In vivo orthotopic tumor xenograft models

All animal studies were approved by the institutional guidelines and those formulated by the European Community for the Use of Experimental Animals. Six week-old BALB/c Female nude mice (Charles River Laboratories) with a mean body weight of 18-20g were used to establish orthotopic xenograft tumor models. The mice were housed and fed under specific pathogen-free conditions.

To produce tumors, renal cancer cells 786-O-luc (VHL^-) were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin-EDTA. Trypsinization was stopped with medium containing 10% FBS, and the cells were washed once in serum-free medium and resuspended in 500μl PBS. Renal orthotopic implantation was carried out by injection of 3×10⁶ 786-O luc cells into the right kidney of athymic nude mice. Seven weeks after the implantation of the xenografts, animals were randomly divided into treated and untreated groups. Mice were treated with vehicle (0.5% Tween 80), GDC-0941 (100mg/kg), Saracatinib (10mg/kg), or their combination by force-feeding, three times a week. Mice were weighed once a week to monitor their health and tumor growth was measured by imaging luminescence of 786-O-luc cells (IVIS).

A PDX model RCC-10-B was generated by Xentech, (Paris France). Briefly, tumor fragments (30mm³) were grafted in the inter-scapular subcutaneous tissue, one fragment per mouse. Tumors are metastasis in the lymph node of Clear cell/granular kidney Carcinoma. The status of VHL gene was determined as mutated (E160fs14aa (chr3-10191481-G->GA- (frameshift insertion) for RCC10B.