A Novel Therapeutic Approach: Notch Inhibition Enhances Radiotherapy and Checkpoint Blockade Therapy via Reprogramming of the Tumor Microenvironment

BackgroundHigh-dose radiotherapy (HDRT) is highly immunogenic, promoting tumor antigen release and enhancing responses to immune checkpoint inhibitor (ICI). However, HDRT also induces an immunosuppressive tumor microenvironment (TME) that limits therapeutic benefit. The Notch signaling pathway is a key regulator of immune and stromal cell function. We hypothesized that pharmacologic inhibition of Notch signaling with a {gamma}-secretase inhibitor (GSI), AL101, would overcome radiation-induced immunosuppression and synergistically enhance antitumor efficacy when combined with HDRT and anti-PD-1(aPD-1) therapy. MethodsSyngeneic neuroblastoma (9464D) and triple-negative breast cancer (EO771) tumors were established in C57BL/6 mice and athymic nude mice. Mice received GSI AL101 (6.5{square}mg/kg daily x10), a single 12{square}Gy dose of RT (Day 3), and aPD-1 antibody (200{square}{micro}g, Days 0, 3, and 6) for triple combination therapy. A subset of mice were sacrificed on Day 10 for mechanistic studies; the remainder were followed for survival and were euthanized when tumors reached 1.5{square}cm3. Tumors were analyzed by spectral flow cytometry and single-cell RNA sequencing (scRNA-seq). Histological image analysis of lung metastases was performed to quantify metastasis area relative to total lung area. ResultsThe triple combination of RT + GSI+ aPD-1 resulted in synergistic and durable tumor growth inhibition and significantly prolonged survival in both 9464D and EO771 models. In contrast, neither GSI nor aPD-1 treatment alone significantly impacted tumor growth. Dual therapy with RT + GSI modestly improved survival compared with RT alone. The triple combination regimen also markedly reduced lung metastases at the survival endpoint in EO771-bearing mice. These effects were abrogated in athymic nude mice, confirming an immune-dependent mechanism. ScRNA-seq revealed that RT alone increased exhausted T cells and immunosuppressive M2-like macrophages, whereas triple therapy reversed these effects, significantly reducing exhausted T cells while increasing activated CD8 T cells, M1-like macrophages, and monocytic myeloid-derived suppressor cells (M-MDSC). Moreover, triple therapy also restored CD103 dendritic cells--specialized for antigen cross-presentation--within the TME. Spectral flow cytometry corroborated these findings, demonstrating that GSI combined with RT + aPD-1 reprograms the TME toward an immunostimulatory state. ConclusionsThis study demonstrates the novel repurposing of GSI therapy to remodel the immunosuppressive TME induced by radiation and enhance the efficacy of HDRT combined with ICI. Triple therapy activates CD8 T cells, reduces Tregs and exhausted T cells, restores cross-presenting dendritic cells, and reverses myeloid-driven suppression, supporting Notch blockade as a promising immuno-radiotherapeutic strategy with strong translational potential.