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

Background High-dose radiotherapy (RT) in cancer is immunogenic but also induces an immunosuppressive tumor microenvironment (TME) that limits the efficacy of immune checkpoint inhibitors (ICIs). Overcoming this immunosuppressive barrier is therefore critical to unlocking the full potential of RT-ICI combinations. As Notch signaling regulates tumor microenvironment, we hypothesized that the γ-secretase inhibitor (GSI), AL101, would suppress radiation-induced immunosuppression and enhance antitumor efficacy when combined with RT and anti-PD-1 (aPD-1) therapy. Methods Syngeneic neuroblastoma (9464D) and triple-negative breast cancer (EO771) tumors were established in C57BL/6, macrophage-depleted C57BL/6 mice, or athymic mice. Mice received 12 Gy RT (day 3), AL101 (6.5 mg/kg daily, day 0-9), and aPD-1 (days 0, 3, 6). Tumors were analyzed by spectral flow cytometry and single-cell RNA sequencing (scRNA-seq), and lung metastases were evaluated histologically. Results The triple combination of RT, aPD-1, and GSI produced durable tumor growth inhibition and significantly prolonged survival in both models, with median survival more than doubled compared to all other treatment groups. Triple therapy also markedly reduced lung metastases in EO771 mice. These effects were abrogated in athymic nude mice and macrophage-depleted immunocompetent mice, consistent with an immune-dependent mechanism. Based on scRNA-seq and spectral flow cytometry analysis, RT alone increased exhausted T cells and immunosuppressive macrophages, while triple therapy reversed these effects, including expansion of activated CD8⁺ T cells, reduction of Tregs and exhausted T cells, restoration of cross-presenting CD103⁺ dendritic cells, and reprogramming of myeloid cells toward a proinflammatory state. Conclusions GSI remodels the RT-induced immunosuppressive TME and potentiates the efficacy of RT + ICI. GSI combined with RT+aPD-1 reprograms the TME toward an immunostimulatory state and supports GSI as a promising immuno-radiotherapeutic strategy with strong translational potential.