Korean J Urol.  2011 May;52(5):327-334.

Combined Treatment with Anticancer Vaccine Using Genetically Modified Endothelial Cells and Imatinib in Bladder Cancer

Affiliations
  • 1Department of Urology, Seoul National University College of Medicine, Seoul, Korea. mdrafael@snu.ac.kr

Abstract

PURPOSE
We sought to maximize the antitumor effect of an anticancer vaccine based on genetically modified endothelial cells by combining it with the platelet-derived growth factor receptor inhibitor imatinib.
MATERIALS AND METHODS
Human umbilical vein endothelial cells (HUVECs) were infected with 10 MOI of Ad-CMV-mGMCSF to make anticancer vaccines. One million mouse bladder cancer cells (MBT-2) were subcutaneously inoculated in C3H mice. The experimental groups included the following: Group 1 (phosphate-buffered saline), Group 2 (anticancer vaccine and GM-CSF), Group 3 (imatinib), and Group 4 (anticancer vaccine, GM-CSF, and imatinib). Tumor growth and body weight were measured weekly. At 4 weeks, the tumors were immunostained with anti-CD31, and microvessel density (MVD) was measured. To evaluate the immunological mechanism of each treatment, flow cytometry analysis of activated CD4 and CD8 cells was performed.
RESULTS
At 4 weeks, the mean body weight of each group, excluding the extracted tumor weight, was not significantly different. Since week 3, the mean tumor volume in Group 4 was the smallest among the treatment groups (p<0.05), and a synergistic suppressive effect on tumor volume was observed in Group 4. The MVD in Group 4 was the most suppressed among the treatment groups (p<0.05), and a synergistic anti-angiogenic effect was observed. Flow cytometry analysis revealed that activated CD4+ and CD8+ cells increased in Group 2 and decreased in Group 3 compared with the other groups.
CONCLUSIONS
The combination of genetically modified endothelial cell vaccines and imatinib showed a synergistic antiangiogenic effect in bladder cancer.

Keyword

Immunotherapy; Platelet-derived growth factor; Urinary bladder neoplasms

MeSH Terms

Animals
Benzamides
Body Weight
Endothelial Cells
Flow Cytometry
Granulocyte-Macrophage Colony-Stimulating Factor
Human Umbilical Vein Endothelial Cells
Immunotherapy
Mice
Mice, Inbred C3H
Microvessels
Piperazines
Platelet-Derived Growth Factor
Pyrimidines
Receptors, Platelet-Derived Growth Factor
Tumor Burden
Urinary Bladder
Urinary Bladder Neoplasms
Vaccines
Imatinib Mesylate
Benzamides
Granulocyte-Macrophage Colony-Stimulating Factor
Piperazines
Platelet-Derived Growth Factor
Pyrimidines
Receptors, Platelet-Derived Growth Factor
Vaccines

Figure

  • FIG. 1 Mean body weights of mice during the treatment period. After 2 weeks of treatment, the mean body weights in Groups 2, 3, and 4 were less than in Group 1 (p<0.05).

  • FIG. 2 Mean body weights of mice without the mass of the extracted tumor at week 4. When comparing body weights without the mass of the extracted tumor at week 4, no significant differences were observed among the treatment groups (p=0.412).

  • FIG. 3 Mean tumor volumes during the treatment period. At weeks 3 and 4 of the treatment period, the mean tumor volume in Groups 3 and 4 was significantly lower than in Group 1 (p<0.05). In Group 4, the mean tumor volume was the smallest among the treatment groups, and the differences were all statistically significant compared with the other groups (p<0.05). The combination index of Group 4 at week 4 was 0.87.

  • FIG. 4 Gross subcutaneous MBT-2 tumor in mice. These pictures were taken at week 4. The tumor size in Group 4 was the smallest among the treatment groups (p<0.05).

  • FIG. 5 Flow cytometry analysis of (A) helper T-cells (CD4+ cells) and (B) cytotoxic T-cells (CD8+ cells). The dots in the upper right quadrant represent the number of activated T-cells. More activated helper and cytotoxic T-cells were observed in Groups 2 and 4 than in Group 1. However, in Group 3, which was treated with imatinib, fewer activated helper and cytotoxic T-cells were observed compared with Groups 1 and 2. In Group 4, which was treated with vaccine and imatinib, activated helper T-cells and cytotoxic T-cells were relatively low compared with Group 2.

  • FIG. 6 Analysis of microvessel density (MVD) of MBT-2 tumor. The differences between groups were statistically significant (p<0.001). The combination index was 0.43 with the combination therapy (Group 4).

  • FIG. 7 Photomicrographs depicting tumor microvessels (CD31 immunohistochemical staining, ×200). After immunohistochemical staining with CD31, the microvessels were stained brown. Microvessels decreased markedly in Group 4, moderately in Group 3, and slightly in Group 2.


Reference

1. von der Maase H, Hansen SW, Roberts JT, Dogliotti L, Oliver T, Moore MJ, et al. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol. 2000. 18:3068–3077.
2. Igawa M, Ohkuchi T, Ueki T, Ueda M, Okada K, Usui T. Usefulness and limitations of methotrexate, vinblastine, doxorubicin and cisplatin for the treatment of advanced urothelial cancer. J Urol. 1990. 144:662–665.
3. Böhle A, Brandau S. Immune mechanisms in bacillus Calmette-Guerin immunotherapy for superficial bladder cancer. J Urol. 2003. 170:964–969.
4. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol. 2002. 3:991–998.
5. Wei YQ, Wang QR, Zhao X, Yang L, Tian L, Lu Y, et al. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat Med. 2000. 6:1160–1166.
6. Plum SM, Holaday JW, Ruiz A, Madsen JW, Fogler WE, Fortier AH. Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor development. Vaccine. 2000. 19:1294–1303.
7. Seavey MM, Maciag PC, Al-Rawi N, Sewell D, Paterson Y. An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a mouse model. J Immunol. 2009. 182:5537–5546.
8. Jeong H, Kwak C, Park MS, Lee SE. Novel anticancer vaccine using genetically modified endothelial cells. Korean J Urol. 2004. 45:69–76.
9. Kassouf W, Dinney CP, Brown G, McConkey DJ, Diehl AJ, Bar-Eli M, et al. Uncoupling between epidermal growth factor receptor and downstream signals defines resistance to the antiproliferative effect of Gefitinib in bladder cancer cells. Cancer Res. 2005. 65:10524–10535.
10. Yoon CY, Lee JS, Kim BS, Jeong SJ, Hong SK, Byun SS, et al. Sunitinib malate synergistically potentiates anti-tumor effect of gemcitabine in human bladder cancer cells. Korean J Urol. 2011. 52:55–63.
11. Wallerand H, Reiter RR, Ravaud A. Molecular targeting in the treatment of either advanced or metastatic bladder cancer or both according to the signalling pathways. Curr Opin Urol. 2008. 18:524–532.
12. Forsberg K, Valyi-Nagy I, Heldin CH, Herlyn M, Westermark B. Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB. Proc Natl Acad Sci U S A. 1993. 90:393–397.
13. Shao ZM, Nguyen M, Barsky SH. Human breast carcinoma desmoplasia is PDGF initiated. Oncogene. 2000. 19:4337–4345.
14. Gasparini G, Harris AL. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol. 1995. 13:765–782.
15. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984. 22:27–55.
16. Coley WB. The treatment of inoperable sarcoma by bacterial toxins (the mixed toxins of the streptococcus erysipelas and the bacillus prodigiosus). Proc R Soc Med. 1910. 3:1–48.
17. Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay Nel H, et al. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin Cancer Res. 2006. 12:465–472.
18. Ruffini E, Asioli S, Filosso PL, Lyberis P, Bruna MC, Macri L, et al. Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg. 2009. 87:365–371.
19. Koos D, Josephs SF, Alexandrescu DT, Chan RC, Ramos F, Bogin V, et al. Tumor vaccines in 2010: need for integration. Cell Immunol. 2010. 263:138–147.
20. Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible for angiogenesis. J Exp Med. 1971. 133:275–288.
21. Witmer-Pack MD, Olivier W, Valinsky J, Schuler G, Steinman RM. Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J Exp Med. 1987. 166:1484–1498.
22. Reali E, Canter D, Zeytin H, Schlom J, Greiner JW. Comparative studies of Avipox-GM-CSF versus recombinant GM-CSF protein as immune adjuvants with different vaccine platforms. Vaccine. 2005. 23:2909–2921.
23. Serafini P, Carbley R, Noonan KA, Tan G, Bronte V, Borrello I. High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through the recruitment of myeloid suppressor cells. Cancer Res. 2004. 64:6337–6343.
24. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther. 2000. 295:139–145.
25. Black PC, Agarwal PK, Dinney CP. Targeted therapies in bladder cancer--an update. Urol Oncol. 2007. 25:433–438.
26. Seggewiss R, Loré K, Greiner E, Magnusson MK, Price DA, Douek DC, et al. Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner. Blood. 2005. 105:2473–2479.
27. Dietz AB, Souan L, Knutson GJ, Bulur PA, Litzow MR, Vuk-Pavlovic S. Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo. Blood. 2004. 104:1094–1099.
28. Baxevanis CN, Perez SA, Papamichail M. Combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies for cancer therapy. Cancer Immunol Immunother. 2009. 58:317–324.
29. Wrzesinski C, Paulos CM, Gattinoni L, Palmer DC, Kaiser A, Yu Z, et al. Hematopoietic stem cells promote the expansion and function of adoptively transferred antitumor CD8 T cells. J Clin Invest. 2007. 117:492–501.
30. Larmonier N, Janikashvili N, LaCasse CJ, Larmonier CB, Cantrell J, Situ E, et al. Imatinib mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances active immunotherapy against BCR-ABL-tumors. J Immunol. 2008. 181:6955–6963.
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