\): p. 5671-7)] TJ ET BT 26.250 94.622 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 94.622 Td /F1 9.8 Tf [(Paoloni, M. and C. Khanna, Translation of new cancer treatments from pet dogs to humans. Nat Rev Cancer, 2008. 8\(2\): p. )] TJ ET BT 26.250 82.717 Td /F1 9.8 Tf [(147-56)] TJ ET BT 26.250 63.312 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 63.312 Td /F1 9.8 Tf [(Porrello, A., P. Cardelli, and E.P. Spugnini, Oncology of companion animals as a model for humans. an overview of tumor )] TJ ET BT 26.250 51.408 Td /F1 9.8 Tf [(histotypes. J Exp Clin Cancer Res, 2006. 25\(1\): p. 97-105)] TJ ET Q q 15.000 41.527 577.500 735.473 re W n q 405.000 0 0 452.250 26.250 324.750 cm /I10 Do Q 0.271 0.267 0.267 rg BT 26.250 307.726 Td /F4 9.8 Tf [(Supplementary Fig 1)] TJ ET BT 122.151 307.726 Td /F1 9.8 Tf [(: Multiple amino acid sequence alignments on CEARs of different species using ClustalW.The following )] TJ ET BT 26.250 295.821 Td /F1 9.8 Tf [(color code was used: red, identical amino acids; green, strongly similar amino acids; blue, weakly similar amino acids; black, )] TJ ET BT 26.250 283.917 Td /F1 9.8 Tf [(non-conserved amino acids.)] TJ ET BT 26.250 247.314 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 219.860 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 219.860 Td /F1 9.8 Tf [(Paoloni, M.C., et al., Rapamycin pharmacokinetic and pharmacodynamic relationships in osteosarcoma: a comparative )] TJ ET BT 26.250 207.955 Td /F1 9.8 Tf [(oncology study in dogs. PLoS One, 2010. 5\(6\): p. e11013)] TJ ET BT 26.250 188.550 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 188.550 Td /F1 9.8 Tf [(Paoloni, M.C., et al., Launching a novel preclinical infrastructure: comparative oncology trials consortium directed therapeutic )] TJ ET BT 26.250 176.646 Td /F1 9.8 Tf [(targeting of TNFalpha to cancer vasculature. PLoS One, 2009. 4\(3\): p. e4972)] TJ ET BT 26.250 157.241 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 157.241 Td /F1 9.8 Tf [(Gordon, I., et al., The Comparative Oncology Trials Consortium: using spontaneously occurring cancers in dogs to inform the )] TJ ET BT 26.250 145.336 Td /F1 9.8 Tf [(cancer drug development pathway. PLoS Med, 2009. 6\(10\): p. e1000161)] TJ ET BT 26.250 125.931 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 125.931 Td /F1 9.8 Tf [(Khanna, C., et al., Guiding the optimal translation of new cancer treatments from canine to human cancer patients. Clin )] TJ ET BT 26.250 114.027 Td /F1 9.8 Tf [(Cancer Res, 2009. 15\(18\): p. 5671-7)] TJ ET BT 26.250 94.622 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 94.622 Td /F1 9.8 Tf [(Paoloni, M. and C. Khanna, Translation of new cancer treatments from pet dogs to humans. Nat Rev Cancer, 2008. 8\(2\): p. )] TJ ET BT 26.250 82.717 Td /F1 9.8 Tf [(147-56)] TJ ET BT 26.250 63.312 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 63.312 Td /F1 9.8 Tf [(Porrello, A., P. Cardelli, and E.P. Spugnini, Oncology of companion animals as a model for humans. an overview of tumor )] TJ ET BT 26.250 51.408 Td /F1 9.8 Tf [(histotypes. J Exp Clin Cancer Res, 2006. 25\(1\): p. 97-105)] TJ ET Q q 15.000 41.527 577.500 735.473 re W n q 405.000 0 0 452.250 26.250 324.750 cm /I10 Do Q 0.271 0.267 0.267 rg BT 26.250 307.726 Td /F4 9.8 Tf [(Supplementary Fig 1)] TJ ET BT 122.151 307.726 Td /F1 9.8 Tf [(: Multiple amino acid sequence alignments on CEARs of different species using ClustalW.The following )] TJ ET BT 26.250 295.821 Td /F1 9.8 Tf [(color code was used: red, identical amino acids; green, strongly similar amino acids; blue, weakly similar amino acids; black, )] TJ ET BT 26.250 283.917 Td /F1 9.8 Tf [(non-conserved amino acids.)] TJ ET BT 26.250 247.314 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 219.860 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 219.860 Td /F1 9.8 Tf [(Paoloni, M.C., et al., Rapamycin pharmacokinetic and pharmacodynamic relationships in osteosarcoma: a comparative )] TJ ET BT 26.250 207.955 Td /F1 9.8 Tf [(oncology study in dogs. PLoS One, 2010. 5\(6\): p. e11013)] TJ ET BT 26.250 188.550 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 188.550 Td /F1 9.8 Tf [(Paoloni, M.C., et al., Launching a novel preclinical infrastructure: comparative oncology trials consortium directed therapeutic )] TJ ET BT 26.250 176.646 Td /F1 9.8 Tf [(targeting of TNFalpha to cancer vasculature. PLoS One, 2009. 4\(3\): p. e4972)] TJ ET BT 26.250 157.241 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 157.241 Td /F1 9.8 Tf [(Gordon, I., et al., The Comparative Oncology Trials Consortium: using spontaneously occurring cancers in dogs to inform the )] TJ ET BT 26.250 145.336 Td /F1 9.8 Tf [(cancer drug development pathway. PLoS Med, 2009. 6\(10\): p. e1000161)] TJ ET BT 26.250 125.931 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 125.931 Td /F1 9.8 Tf [(Khanna, C., et al., Guiding the optimal translation of new cancer treatments from canine to human cancer patients. Clin )] TJ ET BT 26.250 114.027 Td /F1 9.8 Tf [(Cancer Res, 2009. 15\(18\): p. 5671-7)] TJ ET BT 26.250 94.622 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 94.622 Td /F1 9.8 Tf [(Paoloni, M. and C. Khanna, Translation of new cancer treatments from pet dogs to humans. Nat Rev Cancer, 2008. 8\(2\): p. )] TJ ET BT 26.250 82.717 Td /F1 9.8 Tf [(147-56)] TJ ET BT 26.250 63.312 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 63.312 Td /F1 9.8 Tf [(Porrello, A., P. Cardelli, and E.P. Spugnini, Oncology of companion animals as a model for humans. an overview of tumor )] TJ ET BT 26.250 51.408 Td /F1 9.8 Tf [(histotypes. 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Zt{>kmXyWhvId _Guj~Anah6ĠGpu;!u@ EzIWS~ҼK85x/.5ᴋP[b$(vއwg:_i[H KҬ/1,!R܏-it&ǁ$}CQ6M5^H(9,D>a\e_sgbXVXk'Tg⾍,άsj3l]I;vz$?eԢ1xhԤ!_i9ȱ0wļ95h5a=][j2s E%%/-#@B%v@jucys=Ƨ? rwN|PI2ݫ|mވMGRExm楣F K#}ݯ/eپ&9 fS@ƝZ؉t]fWv-i#[#dÆ+ BM2w6HzW^vUlVXty˪ݳ$Ӂ,!@dٹ^мYu=nZO7ƼG]ܲK+=_B֥{8;ʙ7m#r98Ҽ:[GtH\uRj1 1 H6 7i:]K5 6}"جSk ||Tsw)M(=r]]v]l@&q*NS;IʎHȢ=-z}]Ll`gsbh9'2kĭ't՝o;-B(3ڛ\VZm1IsZwmkYa"կ?n"+_Ychdpqong[=˸^#kdծ;5+$ӴKT|kO,nQ##Gۉ!qLH]rCuC\2EZiYf88,9摙k63Y'>|+nv,ܱ''*v;S}^ʖ`2F>νk _^Gݗ5Ii8Mg\sFJR0n [W:A-Ae,N,C2щ D 64N_i~Ljm.Q.lc}%abۅI!!'TWZþ5V.s@jډC.r#ҡA%@q X^-].I &kZ_YD k\bdWK+k}I_8_]ɋKĞ.[?x HFl rmI_[o Gs/̖j%̯#)cs,xF&Gou-Yt %|UtM JӭL5?:VY$_>k.K7u85gnts7ʲ1*ɧXxGU&ŷ`5[Rxa}DZ]d-|(쁋~s>^[{.5XBuXhL٘wv 2kΑwvZM~85%?dT's,mbFtjtf<]w*yLrBrpnh|5HF5]0\\B녞MXUuPY v"2|g1$OShׇY_ti{?t6:.֗~T@} U픚o_CSkQb/u`Sgsēцa^7*R'v{ o&J> endobj 409 0 obj << /Length 22430 >> stream 0.271 0.267 0.267 rg q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Vail, D.M. and E.G. MacEwen, Spontaneously occurring tumors of companion animals as models for human cancer. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Invest, 2000. 18\(8\): p. 781-92)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Misdorp, W., Tumors of the Mammary Gland, in Tumors in Domestic Animals, D.J. Meuten, Editor. 2002, Iowa State Press: )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Ames, Iowa. p. 575-606)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Dobson, J.M., et al., Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Anim Pract, 2002. 43\(6\): p. 240-6)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Egenvall, A., et al., Incidence of and survival after mammary tumors in a population of over 80,000 insured female dogs in )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(Sweden from 1995 to 2002. Prev Vet Med, 2005. 69\(1-2\): p. 109-27)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sorenmo, K., Canine mammary gland tumors. Vet Clin North Am Small Anim Pract, 2003. 33\(3\): p. 573-96)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(Dorn, C.R., et al., Survey of animal neoplasms in Alameda and Contra Costa Counties, California. I. Methodology and )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(description of cases. J Natl Cancer Inst, 1968. 40\(2\): p. 295-305)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Adams, G.P. and L.M. Weiner, Monoclonal antibody therapy of cancer. Nat Biotechnol, 2005. 23\(9\): p. 1147-57)] TJ ET BT 26.250 564.619 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 564.619 Td /F1 9.8 Tf [(Carter, P., Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer, 2001. 1\(2\): p. 118-29)] TJ ET BT 26.250 545.214 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 545.214 Td /F1 9.8 Tf [(Bajenova, O., et al., Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its )] TJ ET BT 26.250 533.310 Td /F1 9.8 Tf [(function as a carcinoembryonic antigen receptor. Exp Cell Res, 2003. 291\(1\): p. 228-41)] TJ ET BT 26.250 513.905 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 513.905 Td /F1 9.8 Tf [(Laguinge, L., et al., Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells. Anticancer Res, )] TJ ET BT 26.250 502.000 Td /F1 9.8 Tf [(2005. 25\(1A\): p. 23-31)] TJ ET BT 26.250 482.595 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 482.595 Td /F1 9.8 Tf [(Ordonez, C., et al., GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through )] TJ ET BT 26.250 470.691 Td /F1 9.8 Tf [(enhanced integrin alpha5beta1-fibronectin interaction. J Cell Physiol, 2007. 210\(3\): p. 757-65)] TJ ET BT 26.250 451.286 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 451.286 Td /F1 9.8 Tf [(Blumenthal, R.D., H.J. Hansen, and D.M. Goldenberg, Inhibition of adhesion, invasion, and metastasis by antibodies )] TJ ET BT 26.250 439.381 Td /F1 9.8 Tf [(targeting CEACAM6 \(NCA-90\) and CEACAM5 \(Carcinoembryonic Antigen\). Cancer Res, 2005. 65\(19\): p. 8809-17)] TJ ET BT 26.250 419.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 419.976 Td /F1 9.8 Tf [(Berinstein, N.L., Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review. J Clin Oncol, 2002. )] TJ ET BT 26.250 408.072 Td /F1 9.8 Tf [(20\(8\): p. 2197-207)] TJ ET BT 26.250 388.667 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 388.667 Td /F1 9.8 Tf [(Uchino, J., et al., Tumor targeting carboxylesterase fused with anti-CEA scFv improve the anticancer effect with a less toxic )] TJ ET BT 26.250 376.762 Td /F1 9.8 Tf [(dose of irinotecan. Cancer Gene Ther, 2008. 15\(2\): p. 94-100)] TJ ET BT 26.250 357.357 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 357.357 Td /F1 9.8 Tf [(Jia, F., T.D. Shelton, and M.R. Lewis, Preparation, characterization, and biological evaluation of a streptavidin-chimeric )] TJ ET BT 26.250 345.453 Td /F1 9.8 Tf [(t84.66 conjugate for antibody pretargeting. Cancer Biother Radiopharm, 2007. 22\(5\): p. 654-64)] TJ ET BT 26.250 326.048 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 326.048 Td /F1 9.8 Tf [(Perez, L., et al., A multivalent recombinant antibody fragment specific for carcinoembryonic antigen. Biotechnol Appl )] TJ ET BT 26.250 314.143 Td /F1 9.8 Tf [(Biochem, 2006. 43\(Pt 1\): p. 39-48)] TJ ET BT 26.250 294.738 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 294.738 Td /F1 9.8 Tf [(Stein, R. and D.M. Goldenberg, A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits )] TJ ET BT 26.250 282.834 Td /F1 9.8 Tf [(tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy. Mol Cancer Ther, 2004. )] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(3\(12\): p. 1559-64)] TJ ET BT 26.250 251.524 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 251.524 Td /F1 9.8 Tf [(Blumenthal, R.D., et al., Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a )] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(human colonic carcinoma xenograft. Cancer Immunol Immunother, 2005. 54\(4\): p. 315-27)] TJ ET BT 26.250 220.215 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 220.215 Td /F1 9.8 Tf [(Yazaki, P.J., et al., Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Eng Des Sel, )] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(2004. 17\(5\): p. 481-9)] TJ ET BT 26.250 188.905 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 188.905 Td /F1 9.8 Tf [(Alves, P.M., et al., Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and )] TJ ET BT 26.250 177.000 Td /F1 9.8 Tf [(colorectal carcinoma patients. Cancer Immunol Immunother, 2007. 56\(11\): p. 1795-805)] TJ ET BT 26.250 157.596 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 157.596 Td /F1 9.8 Tf [(Hou, Y., B. Kavanagh, and L. Fong, Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a )] TJ ET BT 26.250 145.691 Td /F1 9.8 Tf [(tumor-associated antigen. J Immunol, 2008. 180\(3\): p. 1526-34)] TJ ET BT 26.250 126.286 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 126.286 Td /F1 9.8 Tf [(Kavanagh, B., et al., Vaccination of metastatic colorectal cancer patients with matured dendritic cells loaded with multiple )] TJ ET BT 26.250 114.381 Td /F1 9.8 Tf [(major histocompatibility complex class I peptides. J Immunother, 2007. 30\(7\): p. 762-72)] TJ ET BT 26.250 94.977 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 94.977 Td /F1 9.8 Tf [(Loisel-Meyer, S., et al., Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing )] TJ ET BT 26.250 83.072 Td /F1 9.8 Tf [(tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection. Mol Cancer Ther, 2009. )] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(8\(3\): p. 692-702)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Horig, H., et al., Strategies for cancer therapy using carcinoembryonic antigen vaccines. Expert Rev Mol Med, 2000. 2\(3\): p. )] TJ ET Q q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Vail, D.M. and E.G. MacEwen, Spontaneously occurring tumors of companion animals as models for human cancer. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Invest, 2000. 18\(8\): p. 781-92)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Misdorp, W., Tumors of the Mammary Gland, in Tumors in Domestic Animals, D.J. Meuten, Editor. 2002, Iowa State Press: )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Ames, Iowa. p. 575-606)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Dobson, J.M., et al., Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Anim Pract, 2002. 43\(6\): p. 240-6)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Egenvall, A., et al., Incidence of and survival after mammary tumors in a population of over 80,000 insured female dogs in )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(Sweden from 1995 to 2002. Prev Vet Med, 2005. 69\(1-2\): p. 109-27)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sorenmo, K., Canine mammary gland tumors. Vet Clin North Am Small Anim Pract, 2003. 33\(3\): p. 573-96)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(Dorn, C.R., et al., Survey of animal neoplasms in Alameda and Contra Costa Counties, California. I. Methodology and )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(description of cases. J Natl Cancer Inst, 1968. 40\(2\): p. 295-305)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Adams, G.P. and L.M. Weiner, Monoclonal antibody therapy of cancer. Nat Biotechnol, 2005. 23\(9\): p. 1147-57)] TJ ET BT 26.250 564.619 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 564.619 Td /F1 9.8 Tf [(Carter, P., Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer, 2001. 1\(2\): p. 118-29)] TJ ET BT 26.250 545.214 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 545.214 Td /F1 9.8 Tf [(Bajenova, O., et al., Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its )] TJ ET BT 26.250 533.310 Td /F1 9.8 Tf [(function as a carcinoembryonic antigen receptor. Exp Cell Res, 2003. 291\(1\): p. 228-41)] TJ ET BT 26.250 513.905 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 513.905 Td /F1 9.8 Tf [(Laguinge, L., et al., Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells. Anticancer Res, )] TJ ET BT 26.250 502.000 Td /F1 9.8 Tf [(2005. 25\(1A\): p. 23-31)] TJ ET BT 26.250 482.595 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 482.595 Td /F1 9.8 Tf [(Ordonez, C., et al., GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through )] TJ ET BT 26.250 470.691 Td /F1 9.8 Tf [(enhanced integrin alpha5beta1-fibronectin interaction. J Cell Physiol, 2007. 210\(3\): p. 757-65)] TJ ET BT 26.250 451.286 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 451.286 Td /F1 9.8 Tf [(Blumenthal, R.D., H.J. Hansen, and D.M. Goldenberg, Inhibition of adhesion, invasion, and metastasis by antibodies )] TJ ET BT 26.250 439.381 Td /F1 9.8 Tf [(targeting CEACAM6 \(NCA-90\) and CEACAM5 \(Carcinoembryonic Antigen\). Cancer Res, 2005. 65\(19\): p. 8809-17)] TJ ET BT 26.250 419.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 419.976 Td /F1 9.8 Tf [(Berinstein, N.L., Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review. J Clin Oncol, 2002. )] TJ ET BT 26.250 408.072 Td /F1 9.8 Tf [(20\(8\): p. 2197-207)] TJ ET BT 26.250 388.667 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 388.667 Td /F1 9.8 Tf [(Uchino, J., et al., Tumor targeting carboxylesterase fused with anti-CEA scFv improve the anticancer effect with a less toxic )] TJ ET BT 26.250 376.762 Td /F1 9.8 Tf [(dose of irinotecan. Cancer Gene Ther, 2008. 15\(2\): p. 94-100)] TJ ET BT 26.250 357.357 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 357.357 Td /F1 9.8 Tf [(Jia, F., T.D. Shelton, and M.R. Lewis, Preparation, characterization, and biological evaluation of a streptavidin-chimeric )] TJ ET BT 26.250 345.453 Td /F1 9.8 Tf [(t84.66 conjugate for antibody pretargeting. Cancer Biother Radiopharm, 2007. 22\(5\): p. 654-64)] TJ ET BT 26.250 326.048 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 326.048 Td /F1 9.8 Tf [(Perez, L., et al., A multivalent recombinant antibody fragment specific for carcinoembryonic antigen. Biotechnol Appl )] TJ ET BT 26.250 314.143 Td /F1 9.8 Tf [(Biochem, 2006. 43\(Pt 1\): p. 39-48)] TJ ET BT 26.250 294.738 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 294.738 Td /F1 9.8 Tf [(Stein, R. and D.M. Goldenberg, A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits )] TJ ET BT 26.250 282.834 Td /F1 9.8 Tf [(tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy. Mol Cancer Ther, 2004. )] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(3\(12\): p. 1559-64)] TJ ET BT 26.250 251.524 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 251.524 Td /F1 9.8 Tf [(Blumenthal, R.D., et al., Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a )] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(human colonic carcinoma xenograft. Cancer Immunol Immunother, 2005. 54\(4\): p. 315-27)] TJ ET BT 26.250 220.215 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 220.215 Td /F1 9.8 Tf [(Yazaki, P.J., et al., Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Eng Des Sel, )] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(2004. 17\(5\): p. 481-9)] TJ ET BT 26.250 188.905 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 188.905 Td /F1 9.8 Tf [(Alves, P.M., et al., Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and )] TJ ET BT 26.250 177.000 Td /F1 9.8 Tf [(colorectal carcinoma patients. Cancer Immunol Immunother, 2007. 56\(11\): p. 1795-805)] TJ ET BT 26.250 157.596 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 157.596 Td /F1 9.8 Tf [(Hou, Y., B. Kavanagh, and L. Fong, Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a )] TJ ET BT 26.250 145.691 Td /F1 9.8 Tf [(tumor-associated antigen. J Immunol, 2008. 180\(3\): p. 1526-34)] TJ ET BT 26.250 126.286 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 126.286 Td /F1 9.8 Tf [(Kavanagh, B., et al., Vaccination of metastatic colorectal cancer patients with matured dendritic cells loaded with multiple )] TJ ET BT 26.250 114.381 Td /F1 9.8 Tf [(major histocompatibility complex class I peptides. J Immunother, 2007. 30\(7\): p. 762-72)] TJ ET BT 26.250 94.977 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 94.977 Td /F1 9.8 Tf [(Loisel-Meyer, S., et al., Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing )] TJ ET BT 26.250 83.072 Td /F1 9.8 Tf [(tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection. Mol Cancer Ther, 2009. )] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(8\(3\): p. 692-702)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Horig, H., et al., Strategies for cancer therapy using carcinoembryonic antigen vaccines. Expert Rev Mol Med, 2000. 2\(3\): p. )] TJ ET Q q 15.000 37.477 577.500 739.523 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 759.976 Td /F1 9.8 Tf [(Vail, D.M. and E.G. MacEwen, Spontaneously occurring tumors of companion animals as models for human cancer. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Invest, 2000. 18\(8\): p. 781-92)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 728.667 Td /F1 9.8 Tf [(Misdorp, W., Tumors of the Mammary Gland, in Tumors in Domestic Animals, D.J. Meuten, Editor. 2002, Iowa State Press: )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(Ames, Iowa. p. 575-606)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 697.357 Td /F1 9.8 Tf [(Dobson, J.M., et al., Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Anim Pract, 2002. 43\(6\): p. 240-6)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Egenvall, A., et al., Incidence of and survival after mammary tumors in a population of over 80,000 insured female dogs in )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(Sweden from 1995 to 2002. Prev Vet Med, 2005. 69\(1-2\): p. 109-27)] TJ ET BT 26.250 634.738 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 634.738 Td /F1 9.8 Tf [(Sorenmo, K., Canine mammary gland tumors. Vet Clin North Am Small Anim Pract, 2003. 33\(3\): p. 573-96)] TJ ET BT 26.250 615.333 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 615.333 Td /F1 9.8 Tf [(Dorn, C.R., et al., Survey of animal neoplasms in Alameda and Contra Costa Counties, California. I. Methodology and )] TJ ET BT 26.250 603.429 Td /F1 9.8 Tf [(description of cases. J Natl Cancer Inst, 1968. 40\(2\): p. 295-305)] TJ ET BT 26.250 584.024 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 584.024 Td /F1 9.8 Tf [(Adams, G.P. and L.M. Weiner, Monoclonal antibody therapy of cancer. Nat Biotechnol, 2005. 23\(9\): p. 1147-57)] TJ ET BT 26.250 564.619 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 564.619 Td /F1 9.8 Tf [(Carter, P., Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer, 2001. 1\(2\): p. 118-29)] TJ ET BT 26.250 545.214 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 545.214 Td /F1 9.8 Tf [(Bajenova, O., et al., Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its )] TJ ET BT 26.250 533.310 Td /F1 9.8 Tf [(function as a carcinoembryonic antigen receptor. Exp Cell Res, 2003. 291\(1\): p. 228-41)] TJ ET BT 26.250 513.905 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 513.905 Td /F1 9.8 Tf [(Laguinge, L., et al., Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells. Anticancer Res, )] TJ ET BT 26.250 502.000 Td /F1 9.8 Tf [(2005. 25\(1A\): p. 23-31)] TJ ET BT 26.250 482.595 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 482.595 Td /F1 9.8 Tf [(Ordonez, C., et al., GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through )] TJ ET BT 26.250 470.691 Td /F1 9.8 Tf [(enhanced integrin alpha5beta1-fibronectin interaction. J Cell Physiol, 2007. 210\(3\): p. 757-65)] TJ ET BT 26.250 451.286 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 451.286 Td /F1 9.8 Tf [(Blumenthal, R.D., H.J. Hansen, and D.M. Goldenberg, Inhibition of adhesion, invasion, and metastasis by antibodies )] TJ ET BT 26.250 439.381 Td /F1 9.8 Tf [(targeting CEACAM6 \(NCA-90\) and CEACAM5 \(Carcinoembryonic Antigen\). Cancer Res, 2005. 65\(19\): p. 8809-17)] TJ ET BT 26.250 419.976 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 419.976 Td /F1 9.8 Tf [(Berinstein, N.L., Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review. J Clin Oncol, 2002. )] TJ ET BT 26.250 408.072 Td /F1 9.8 Tf [(20\(8\): p. 2197-207)] TJ ET BT 26.250 388.667 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 388.667 Td /F1 9.8 Tf [(Uchino, J., et al., Tumor targeting carboxylesterase fused with anti-CEA scFv improve the anticancer effect with a less toxic )] TJ ET BT 26.250 376.762 Td /F1 9.8 Tf [(dose of irinotecan. Cancer Gene Ther, 2008. 15\(2\): p. 94-100)] TJ ET BT 26.250 357.357 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 357.357 Td /F1 9.8 Tf [(Jia, F., T.D. Shelton, and M.R. Lewis, Preparation, characterization, and biological evaluation of a streptavidin-chimeric )] TJ ET BT 26.250 345.453 Td /F1 9.8 Tf [(t84.66 conjugate for antibody pretargeting. Cancer Biother Radiopharm, 2007. 22\(5\): p. 654-64)] TJ ET BT 26.250 326.048 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 326.048 Td /F1 9.8 Tf [(Perez, L., et al., A multivalent recombinant antibody fragment specific for carcinoembryonic antigen. Biotechnol Appl )] TJ ET BT 26.250 314.143 Td /F1 9.8 Tf [(Biochem, 2006. 43\(Pt 1\): p. 39-48)] TJ ET BT 26.250 294.738 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 294.738 Td /F1 9.8 Tf [(Stein, R. and D.M. Goldenberg, A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits )] TJ ET BT 26.250 282.834 Td /F1 9.8 Tf [(tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy. Mol Cancer Ther, 2004. )] TJ ET BT 26.250 270.929 Td /F1 9.8 Tf [(3\(12\): p. 1559-64)] TJ ET BT 26.250 251.524 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 251.524 Td /F1 9.8 Tf [(Blumenthal, R.D., et al., Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a )] TJ ET BT 26.250 239.619 Td /F1 9.8 Tf [(human colonic carcinoma xenograft. Cancer Immunol Immunother, 2005. 54\(4\): p. 315-27)] TJ ET BT 26.250 220.215 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 220.215 Td /F1 9.8 Tf [(Yazaki, P.J., et al., Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Eng Des Sel, )] TJ ET BT 26.250 208.310 Td /F1 9.8 Tf [(2004. 17\(5\): p. 481-9)] TJ ET BT 26.250 188.905 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 188.905 Td /F1 9.8 Tf [(Alves, P.M., et al., Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and )] TJ ET BT 26.250 177.000 Td /F1 9.8 Tf [(colorectal carcinoma patients. Cancer Immunol Immunother, 2007. 56\(11\): p. 1795-805)] TJ ET BT 26.250 157.596 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 157.596 Td /F1 9.8 Tf [(Hou, Y., B. Kavanagh, and L. Fong, Distinct CD8+ T cell repertoires primed with agonist and native peptides derived from a )] TJ ET BT 26.250 145.691 Td /F1 9.8 Tf [(tumor-associated antigen. J Immunol, 2008. 180\(3\): p. 1526-34)] TJ ET BT 26.250 126.286 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 126.286 Td /F1 9.8 Tf [(Kavanagh, B., et al., Vaccination of metastatic colorectal cancer patients with matured dendritic cells loaded with multiple )] TJ ET BT 26.250 114.381 Td /F1 9.8 Tf [(major histocompatibility complex class I peptides. J Immunother, 2007. 30\(7\): p. 762-72)] TJ ET BT 26.250 94.977 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 94.977 Td /F1 9.8 Tf [(Loisel-Meyer, S., et al., Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing )] TJ ET BT 26.250 83.072 Td /F1 9.8 Tf [(tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection. Mol Cancer Ther, 2009. )] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(8\(3\): p. 692-702)] TJ ET BT 26.250 51.762 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 51.762 Td /F1 9.8 Tf [(Horig, H., et al., Strategies for cancer therapy using carcinoembryonic antigen vaccines. Expert Rev Mol Med, 2000. 2\(3\): p. )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(11)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 410 0 obj << /Type /Page /Parent 3 0 R /Contents 411 0 R >> endobj 411 0 obj << /Length 22106 >> stream 0.271 0.267 0.267 rg q 15.000 49.381 577.500 727.619 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(1-24)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Bramswig, K.H., et al., Immunization with mimotopes prevents growth of carcinoembryonic antigen positive tumors in )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(BALB/c mice. Clin Cancer Res, 2007. 13\(21\): p. 6501-8)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Facciabene, A., et al., Vectors encoding carcinoembryonic antigen fused to the B subunit of heat-labile enterotoxin elicit )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(antigen-specific immune responses and antitumor effects. Vaccine, 2007. 26\(1\): p. 47-58)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Smith, B.F., et al., Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(carcinoembryonic antigen. Gene Ther, 1998. 5\(7\): p. 865-8)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Rabanal, R., et al., Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(antitrypsin in carcinomas of the exocrine pancreas of the dog. Res Vet Sci, 1992. 52\(2\): p. 217-23)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Martin de las Mulas, J., et al., Immunohistochemical evaluation of canine primary liver carcinomas: distribution of alpha-)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(fetoprotein, carcinoembryonic antigen, keratins and vimentin. Res Vet Sci, 1995. 59\(2\): p. 124-7)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Cantile, C., et al., Pathological and immunohistochemical studies of choroid plexus carcinoma of the dog. J Comp Pathol, )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(2002. 126\(2-3\): p. 183-93)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Radi, Z.A., D.L. Miller, and M.E. Hines, 2nd, Rete testis mucinous adenocarcinoma in a dog. Vet Pathol, 2004. 41\(1\): p. 75-8)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Ohuchi, N., et al., Differential expression of carcinoembryonic antigen in early gastric adenocarcinomas versus benign )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(gastric lesions defined by monoclonal antibodies reactive with restricted antigen epitopes. Cancer Res, 1987. 47\(13\): p. 3565-71)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Chhieng, D.C., et al., Expression of CEA, Tag-72, and Lewis-Y antigen in primary and metastatic lesions of ovarian )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(carcinoma. Hum Pathol, 2003. 34\(10\): p. 1016-21)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Kass, E.S., et al., Carcinoembryonic antigen as a target for specific antitumor immunotherapy of head and neck cancer. )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Cancer Research, 2002. 62\(17\): p. 5049-57)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Tendler, A., H.L. Kaufman, and A.S. Kadish, Increased carcinoembryonic antigen expression in cervical intraepithelial )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(neoplasia grade 3 and in cervical squamous cell carcinoma. Hum Pathol, 2000. 31\(11\): p. 1357-62)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Irvine, K. and J. Schlom, Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72. Cancer Immunol Immunother, 1993. )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(36\(5\): p. 281-92)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Gonzales, N.R., et al., SDR grafting of a murine antibody using multiple human germline templates to minimize its )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(immunogenicity. Mol Immunol, 2004. 41\(9\): p. 863-72)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Kammerer, R. and W. Zimmermann, Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(antigen families. BMC Biol, 2010. 8: p. 12)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Tamura, K., et al., MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. Mol Biol Evol, 2007. )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(24\(8\): p. 1596-9)] TJ ET BT 26.250 278.429 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 278.429 Td /F1 9.8 Tf [(Kumar, S., et al., MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief )] TJ ET BT 26.250 266.524 Td /F1 9.8 Tf [(Bioinform, 2008. 9\(4\): p. 299-306)] TJ ET BT 26.250 247.119 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 247.119 Td /F1 9.8 Tf [(Jones, D.T., W.R. Taylor, and J.M. Thornton, The rapid generation of mutation data matrices from protein sequences. )] TJ ET BT 26.250 235.215 Td /F1 9.8 Tf [(Comput Appl Biosci, 1992. 8\(3\): p. 275-82)] TJ ET BT 26.250 215.810 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 215.810 Td /F1 9.8 Tf [(Tamura, K. and M. Nei, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in )] TJ ET BT 26.250 203.905 Td /F1 9.8 Tf [(humans and chimpanzees. Mol Biol Evol, 1993. 10\(3\): p. 512-26)] TJ ET BT 26.250 184.500 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 184.500 Td /F1 9.8 Tf [(Voges, M., et al., CEACAM1 recognition by bacterial pathogens is species-specific. BMC Microbiol, 2010. 10:)] TJ ET BT 26.250 165.096 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 165.096 Td /F1 9.8 Tf [(Yamamoto, Y., et al., Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma. Biochem )] TJ ET BT 26.250 153.191 Td /F1 9.8 Tf [(Biophys Res Commun, 2005. 333\(1\): p. 223-9)] TJ ET BT 26.250 133.786 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 133.786 Td /F1 9.8 Tf [(Kammerer, R., et al., Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-)] TJ ET BT 26.250 121.881 Td /F1 9.8 Tf [(related immune receptors in the dog. BMC Evol Biol, 2007. 7: p. 196)] TJ ET BT 26.250 102.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 102.477 Td /F1 9.8 Tf [(Samsen, A., et al., DC-SIGN and SRCL bind glycans of carcinoembryonic antigen \(CEA\) and CEA-related cell adhesion )] TJ ET BT 26.250 90.572 Td /F1 9.8 Tf [(molecule 1 \(CEACAM1\): recombinant human glycan-binding receptors as analytical tools. Eur J Cell Biol, 2010. 89\(1\): p. 87-94)] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 71.167 Td /F1 9.8 Tf [(Nazato, D.M., et al., Prognostic value of carcinoembryonic antigen distribution in tumor tissue of colorectal carcinoma. Arq )] TJ ET BT 26.250 59.262 Td /F1 9.8 Tf [(Gastroenterol, 2009. 46\(1\): p. 26-31)] TJ ET Q q 15.000 49.381 577.500 727.619 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(1-24)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Bramswig, K.H., et al., Immunization with mimotopes prevents growth of carcinoembryonic antigen positive tumors in )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(BALB/c mice. Clin Cancer Res, 2007. 13\(21\): p. 6501-8)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Facciabene, A., et al., Vectors encoding carcinoembryonic antigen fused to the B subunit of heat-labile enterotoxin elicit )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(antigen-specific immune responses and antitumor effects. Vaccine, 2007. 26\(1\): p. 47-58)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Smith, B.F., et al., Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(carcinoembryonic antigen. Gene Ther, 1998. 5\(7\): p. 865-8)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Rabanal, R., et al., Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(antitrypsin in carcinomas of the exocrine pancreas of the dog. Res Vet Sci, 1992. 52\(2\): p. 217-23)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Martin de las Mulas, J., et al., Immunohistochemical evaluation of canine primary liver carcinomas: distribution of alpha-)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(fetoprotein, carcinoembryonic antigen, keratins and vimentin. Res Vet Sci, 1995. 59\(2\): p. 124-7)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Cantile, C., et al., Pathological and immunohistochemical studies of choroid plexus carcinoma of the dog. J Comp Pathol, )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(2002. 126\(2-3\): p. 183-93)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Radi, Z.A., D.L. Miller, and M.E. Hines, 2nd, Rete testis mucinous adenocarcinoma in a dog. Vet Pathol, 2004. 41\(1\): p. 75-8)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Ohuchi, N., et al., Differential expression of carcinoembryonic antigen in early gastric adenocarcinomas versus benign )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(gastric lesions defined by monoclonal antibodies reactive with restricted antigen epitopes. Cancer Res, 1987. 47\(13\): p. 3565-71)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Chhieng, D.C., et al., Expression of CEA, Tag-72, and Lewis-Y antigen in primary and metastatic lesions of ovarian )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(carcinoma. Hum Pathol, 2003. 34\(10\): p. 1016-21)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Kass, E.S., et al., Carcinoembryonic antigen as a target for specific antitumor immunotherapy of head and neck cancer. )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Cancer Research, 2002. 62\(17\): p. 5049-57)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Tendler, A., H.L. Kaufman, and A.S. Kadish, Increased carcinoembryonic antigen expression in cervical intraepithelial )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(neoplasia grade 3 and in cervical squamous cell carcinoma. Hum Pathol, 2000. 31\(11\): p. 1357-62)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Irvine, K. and J. Schlom, Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72. Cancer Immunol Immunother, 1993. )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(36\(5\): p. 281-92)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Gonzales, N.R., et al., SDR grafting of a murine antibody using multiple human germline templates to minimize its )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(immunogenicity. Mol Immunol, 2004. 41\(9\): p. 863-72)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Kammerer, R. and W. Zimmermann, Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(antigen families. BMC Biol, 2010. 8: p. 12)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Tamura, K., et al., MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. Mol Biol Evol, 2007. )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(24\(8\): p. 1596-9)] TJ ET BT 26.250 278.429 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 278.429 Td /F1 9.8 Tf [(Kumar, S., et al., MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief )] TJ ET BT 26.250 266.524 Td /F1 9.8 Tf [(Bioinform, 2008. 9\(4\): p. 299-306)] TJ ET BT 26.250 247.119 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 247.119 Td /F1 9.8 Tf [(Jones, D.T., W.R. Taylor, and J.M. Thornton, The rapid generation of mutation data matrices from protein sequences. )] TJ ET BT 26.250 235.215 Td /F1 9.8 Tf [(Comput Appl Biosci, 1992. 8\(3\): p. 275-82)] TJ ET BT 26.250 215.810 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 215.810 Td /F1 9.8 Tf [(Tamura, K. and M. Nei, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in )] TJ ET BT 26.250 203.905 Td /F1 9.8 Tf [(humans and chimpanzees. Mol Biol Evol, 1993. 10\(3\): p. 512-26)] TJ ET BT 26.250 184.500 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 184.500 Td /F1 9.8 Tf [(Voges, M., et al., CEACAM1 recognition by bacterial pathogens is species-specific. BMC Microbiol, 2010. 10:)] TJ ET BT 26.250 165.096 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 165.096 Td /F1 9.8 Tf [(Yamamoto, Y., et al., Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma. Biochem )] TJ ET BT 26.250 153.191 Td /F1 9.8 Tf [(Biophys Res Commun, 2005. 333\(1\): p. 223-9)] TJ ET BT 26.250 133.786 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 133.786 Td /F1 9.8 Tf [(Kammerer, R., et al., Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-)] TJ ET BT 26.250 121.881 Td /F1 9.8 Tf [(related immune receptors in the dog. BMC Evol Biol, 2007. 7: p. 196)] TJ ET BT 26.250 102.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 102.477 Td /F1 9.8 Tf [(Samsen, A., et al., DC-SIGN and SRCL bind glycans of carcinoembryonic antigen \(CEA\) and CEA-related cell adhesion )] TJ ET BT 26.250 90.572 Td /F1 9.8 Tf [(molecule 1 \(CEACAM1\): recombinant human glycan-binding receptors as analytical tools. Eur J Cell Biol, 2010. 89\(1\): p. 87-94)] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 71.167 Td /F1 9.8 Tf [(Nazato, D.M., et al., Prognostic value of carcinoembryonic antigen distribution in tumor tissue of colorectal carcinoma. Arq )] TJ ET BT 26.250 59.262 Td /F1 9.8 Tf [(Gastroenterol, 2009. 46\(1\): p. 26-31)] TJ ET Q q 15.000 49.381 577.500 727.619 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(1-24)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Bramswig, K.H., et al., Immunization with mimotopes prevents growth of carcinoembryonic antigen positive tumors in )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(BALB/c mice. Clin Cancer Res, 2007. 13\(21\): p. 6501-8)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(Facciabene, A., et al., Vectors encoding carcinoembryonic antigen fused to the B subunit of heat-labile enterotoxin elicit )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(antigen-specific immune responses and antitumor effects. Vaccine, 2007. 26\(1\): p. 47-58)] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(33.)] TJ ET BT 43.553 685.452 Td /F1 9.8 Tf [(Smith, B.F., et al., Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human )] TJ ET BT 26.250 673.548 Td /F1 9.8 Tf [(carcinoembryonic antigen. Gene Ther, 1998. 5\(7\): p. 865-8)] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(34.)] TJ ET BT 43.553 654.143 Td /F1 9.8 Tf [(Rabanal, R., et al., Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-)] TJ ET BT 26.250 642.238 Td /F1 9.8 Tf [(antitrypsin in carcinomas of the exocrine pancreas of the dog. Res Vet Sci, 1992. 52\(2\): p. 217-23)] TJ ET BT 26.250 622.833 Td /F1 9.8 Tf [(35.)] TJ ET BT 43.553 622.833 Td /F1 9.8 Tf [(Martin de las Mulas, J., et al., Immunohistochemical evaluation of canine primary liver carcinomas: distribution of alpha-)] TJ ET BT 26.250 610.929 Td /F1 9.8 Tf [(fetoprotein, carcinoembryonic antigen, keratins and vimentin. Res Vet Sci, 1995. 59\(2\): p. 124-7)] TJ ET BT 26.250 591.524 Td /F1 9.8 Tf [(36.)] TJ ET BT 43.553 591.524 Td /F1 9.8 Tf [(Cantile, C., et al., Pathological and immunohistochemical studies of choroid plexus carcinoma of the dog. J Comp Pathol, )] TJ ET BT 26.250 579.619 Td /F1 9.8 Tf [(2002. 126\(2-3\): p. 183-93)] TJ ET BT 26.250 560.214 Td /F1 9.8 Tf [(37.)] TJ ET BT 43.553 560.214 Td /F1 9.8 Tf [(Radi, Z.A., D.L. Miller, and M.E. Hines, 2nd, Rete testis mucinous adenocarcinoma in a dog. Vet Pathol, 2004. 41\(1\): p. 75-8)] TJ ET BT 26.250 540.810 Td /F1 9.8 Tf [(38.)] TJ ET BT 43.553 540.810 Td /F1 9.8 Tf [(Ohuchi, N., et al., Differential expression of carcinoembryonic antigen in early gastric adenocarcinomas versus benign )] TJ ET BT 26.250 528.905 Td /F1 9.8 Tf [(gastric lesions defined by monoclonal antibodies reactive with restricted antigen epitopes. Cancer Res, 1987. 47\(13\): p. 3565-71)] TJ ET BT 26.250 509.500 Td /F1 9.8 Tf [(39.)] TJ ET BT 43.553 509.500 Td /F1 9.8 Tf [(Chhieng, D.C., et al., Expression of CEA, Tag-72, and Lewis-Y antigen in primary and metastatic lesions of ovarian )] TJ ET BT 26.250 497.595 Td /F1 9.8 Tf [(carcinoma. Hum Pathol, 2003. 34\(10\): p. 1016-21)] TJ ET BT 26.250 478.191 Td /F1 9.8 Tf [(40.)] TJ ET BT 43.553 478.191 Td /F1 9.8 Tf [(Kass, E.S., et al., Carcinoembryonic antigen as a target for specific antitumor immunotherapy of head and neck cancer. )] TJ ET BT 26.250 466.286 Td /F1 9.8 Tf [(Cancer Research, 2002. 62\(17\): p. 5049-57)] TJ ET BT 26.250 446.881 Td /F1 9.8 Tf [(41.)] TJ ET BT 43.553 446.881 Td /F1 9.8 Tf [(Tendler, A., H.L. Kaufman, and A.S. Kadish, Increased carcinoembryonic antigen expression in cervical intraepithelial )] TJ ET BT 26.250 434.976 Td /F1 9.8 Tf [(neoplasia grade 3 and in cervical squamous cell carcinoma. Hum Pathol, 2000. 31\(11\): p. 1357-62)] TJ ET BT 26.250 415.572 Td /F1 9.8 Tf [(42.)] TJ ET BT 43.553 415.572 Td /F1 9.8 Tf [(Irvine, K. and J. Schlom, Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to )] TJ ET BT 26.250 403.667 Td /F1 9.8 Tf [(tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72. Cancer Immunol Immunother, 1993. )] TJ ET BT 26.250 391.762 Td /F1 9.8 Tf [(36\(5\): p. 281-92)] TJ ET BT 26.250 372.357 Td /F1 9.8 Tf [(43.)] TJ ET BT 43.553 372.357 Td /F1 9.8 Tf [(Gonzales, N.R., et al., SDR grafting of a murine antibody using multiple human germline templates to minimize its )] TJ ET BT 26.250 360.453 Td /F1 9.8 Tf [(immunogenicity. Mol Immunol, 2004. 41\(9\): p. 863-72)] TJ ET BT 26.250 341.048 Td /F1 9.8 Tf [(44.)] TJ ET BT 43.553 341.048 Td /F1 9.8 Tf [(Kammerer, R. and W. Zimmermann, Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic )] TJ ET BT 26.250 329.143 Td /F1 9.8 Tf [(antigen families. BMC Biol, 2010. 8: p. 12)] TJ ET BT 26.250 309.738 Td /F1 9.8 Tf [(45.)] TJ ET BT 43.553 309.738 Td /F1 9.8 Tf [(Tamura, K., et al., MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. Mol Biol Evol, 2007. )] TJ ET BT 26.250 297.834 Td /F1 9.8 Tf [(24\(8\): p. 1596-9)] TJ ET BT 26.250 278.429 Td /F1 9.8 Tf [(46.)] TJ ET BT 43.553 278.429 Td /F1 9.8 Tf [(Kumar, S., et al., MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief )] TJ ET BT 26.250 266.524 Td /F1 9.8 Tf [(Bioinform, 2008. 9\(4\): p. 299-306)] TJ ET BT 26.250 247.119 Td /F1 9.8 Tf [(47.)] TJ ET BT 43.553 247.119 Td /F1 9.8 Tf [(Jones, D.T., W.R. Taylor, and J.M. Thornton, The rapid generation of mutation data matrices from protein sequences. )] TJ ET BT 26.250 235.215 Td /F1 9.8 Tf [(Comput Appl Biosci, 1992. 8\(3\): p. 275-82)] TJ ET BT 26.250 215.810 Td /F1 9.8 Tf [(48.)] TJ ET BT 43.553 215.810 Td /F1 9.8 Tf [(Tamura, K. and M. Nei, Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in )] TJ ET BT 26.250 203.905 Td /F1 9.8 Tf [(humans and chimpanzees. Mol Biol Evol, 1993. 10\(3\): p. 512-26)] TJ ET BT 26.250 184.500 Td /F1 9.8 Tf [(49.)] TJ ET BT 43.553 184.500 Td /F1 9.8 Tf [(Voges, M., et al., CEACAM1 recognition by bacterial pathogens is species-specific. BMC Microbiol, 2010. 10:)] TJ ET BT 26.250 165.096 Td /F1 9.8 Tf [(50.)] TJ ET BT 43.553 165.096 Td /F1 9.8 Tf [(Yamamoto, Y., et al., Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma. Biochem )] TJ ET BT 26.250 153.191 Td /F1 9.8 Tf [(Biophys Res Commun, 2005. 333\(1\): p. 223-9)] TJ ET BT 26.250 133.786 Td /F1 9.8 Tf [(51.)] TJ ET BT 43.553 133.786 Td /F1 9.8 Tf [(Kammerer, R., et al., Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-)] TJ ET BT 26.250 121.881 Td /F1 9.8 Tf [(related immune receptors in the dog. BMC Evol Biol, 2007. 7: p. 196)] TJ ET BT 26.250 102.477 Td /F1 9.8 Tf [(52.)] TJ ET BT 43.553 102.477 Td /F1 9.8 Tf [(Samsen, A., et al., DC-SIGN and SRCL bind glycans of carcinoembryonic antigen \(CEA\) and CEA-related cell adhesion )] TJ ET BT 26.250 90.572 Td /F1 9.8 Tf [(molecule 1 \(CEACAM1\): recombinant human glycan-binding receptors as analytical tools. Eur J Cell Biol, 2010. 89\(1\): p. 87-94)] TJ ET BT 26.250 71.167 Td /F1 9.8 Tf [(53.)] TJ ET BT 43.553 71.167 Td /F1 9.8 Tf [(Nazato, D.M., et al., Prognostic value of carcinoembryonic antigen distribution in tumor tissue of colorectal carcinoma. Arq )] TJ ET BT 26.250 59.262 Td /F1 9.8 Tf [(Gastroenterol, 2009. 46\(1\): p. 26-31)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(12)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Tree of Life)] TJ ET Q endstream endobj 412 0 obj << /Type /Page /Parent 3 0 R /Contents 413 0 R >> endobj 413 0 obj << /Length 4037 >> stream 0.271 0.267 0.267 rg q 15.000 636.762 577.500 140.238 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Guo, K., et al., Monoclonal antibodies target intracellular PRL phosphatases to inhibit cancer metastases in mice. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Biol Ther, 2008. 7\(5\): p. 750-7)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Avrameas, A., et al., Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(and intranuclear translocation of macromolecules. Proc Natl Acad Sci U S A, 1998. 95\(10\): p. 5601-6)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Joller, N., et al., Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting. Proc Natl )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Acad Sci U S A, 2010)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Bajenova, O.V., et al., Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells. J )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(Biol Chem, 2001. 276\(33\): p. 31067-73)] TJ ET Q q 15.000 636.762 577.500 140.238 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Guo, K., et al., Monoclonal antibodies target intracellular PRL phosphatases to inhibit cancer metastases in mice. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Biol Ther, 2008. 7\(5\): p. 750-7)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Avrameas, A., et al., Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(and intranuclear translocation of macromolecules. Proc Natl Acad Sci U S A, 1998. 95\(10\): p. 5601-6)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Joller, N., et al., Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting. Proc Natl )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Acad Sci U S A, 2010)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Bajenova, O.V., et al., Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells. J )] TJ ET BT 26.250 654.143 Td /F1 9.8 Tf [(Biol Chem, 2001. 276\(33\): p. 31067-73)] TJ ET Q q 15.000 636.762 577.500 140.238 re W n 0.271 0.267 0.267 rg BT 26.250 759.976 Td /F1 9.8 Tf [(54.)] TJ ET BT 43.553 759.976 Td /F1 9.8 Tf [(Guo, K., et al., Monoclonal antibodies target intracellular PRL phosphatases to inhibit cancer metastases in mice. Cancer )] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(Biol Ther, 2008. 7\(5\): p. 750-7)] TJ ET BT 26.250 728.667 Td /F1 9.8 Tf [(55.)] TJ ET BT 43.553 728.667 Td /F1 9.8 Tf [(Avrameas, A., et al., Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic )] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(and intranuclear translocation of macromolecules. Proc Natl Acad Sci U S A, 1998. 95\(10\): p. 5601-6)] TJ ET BT 26.250 697.357 Td /F1 9.8 Tf [(56.)] TJ ET BT 43.553 697.357 Td /F1 9.8 Tf [(Joller, N., et al., Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting. Proc Natl )] TJ ET BT 26.250 685.452 Td /F1 9.8 Tf [(Acad Sci U S A, 2010)] TJ ET BT 26.250 666.048 Td /F1 9.8 Tf [(57.)] TJ ET BT 43.553 666.048 Td /F1 9.8 Tf [(Bajenova, O.V., et al., Heterogeneous RNA-binding protein M4 is a receptor for carcinoembryonic antigen in Kupffer cells. 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