Single-cell RNA sequencing on TILs from 4T1 mouse model treated with STING agonist deliveredy by PEG biopsy marker shows activation of antigen presenting cells and macrophages but does not show T cell clonality.

To better characterize the TME effects of ADU-S100 delivered by the PEG, we performed single cell RNA sequencing (scRNA-seq) on CD45+ immune cells from 4T1 tumors 14 days after treatment. We found that antigen presenting cell (APC) and macrophage clusters exhibited robust upregulation of IFN-induced genes by ADU-S100, as well as significant enrichment for hallmark IFN gene signatures. To explore whether immune escape could be secondary to a failure of STING agonist to induce T-cell clonality and expansion, we further performed T-cell receptor sequencing (TCR-seq) on these CD45+ immune cells from animals treated with PEG markers with PBS or 100 µg ADU-S100. We then analyzed the frequency of unique clonotypes that were expanded following PBS versus ADU-S100 treatment. In both groups, there was no evidence for clonotype expansion. Overall design: Balb/c mice were inoculated with 100K 4T1 cells followed by PEG with PBS or 100 µg ADU-S100 implantation on Day 6 when tumor volumes reached 75 mm3. On Day 14 after PEG implantation, tumors were harvested and pooled (n=4 per group). We then sorted CD45+ cells by Fluorescence-activated cell sorting (FACS) and performed scRNAseq/TCRseq.