Abstract SY36-02: Interrogating how the dialogue between immune cells and fibroblasts impacts radiotherapy outcomes

Abstract Background: Increased stromal fibroblasts in tumors is associated with inferior radiotherapy responses, however, we lack a mechanistic understanding of how fibroblast reprogramming contributes to radiotherapy outcomes. Methods: Radiotherapy (8Gy) was applied to 5555 BRAF mutant melanoma flank tumors grown in WT and RAG1 KO Bl/6 mice. Tumors were harvested at early (1 week post radiation) and late timepoints. 1D11, which inhibits all three isoforms of TGFβ, or the relevant isotype control, was administered from 24 hours prior to irradiation for one week. Multiplex immunohistochemistry and flow cytometry evaluated biological changes following treatment. A Cre-inducible murine model of podoplanin (PDPN) knockout was used to evaluate the specific role of PDPN expression in fibroblasts. NanoString GeoMx spatial profiling was used to evaluate transcriptional changes in different tumour regions. Results: An extensive peri-tumoral expansion of PDPN+ fibroblasts was observed after radiation in WT mice but not in RAG1 KO mice where tumours grew more rapidly. Radiation induced entry of CD8+ T cells which remained sequestered at the fibroblast/tumor cell interface, with minimal entry into the tumor core. Upregulation of inflammatory signaling was identified in PDPN+ fibroblasts at this interface with tumor cells but not elsewhere. The addition of 1D11 to irradiation promoted T cell entry into the tumor core however a paradoxical increase in TGFβ signaling was observed specifically in PDPN fibroblasts with 1D11. When PDPN knockout was induced shortly before irradiation, a higher rate of spontaneous tumor rejection was observed. The PDPN+ fibroblast phenotype observed in the above pre-clinical models is also seen across a range of human tumors both pre- and post-radiation. Conclusion: Rapid stromal reprogramming after irradiation, involving PDPN+ fibroblast expansion and associated inflammatory signaling, restricts T cell entry into tumors which appears to compromise tumor control Citation Format: Anna Wilkins. Interrogating how the dialogue between immune cells and fibroblasts impacts radiotherapy outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr SY36-02.