Abstract 2194: Patient-derived triple negative breast cancer organoids show heterogeneous radiation-induced immune signaling

Abstract Only a minority of triple-negative breast cancer (TNBC) patients respond to immune checkpoint inhibitors (ICI). Radiation therapy (RT) increases ICI responses in preclinical TNBC models and in some patients1, 2, but there are no biomarkers to predict RT benefit. TNBC comprises multiple subtypes and we hypothesized that patient-derived tumor organoids (PDOs) representative of such heterogeneity could be used to study RT-induced cancer cell-intrinsic responses.Two PDOs molecularly classified as Luminal Androgen Receptor (LAR) and Basal-Like 1 (BL1) TNBC subtypes3 were mock-treated or irradiated with 3 daily doses of 8Gy and harvested 24 hours post-RT. Differential gene expression analysis (DESeq2), Gene Set Enrichment Analysis (GSEA), and Qiagen’s Ingenuity Pathway Analysis® were performed. Protein expression was assessed by western blot and flow cytometry. Healthy donors CD14+ monocytes were matured in M-CSF for one day prior to culture with PDO. RT upregulated multiple pro-inflammatory pathways (padj < 0.05), including TNFα via NF-κβ, antigen presentation via MHC-I and type I interferon (IFN-I), in the LAR PDO. In contrast, in the BL1 PDO only TNFα via NF-κβ was upregulated by RT, while IFN-I and antigen presentation pathways were not. RT also upregulated genes with immunosuppressive functions (CD73 and TGFB1) in the LAR PDO, and Pathway Analysis identified TGFB1 as a key upstream regulator (p = 7.33 x 10-17). Epithelial to mesenchymal transition (EMT) pathway was induced by RT in both PDOs. Flow cytometry confirmed RT-induced increase in MHC-I and CD73 expression in the LAR PDO, while only CD73 was upregulated in the BL1 PDO. Given the central role of IFN-I induction via cGAS/STING in RT-induced anti-tumor immune responses4, we investigated cGAS and STING expression by the two PDOs. Western blot revealed suppressed expression of STING in the BL1 PDO. STING repression induces resistance to ICI in mouse models of TNBC and was shown to be mediated by MYC amplification in human TNBC5. Consistently, the BL1 PDO was derived from a tumor with MYC amplification. Finally, macrophages co-cultured with the LAR PDO showed reduced MHC-II and CD86 expression, an effect that was reversed when the PDO was treated with RT before co-culture. Our data support the use of PDOs that reflect the heterogeneity of human TNBC as models to study the immunomodulatory effects of RT. Findings show differential induction of key pro-immunogenic mediators by RT in different PDOs and suggest that CD73 is an actionable target to improve RT-induced ICI responses6. Understanding the multifaceted effects of RT using a PDO-based platform may enable a precision use of RT that is tailored to the specific characteristics of a given patient TNBC. Supported by BCRF 23-053 & DOD W81XWH-21-2-0034. DS is the recipient of a Burroughs Welcome Physician-Scientist Award. PMID: 1. 37620372 2. 38194915 3. 27310713 4. 32161398 5. 35561311 6. 32047024 Citation Format: Diego Sialer, Xiaoxue Deng, Maud Charpentier, Neil Rupchand, Marvin Campos, Jenna Moyer, Wen Shen, Olivier Elemento, Doron Betel, Silvia Formenti, Sandra Demaria. Patient-derived triple negative breast cancer organoids show heterogeneous radiation-induced immune signaling [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 2194.