1120 - Target single-strand DNA (ssDNA) Sensing Signal to Potentiate Radiation-Induced Anti-Tumor Immunoactivation

Purpose/Objective(s): Radiotherapy serves as a “game changer” of the tumor immune microenvironment, with one of its principal immunostimulatory mechanisms being the activation of cGAS-STING pathway via double-strand DNA (dsDNA). However, evidences indicate that low-dose radiotherapy (LDRT) predominantly induces single-strand DNA (ssDNA) damage, with ssDNA accumulation exceeding dsDNA lesions. Despite its abundance, the immunologic potential of ssDNA remains largely unexplored, particularly regarding its capacity to initiate innate immune signaling. This project aimed to systematically investigate the immunostimulatory properties of LDRT-induced ssDNA accumulation, with specific focus on delineating the downstream signaling of anti-tumor immunoactivation mediated by ssDNA sensing.

Materials/Methods: To confirm SLFN11 as the cognate sensor for ssDNA, Mass Spectrometry (MS) and Co-Immunoprecipitation (Co-IP) was employed in HEK-293T cells transfected with biotinylated ssDNA probes. The radiobiological impact of ssDNA recognition via SLFN11 was evaluated through functional assays, including Colony Formation assay and CCK-8 proliferation assay. Molecular characterization of SLFN11-mediated immunogenicity was performed using RT-qPCR and Western Blot. In order to validate the enhanced radiosensitivity in vivo, LDRT (2 Gy × 5 Fractions) was employed on C57BL/6J mice injected with Vector control tumors and Slfn9 (the homologue of SLFN11 in mice) overexpressing tumors. The tumor immune microenvironment was comprehensively profiled through scRNA-seq to explore the corresponding immune cell populations.

Results: Quantitative analysis revealed a remarkably increase of ssDNA content in tumor cells post-radiation, exceeding that of dsDNA lesions. Experiments employing MS and Co-IP confirmed the specific binding affinity between SLFN11 and ssDNA, establishing SLFN11 as the cognate sensor for radiation-induced ssDNA. In vitro functional characterization in tumor cells revealed that fostering ssDNA recognition by SLFN11 overexpression potentiated radiotherapy-induced pyroptosis, with activation of AIM2-Caspase1-GSDMD axis. Furthermore, an augmented tumor immunogenicity was observed as evidenced by elevated release of HMGB1, ATP and Calreticulin, along with upregulated expression of pro-inflammatory cytokines. In vivo studies demonstrated that Slfn9-overexpressing tumors exhibited a significant immune activation even with LDRT, particularly characterized by increased infiltration of activated dendritic cells and cytotoxic CD8⁺ T lymphocytes, resulting in superior therapeutic efficacy of LDRT.

Conclusion: Enhanced recognition of ssDNA through SLFN11-mediated sensing not only triggers tumor cell pyroptosis but also reshapes the tumor microenvironment through improved dendritic cell maturation and cytotoxic T lymphocyte infiltration, providing a promising perspective for the combination of LDRT and immunotherapy.