Tumor cell response to bevacizumab single agent therapy in vitro

Abstract

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.

Figure 1

Expression of VEGF receptors and hypoxic VEGF mRNA induction in tumor cells. (A) Protein expression of VEGFR1, VEGFR2 and NRP1 was determined in tumor cells and HUVECs under normoxia and after 24 hours of hypoxia with or without bevacizumab. Vinculin was used as a loading control. (B) Cell surface expression of VEGFR2 and NRP1 as analyzed by flow cytometry. Unstained cells cultured under normoxic conditions were used as a control. (C) Quantification of VEGFR2⁺ and NRP1⁺ cell surface expression. (D) Relative change of VEGFA mRNA expression under hypoxia versus normoxic controls. (E) Relative change of GLUT1 mRNA expression under hypoxia versus normoxic controls. n.d. = not done.

Figure 2

Gene expression analysis in bevacizumab treated tumor cells. Change in relative expression of (A)VEGFA isoforms, (B)VEGFR1, (C)NRP1 and (D)VEGFR2 in bevacizumab treated cells after 24 hours of hypoxia versus untreated hypoxic cells. Only cell lines with detectable expression are included. * indicates HUVECs were in addition stimulated with rhVEGF in the absence of bevacizumab and normalized against untreated controls.

Figure 3

Tumor cell survival after bevacizumab treatment. (A, B) Levels of apoptosis were determined in bevacizumab treated cells after 48 hours by western blot analysis using an antibody against cleaved PARP. ß-Actin served as a loading control. As a positive control all cell lines were treated with staurosporine (0.15 μM) for 24 hours to induce apoptosis, an example of KM12 (CRC) and HUVEC is shown. (C) Quantification of cellular sub G1 fraction after 48 hours of bevacizumab treatment. Cells were stained with propidium iodide and analyzed by flow cytometry. Averaged data from three experiments are shown.

Figure 4

Tumor cell proliferation and migration analysis after bevacizumab treatment. (A, B) Proliferation of bevacizumab treated cells as a percentage of control. Cells were cultured under hypoxia and serum starved conditions for 72 hours. For comparison HUVEC were stimulated with rhVEGF and treated with bevacizumab. (C, D) Migration of bevacizumab treated versus untreated cells after 24 or 6 hours of hypoxia. HUVEC were again treated with rhVEGF.