Tumor cell response to bevacizumab single agent therapy in vitro

Cancer Cell International

Shannon Graver¹  Melanie Hein² 

[1] Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany;Developmental Biochemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany

关键词: In vitro;    Hypoxia;    VEGFA;    Tumor angiogenesis;    NCI-60;    Bevacizumab;   

Others  :  792860
DOI  :  10.1186/1475-2867-13-94

 received in 2013-07-30, accepted in 2013-09-05,  发布年份 2013

【 摘 要 】

Background

Angiogenesis represents a highly multi-factorial and multi-cellular complex (patho-) physiologic event involving endothelial cells, tumor cells in malignant conditions, as well as bone marrow derived cells and stromal cells. One main driver is vascular endothelial growth factor (VEGFA), which is known to interact with endothelial cells as a survival and mitogenic signal. The role of VEGFA on tumor cells and /or tumor stromal cell interaction is less clear. Condition specific (e.g. hypoxia) or tumor specific expression of VEGFA, VEGF receptors and co-receptors on tumor cells has been reported, in addition to the expression on the endothelium. This suggests a potential paracrine/autocrine loop that could affect changes specific to tumor cells.

Methods

We used the monoclonal antibody against VEGFA, bevacizumab, in various in vitro experiments using cell lines derived from different tumor entities (non small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC) and renal cell carcinoma (RCC)) in order to determine if potential VEGFA signaling could be blocked in tumor cells. The experiments were done under hypoxia, a major inducer of VEGFA and angiogenesis, in an attempt to mimic the physiological tumor condition. Known VEGFA induced endothelial biological responses such as proliferation, migration, survival and gene expression changes were evaluated.

Results

Our study was able to demonstrate expression of VEGF receptors on tumor cells as well as hypoxia regulated angiogenic gene expression. In addition, there was a cell line specific effect in tumor cells by VEGFA blockade with bevacizumab in terms of proliferation; however overall, there was a limited measurable consequence of bevacizumab therapy detected by migration and survival.

Conclusion

The present study showed in a variety of in vitro experiments with several tumor cell lines from different tumor origins, that by blocking VEGFA with bevacizumab, there was a limited autocrine or cell-autonomous function of VEGFA signaling in tumor cells, when evaluating VEGFA induced downstream outputs known in endothelial cells.

【 授权许可】

   
2013 Hein and Graver; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86:353-364.
• [2]Hockel M, Vaupel P: Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001, 93:266-276.
• [3]Harris AL: Hypoxia–a key regulatory factor in tumour growth. Nat Rev Cancer 2002, 2:38-47.
• [4]Ferrara N, Kerbel RS: Angiogenesis as a therapeutic target. Nature 2005, 438:967-974.
• [5]Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med 1971, 285:1182-1186.
• [6]Ferrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004, 3:391-400.
• [7]Ellis LM, Hicklin DJ: VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 2008, 8:579-591.
• [8]Jubb AM, Harris AL: Biomarkers to predict the clinical efficacy of bevacizumab in cancer. Lancet Oncol 2010, 11:1172-1183.
• [9]Smith NR, Baker D, James NH, Ratcliffe K, Jenkins M, et al.: Vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3 are localized primarily to the vasculature in human primary solid cancers. Clin Cancer Res 2010, 16:3548-3561.
• [10]Claesson-Welsh L: Blood vessels as targets in tumor therapy. Ups J Med Sci 2012, 117:178-186.
• [11]Amaya H, Tanigawa N, Lu C, Matsumura M, Shimomatsuya T, et al.: Association of vascular endothelial growth factor expression with tumor angiogenesis, survival and thymidine phosphorylase/platelet-derived endothelial cell growth factor expression in human colorectal cancer. Cancer Lett 1997, 119:227-235.
• [12]Duff SE, Jeziorska M, Rosa DD, Kumar S, Haboubi N, et al.: Vascular endothelial growth factors and receptors in colorectal cancer: implications for anti-angiogenic therapy. Eur J Cancer 2006, 42:112-117.
• [13]Seto T, Higashiyama M, Funai H, Imamura F, Uematsu K, et al.: Prognostic value of expression of vascular endothelial growth factor and its flt-1 and KDR receptors in stage I non-small-cell lung cancer. Lung Cancer 2006, 53:91-96.
• [14]Kajita T, Ohta Y, Kimura K, Tamura M, Tanaka Y, et al.: The expression of vascular endothelial growth factor C and its receptors in non-small cell lung cancer. Br J Cancer 2001, 85:255-260.
• [15]Donnem T, Al-Saad S, Al-Shibli K, Delghandi MP, Persson M, et al.: Inverse prognostic impact of angiogenic marker expression in tumor cells versus stromal cells in non small cell lung cancer. Clin Cancer Res 2007, 13:6649-6657.
• [16]Ryden L, Linderholm B, Nielsen NH, Emdin S, Jonsson PE, et al.: Tumor specific VEGF-a and VEGFR2/KDR protein are co-expressed in breast cancer. Breast Cancer Res Treat 2003, 82:147-154.
• [17]Ghosh S, Sullivan CA, Zerkowski MP, Molinaro AM, Rimm DL, et al.: High levels of vascular endothelial growth factor and its receptors (VEGFR-1, VEGFR-2, neuropilin-1) are associated with worse outcome in breast cancer. Hum Pathol 2008, 39:1835-1843.
• [18]Wulfing P, Kersting C, Buerger H, Mattsson B, Mesters R, et al.: Expression patterns of angiogenic and lymphangiogenic factors in ductal breast carcinoma in situ. Br J Cancer 2005, 92:1720-1728.
• [19]Decaussin M, Sartelet H, Robert C, Moro D, Claraz C, et al.: Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol 1999, 188:369-377.
• [20]Fan F, Wey JS, McCarty MF, Belcheva A, Liu W, et al.: Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene 2005, 24:2647-2653.
• [21]Wang ES, Teruya-Feldstein J, Wu Y, Zhu Z, Hicklin DJ, et al.: Targeting autocrine and paracrine VEGF receptor pathways inhibits human lymphoma xenografts in vivo. Blood 2004, 104:2893-2902.
• [22]Brutsch R, Liebler SS, Wustehube J, Bartol A, Herberich SE, et al.: Integrin cytoplasmic domain-associated protein-1 attenuates sprouting angiogenesis. Circ Res 2010, 107:592-601.
• [23]Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65:55-63.
• [24]Liang CC, Park AY, Guan JL: In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007, 2:329-333.
• [25]Schneider CA, Rasband WS, Eliceiri KW: NIH image to ImageJ: 25 years of image analysis. Nat Methods 2012, 9:671-675.
• [26]Shankavaram UT, Reinhold WC, Nishizuka S, Major S, Morita D, et al.: Transcript and protein expression profiles of the NCI-60 cancer cell panel: an integromic microarray study. Mol Cancer Ther 2007, 6:820-832.
• [27]Carneiro A, Falcao M, Pirraco A, Milheiro-Oliveira P, Falcao-Reis F, et al.: Comparative effects of bevacizumab, ranibizumab and pegaptanib at intravitreal dose range on endothelial cells. Exp Eye Res 2009, 88:522-527.
• [28]Wang Y, Fei D, Vanderlaan M, Song A: Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro. Angiogenesis 2004, 7:335-345.
• [29]de Santa C, Pau E, Arias FC, Caso Pelaez E, Munoz Molina GM, Sanchez Hernandez I, et al.: Prognostic significance of the expression of vascular endothelial growth factors A, B, C, and D and their receptors R1, R2, and R3 in patients with nonsmall cell lung cancer. Cancer 2009, 115:1701-1712.
• [30]Barr MP, Bouchier-Hayes DJ, Harmey JJ: Vascular endothelial growth factor is an autocrine survival factor for breast tumour cells under hypoxia. Int J Oncol 2008, 32:41-48.
• [31]Bae D, Lu S, Taglienti CA, Mercurio AM: Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2. J Biol Chem 2008, 283:28074-28080.
• [32]Koch S, Tugues S, Li X, Gualandi L, Claesson-Welsh L: Signal transduction by vascular endothelial growth factor receptors. Biochem J 2011, 437:169-183.
• [33]Wey JS, Fan F, Gray MJ, Bauer TW, McCarty MF, et al.: Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer 2005, 104:427-438.
• [34]Fan F, Samuel S, Gaur P, Lu J, Dallas NA, et al.: Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration. Br J Cancer 2011, 104:1270-1277.
• [35]Lee TH, Seng S, Sekine M, Hinton C, Fu Y, et al.: Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1. PLoS Med 2007, 4:e186.
• [36]Selvakumaran M, Yao KS, Feldman MD, O'Dwyer PJ: Antitumor effect of the angiogenesis inhibitor bevacizumab is dependent on susceptibility of tumors to hypoxia-induced apoptosis. Biochem Pharmacol 2008, 75:627-638.
• [37]Poindessous V, Ouaret D, El Ouadrani K, Battistella A, Megalophonos VF, et al.: EGFR- and VEGF(R)-targeted small molecules show synergistic activity in colorectal cancer models refractory to combinations of monoclonal antibodies. Clin Cancer Res 2011, 17:6522-6530.
• [38]Shi W, Siemann DW: Simultaneous targeting of VEGF message and VEGF receptor signaling as a therapeutic anticancer approach. Anticancer Res 2004, 24:213-218.
• [39]Ortholan C, Durivault J, Hannoun-Levi JM, Guyot M, Bourcier C, et al.: Bevacizumab/docetaxel association is more efficient than docetaxel alone in reducing breast and prostate cancer cell growth: a new paradigm for understanding the therapeutic effect of combined treatment. Eur J Cancer 2010, 46:3022-3036.
• [40]Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL, et al.: Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004, 10:145-147.