脂质体阿霉素增强口腔鳞状细胞癌的抗肿瘤免疫应答

Resistance to cisplatin and low response rates to immune checkpoint blockade (ICB) limit survival outcomes in oral cavity squamous cell carcinoma (OCSCC). Anthracyclines, including liposomal doxorubicin (Doxil®), possess immunomodulatory properties and can induce immunogenic cell death (ICD), providing a rationale for combination with radiation and immunotherapy. We therefore hypothesized that Doxil would enhance anti-tumor immunity in multimodal treatment of OCSCC. To test this hypothesis, we evaluated anti-tumor efficacy and modulation of the tumor immune microenvironment (TIME) using HPV-negative (MOC2, ROC1) and HPV-positive (mEER) syngeneic murine models of OCSCC. C57BL/6 mice bearing subcutaneous flank tumors were treated with radiation therapy (RT; 8 Gy × 3 fractions), Doxil (5 mg/kg IV weekly × 4), and anti-CTLA-4 (100 µg IP twice weekly × 4), alone or in combination. For immune profiling, tumors were harvested approximately 21 days post-inoculation after mice had received one dose of Doxil, one fraction of RT, and/or two doses of anti-CTLA-4, then analyzed using full-spectrum flow cytometry and single nuclear RNA sequencing (snRNA-seq). Doxil demonstrated modest single-agent activity in both HPV-positive and HPV-negative models but showed enhanced efficacy when combined with RT and/or anti-CTLA-4. The greatest therapeutic benefit was observed with the triple combination of Doxil, RT, and anti-CTLA-4 (DRC), which resulted in complete response rates of 43.5-69.6% and prolonged median survival of up to 173 days in the mEER model. Flow cytometric analysis of the TIME of Doxil- and DRC-treated mice revealed a 1.9- to 7.6-fold decrease in regulatory T cells and 1.4- to 1.7-fold decrease in proliferating M2-like macrophages. snRNA-seq demonstrated increased expression of ICD-associated genes in Doxil- or DRC-treated tumor cells. Notably, DRC treatment induced robust upregulation of Cxcl10 , Cxcl9 , and Ccl5 with mean expression fold changes of 5.6, 26.8, and 4.7, respectively. These tumor-intrinsic changes were associated with a 3.1- and 3.4-fold increase in mean Cxcr3 expression on CD8 + T cells in Doxil- and DRC-treated tumors, respectively. Doxil and DRC treatment also increased proportions of activated dendritic cells. Additionally, snRNA-seq analysis demonstrated approximately 50% and 75% reductions in mean expression of epithelial-to-mesenchymal transition (EMT)-related genes including Vim , Sparc , and Spp1 in Doxil- and DRC-treated tumor cells, respectively, suggesting Doxil may inhibit EMT. In summary, combining Doxil with radiation and CTLA-4 blockade induces ICD, reshapes the TIME toward a pro-inflammatory and antigen-presenting state, and promotes durable tumor control in both HPV-positive and HPV-negative OCSCC models. These findings support the translational potential of incorporating liposomal doxorubicin into multimodal immunotherapeutic strategies for OCSCC.