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. 2011 Apr 12;104(8):1270-7.
doi: 10.1038/bjc.2011.81. Epub 2011 Mar 15.

Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration

F Fan  1 , S Samuel, P Gaur, J Lu, N A Dallas, L Xia, D Bose, V Ramachandran, L M Ellis
Affiliations

Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration

F Fan et al. Br J Cancer. .

Abstract

Background: Bevacizumab (Bev), a monoclonal antibody to vascular endothelial growth factor (VEGF), is used in combination with chemotherapy for the treatment of metastatic colorectal cancer (CRC). The effects of Bev on angiogenesis have been well described, but the direct effect of Bev on tumour cells is unknown. This study was carried out to determine the molecular and phenotypic changes in CRC cells after chronic Bev exposure in vitro.

Methods: Human CRC cell lines were chronically exposed (3 months) to Bev in vitro to develop Bev-adapted (Bev-A) cell lines. Vascular endothelial growth factor family members were determined by reverse transcription-polymerase chain reaction and western blotting. Migration and invasion was determined using standard in vitro assays. Intravenous injection of tumour cells was carried out to evaluate metastatic potential in mice.

Results: Bevacizumab-adapted cells were found to be more migratory and invasive than control cells (P<0.001). Bevacizumab-adapted cells showed higher levels of VEGF-A, -B, -C, placental growth factor (PlGF), VEGF receptor-1 (VEGFR-1) and phosphorylation of VEGFR-1. Furthermore, treatment with SU5416, a VEGFR protein tyrosine kinase inhibitor, led to significantly decreased cell migration in vitro (P<0.001). Bevacizumab-adapted cells were more metastatic in vivo (P<0.05).

Conclusion: Chronic exposure of CRC cells to Bev (1) increased expression of VEGF-A, -B, -C, PlGF, VEGFR-1 and VEGFR-1 phosphorylation, (2) increased tumour cell migration and invasion, and (3) metastatic potential in vivo. Our study shows the functional significance of autocrine VEGF signalling in CRC cells.

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Figures

Figure 1
Figure 1
Effect of chronic bevacizumab exposure on CRC cells VEGF family profile. (A and B) RT–PCR shows that cells under Bev treatment have increased VEGFR-1, -3 and NRP-1 expression in both HCT116 and SW480 cells. (C and D) Western blot confirmed that in both HCT116 and SW480 cells, chronic bevacizumab exposure increased phosphorylation and total level of VEGFR-1 expression. Vascular endothelial growth factor receptor-3 and NRP-1, -2 remained unchanged in HCT116 cells. Neuropilin-1 was increased and VEGFR-3 and NRP-2 remained unchanged in SW480 cells. (E and F) Conditioned medium was collected and western blot performed in both HCT116 and SW480 cell. Cells under bevacizumab exposure had increased VEGF-A, -C and PlGF expression. Vascular endothelial growth factor-B was increased in HCT116 cells and remained unchanged in SW480 cells.
Figure 2
Figure 2
Effect of chronic bevacizumab exposure on CRC cell migration. (A and B) Using Boyden chamber migration assays, both HCT116/Bev-A (A) and SW480/Bev-A (B) cells showed a two- to three-fold increase in migration compared with that of control cells (P<0.001, P<0.0001, respectively). (C and D) Using the scratch assay, both HCT116/Bev-A (C) and SW480/Bev-A (D) cells showed an increase in migration compared with control cells (76 vs 43% 80 vs 29%, respectively). Wound closure was more complete in the Bev-A cells than in control cells.
Figure 3
Figure 3
Effect of chronic bevacizumab exposure on CRC cell invasion. (A) Using modified Boyden chamber assays, HCT116/Bev-A showed a three- to four-fold increase in invasion compared with the HCT116/control cells (P<0.001). (B) SW480/Bev-A showed a four- to five-fold increase in invasion compared with the SW480/control cells (P<0.0004).
Figure 4
Figure 4
Effect of chronic bevacizumab exposure on CRC cell migration under SU5416 treatment. SU5416 treatment led to decreased cell migration in HCT116/Bev-A cells determined by in vitro wound healing/migration assay (A) and the modified Boyden chamber assay (B) (P<0.0002). (C) Western blots showed a decrease in VEGFR-1 phosphorylation with SU5416 treatment in HCT116/Bev-A compared with control. Vinculin served as a loading control. (D–F) These results were further confirmed in the second cell line, SW480/Bev-A cells (P<0.001).
Figure 5
Figure 5
Effect of chronic bevacizumab exposure on metastatic potential of CRC cells in vivo. (A) The mice injected with HCT116/Bev-A cells showed significantly higher luciferase activity (∼10-fold higher) compared to the mice injected with control cells (P<0.005). Images of the whole animal and harvested organs revealed that luciferase activity was higher from organs harvested in mice injected with the HCT116/Bev-A cells (lower panel). (B) There was a significant increase in the mean number of metastases in the Bev-A group compared with the control group (P<0.02, red hash mark represents the mean). (C) Bev-A cells injected intravenously led to a significant increase in average tumour volume per mouse compared with control cells (P<0.01, red hash mark represents the mean). The colour reproduction of this figure is available on the html full text version of the manuscript
See this image and copyright information in PMC

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