Abstract 2569: Cancer associated fibroblast dependency in a model of muscle invasive bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy is crucial, especially in diseases like bladder cancer where the PI3K/AKT/mTOR pathway is widely activated but untargeted. We have developed an immunocompetent, CRISPR-edited bladder cancer mouse model that recapitulates common PI3K/AKT/mTOR mutations. Through targeting generated PI3K/AKT/mTOR mutations, we hope to establish rationale for clinical investigation of PI3K/AKT/mTOR-directed therapy. Our mouse model displays a dependency on Cancer Associated Fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo, facilitating engraftment and tumor progression. Given the observed in vivo CAF dependency, we aim to understand the mechanisms of immune modulation between CAFs and the tumor microenvironment. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 LSL-Cas9/GFP transgenic mice to develop organoids. These organoids were co-edited for Trp53 knockout and either Rb1 knockout or 9p21 (homologous to mouse 4q) large deletion. Further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib, while a Pikc3a H1047R hotspot mutation sensitized organoids up to 2000-fold to the Pik3 inhibitor pictilisib. Despite successful engraftment into mouse bladder, these edited cells regressed before tumor formation. We have since demonstrated that co-injecting edited organoids with skin cancer or bladder cancer derived-CAFs yielded palpable subcutaneous and orthotopic tumors. To further understand the mechanisms of immune suppression in vivo, we performed a medium throughput cytokine array with a panel of 16 mouse adaptive immune markers. Cytokine analysis revealed that CAFs secreted interlukin-6 in the presence of organoids which themselves secreted GM-CSF. This combination is well-known to be pro-tumorigenic, facilitating recruitment of M2 macrophages which we also observe in in vivo tumors. Further, CAFs activate TGF-β pathway as demonstrated by phosphorylation of SMAD2/3. Taken together we believe that CAFs, through GM-CSF/IL-6 secretion along with TGF Beta pathway activation, regulate the TME to create a suitable environment for tumor engraftment. Conclusions: Our research demonstrates the importance of CAFs in bladder cancer progression. Through further examining the mechanistic interplays between CAFs and immunoregulation, we can gain a better understanding of the main drivers and potential targets in bladder tumorigenesis. Citation Format: Henkel Valentine, Uttam Satyal, Alexander Metz, Rhea Arya, Tahmina Hague, Laura Bukavina, Philip H. Abbosh. Cancer associated fibroblast dependency in a model of muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2569.