INVESTIGATING THE ROLE OF STING SIGNALLING IN IMMUNE AND NON-IMMUNE CELLULAR COMPARTMENTS ON SURVIVAL PHENOTYPES IN THE KP MODEL OF UNDIFFERENTIATED PLEOMORPHIC SARCOMA

In recent studies of the murine KP model of undifferentiated pleomorphic sarcoma (UPS), intratumoral (IT) activation of the STimulator of INterferon Genes (STING) pathway using the murine specific STING agonist, DMXAA, and translational STING agonists E7766 and CDN have demonstrated durable tumor clearance. Although IT STING therapy can stimulate KP UPS tumor clearance, further investigations are required to understand which cell types in the UPS tumor microenvironment contribute to STING signaling and resultant survival outcomes. To investigate this, we sought to 1) characterize STING expression in KP UPS cells and the consequences of STING ablation in UPS cells and 2) identify immune populations that engage in STING signaling in the KP UPS tumor microenvironment following therapy using single cell RNA sequencing (scRNAseq). To assess the importance of UPS cell STING expression in the context of tumor clearance following STING therapy, CRISPR-Cas9 was used to knockout the Sting1 gene from our UPS cell line. STING knockout UPS cells were engrafted in C57Bl/6 mice and treated intratumorally with DMXAA or E7766 and monitored over time. To investigate which cells in the UPS-TME engage in STING signaling following therapy, scRNAseq was performed on wild-type KP UPS tumors 6hrs and 1-week following treatment with CDN, E7766, or DMXAA. SingleR was used to identify single cell clusters and the AddModule tool was used to generate interferon stimulated gene (ISG) scores as a measure of STING signaling at the single cell level. Sting1 was successfully knocked out of the UPS cell line (Figure1A). In the absence of tumor STING expression, both DMXAA and E7766 continue to exert similar anti-tumor clearance frequencies to the wildtype condition indicating that UPS cell STING expression is not required to observe tumor clearance phenotypes in this model (Figure1A). Next, we sought to identify which non-malignant cell types in the UPS TME engage in STING signaling following stimulation with DMXAA, CDN, or E7766. Comparing annotated cell clusters among treatments, neutrophils emerged as the most abundant cell type in DMXAA and E7766 treated tumors at 6hrs, and 1-week post-therapy compared to CDN and control (Figure1B). Moreover, ISG scores were most significantly elevated in myeloid lineage cells (Figure2A). Upon stratifying ISG scores among treatments and timepoints, it is evident that; DMXAA and E7766, appear to engage prolonged ISG score elevation relative to CDN and controls across several myeloid cell types (Figure2B). We have established that UPS cell STING expression is not required to observe tumor clearance phenotypes in the KP model following STING therapy and that myeloid cells appear to be abundantly involved in ISG signaling in the TME following agonist therapy. Further, E7766 and DMXAA conditions maintain ISG elevation over time in all myeloid cell types at 1-week post treatment, a feature not observed in the CDN treatment condition. It is possible that STING signaling in myeloid cells is critical to tumor clearance. To investigate this, mouse studies are currently underway in which STING can be selectively ablated from myeloid cells. The present study and ongoing investigations will be key for defining in which cell types STING signaling in the UPS TME must occur for promoting anti-tumor outcomes, potentially informing which UPS patients may be more favourable candidates for STING therapy based on TME composition. For any figures or tables, please contact authors directly.