Abstract PS3-12-16: Reprogramming the Tumor Microenvironment in TNBC: A Novel Strategy Combining Notch Inhibition with Radiotherapy and Checkpoint Blockade

Abstract Background: High-dose radiotherapy (HDRT) is immunogenic, promoting tumor antigen release and enhancing immune checkpoint inhibitor (ICI) efficacy. However, HDRT also induces an immunosuppressive tumor microenvironment (TME), limiting the therapeutic response of combined RT + ICI. The Notch signaling pathway is a critical regulator of immune and stromal cell behavior. We hypothesized that inhibiting Notch signaling using a γ-secretase inhibitor (GSI) AL101 would overcome radiation-induced immunosuppression and synergize with HDRT and αPD-1 therapy in triple-negative breast cancer (TNBC). Methods: EO771 murine TNBC tumors were established in syngeneic C57BL/6 mice and athymic nude mice. Mice received GSI AL101 (6.5 mg/kg daily ×10), a single 12 Gy dose of HDRT (Day 3), and αPD-1 antibody (200 µg, Days 0, 3, and 6). A group of mice were sacrificed on Day 10 for mechanistic analyses; others were followed for survival and euthanized when tumors reached 1.5 cm3. Tumors were analyzed by spectral flow cytometry for immune cell subsets. Lung metastasis were evaluated by histology image analysis for metastasis/total lung area using HALO image analysis. Results: Neither GSI nor αPD-1 monotherapy inhibited EO771 tumor growth (median survival: control 14d vs GSI 18d and αPD-1 15d, p>0.05). GSI + HDRT modestly prolonged survival compared to RT alone (41d vs 35d, p < 0.05). Triple therapy (GSI + HDRT + αPD-1) resulted in durable tumor growth control and improved survival (98.5d, p