14Influence of sex on immunosurveillance and metastatic tropism in novel mouse models of clear cell renal cell carcinoma

Abstract Background Sex bias in renal cell carcinoma (RCC) is marked by males having twice the incidence and mortality rates of females, along with larger, higher-grade tumors and greater metastatic burden. Several hormonal, genetic, and behavioral reasons have been identified, however, the role that the immune system plays in this remains unclear. This is in part due to the historical lack of relevant and reliable animal models that recapitulate human disease. We therefore sought to utilize two newly developed syngeneic ccRCC mouse models to first establish and characterize the features of mouse ccRCC sex bias, and subsequently to determine whether a strong immunological basis exists. Methods Because of the known heterogeneity in ccRCC, we evaluated these sex-specific differences in both an immune checkpoint blockade (ICB)-resistant (Vhl-/- p53-/- Rb1-/- Myc-overexpression) & an ICB-responsive (Vhl-/- Cdkn2a/2b-/- Bap1-/-) model developed by our lab. Tumor cells were orthotopically implanted by sub-renal capsular injection into the kidneys of male and female WT or immune-deficient (ID, NSG- NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, and tumor growth was longitudinally monitored using ultrasound imaging. In addition, metastatic derivatives of these cells lines were injected intravenously. At necropsy, liver and lung metastases were quantified and immune populations in the tumor and peripheral blood were analyzed by flow cytometry. Results WT females developed smaller primary tumors with lower penetrance compared to WT males, while ID mice showed no sex differences, indicating that the gender bias is dependent on the immune system. Flow cytometry revealed females had more circulating T-cells. Interestingly in tumors, CD8+ T-cells in the ICB-resistant model correlated with larger tumors (males), while in the ICB-responsive model, they correlated with smaller tumors (females), suggesting CD8+ T-cells can have pro-tumor/anti-tumor effects depending on the microenvironment. Metastases were observed exclusively in ID mice in both models. However, while the ICB-responsive model had a higher incidence of lung metastases in females and no sex-related differences in liver metastases, male mice injected with ICB-resistant cells had higher incidence of both lung and liver metastases. This suggests a critical role of immune surveillance in restricting both liver and lung ccRCC metastases; gender specific organ tropism was model-dependent. Given that WT mice did not develop metastases, metastatic derivatives of the two lines were used to evaluate metastatic seeding in an immunocompetent setting. In both models, WT females had significantly fewer liver metastases compared to males. In the ICB-responsive model, this correlated with elevated levels of ILC1s in the liver tumors. In the lung, both models exhibited distinct phenotypes: males developed more lung masses in the ICB-resistant model, whereas females showed a higher burden in the ICB-responsive model. Males with fewer lung metastases had significantly greater populations of monocytes, DCs, and T-cells, compared to females having a greater abundance of macrophages, suggesting different sex- dependent mechanisms for immunosurveillance in the lung. Conclusions Overall, our findings highlight deterministic sex-related immunologic differences in immune surveillance and metastatic tropism in ccRCC and support the need for further mechanistic studies.