晚期卵巢癌建模及验证用于转基因表达和肿瘤监测的溶瘤腺病毒工具

Purpose : High-grade serous ovarian cancer (HGSOC) is characterized by profound immune exclusion and peritoneal spread, hypothesized to be orchestrated by the TGF-beta and GM-CSF cytokines. We established an immunocompetent murine model of advanced HGSOC and evaluated the tumor selectivity and transgene delivery efficiency of hTERT-driven oncolytic adenoviruses encoding GM-CSF and/or a soluble TGF-beta receptor Fc fusion (sTGFbetaR-Fc). Experimental Procedures : A syngeneic orthotopic model of advanced HGSOC was established by intraperitoneally injecting luciferase-labeled MOE-KRAS/PTEN (MKP) cells into immunocompetent FVB/N mice. Tumor burden was monitored longitudinally by bioluminescence imaging (BLI). Four hTERT-driven oncolytic adenoviruses (Ad.Null, Ad.GM-CSF [Ad.GM], Ad.sTGFbetaR [Ad.sT], Ad.sTGFbetaR.GM-CSF [Ad.sT.GM]) were evaluated in vitro for infectivity, cytolytic activity (Incucyte/caspase 3/7), and transgene expression (flow cytometry) in MKP tumor cells, NIH 3T3 fibroblasts, and 3T3-derived cancer-associated fibroblasts (CAFs). Results : Serial BLI showed consistent peritoneal tumor engraftment and progression by day 26 (n = 14) confirming model fidelity for late-stage disease. In vitro, IncuCyte live-cell imaging revealed strong, selective apoptosis in MKP cells following oncolytic infection, while NIH 3T3 fibroblasts and CAFs (non-tumor controls) showed minimal response. Apoptotic activity in MKP cells rose sharply at 30-35 hours and peaked at 45 hours post-infection (>60-80% fold increase in caspase3/7+ area over untreated but minimal apoptotic activity in 3T3 and CAFs (<8-fold over untreated). Notably, among virus constructs, Ad.GM and Ad. Null induced the most rapid and robust apoptosis, whereas Ad. sT and Ad.sT.GM showed significantly delayed and reduced effects (Ad.GM vs Ad. Null: p = 0.0042; Ad. sT vs Ad.GM or Ad. Null: p < 0.0001; 2-way ANOVA with Sidak's test). Transgene expression was quantified by flow cytometry following 48-hour infection. Ad.GM and Ad.sT.GM treatments produced GM-CSF positive cells (2.25% and 1.04% gated, mean fluorescence intensity 6217.54 and 5508.2 respectively) while Ad.sT and Ad.sT.GM treatments produced sTGFbetaR-Fc positive cells (8.04% and 9.75% gated, mean fluorescence intensity 85092.63 and 84097.65 respectively); live cell frequencies decreased in treated groups (61.45-67.83%) compared to untreated (96.32%) controls. These results demonstrate selective transgene expression and cytotoxicity in tumor cells achieved by the engineered oncolytic constructs. Conclusions : These data confirm the fidelity for advanced-stage HGSOC model and establish oncolytic adenoviral platforms as effective mechanistic tools-functioning both as cytolytic agents and as delivery vehicles for functional transgenes-to mechanistically dissect TGF-beta and GM-CSF signaling in ovarian cancer.