Transient high-level DNA release mediated by cryo-thermal therapy promoted myeloid cell hyperactivation to induce...

Tumor patients often exhibit limited responses to immunotherapy owing to the low immunogenicity and immunosuppressive environment of tumors. Our previous studies revealed that cryo-thermal therapy caused tumor cell rupture due to mechanical compression, notably causing the release of a substantial amount of DAMPs (danger-associated molecular patterns), such as heat shock protein 70, calreticulin and high-mobility group box protein 1; the release of these DAMPs increased myeloid cell maturation, thereby reshaping the systemic immune environment and ultimately inducing durable CD4⁺ T helper type 1 (Th1) cell-dominated antitumor immunity. In fact, under conditions of mechanical stress and rapid temperature changes, the disruption of tumor cells caused by cryo-thermal therapy results in extensive deoxyribonucleic acid (DNA) damage and the rapid release of substantial amounts of DNA. Consequently, tumor-derived DNA, which potently activates innate immunity by engaging multiple DNA sensors, plays a pivotal role in orchestrating antitumor immunity. We hypothesized that cryo-thermal therapy induces the transient release of high levels of DNA, which modulates CD11b⁺ myeloid cell function, subsequently influencing CD4⁺ Th1-cell dominated antitumor immune responses. In this study, a B16F10 melanoma model was established, and DNA concentrations were measured at different time points after cryo-thermal therapy. Deoxyribonuclease I (DNase I) was subsequently administered immediately following cryo-thermal therapy to deplete extracellular DNA, allowing an investigation of the role of DNA in regulating CD11b⁺ myeloid cell function and CD4⁺ T cell differentiation. The phenotype and function of CD11b⁺ myeloid cells and CD4⁺ T cells were assessed by flow cytometry, RNA sequencing, and cell culture in vitro. Our studies confirmed that cryo-thermal therapy triggered a transient release of high levels of DNA, which was internalized by CD11b⁺ myeloid cells via C-type lectin receptors and subsequently sensed by inflammasomes. Then, the intracellular sensing of DNA induced the production of the mature form of interleukin (IL)-18, ultimately promoting the Th1 differentiation of CD4⁺ T cells. This study highlights the pivotal role of DNA release after cryo-thermal therapy in driving CD4⁺ Th1 cell-dominant antitumor immunity.