Tumor CD45+ cells and spleen LY6G+ PMNs isolated from Hdc-GFP mice with subcutaneous ACKP syngeneic GC line-established tumors and treated with TFF2-MSA and/or anti-PD-1 therapies.

We designed a novel TFF2-MSA peptide as a CXCR4 partial agonist, and used it either alone or in combination with anti-PD-1 to treat established ACKP tumors. PMNs are known to be highly heterogenous, thus these tumors are grown in Hdc-GFP host mice to trace Hdc+ PMN subsets. We then used single cell-RNA sequencing (scRNA-seq) to elucidate the effect of TFF2-MSA on tumor and spleen PMNs, and more broadly the tumor microenvironment. Overall design: The CD45+ cells were isolated with subcutaneous syngeneic ACKP tumors following 3 cycles of treatment with vehicle, TFF2-MSA, anti-PD-1, or their combination. To detect the systemic effect of TFF2-MSA on PMNs, LY6G+ PMNs were isolated from the spleens of ACKP tumor-bearing mice following the vehicle, TFF2-MSA, and TFF2-MSA plus anti-PD-1 treatments. A spleen PMN sample from tumor-free mice was included to identify tumor-induced effects on PMNs. To increase the visibility of PMNs in scRNA-seq, given their low RNA content, we employed a strategy of mixing Ly6G+ tumor PMNs with total CD45+ cells isolated from the same tumors at a 1:2 ratio during sample preparation for scRNA-seq analysis.