Alternative pathways to the vascular endothelial grow factor (VEGF) such as hepatocyte growth factor or HGF/c-met are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. inhibitor crizotinib (25 mg/kg 1 or combination. We further tested this drug combination in a human ccRCC AS 602801 patient derived xenograft RP-R-01 in both VEGF-targeted therapy sensitive and resistant models. To evaluate the resistance phenotype we established RP-R-01 sunitinib resistant model by continuous sunitinib treatment (60 mg/kg 1 of RP-R-01 bearing-mice. Treatment with single agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model despite also a significant increase of c-met expression and phosphorylation in the sunitinib resistant tumors. In contrast axitinib treatment was effective AS 602801 in inhibiting angiogenesis and tumor growth in both models with its anti-tumor effect significantly increased by the combined treatment with crizotinib independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC both in first and second line setting. formulations axitinib was prepared in 5% carboxymethylcellulose solution and crizotinib was dissolved in water by pH adjustment to a value between 3.5 and 4. Drugs were administered by oral AS 602801 gavage (or PO). The experimental groups were the following: vehicle (5% carboxymethylcellulose 2 5 PO) axitinib (36 mg/kg 2 5 PO) crizotinib (25 mg/kg 1 5 PO) axitinib plus crizotinib combination (same schedule and concentration as in single agent groups). Treatments were administered as follows: four weeks (786-O one month endpoint) six weeks (RP-R-01 sunitinib resistant) ten weeks (RP-R-01 sunitinib sensitive) or up to 15 weeks (786-O survival). Mouse body weight and tumor caliper measurements were taken weekly. No overt signs of toxicity were observed in any treatment group (i.e. significant weight loss or diarrhea). Xenograft models and treatment protocol Immunodeficient SCID male mice purchased from Roswell Park Cancer Institute (RPCI) were utilized for these studies and all procedures were approved by the Institute Animal Care and Use Committee (IACUC). Mice were kept in a temperature controlled room on a 12/12 hours light/dark schedule with food and water only in order to maintain the heterogeneity of the primary tumor. Short term study: mice (3/group) were implanted subcutaneously in the flank area with ~4 mm2 size RP-R-01 tumor pieces. Treatment started when average tumor dimension reached ~35 mm2: mice were randomized in the above-mentioned experimental groups and treated for either 2 or 7 days. Since previous studies performed in our lab showed good anti-tumor efficacy of sunitinib in this model we implanted mice (8/group) subcutaneously with RP-R-01 tumor pieces as described above as models of sunitinib sensitive human ccRCC. Treatment started when average tumor dimension reached ~50 mm2. In order to establish a sunitinib resistant model we implanted 35 mice subcutaneously in the flank area with ~4-5 mm2 size RP-R-01 tumor pieces and approximately 6 weeks later when tumors reached an average size of ~25 mm2 mice were treated with sunitinib (60 mg/kg 5 PO). We defined resistant tumors when they reached doubled size upon treatment (~50 Itgb8 mm2). Thereafter mice were divided into homogenous groups (7 mice/group) as determined by caliper measurements and randomized to the above-mentioned experimental groups. Mice in all experiments have been sacrificed between 12 and 18 hours post last treatment. mmunohistochemistry Tissues AS 602801 were fixed for 24 hours in AS 602801 10% neutral buffered formalin (c-met E-cadherin and Ki67) or zinc fixative (CD31) paraffin embedded and cut at 4 ��m placed on charged slides and dried at 60��C for one hour. Slides were cooled to room temperature deparaffinized in xylene and rehydrated using graded alcohols. Antigen unmasking was heat-mediated in citrate buffer (pH 6.0) and followed by a 20 minutes cool down. Endogenous peroxidases were quenched with 3% H2O2 for 10 minutes and washed with PBS-Tween20 0.1%. Slides were then blocked for one hour with PBS 1% BSA (bovine serum albumin) and incubated overnight in primary antibodies: mouse CD31 (1:100 550274 BD Pharmingen) c-met (1:300 8198 Cell Signaling) E-cadherin (1:400 3195 Cell AS 602801 Signaling) or Ki67 (1:500 Thermo Scientific RM-9106). Sections were then.