Combination Radiation Therapy and Selective TLR9 Agonist Improves Local Control in a Murine Model of HPV-Related HNSCC

Abstract

<h3>Purpose/Objective(s)</h3> As novel immunotherapies emerge, it has become increasingly evident that personalized treatment combinations are necessary to improve outcomes in head and neck squamous cell carcinomas (HNSCC). Toll-like receptors (TLR) play a critical role in the activation of professional antigen presenting cells (APC). Given the increasing appreciation of the role of APC and B-cells in HPV+ HNSCC, we sought to evaluate the therapeutic role of selective TLR agonists in combination with radiation therapy (RT). <h3>Materials/Methods</h3> For human tumor RNA sequencing studies, we analyzed data from the TCGA and a separate independent validation cohort of HNSCC patients, cumulatively totaling nearly 600 patients. Single cell RNA sequencing (scRNA-seq) databases were analyzed using Seurat v4.0. For murine studies, wild type C3H mice were injected subcutaneously with 5 × 10⁵ cells of AT-84-E7-OVA syngeneic squamous cell carcinoma into the right flank. TLR agonists were administered intratumorally in 25 uL per published schedules for: TLR4 (MPLA-SM, 1 mg/ml), TLR7 (1V270, 2 mg/ml), and TLR9 (ODN 1826, 2 mg/ml). Control mice received PBS. Mice receiving RT were treated with 12 Gy in 1 fraction. Cell surface markers in the tumor draining lymph node (TDLN) and tumor were analyzed by flow cytometry. <h3>Results</h3> Treatment with TLR4, TLR7, and TLR9 agonists decreased the size of the primary tumor in our murine model of HPV+ HNSCC, with a B cell selective TLR9 agonist exhibiting the greatest therapeutic benefit. When analyzing transcriptomes of HPV- tumors, we found no correlation between high TLR4/7/9 expression and outcomes, however, in HPV+ tumors, among TLRs 4/7/9, only TLR9 had a significant correlation with overall survival (HR 0.38, P = 0.008). In an independent cohort of HPV+ HNSCC patients, the correlation between OS and high TLR9 expression was maintained. scRNA-seq of human HNSCC TILs revealed heterogeneity in TLR9 expression among various immune cells. In the TDLN, TLR9 agonist were shown to decrease the relative proportion of B cells and trended towards increased proportions of CD4+ T cells, which had decreased expression of CD44. In comparison, in the tumor, we observed increased CD8+ T cell infiltration but decreased proportions of B cells with TLR9 agonists. When evaluating TLR9 agonists in conjunction with RT, we found that the combination treatment led to the largest therapeutic benefit. <h3>Conclusion</h3> In HPV+ HNSCC, transcriptional analysis revealed a significant correlation between high TLR9 expression and overall survival. Concordantly, the combination of RT and TLR9 agonists lead to a significant reduction in the primary tumor, which may be driven by dynamic changes in B cell phenotype or migration patterns and CD8+ T cell tumor infiltration. These findings support the further investigation into the use of TLR9 agonists in combination with radiation therapy for the treatment of HPV+ HNSCC.