Interferon-Gamma Receptor Signaling Plays an Important Role in Restraining Murine Ovarian Tumor Progression

Abstract

Immune cell-derived cytotoxic pathways have been implicated in antitumor immune responses. The goal of this study is to characterize how these cytotoxic pathways influence ovarian cancer development. We have utilized the TgMISIIR-TAg transgenic mouse model which expresses the transforming SV40 TAg in the ovary, leading to spontaneous development of ovarian tumors that closely mimic human epithelial ovarian cancer. To test how perforin (Prf1), granzyme B (GzmB) and interferon-gamma (IFNg) impact tumor occurrence and progression, we bred the TgMISIIR-TAg transgene into Prf1^-/-, GzmB^-/-, and IFNgR1^-/- mice. The transgenic females developed peritoneal tumors at 9-15 weeks and succumbed at 184 ± 37 days of age with 100% penetrance (n=41). Knockout of these cytotoxic genes does not affect tumor occurrence. However, loss of function in the IFNg signaling pathway significantly expedited tumor progression with all of the IFNg R1^-/- TgMISIIR-TAg females succumbing to tumor outgrowth at 167 ± 27 days of age (p=0.0074, n=24). In contrast, loss of function of Prf1 or GzmB did not significantly impact tumor progression and host survival. Since tumor cells in the IFNg R1^-/- TgMISIIR-TAg mice are IFNg R1 deficient, we used the implantable MOSEC (mouse ovarian surface epithelial cell) tumor line to validate that IFNg R signaling in host immune cells but not in tumor cells impacts tumor progression. Indeed, when the IFNg -responsive MOSEC cells were inoculated, IFNg R1^-/- mice exhibited significantly higher tumor burden compared to WT mice. Furthermore, a MOSEC-splenocyte co-culture system confirmed that IFNg R1^-/- immune cells were less effective than WT immune cells in controlling MOSEC tumor growth in vitro. Together, these results indicate that the IFNg R signaling pathway plays an important role in restraining murine ovarian tumor progression.

Keywords: Cytotoxic pathways; Granzyme B (GzmB); Interferon-gamma (IFNg); Ovarian cancer; Perforin (Prf1); Tumor immunity.

Figure 1. Global loss of function of IFNg R1, but not Prf1 and GzmB, accelerates ovarian tumor progression driven by the TgMISIIR-TAg transgene. TgMISIIR-TAg transgene was bred into strain-matched IFNg R1^−/−, Prf1^−/−, GzmB^−/− mice, and tumor development and mouse survival were monitored as described in Materials and Methods. Summarized data are shown as Kaplan-Meier survival curves of IFNg R1^−/− TgMISIIR- TAg mice

(A), Prf1^−/− TgMISIIR-TAg mice (B), and GzmB^−/− TgMISIIR-TAg mice. (C) bearing ovarian tumors analyzed against TgMISIIR-TAg mice. Mantel-Cox test was performed to determine statistical significance.

Figure 2. IFNg R1^−/− mice control MOSEC tumor growth less efficiently than WT mice

(A) MOSEC tumor cells are responsive to IFN. Different doses (1000, 2000, and 4000) of luciferase-expressing MOSEC tumor cells were cultured with various doses (1ng, 10ng, and 100ng) of recombinant IFNg in 48-well plates with a total volume of 1ml media in each well for 92 hours, and then 20 μl D-Luciferin (15mg/ml) was added into each well, and bioluminescence imaging was performed to measure tumor burden. Two-tailed t-tests were performed to determine statistical significance. (B) WT and IFNg R1^−/− mice were injected intraperitoneally with 2.5 × 10⁶ luciferase-expressing MOSEC tumor cells, and tumor burden was measured by bioluminescence imaging. Summary data are shown as mean ± SD, with 9 mice in each group. Two-way ANOVA was performed to determine statistical significance.

Figure 3

IFNg R1^−/− spleen cells have diminished ability to control MOSEC tumor growth in vitro compared to WT spleen cells 2 × 10⁶ spleen cells isolated from WT and IFNg R1^−/− mice were mixed with different doses (1000, 2000, and 4000) of luciferase-expressing MOSEC tumor cells, and co-cultured in 48-well plates with a total volume of 1ml media in each well. Tumor cells were also cultured with media only without spleen cells as a negative control. 92 hours later, 20 μl D-Luciferin (15mg/ml) was added into each well, and bioluminescence imaging was performed to measure tumor burden. Two-tailed t-tests were performed to determine statistical significance.