Enhancing radiotherapy response via intratumoral injection of a TLR9 agonist in autochthonous murine sarcomas

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Abstract

Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell–dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.

Authors

Chang Su, Collin L. Kent, Matthew Pierpoint, Warren Floyd, Lixia Luo, Nerissa T. Williams, Yan Ma, Brian Peng, Alexander L. Lazarides, Ajay Subramanian, Jonathon E. Himes, Vincent M. Perez, Rosa D. Hernansaiz-Ballesteros, Kimberly E. Roche, Jennifer L. Modliszewski, Sara R. Selitsky, Mari L. Shinohara, Amy J. Wisdom, Everett J. Moding, Yvonne M. Mowery, David G. Kirsch