the positive ratio of ssDNA staining was only 4.3% when xenografts from IL13RA2-transfected 786-O cells were treated with sunitinib compared with 0.8% when treated with vehicle. Thus, xenografts from IL13RA2-expressing 786-O cells were more likely to escape from apoptosis by sunitinib treatment than mock-transfected 786-O cells. As for xenograft tumors derived from Caki-1 cells, the number of apoptotic tumor cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741226 indicated by ssDNA immunopositivity was not significantly increased by sunitinib treatment compared with vehicle PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1974422 in scramble shRNA-infected cells. However, in those derived from Caki-1 shIL13RA2 cells, apoptosis was significantly induced by sunitinib treatment compared with vehicle only. Finally, we examined if the inhibition of apoptosis regulated the sensitivity and development of sunitinib resistance in our primary xenograft model KURC1. We first examined the number of ssDNA-positive cells in both xenografts. Sunitinib treatment significantly increased the 9 / 20 IL13RA2 and Resistance to Sunitinib in ccRCC 10 / 20 IL13RA2 and Resistance to Sunitinib in ccRCC Fig 3. Overexpression of IL13RA2 leads to acquired resistance to sunitinib and shRNA-mediated IL13RA2 knockdown induces sensitivity to sunitinib. Immunoblot analysis of 786-O subclones infected with retrovirus encoding mock or WT IL13RA2. Whole cell PCI-32765 extracts were immunoblotted using the indicated antibodies. Sequential changes in subcutaneous xenograft tumors from 786-O subclones infected with mock or WT IL13RA2 treated with sunitinib and vehicle. Each time point represents the mean SE of tumor volume in each group. The difference in tumor size between the treatment group and control was statistically significant in 786-O-mock cells but not statistically significant in 786-O-IL13RA2 cells. The horizontal arrow bars indicate the periods of sunitinib administration. Immunoblot analysis of Caki-1 subclones infected with lentivirus encoding scrambled or IL13RA2 shRNA. Whole cell extracts were immunoblotted using the indicated antibodies. Sequential changes of subcutaneous xenograft tumors from a Caki-1 subclone infected with scrambled or IL13RA2 shRNA treated with sunitinib and vehicle. Each time point represents the mean SE of tumor volume in each group. Day 0 is the first day of sunitinib administration 4 weeks after transplantation. The difference in tumor size between the treatment group and control was not significant in Caki-1-sh-scrambled cells but statistically significant in Caki-1-sh-IL13RA2 cells. The arrow bars indicate the period of sunitinib administration. doi:10.1371/journal.pone.0130980.g003 number of ssDNA-positive apoptotic cells in sunitinib-sensitive in KURC1 tumors at day 30. In contrast, in KURC1 sunitinib-resistant tumors at day 50, the number of apoptotic cells decreased to a level almost comparable to that of vehicle-treated cells at day 50. We next measured MVD in each xenograft. MVD was reduced after the treatment of sunitinib at day 30 or 50 in KURC1 xenograft tumors, irrespective of sunitinib sensitivity. In order to estimate the total number of tumor vessels, MDV multiply the corresponding tumor volume. According to calculations, the ratio of number of vessels in control tumor at day 50, sensitive tumor at day 30, and resistant tumor at day 50 were 17, 1, and 3, respectively. Indeed, the ratio of them in p5 control tumor at day 50 and p5 resistant tumor at day 50 were estimated 2 and 1. These observations implicated total number of tumor vess