Angiogenesis inhibitors are widely used for treatment of metastastic colorectal cancer. However, no predictive biomarkers are currently available for patient selection. Besides the tumor-associated endothelial cells, VEGF-signaling inhibitors target CRC cells that express VEGF as well as functional VEGFR1 receptors thereby mediating both paracrine and autocrine VEGF-signaling. The aims of this work is i) to establish the direct influence of aflibercept (Zaltrap®) that inhibits all three VEGFR1 ligands (VEGF-A, VEGF-B and PlGF) on CRC cells, ii) to identify tumor phenotypes associated with resistance to aflibercept in vitro and, iii) to extend these findings to human xenograft models.
A panel of 12 well-characterized CRC cell lines was used to establish the influence of VEGFR1 stimulation (VEGF-A, VEGF-B, PlGF) and inhibition (aflibercept) on VEGF-mediated tumor cell migration. Expression of VEGF ligands and receptors was determined by qRT-PCR and ELISA assays. The in vivo influence of aflibercept was determined in human xenograft models and complemented by IHC and Western blot analysis.
Aflibercept inhibited the migration of most CRC cells under both normoxia and hypoxia including the highly sensitive HCT-116 cells. In contrast, LS174T cells did not respond to either aflibercept or to purified VEGF ligands. These cells expressed high levels of VEGF ligands and HIF2alpha, even under normoxia. Accordingly, aflibercept showed pronounced in vivo activity toward HCT-116 xenografts with 75% tumor growth inhibition but only 40% tumor growth inhibition toward LS174T tumors, compared to the corresponding vehicle controls. A similar trend was observed for bevacizumab with 41% tumor growth inhibition for HCT-116 and 32% inhibition for LS174T xenografts. IHC analysis of LS174T xenografts confirmed the strong expression of HIF2alpha and VEGF ligands as well as a modest inhibitory effect on the tumor-associated endothelia cells.
We here report that aflibercept has direct antimigratory effects on most CRC cells. Strong expression of HIF-2alpha and VEGF ligands was accompanied by aflibercept resistance in vitro as in vivo.
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INSERM U938 and Université Pierre et Marie Curie (UPMC), Sorbonne Universités, Paris, France
A.K. Larsen: This work was supported in part by Sanofi Genzyme. E. Dochy: Employed by Sanofi-Genzyme. A. de Gramont: This research is in part supported by Sanofi. All other authors have declared no conflicts of interest.