NeuroMolecular Medicine

, Volume 16, Issue 4, pp 752–771 | Cite as

Direct Effect of Bevacizumab on Glioblastoma Cell Lines In Vitro

  • Thomas SimonEmail author
  • Bérénice Coquerel
  • Alexandre Petit
  • Yusra Kassim
  • Elise Demange
  • Didier Le Cerf
  • Valérie Perrot
  • Jean-Pierre Vannier
Original Paper


Bevacizumab is a humanized monoclonal antibody directed against the pro-angiogenic factor vascular and endothelial growth factor-A (VEGF-A) used in the treatment of glioblastomas. Although most patients respond initially to this treatment, studies have shown that glioblastomas eventually recur. Several non-mutually exclusive theories based on the anti-angiogenic effect of bevacizumab have been proposed to explain these mechanisms of resistance. In this report, we studied whether bevacizumab can act directly on malignant glioblastoma cells. We observe changes in the expression profiles of components of the VEGF/VEGF-R pathway and in the response to a VEGF-A stimulus following bevacizumab treatment. In addition, we show that bevacizumab itself acts on glioblastoma cells by activating the Akt and Erks survival signaling pathways. Bevacizumab also enhances proliferation and invasiveness of glioblastoma cells in hyaluronic acid hydrogel. We propose that the paradoxical effect of bevacizumab on glioblastoma cells could be due to changes in the VEGF-A-dependent autocrine loop as well as in the intracellular survival pathways, leading to the enhancement of tumor aggressiveness. Investigation of how bevacizumab interacts with glioblastoma cells and the resulting downstream signaling pathways will help targeting populations of resistant glioblastoma cells.


Anti-angiogenic therapies Autocrine loop Brain extracellular matrix Glioblastoma VEGF-A 



Central nervous system


Extracellular matrix


Hyaluronic acid


Immunoglobulin G1


Placental growth factor


Vascular and endothelial growth factor



The authors would like to thank Catherine Buquet, Wiem Khelil, Laure Klosek, and Elisabeth Legrand for their technical help. The authors are very grateful to Dr. Flore Gouel and Pr. Isabelle Dubus for fruitful discussions. T. Simon is recipient of a fellowship from the “Conseil Régional de Haute-Normandie.” A. Petit is recipient of a fellowship from “Ministère de l’Enseignement supérieur et de la Recherche”.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Cao, Y., Zhong, W., & Sun, Y. (2009). Improvement of antiangiogenic cancer therapy by understanding the mechanisms of angiogenic factor interplay and drug resistance. Seminars in Cancer Biology, 19(5), 338–343. doi: 10.1016/j.semcancer.2009.05.001.PubMedCrossRefGoogle Scholar
  2. Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., & Kettenmann, H. (2012). The brain tumor microenvironment. Glia, 60(3), 502–514.PubMedCrossRefGoogle Scholar
  3. Chauzy, C., Delpech, B., Olivier, A., Bastard, C., Girard, N., Courel, M. N., Creissard, P. (1992). Establishment and characterisation of a human glioma cell line. European journal of cancer (Oxford, England: 1990), 28A(6–7), 1129–1134.Google Scholar
  4. Coquerel, B., Poyer, F., Torossian, F., Dulong, V., Bellon, G., Dubus, I., et al. (2009). Elastin-derived peptides: Matrikines critical for glioblastoma cell aggressiveness in a 3-D system. Glia, 57(16), 1716–1726. doi: 10.1002/glia.20884.PubMedCrossRefGoogle Scholar
  5. David, L., Dulong, V., Le Cerf, D., Cazin, L., Lamacz, M., & Vannier, J.-P. (2008). Hyaluronan hydrogel: An appropriate three-dimensional model for evaluation of anticancer drug sensitivity. Acta Biomaterialia, 4(2), 256–263. doi: 10.1016/j.actbio.2007.08.012.PubMedCrossRefGoogle Scholar
  6. David, L., Dulong, V., Le Cerf, D., Chauzy, C., Norris, V., Delpech, B., et al. (2004). Reticulated hyaluronan hydrogels: A model for examining cancer cell invasion in 3D. Matrix Biology: Journal of the International Society for Matrix Biology, 23(3), 183–193. doi: 10.1016/j.matbio.2004.05.005.CrossRefGoogle Scholar
  7. De Groot, J. F., Fuller, G., Kumar, A. J., Piao, Y., Eterovic, K., Ji, Y., et al. (2010). Tumor invasion after treatment of glioblastoma with bevacizumab: Radiographic and pathologic correlation in humans and mice. Neuro-oncology, 12(3), 233–242. doi: 10.1093/neuonc/nop027.PubMedCentralPubMedCrossRefGoogle Scholar
  8. DeAngelis, L. M. (2001). Brain tumors. The New England journal of medicine, 344(2), 114–123. doi: 10.1056/NEJM200101113440207.PubMedCrossRefGoogle Scholar
  9. Ellis, L. M., & Hicklin, D. J. (2008). Pathways mediating resistance to vascular endothelial growth factor-targeted therapy. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 14(20), 6371–6375. doi: 10.1158/1078-0432.CCR-07-5287.CrossRefGoogle Scholar
  10. Fan, F., Samuel, S., Gaur, P., Lu, J., Dallas, N. A., Xia, L., et al. (2011). Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration. British Journal of Cancer, 104(8), 1270–1277. doi: 10.1038/bjc.2011.81.PubMedCentralPubMedCrossRefGoogle Scholar
  11. Ferrara, N. (2009). VEGF-A: A critical regulator of blood vessel growth. European Cytokine Network, 20(4), 158–163. doi: 10.1684/ecn.2009.0170.PubMedGoogle Scholar
  12. Ferrara, N. (2010). Binding to the extracellular matrix and proteolytic processing: Two key mechanisms regulating vascular endothelial growth factor action. Molecular Biology of the Cell, 21(5), 687–690. doi: 10.1091/mbc.E09-07-0590.PubMedCentralPubMedCrossRefGoogle Scholar
  13. Ferrara, N., Hillan, K. J., & Novotny, W. (2005). Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochemical and Biophysical Research Communications, 333(2), 328–335. doi: 10.1016/j.bbrc.2005.05.132.PubMedCrossRefGoogle Scholar
  14. Fischer, C., Jonckx, B., Mazzone, M., Zacchigna, S., Loges, S., Pattarini, L., et al. (2007). Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell, 131(3), 463–475. doi: 10.1016/j.cell.2007.08.038.PubMedCrossRefGoogle Scholar
  15. Folkman, J. (1971). Tumor angiogenesis: Therapeutic implications. The New England Journal of Medicine, 285(21), 1182–1186. doi: 10.1056/NEJM197111182852108.PubMedCrossRefGoogle Scholar
  16. Friedman, H. S., Prados, M. D., Wen, P. Y., Mikkelsen, T., Schiff, D., Abrey, L. E., et al. (2009). Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, 27(28), 4733–4740. doi: 10.1200/JCO.2008.19.8721.CrossRefGoogle Scholar
  17. Galas, L., Garnier, M., & Lamacz, M. (2000). Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na+ channel. Molecular and Cellular Endocrinology, 170(1–2), 197–209.PubMedCrossRefGoogle Scholar
  18. Grau, S., Thorsteinsdottir, J., von Baumgarten, L., Winkler, F., Tonn, J.-C., & Schichor, C. (2011). Bevacizumab can induce reactivity to VEGF-C and -D in human brain and tumour derived endothelial cells. Journal of Neuro-oncology, 104(1), 103–112. doi: 10.1007/s11060-010-0480-6.PubMedCrossRefGoogle Scholar
  19. Hoelzinger, D. B., Demuth, T., & Berens, M. E. (2007). Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment. Journal of the National Cancer Institute, 99(21), 1583–1593. doi: 10.1093/jnci/djm187.PubMedCrossRefGoogle Scholar
  20. Hong, X., Jiang, F., Kalkanis, S. N., Zhang, Z. G., Zhang, X.-P., DeCarvalho, A. C., et al. (2006). SDF-1 and CXCR4 are up-regulated by VEGF and contribute to glioma cell invasion. Cancer Letters, 236(1), 39–45. doi: 10.1016/j.canlet.2005.05.011.PubMedCrossRefGoogle Scholar
  21. Jackson, A. P., Timmerman, M. P., Bagshaw, C. R., & Ashley, C. C. (1987). The kinetics of calcium binding to fura-2 and indo-1. FEBS Letters, 216(1), 35–39.PubMedCrossRefGoogle Scholar
  22. Keunen, O., Johansson, M., Oudin, A., Sanzey, M., Rahim, S. A. A., Fack, F., et al. (2011). Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proceedings of the National Academy of Sciences of the United States of America, 108(9), 3749–3754. doi: 10.1073/pnas.1014480108.PubMedCentralPubMedCrossRefGoogle Scholar
  23. Knizetova, P., Darling, J. L., & Bartek, J. (2008a). Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy. Journal of Cellular and Molecular Medicine, 12(1), 111–125. doi: 10.1111/j.1582-4934.2007.00153.x.PubMedCrossRefGoogle Scholar
  24. Knizetova, P., Ehrmann, J., Hlobilkova, A., Vancova, I., Kalita, O., Kolar, Z., & Bartek, J. (2008b). Autocrine regulation of glioblastoma cell cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay. Cell cycle (Georgetown, Tex.), 7(16), 2553–2561.Google Scholar
  25. Kwiatkowska, A., & Symons, M. (2013). Signaling determinants of glioma cell invasion. Advances in Experimental Medicine and Biology, 986, 121–141. doi: 10.1007/978-94-007-4719-7_7.PubMedCrossRefGoogle Scholar
  26. Lee, S., Jilani, S. M., Nikolova, G. V., Carpizo, D., & Iruela-Arispe, M. L. (2005). Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. The Journal of Cell Biology, 169(4), 681–691. doi: 10.1083/jcb.200409115.PubMedCentralPubMedCrossRefGoogle Scholar
  27. Lee, J., Lee, J., Yu, H., Choi, K., & Choi, C. (2011). Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival. Cancer Letters, 309(2), 145–150. doi: 10.1016/j.canlet.2011.05.026.PubMedCrossRefGoogle Scholar
  28. Lucio-Eterovic, A. K., Piao, Y., & de Groot, J. F. (2009). Mediators of glioblastoma resistance and invasion during antivascular endothelial growth factor therapy. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 15(14), 4589–4599. doi: 10.1158/1078-0432.CCR-09-0575.CrossRefGoogle Scholar
  29. Mahesparan, R., Read, T.-A., Lund-Johansen, M., Skaftnesmo, K. O., Bjerkvig, R., & Engebraaten, O. (2003). Expression of extracellular matrix components in a highly infiltrative in vivo glioma model. Acta Neuropathologica, 105(1), 49–57. doi: 10.1007/s00401-002-0610-0.PubMedGoogle Scholar
  30. Masood, R., Cai, J., Zheng, T., Smith, D. L., Hinton, D. R., & Gill, P. S. (2001). Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors. Blood, 98(6), 1904–1913.PubMedCrossRefGoogle Scholar
  31. Mellinghoff, I. K., Lassman, A. B., & Wen, P. Y. (2011). Signal transduction inhibitors and antiangiogenic therapies for malignant glioma. Glia, 59(8), 1205–1212. doi: 10.1002/glia.21137.PubMedCrossRefGoogle Scholar
  32. Miletic, H., Niclou, S. P., Johansson, M., & Bjerkvig, R. (2009). Anti-VEGF therapies for malignant glioma: Treatment effects and escape mechanisms. Expert Opinion on Therapeutic Targets, 13(4), 455–468. doi: 10.1517/14728220902806444.PubMedCrossRefGoogle Scholar
  33. Moreno Garcia, V., Basu, B., Molife, L. R., & Kaye, S. B. (2012). Combining antiangiogenics to overcome resistance: Rationale and clinical experience. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 18(14), 3750–3761. doi: 10.1158/1078-0432.CCR-11-1275.CrossRefGoogle Scholar
  34. Nakada, M., Nakada, S., Demuth, T., Tran, N. L., Hoelzinger, D. B., & Berens, M. E. (2007). Molecular targets of glioma invasion. Cellular and Molecular Life Sciences: CMLS, 64(4), 458–478. doi: 10.1007/s00018-007-6342-5.PubMedCrossRefGoogle Scholar
  35. Olsson, A.-K., Dimberg, A., Kreuger, J., & Claesson-Welsh, L. (2006). VEGF receptor signalling: In control of vascular function. Nature Reviews Molecular Cell Biology, 7(5), 359–371. doi: 10.1038/nrm1911.PubMedCrossRefGoogle Scholar
  36. Plate, K. H., Scholz, A., & Dumont, D. J. (2012). Tumor angiogenesis and anti-angiogenic therapy in malignant gliomas revisited. Acta Neuropathologica, 124(6), 763–775. doi: 10.1007/s00401-012-1066-5.PubMedCentralPubMedCrossRefGoogle Scholar
  37. Pollo, B. (2012). Pathological classification of brain tumors. The Quarterly Journal of Nuclear Medicine and Molecular Imaging: Official Publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR), [and] Section of the Society of Radiopharmaceutical Chemistry and Biology, 56(2), 103–111.Google Scholar
  38. Rahman, R., Smith, S., Rahman, C., & Grundy, R. (2010). Antiangiogenic therapy and mechanisms of tumor resistance in malignant glioma. Journal of Oncology, 2010, 251231. doi: 10.1155/2010/251231.PubMedCentralPubMedCrossRefGoogle Scholar
  39. Red-Horse, K., Crawford, Y., Shojaei, F., & Ferrara, N. (2007). Endothelium-microenvironment interactions in the developing embryo and in the adult. Developmental Cell, 12(2), 181–194. doi: 10.1016/j.devcel.2007.01.013.PubMedCrossRefGoogle Scholar
  40. Stupp, R., Hegi, M. E., Mason, W. P., van den Bent, M. J., Taphoorn, M. J. B., Janzer, R. C., et al. (2009). Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. The Lancet Oncology, 10(5), 459–466. doi: 10.1016/S1470-2045(09)70025-7.PubMedCrossRefGoogle Scholar
  41. Tabatabai, G., Weller, M., Nabors, B., Picard, M., Reardon, D., Mikkelsen, T., et al. (2010). Targeting integrins in malignant glioma. Targeted Oncology, 5(3), 175–181. doi: 10.1007/s11523-010-0156-3.PubMedCrossRefGoogle Scholar
  42. Takano, S., Mashiko, R., Osuka, S., Ishikawa, E., Ohneda, O., & Matsumura, A. (2010). Detection of failure of bevacizumab treatment for malignant glioma based on urinary matrix metalloproteinase activity. Brain Tumor Pathology, 27(2), 89–94. doi: 10.1007/s10014-010-0271-y.PubMedCrossRefGoogle Scholar
  43. Tate, M. C., & Aghi, M. K. (2009). Biology of angiogenesis and invasion in glioma. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics, 6(3), 447–457. doi: 10.1016/j.nurt.2009.04.001.CrossRefGoogle Scholar
  44. Thompson, E. M., Frenkel, E. P., & Neuwelt, E. A. (2011). The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology, 76(1), 87–93. doi: 10.1212/WNL.0b013e318204a3af.PubMedCentralPubMedCrossRefGoogle Scholar
  45. Tuettenberg, J., Friedel, C., & Vajkoczy, P. (2006). Angiogenesis in malignant glioma: A target for antitumor therapy? Critical Reviews in Oncology/Hematology, 59(3), 181–193. doi: 10.1016/j.critrevonc.2006.01.004.PubMedCrossRefGoogle Scholar
  46. Videira, P. A., Piteira, A. R., Cabral, M. G., Martins, C., Correia, M., Severino, P., et al. (2011). Effects of bevacizumab on autocrine VEGF stimulation in bladder cancer cell lines. Urologia Internationalis, 86(1), 95–101. doi: 10.1159/000321905.PubMedCrossRefGoogle Scholar
  47. Vredenburgh, J. J., Desjardins, A., Herndon, J. E, 2nd, Dowell, J. M., Reardon, D. A., Quinn, J. A., et al. (2007). Phase II trial of bevacizumab and irinotecan in recurrent malignant glioma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 13(4), 1253–1259. doi: 10.1158/1078-0432.CCR-06-2309.CrossRefGoogle Scholar
  48. Wade, A., Robinson, A. E., Engler, J. R., Petritsch, C., James, C. D., & Phillips, J. J. (2013). Proteoglycans and their roles in brain cancer. The FEBS Journal, 280(10), 2399–2417. doi: 10.1111/febs.12109.PubMedCentralPubMedCrossRefGoogle Scholar
  49. Watkins, S., & Sontheimer, H. (2012). Unique biology of gliomas: Challenges and opportunities. Trends in Neurosciences, 35(9), 546–556. doi: 10.1016/j.tins.2012.05.001.PubMedCentralPubMedCrossRefGoogle Scholar
  50. Wen, P. Y., & Kesari, S. (2008). Malignant gliomas in adults. The New England Journal of Medicine, 359(5), 492–507. doi: 10.1056/NEJMra0708126.PubMedCrossRefGoogle Scholar
  51. Xu, T., Chen, J., Lu, Y., & Wolff, J. E. (2010). Effects of bevacizumab plus irinotecan on response and survival in patients with recurrent malignant glioma: A systematic review and survival-gain analysis. BMC Cancer, 10, 252. doi: 10.1186/1471-2407-10-252.PubMedCentralPubMedCrossRefGoogle Scholar
  52. Xu, L., Duda, D. G., di Tomaso, E., Ancukiewicz, M., Chung, D. C., Lauwers, G. Y., et al. (2009). Direct evidence that bevacizumab, an anti-VEGF antibody, up-regulates SDF1alpha, CXCR4, CXCL6, and neuropilin 1 in tumors from patients with rectal cancer. Cancer Research, 69(20), 7905–7910. doi: 10.1158/0008-5472.CAN-09-2099.PubMedCentralPubMedCrossRefGoogle Scholar
  53. Yamagishi, N., Teshima-Kondo, S., Masuda, K., Nishida, K., Kuwano, Y., Dang, D. T., et al. (2013). Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells. BMC Cancer, 13(1), 229. doi: 10.1186/1471-2407-13-229.PubMedCentralPubMedCrossRefGoogle Scholar
  54. Zimmermann, D. R., & Dours-Zimmermann, M. T. (2008). Extracellular matrix of the central nervous system: From neglect to challenge. Histochemistry and Cell Biology, 130(4), 635–653. doi: 10.1007/s00418-008-0485-9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Thomas Simon
    • 1
    • 2
    Email author
  • Bérénice Coquerel
    • 1
    • 2
  • Alexandre Petit
    • 1
    • 2
  • Yusra Kassim
    • 1
    • 2
  • Elise Demange
    • 1
    • 2
    • 3
  • Didier Le Cerf
    • 4
    • 5
  • Valérie Perrot
    • 1
    • 2
  • Jean-Pierre Vannier
    • 1
    • 2
  1. 1.Groupe de Recherche «Micro-Environnement et Renouvellement Cellulaire Intégrés» MERCI UPRES EA 3829, Faculté de Médecine et PharmacieUniversité de RouenRouen CedexFrance
  2. 2.Normandie UniversitéRouen CedexFrance
  3. 3.CelenysRouen CedexFrance
  4. 4.Laboratoire Polymères Biopolymères Surfaces, Faculté des Sciences et des TechniquesUniversité de RouenMont-Saint-Aignan CedexFrance
  5. 5.CNRS UMR 6270 and FR 3038Mont-Saint-Aignan CedexFrance

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