Liposomal doxorubicin potentiates anti-tumor immune response in oral cavity squamous cell carcinoma

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.