Abstract 3000: Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma
Authors: Tijeras-Raballand Annemilai , Santos Célia Dos , Serova Maria , Martinet Matthieu , Faivre Sandrine , Gramont Armand de , Raymond Eric 
Published: May 2015

Abstract
Background. Sunitinib inhibits VEGFR/PDGFR signaling in cancer cells and reduces tumor blood flow, leading to tumor starvation, hypoxia, and necrosis. The aim of this study was to investigate mechanisms associated with adaptive resistance to sunitinib in vitro and in vivo in renal cancer models (RCC).
Materials and Methods: CAKI1 (VHL+) and 786-0 (VHL-) RCC cells were exposed in vitro to stepwise increasing doses of sunitinib for &gt;6 months. In vitro, vascular mimicry was assessed by pseudo-tube formation assay using Matrigel. Pseudo-tube networks were quantified by “angiogenesis analyzer” for Image J, assessing the meshes number. CAKI1 or 786-0 cells were engrafted in nude mice and treated at 60 mg/kg/day of sunitinib until progression.
Results. We confirmed that the VHL pathway was functional in CAKI1 but not in 786-0 cells. In vivo, sunitinib significantly delay tumor growth with a median time-to-tumor progression of 50 days versus 17 days (p&lt;0.001) in CAKI and over 60 days versus 22 days (p&lt;0.01) in 786-0 xenogratfs. In CAKI1 and 786-0, quantitative immunohistochemistry and fluorescence analyses at the time of sunitinib progression showed patterns of angiogenesis recovery with tumor blood vessel sprouting associated with reduction of hypoxic and necrotic areas. In these sunitinib-resistant tumors, cancer cells displayed stem cell and mesenchymal differentiation markers such as CD133 vimentin and CXCR4. Vessels sprouting under sunitinib exposure contained CD10/CD31/von Willebrand co-stained cells embedded in their walls suggesting vascular mimicry. We further investigated whether cancer cell differentiation into endothelial cell was related to chemical inhibition of VEGFR/PDGFR by sunitinib. We first characterized CAKI and 786 cells for VEGFR and PDGFR expression, showing that CAKI expressed high level of PDGFR, whereas 786 expressed low levels of VEGFR and PDGFR. In normally oxygenated culture conditions, 786-0 cells formed spontaneously more meshes than CAKI1 cells (22 versus 0.33, p&lt;0.05). Interestingly, chronic exposure of CAKI1 cells to sunitinib led to a 32-fold increase in the number of meshes (10.6 versus 0.33, p&lt;0.01), which could be related to the inhibition of the PDGFR pathway.
Conclusions. Our data showed that chronic exposure to subtoxic doses of sunitinib might force cancer stem cells to differentiate into endothelial-like cells in vitro and in vivo independently of the VHL status of tumor cells. This phenomenon known as vasculogenic mimicry is associated with poor prognosis in renal cell carcinoma. VHL-mutated 786-0 cells appeared more prompt to form vessels and can spontaneously form meshes in normoxic condition in vitro. Interestingly, this plastic capacity of forming vessels in normoxic condition can be acquired in wild-type VHL CAKI1 when treated chronically with subtoxic doses of sunitinib.
Citation Format: Annemilai Tijeras-Raballand, Célia Dos Santos, Maria Serova, Matthieu Martinet, Sandrine Faivre, Armand de Gramont, Eric Raymond. Angiogenic recovery under chronic exposure to sunitinib is associated with vasculogenic mimicry in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2014-3000