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• W2115183912 abstract "Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction. Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction. Various strategies for cancer vaccines including whole tumor cell vaccines, genetically modified tumor vaccines, dendritic cell vaccines, and peptide and protein vaccines have been developed to induce tumor-specific immune response against autologous malignant cells (1Boon T. Coulie P.G. Van den Eynde B. Immunol. Today. 1997; 18: 267-268Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 2Rosenberg S.A. Immunol. Today. 1997; 18: 175-178Abstract Full Text PDF PubMed Scopus (379) Google Scholar). Active specific immunotherapies with cancer vaccines based on tumor antigens represent very promising approaches for cancer therapy (1Boon T. Coulie P.G. Van den Eynde B. Immunol. Today. 1997; 18: 267-268Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 2Rosenberg S.A. Immunol. Today. 1997; 18: 175-178Abstract Full Text PDF PubMed Scopus (379) Google Scholar). However, to date, with a few exceptions such as the case with melanoma antigens, there is limited information on the identity and density of antigenic peptides and the cytotoxic T lymphocyte (CTL) epitopes presented by human solid tumors (1Boon T. Coulie P.G. Van den Eynde B. Immunol. Today. 1997; 18: 267-268Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 2Rosenberg S.A. Immunol. Today. 1997; 18: 175-178Abstract Full Text PDF PubMed Scopus (379) Google Scholar). In addition, most of the identified tumor antigens are self-molecules (1Boon T. Coulie P.G. Van den Eynde B. Immunol. Today. 1997; 18: 267-268Abstract Full Text PDF PubMed Scopus (500) Google Scholar, 2Rosenberg S.A. Immunol. Today. 1997; 18: 175-178Abstract Full Text PDF PubMed Scopus (379) Google Scholar, 3Gouttefangeas C. Rammensee H.G. Nat. Biotechnol. 2000; 18: 491-492Crossref PubMed Scopus (7) Google Scholar). As expected, the reaction of the host toward these self-molecules may show immune tolerance to them if the host is immunized with the vaccines based on these self-molecules (4Grossman Z. Paul W.E. Semin. Immunol. 2000; 12: 197-203Crossref PubMed Scopus (100) Google Scholar, 5Kang L. Tomonori I. Marzena S. Yukino K. Kayo I. Steinman R.M. J. Exp. Med. 2002; 196: 1091-1097Crossref PubMed Scopus (405) Google Scholar, 6Anderton S.M. Viner N.J. Matharu P. Lowrey P.A. Wraith D.C. Nat. Immunol. 2002; 3: 175-181Crossref PubMed Scopus (82) Google Scholar, 7Sigmund K. Nowak M.A. Nature. 2001; 414: 403-405Crossref PubMed Scopus (6) Google Scholar, 8Shortman K. Heath W.R. Nat. Immunol. 2001; 2: 988-989Crossref PubMed Scopus (95) Google Scholar). Efforts are therefore continuing to develop new strategy for cancer vaccines. The generation of new blood vessels, or angiogenesis, is a complex multistep process that includes endothelial proliferation, migration and differentiation, degradation of extracellular matrix, etc. (9Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 10Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar). The complexity of the angiogenic process suggests the existence of multiple controls in the system that can be temporarily turned on and off (9Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 10Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar). Angiogenesis is important for normal embryonic development and the development of pathologic conditions such as cancer, rheumatoid arthritis, retinopathies, etc. (9Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 10Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar, 11Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7475) Google Scholar). Several lines of direct and indirect evidence indicate that the growth and persistence of solid tumors and their metastasis are angiogenesis-dependent (9Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 10Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar, 11Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7475) Google Scholar, 12Ferrara N. Alitalo K. Nat. Med. 1999; 5: 1359-1364Crossref PubMed Scopus (916) Google Scholar, 13Ding L. Donate F. Parry G.C. Guan X. Maher P. Levin E.G. J. Biol. Chem. 2002; 277: 31056-31061Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 14Folkman J. Nat. Med. 1996; 2: 167-168Crossref PubMed Scopus (268) Google Scholar). As a strategy for cancer therapy, antiangiogenic therapy attempts to stop new vessels from forming around a tumor and break up the existing network of abnormal capillaries that feed the cancerous mass (15St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1646) Google Scholar, 16Cheng W.F. Hung C.F. Chai C.Y. Hsu K.F. He L.M. Ling M. Wu T.C. J. Clin. Invest. 2001; 108: 669-678Crossref PubMed Scopus (247) Google Scholar, 17Barinaga M. Science. 2000; 288: 245Crossref PubMed Google Scholar, 18Marx J. Science. 2000; 289: 1121-1122Crossref PubMed Google Scholar). Endothelial cells in the angiogenic vessels within solid tumors express proteins on their surface that are absent or barely detectable in the normal quiescent vascular endothelium, including certain angiogenic growth factors and their receptors such as basic fibroblast growth factor (bFGF) 1The abbreviations used are: bFGF, basic fibroblast growth factor; FGFR-1, fibroblast growth factor receptor-1; HUVEC, human umbilical vein endothelial cells; pmFR1, plasmid DNA encoding homologous mouse FGFR-1; pxFR1, plasmid DNA encoding homologous Xenopus FGFR-1; e-p, empty pcDNA3.1 (+) vector; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; Meth A, methylcholanthrene-induced (fibrosarcoma); FITC, fluorescein isothiocyanate. and its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1), among others (9Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 10Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar, 11Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7475) Google Scholar, 12Ferrara N. Alitalo K. Nat. Med. 1999; 5: 1359-1364Crossref PubMed Scopus (916) Google Scholar, 13Ding L. Donate F. Parry G.C. Guan X. Maher P. Levin E.G. J. Biol. Chem. 2002; 277: 31056-31061Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 14Folkman J. Nat. Med. 1996; 2: 167-168Crossref PubMed Scopus (268) Google Scholar, 15St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1646) Google Scholar). The breaking of immune tolerance against important molecules such as some receptors associated with angiogenesis on autologous angiogenic endothelial cells should be a useful approach for cancer therapy by active immunity. However, the immunity to the self-molecules on angiogenic vessels is presumably difficult to elicit by using autologous or syngeneic protein molecules as vaccine because of the immune tolerance acquired during the development of the immune system (4Grossman Z. Paul W.E. Semin. Immunol. 2000; 12: 197-203Crossref PubMed Scopus (100) Google Scholar, 5Kang L. Tomonori I. Marzena S. Yukino K. Kayo I. Steinman R.M. J. Exp. Med. 2002; 196: 1091-1097Crossref PubMed Scopus (405) Google Scholar, 6Anderton S.M. Viner N.J. Matharu P. Lowrey P.A. Wraith D.C. Nat. Immunol. 2002; 3: 175-181Crossref PubMed Scopus (82) Google Scholar, 7Sigmund K. Nowak M.A. Nature. 2001; 414: 403-405Crossref PubMed Scopus (6) Google Scholar, 8Shortman K. Heath W.R. Nat. Immunol. 2001; 2: 988-989Crossref PubMed Scopus (95) Google Scholar). Many genes were highly conserved during the evolutionary process, which was characterized by varying degrees of gene similarity among different species (19Kornberg T.B. Krasnow M.A. Science. 2000; 287: 2218-2220Crossref PubMed Scopus (68) Google Scholar, 20Hedges S.B. Kumar S. Science. 2002; 297: 1283-1285Crossref PubMed Google Scholar, 21Mural R.J. Adams M.D. Myers E.W. Smith H.O. Gabor Miklos G.L. Wides R. Halpern A. Li P.W. Sutton G.G. Nadeau J. Salzberg S.L. Holt R.A. Kodira C.D. Lu F. Chen L. Deng Z. Evangelista C.C. Gan W. Heiman T.J. Li J. Li Z. Merkulov G.V. Milshina N.V. Naik A.K. Qi R. Chris Shue B. Wang A. Wang J. Wang X. Yan X. Ye J. Yooseph S. Zhao Q. Zheng L. Zhu S.C. Biddick K. Bolanos R. Delcher A.L. Dew I.M. Fasulo D. Flanigan M.J. Huson D.H. Kravitz S.A. Miller J.R. Mobarry C.M. Reinert K. Remington K.A. Zhang Q. Zheng X.H. Nusskern D.R. Lai Z. Lei Y. Zhong W. Yao A. Guan P. Ji R.R. Gu Z. Wang Z.Y. Zhong F. Xiao C. Chiang C. Yandell M. Wortman J.R. Amanatides P.G. Hladun S.L. Pratts E.C. Johnson J.E. Dodson K.L. Woodford K.J. Evans C.A. Gropman B. Rusch D.B. Venter E. Wang M. Smith T.J. Houck J.T. Tompkins D.E. Haynes C. Jacob D. Chin S.H. Allen D.R. Dahlke C.E. Sanders R. Li K. Liu X. Levitsky A.A. Majoros W.H. Chen Q. Xia A.C. Lopez J.R. Donnelly M.T. Newman M.H. Glodek A. Kraft C.L. Nodell F. Ali H. An D. Baldwin-Pitts K.Y. Beeson S. Cai M. Carnes A. Carver P.M. Caulk M. Center A. Chen Y. Cheng M.L. Coyne M.D. Crowder M. Danaher S. Davenport L.B. Desilets R. Dietz S.M. Doup L. Dullaghan P. Ferriera S. Fosler C.R. Gire H.C. Gluecksmann A. Gocayne J.D. Gray J. Hart B. Haynes J. Hoover J. Howland T. Ibegwam C. Jalali M. Johns D. Kline L. Ma D.S. MacCawley S. Magoon A. Mann F. May D. McIntosh T.C. Mehta S. Moy L. Moy M.C. Murphy B.J. Murphy S.D. Nelson K.A. Nuri Z. Parker K.A. Prudhomme A.C. Puri V.N. Qureshi H. Raley J.C. Reardon M.S. Regier M.A. Rogers Y.C. Romblad D.L. Schutz J. Scott J.L. Scott R. Sitter C.D. Smallwood M. Sprague A.C. Stewart E. Strong R.V. Suh E. Sylvester K. Thomas R. Ni Tint N. Tsonis C. Wang G. Wang G. Williams M.S. Williams S.M. Windsor S.M. Wolfe K. Wu M.M. Zaveri J. Chaturvedi K. Gabrielian A.E. Ke Z. Sun J. Subramanian G. Venter J.C. Science. 2002; 296: 1661-1671Crossref PubMed Scopus (295) Google Scholar). Many counterparts of the genes of human and mouse can be identified from the genome sequence of Drosophila melanogaster and other species such as Xenopus laevis (19Kornberg T.B. Krasnow M.A. Science. 2000; 287: 2218-2220Crossref PubMed Scopus (68) Google Scholar, 20Hedges S.B. Kumar S. Science. 2002; 297: 1283-1285Crossref PubMed Google Scholar, 21Mural R.J. Adams M.D. Myers E.W. Smith H.O. Gabor Miklos G.L. Wides R. Halpern A. Li P.W. Sutton G.G. Nadeau J. Salzberg S.L. Holt R.A. Kodira C.D. Lu F. Chen L. Deng Z. Evangelista C.C. Gan W. Heiman T.J. Li J. Li Z. Merkulov G.V. Milshina N.V. Naik A.K. Qi R. Chris Shue B. Wang A. Wang J. Wang X. Yan X. Ye J. Yooseph S. Zhao Q. Zheng L. Zhu S.C. Biddick K. Bolanos R. Delcher A.L. Dew I.M. Fasulo D. Flanigan M.J. Huson D.H. Kravitz S.A. Miller J.R. Mobarry C.M. Reinert K. Remington K.A. Zhang Q. Zheng X.H. Nusskern D.R. Lai Z. Lei Y. Zhong W. Yao A. Guan P. Ji R.R. Gu Z. Wang Z.Y. Zhong F. Xiao C. Chiang C. Yandell M. Wortman J.R. Amanatides P.G. Hladun S.L. Pratts E.C. Johnson J.E. Dodson K.L. Woodford K.J. Evans C.A. Gropman B. Rusch D.B. Venter E. Wang M. Smith T.J. Houck J.T. Tompkins D.E. Haynes C. Jacob D. Chin S.H. Allen D.R. Dahlke C.E. Sanders R. Li K. Liu X. Levitsky A.A. Majoros W.H. Chen Q. Xia A.C. Lopez J.R. Donnelly M.T. Newman M.H. Glodek A. Kraft C.L. Nodell F. Ali H. An D. Baldwin-Pitts K.Y. Beeson S. Cai M. Carnes A. Carver P.M. Caulk M. Center A. Chen Y. Cheng M.L. Coyne M.D. Crowder M. Danaher S. Davenport L.B. Desilets R. Dietz S.M. Doup L. Dullaghan P. Ferriera S. Fosler C.R. Gire H.C. Gluecksmann A. Gocayne J.D. Gray J. Hart B. Haynes J. Hoover J. Howland T. Ibegwam C. Jalali M. Johns D. Kline L. Ma D.S. MacCawley S. Magoon A. Mann F. May D. McIntosh T.C. Mehta S. Moy L. Moy M.C. Murphy B.J. Murphy S.D. Nelson K.A. Nuri Z. Parker K.A. Prudhomme A.C. Puri V.N. Qureshi H. Raley J.C. Reardon M.S. Regier M.A. Rogers Y.C. Romblad D.L. Schutz J. Scott J.L. Scott R. Sitter C.D. Smallwood M. Sprague A.C. Stewart E. Strong R.V. Suh E. Sylvester K. Thomas R. Ni Tint N. Tsonis C. Wang G. Wang G. Williams M.S. Williams S.M. Windsor S.M. Wolfe K. Wu M.M. Zaveri J. Chaturvedi K. Gabrielian A.E. Ke Z. Sun J. Subramanian G. Venter J.C. Science. 2002; 296: 1661-1671Crossref PubMed Scopus (295) Google Scholar). For example, a comparison analysis made in the present study by searching the Swiss Prot data base at the National Center for Biotechnology Information indicates that the Xenopus homologue of FGFR-1 (GenBank™ accession number U24491) is 80 and 74% identical in mouse FGFR-1 (GenBank™ accession number M33760) and human FGFR-1 (GenBank™ accession number M34641), respectively, at the amino acid level (22Musci T.J. Amaya E. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8365-8369Crossref PubMed Scopus (114) Google Scholar, 23Robbie E.P. Peterson M. Amaya E. Musci T.J. Development. 1995; 121: 1775-1785PubMed Google Scholar, 24Reid H.H. Wilk A.F. Bernard O.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1596-1600Crossref PubMed Scopus (172) Google Scholar). In addition, basic fibroblast growth factor has been shown to be one of the most important angiogenic growth factors for angiogenesis through interaction with its high-affinity receptor, FGFR-1 (25Valesky M. Spang A.J. Fisher G.W. Farkas D.L. Becker D. Mol. Med. 2002; 8: 103-112Crossref PubMed Google Scholar, 26Plum S.M. Holaday J.W. Ruiz A. Madsen J.W. Fogler W.E. Fortier A.H. Vaccine. 2000; 19: 1294-1303Crossref PubMed Scopus (85) Google Scholar, 27Guddo F. Fontanini G. Reina C. Vignola A.M. Angeletti A. Bonsignore G. Hum. Pathol. 1999; 30: 788-794Crossref PubMed Scopus (57) Google Scholar, 28Auguste P. Gursel D.B. Lemiere S. Reimers D. Cuevas P. Carceller F. Di Santo J.P. Bikfalvi A. Cancer Res. 2001; 61: 1717-1726PubMed Google Scholar). FGFR-1 is markedly expressed both in endothelial cells and in many different forms of tumor (25Valesky M. Spang A.J. Fisher G.W. Farkas D.L. Becker D. Mol. Med. 2002; 8: 103-112Crossref PubMed Google Scholar, 26Plum S.M. Holaday J.W. Ruiz A. Madsen J.W. Fogler W.E. Fortier A.H. Vaccine. 2000; 19: 1294-1303Crossref PubMed Scopus (85) Google Scholar, 27Guddo F. Fontanini G. Reina C. Vignola A.M. Angeletti A. Bonsignore G. Hum. Pathol. 1999; 30: 788-794Crossref PubMed Scopus (57) Google Scholar, 28Auguste P. Gursel D.B. Lemiere S. Reimers D. Cuevas P. Carceller F. Di Santo J.P. Bikfalvi A. Cancer Res. 2001; 61: 1717-1726PubMed Google Scholar). These findings suggest that FGFR1 plays an important role in tumor angiogenesis and tumor growth, and it may be used as an ideal molecule to explore the feasibility of tumor therapy. The current studies explore the feasibility of immunotherapy of tumors with the plasmid DNA encoding Xenopus FGFR-1 as a vaccine by the breaking of the immune tolerance against FGFR-1 in a cross-reaction between the xenogeneic homologous and self-FGFR-1. To test this concept, we constructed a plasmid DNA encoding Xenopus FGFR-1 (pxFR1). At the same time, the plasmid DNA encoding the corresponding mouse FGFR-1 (pmFR1) and empty vector (e-p) were also constructed and used as controls. The plasmid DNA vaccines were tested for the ability to induce antitumor immune response in three tumor models in mice. Vaccine Preparation—A cDNA clone encoding Xenopus homologous FGFR-1 and mouse FGFR-1 were isolated from by PCR using the Xenopus laevis cDNA library (Clontech) and the mouse skeletal muscle cDNA library (Clontech), respectively. The amplified products were inserted into pT-Adv plasmid (Clontech) and then subcloned into pcDNA 3.1(+) (Invitrogen), which contains a cytomegalovirus promoter. FGFR-1 receptors of Xenopus and mouse inserted into pcDNA 3.1(+) were named pxFR1 and pmFR1, respectively. As a control, pure pcDNA 3.1(+) was used as empty vector. The full-length sequence of Xenopus and mouse FGFR-1 was confirmed by dideoxy method sequence to be identical to those reported previously (22Musci T.J. Amaya E. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8365-8369Crossref PubMed Scopus (114) Google Scholar, 23Robbie E.P. Peterson M. Amaya E. Musci T.J. Development. 1995; 121: 1775-1785PubMed Google Scholar, 24Reid H.H. Wilk A.F. Bernard O.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1596-1600Crossref PubMed Scopus (172) Google Scholar). Plasmids for DNA vaccination were purified by using two rounds of passage over Endo-free columns (Qiagen) as described previously (29Krieg A.M. Wu T. Weeratna R. Efler S.M. Love-Homan L. Yang L. Yi A.K. Short D. Davis H.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12631-12636Crossref PubMed Scopus (358) Google Scholar). The expression of plasmid DNA was confirmed in transfected H22 hepatoma cells by using reverse transcription PCR and a commercially available anti-FGFR-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in Western blotting analysis and ELISA. Tumor Models and Immunization—Meth A fibrosarcoma, H22 hepatoma, and MA782/5S mammary carcinoma models were established in 6-week female BALB/c mice (30Wei Y.Q. Wang Q.R. Zhao X. Yang L. Tian L. Lu Y. Kang B. Lu C.J. Huang M.J. Lou Y.Y. Xiao F. He Q.M. Shu J.M. Xie X.J. Mao Y.Q. Lei S. Luo F. Zhou L.Q. Liu C.E. Zhou H. Jiang Y. Peng F. Yuan L.P. Li Q. Wu Y. Liu J.Y. Nat Med. 2000; 6: 1160-1166Crossref PubMed Scopus (240) Google Scholar). Mice were immunized with different doses (10–200 μg per mouse per injection) of DNA vaccine in normal saline by intramuscular injection in both quadriceps once a week for 4 weeks. Additional control animals were injected with normal saline. 1 × 105 to 1 × 10 7 live tumor cells were then inoculated subcutaneously into mice after the fourth immunization. For an investigation of the therapeutic effect against the established tumors, ten mice in each group were treated with intramuscular injection of the DNA vaccines or normal saline alone once weekly for 4 weeks starting on day 7 after subcutaneous introduction of 1 × 106 live tumor cells. Tumor dimensions were measured with calipers, and tumor volumes were calculated according to the formula width2 × length × 0.52 (31Sauter B.V. Martinet O. Zhang W. Mandeli J. Woo S.L.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4802-4807Crossref PubMed Scopus (245) Google Scholar). All studies involving mice were approved by the institutional Animal Care and Use Committee. Western Blot Analysis—H22 cells were plated in 6-well plates at 2 × 105 cells/well. After incubation for 24 h, the cells were washed with PBS followed by transfection with 2 μg of plasmid (pxFR1, pmFR1 or e-p) and 3 μg of LipofectAMINE (Invitrogen) in serum-free Dulbecco's modified Eagle's medium. After incubation at 37 °C for 5 h, Dulbecco's modified Eagle's medium with 20% fetal bovine serum was added to each well. The transfected cells were collected 48 h after the start of transfection. Western blot analysis was performed as described (30Wei Y.Q. Wang Q.R. Zhao X. Yang L. Tian L. Lu Y. Kang B. Lu C.J. Huang M.J. Lou Y.Y. Xiao F. He Q.M. Shu J.M. Xie X.J. Mao Y.Q. Lei S. Luo F. Zhou L.Q. Liu C.E. Zhou H. Jiang Y. Peng F. Yuan L.P. Li Q. Wu Y. Liu J.Y. Nat Med. 2000; 6: 1160-1166Crossref PubMed Scopus (240) Google Scholar). Briefly, cell lysates were separated by SDS-PAGE. Gels were electro-blotted with Sartoblot onto a polyvinylidene difluoride membrane. The membrane blots were blocked by in 4 °C in 5% nonfat dry milk, washed, and probed with experimental mouse sera. Blots were then washed and incubated with a biotinylated secondary antibody (biotinylated horse anti-mouse IgG or IgM), followed by transfer to Vectastain ABC (Vector Laboratories). Also, the lysates of human umbilical vein endothelial cells (HUVECs) expressing FGFR-1 were analyzed. Immunoglobulin Subclass Response to Xenopus FGFR-1 Immunization in ELISA—The antigen-specific immunoglobulin subclass in the sera was determined by ELISA as described previously (30Wei Y.Q. Wang Q.R. Zhao X. Yang L. Tian L. Lu Y. Kang B. Lu C.J. Huang M.J. Lou Y.Y. Xiao F. He Q.M. Shu J.M. Xie X.J. Mao Y.Q. Lei S. Luo F. Zhou L.Q. Liu C.E. Zhou H. Jiang Y. Peng F. Yuan L.P. Li Q. Wu Y. Liu J.Y. Nat Med. 2000; 6: 1160-1166Crossref PubMed Scopus (240) Google Scholar, 32Blezinger P. Wang J. Gondo M. Quezada A. Mehrens D. French M. Singhal A. Sullivan S. Rolland A. Ralston R. Min W. Nat. Biotechnol. 1999; 17: 343-348Crossref PubMed Scopus (299) Google Scholar). Briefly, H22 cells transfected with pxFR1 and pmFR1 were washed with PBS and lysed by three cycles of freezing and thawing. Microtiter plates were coated with 50 μl of cell lysates (1 × 105 cell equivalents) at 4 °C overnight. The wells were then washed with PBS and blocked with blocking buffer at 37 °C for 2 h. After washing, experimental mouse sera were serially diluted in blocking buffer and added to the ELISA wells. Plates were incubated at 37 °C for 2 h, washed, and then incubated with serially diluted alkaline phosphatase-conjugated antibody against mouse IgG subclass, IgM, or IgA (Zymed Laboratories Inc.). Enzyme activity was measured with an ELISA reader (Bio-Rad). Inhibition of Cell Proliferation in Vitro and Adoptive Transfer in Vivo—Immunoglobulins were purified from the pooled sera derived from the mice at day 7 after the fourth immunization or from control mice by affinity chromatography (CM Affi-Gel blue gel kit; Bio-Rad). The inhibition of bFGF-mediated endothelial cell proliferation was described (28Auguste P. Gursel D.B. Lemiere S. Reimers D. Cuevas P. Carceller F. Di Santo J.P. Bikfalvi A. Cancer Res. 2001; 61: 1717-1726PubMed Google Scholar). Briefly, exponentially growing HUVEC and tumor cells (Meth A, H22, and MA782/5S) were treated with various concentrations (0–300 μg/ml) of immunoglobulins isolated from mice immunized with pxFR1, pmFR1, or e-p in the presence of 3 ng/ml bFGF (Sigma). After 72 h of culture, the number of viable cells was determined by a trypan blue dye exclusion test, and the percentage of inhibition was calculated (30Wei Y.Q. Wang Q.R. Zhao X. Yang L. Tian L. Lu Y. Kang B. Lu C.J. Huang M.J. Lou Y.Y. Xiao F. He Q.M. Shu J.M. Xie X.J. Mao Y.Q. Lei S. Luo F. Zhou L.Q. Liu C.E. Zhou H. Jiang Y. Peng F. Yuan L.P. Li Q. Wu Y. Liu J.Y. Nat Med. 2000; 6: 1160-1166Crossref PubMed Scopus (240) Google Scholar). To assess the efficacy of immunoglobulins in antitumor activity in vivo, the purified immunoglobulins (10–300 mg/kg) were adoptively transferred intravenously 1 day before the mice were challenged with tumor cells, and then the mice were treated twice per week for 3 weeks. As a control, immunoglobulins were adsorbed four times by incubation at 4 °C for 1 h while rocking with endothelial cells expressing FGFR-1 (HUVEC) or with FGFR-1-negative cells (Meth A, H22, and MA782/5S). Depletion of Immune Cell Subsets in Vivo—Immune cell subsets were depleted as described (33Pulendran B. Smith J.L. Caspary G. Brasel K. Pettit D. Maraskovsky E. Maliszewski C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1036-1041Crossref PubMed Scopus (878) Google Scholar, 34Horton H.M. Anderson D. Hernandez P. Barnhart K.M. Norman J.A. Parker S.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1553-1558Crossref PubMed Scopus (114) Google Scholar). Mice were injected intraperitoneally with 500 μg of either the anti-CD4 (clone GK1.5, rat IgG), anti-CD8 (clone 2.43, rat IgG), anti-NK (clone PK136) mAb, or isotype controls 1 day before the immunization and then immunized with 100 μg of plasmid once per week for 3 weeks. Tumor cells were challenged after the fourth immunization. These hybridomas were obtained from the American Type Culture Collection. The depletion of CD4+, CD8+, and NK cells was consistently greater than 98% as determined by flow cytometry (Coulter Elite ESP) (30Wei Y.Q. Wang Q.R. Zhao X. Yang L. Tian L. Lu Y. Kang B. Lu C.J. Huang M.J. Lou Y.Y. Xiao F. He Q.M. Shu J.M. Xie X.J. Mao Y.Q. Lei S. Luo F. Zhou L.Q. Liu C.E. Zhou H. Jiang Y. Peng F. Yuan L.P. Li Q. Wu Y. Liu J.Y. Nat Med. 2000; 6: 1160-1166Crossref PubMed Scopus (240) Google Scholar). Immunohistochemistry—Frozen sections were fixed in acetone and incubated with a stained antibody reactive to CD31 as described (32Blezinger P. Wang J. Gondo M. Quezada A. Mehrens D. French M. Singhal A. Sullivan S. Rolland A. Ralston R. Min W. Nat. Biotechnol. 1999; 17: 343-348Crossref PubMed Scopus (299) Google Scholar). The sections were then stained with labeled streptavidin biotin reagents (Dako). Vessel density was determined counting the microvessels per high power field in the sections as described (32Blezinger P. Wang J. Gondo M. Quezada A. Mehrens D. French M. Singhal A. Sullivan S. Rolland A. Ralston R. Min W. Nat. Biotechnol. 1999; 17: 343-348Crossref PubMed Scopus (299) Google Scholar). To identify the possible deposition of autoantibodies, we stained the sections with goat FITC-conjugated antibody against mouse IgA, IgM, or IgG (Sigma). Slides were examined by fluorescence microscopy. Alginate-encapsulate Tumor Cells Assay—An alginate-encapsulate assay was performed as described (35Hoffmann J. Schirner M. Menrad A. Schneider M.R. Cancer Res. 1997; 57: 3847-3851PubMed Google Scholar). Briefly, mice were treated with 100 μg of pxFR1, pmFR1, e-p, or normal saline alone once a week for 4 weeks. Meth A cells, H22 hepatoma cells, or MA782/5S mammary cancer cells were resuspended in a 1.8% solution of alginate (Sigma) and added dropwise into a solution of 250 mm calcium chloride; alginate beads were formed containing ∼1 × 105 tumor cells per bead. Four beads were then implanted subcutaneously into an incision made on the dorsal side of the immunized mice. After 12 days, mice were injected intravenously with 0.1 ml of a 100 mg/kg FITC-dextran (Sigma) solution. Alginate beads were rapidly removed and photographed 20 min after FITC-dextran injection. The uptake of FITC-dextran was measured as described (35Hoffmann J. Schirner M. Menrad A. Schneider M.R. Cancer Res. 1997; 57: 3847-3851PubMed Google Scholar). Statistical Analysis—For comparison of individual time points, analysis of variance (ANOVA) and an unpaired Student's t test were" @default.
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• W2115183912 title "Inhibition of Tumor Growth with a Vaccine Based on Xenogeneic Homologous Fibroblast Growth Factor Receptor-1 in Mice" @default.
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