SKOV3ip1 cells expressing WT1 − 17AA/− KTS rapidly produced tumors (3/3), and mice injected with the cells were usually dead within 40 days, while mice injected with SKOV3ip1 cells expressing control vector (3/3), WT1 + 17AA/− KTS (3/3), WT1 − 17AA/+ KTS (3/3), or WT1 + 17AA/+ KTS (3/3) developed only small tumors, even after 40 days. Based on these preliminary data, we euthanized mice injected with WT1 − 17AA/− KTS-expressing cells on day 36 and mice injected with cells expressing control vector or the other variants on day 40. The appearances of the mice are shown in Luminespib Figure 1B. Mice injected with cells expressing − 17AA/− KTS showed a significant increase

in body weight gain compared to mice injected CDK inhibitor with cells expressing control vector, + 17AA/− KTS, or − 17AA/+ KTS ( Figure 1C). However, there were no significant differences in abdominal circumference gains among the five groups of mice ( Figure 1D). Interestingly, overexpression of the − 17AA/− KTS splice variant resulted in a significant increase in the volume of ascites, compared with that in mice infected with cells expressing the control vector or other WT1 variants ( Figure 1E). The extent of intra-abdominal dissemination is visually shown in Figure 2A. Massive intra-abdominal dissemination was detected in mice injected with cells expressing the WT1 − 17AA/− KTS

variant. Mice injected with cells expressing the control vector, WT1 + 17AA/− KTS, WT1 − 17AA/+ KTS, or WT1 + 17AA/+ KTS showed

a little intra-abdominal dissemination. Histological analysis of intra-abdominal lesions developed in mice injected with cells expressing the control vector or each variant confirmed the findings of serous Ergoloid adenocarcinoma ( Figure 2B), which was consistent with SKOV3ip1 cells, as described previously [30]. There was no difference in histological findings in cells expressing each of the four WT1 variants ( Figure 2B). Tumors that had disseminated within the abdomen were measured by resected tumor weight ( Figure 2C). Overexpression of − 17AA/− KTS resulted in a significant increase in the disseminated tumor weight, as compared with that in tumors expressing the control vector or + 17AA/+ KTS variant. There were no significant differences in disseminated tumor weights in mice injected with cells expressing the control vector or other variants. Immunoblot analysis showed that WT1 was abundantly expressed in tumors obtained from mice inoculated with cells expressing the four variants (Figure 3A). Absence of WT1 expression was confirmed in tumors from mice inoculated with cells expressing the control vector. Additionally, PCR analysis of RNA extracted from the tumors confirmed the expression of each WT1 variant, including the specific 17AA/KTS insertion/deletion, and the absence of WT1 from the control ( Figure 3B).