Background Head and Neck Squamous Cell Carcinoma (HNSCC), the sixth most common cancer worldwide, is associated with high-risk subtypes of human papillomavirus (HPV), tobacco, and alcohol over-consumption. Standard of care treatments include surgery, radiation or chemoradiation. Five-year survival rates for patients with locoregionally advanced HNSCC have not significantly improved in decades and remain at approximately 60%, driving the need for novel treatment strategies such as immunotherapy. Furthermore, most HNSCC patients treated with single-agent checkpoint inhibitor immunotherapy do not respond, in large part due to the immunosuppressive tumor immune microenvironment (TIME). While checkpoint inhibitors are currently administered systemically, a localized delivery platform that allows controlled release of immunomodulators could potentially maximize effectiveness and limit systemic side effects. Herein, we investigated localized, intratumorally injected multidomain peptide (MDP) hydrogels encapsulating liposomes loaded with a Stimulator of Interferon Genes (STING) agonist, cyclic di-nucleotide (CDN). This was combined with systemic delivery of immune checkpoint inhibitors (ICIs), anti-programmed cell death protein-1 (α-PD-1) and anti-cytotoxic T-lymphocyte-associated antigen-4 (α-CTLA-4), to enhance antigen presentation and reduce immunosuppressive immunocytes in a preclinical model of HNSCC. We hypothesized that the liposomal CDN-MDP hydrogel composites would enhance drug-delivery pharmacokinetics, localizing and extending the release of immunomodulators intratumorally and stimulating anti-tumor effector immunocytes in synergy with systemic delivery of α-PD-1 and α-CTLA-4 to target the immune-excluded TIME in our preclinical model of HNSCC.

Methods ROC1 cell line was maintained as previously published.¹ C57BL/6 mice were injected with the ROC1 cells in the oral maxillary vestibule. ROC1 oral tumor-bearing mice were treated with either single or six intratumoral delivery of CDN, single intratumoral delivery of liposomal CDN-MDP hydrogel composites, or HBSS, and conventional intraperitoneal delivery of α-PD-1 and α-CTLA-4.

Results ROC1 tumor-bearing mice experienced tumor regression and extended survival with six intratumoral doses of CDN. Tumor-bearing mice treated with liposomal CDN-MDP hydrogel composites in combination with systemic injections of α-PD-1 and α-CTLA-4 experienced tumor regression and statistically significant median survival compared to the controls.

Conclusions Our results demonstrate the effectiveness of the liposomal CDN-MDP hydrogel composites to treat an immunosuppressive orthotopic model of oral cancer. In addition, liposomal CDN-MDP hydrogel composites in combination with α-PD-1 and α-CTLA-4 improved immunotherapy responses. Further studies will build upon these results and explore mechanisms of the improved survival.

Acknowledgements AHM is a Ph.D. graduate student at The University of Texas MD Anderson Cancer Center UT Health Houston Graduate School of Biomedical Sciences and a recipient of a Diversity Supplement (R01DE030140) from the National Institutes of Health-National Institute of Dental and Craniofacial Research (NIH-NIDCR). This work was supported by the NIH-NIDCR grants R01DE030140 and R01DE021798.

Reference

1. Shi Y, Xie T, Wang B, Wang R, Cai Y, Yuan B, Gleber-Netto FO, Tian X, Rodriguez-Rosario AE, Osman AA, Wang J, Pickering CR, Ren X, Sikora AG, Myers JN, Rangel R. Mutant p53 drives an immune cold tumor immune microenvironment in oral squamous cell carcinoma. Commun Biol. 2022 Jul 28;5(1):757. doi: 10.1038/s42003-022-03675-4. PMID: 35902768; PMCID: PMC9334280.

Ethics Approval This study was approved by the Animal Welfare Committee and the Center for Laboratory Animal Medicine and Care (CLAMC) at the University of Texas Health Science Center Houston; approval number 24-0029.