Tumor-Initiating Stem Cells Fine-tune the Cell States of Neutrophils to Drive Cancer Relapse from Immunotherapy [Parse scRNAseq]

The highly heterogeneous and plastic nature of tumor-associated neutrophils (TANs) has been recognized, but how different cell states of TANs emerge, evolve, distribute, and impact cancer immunotherapy efficacy remains elusive. Using single-cell RNA sequencing, spatial transcriptomic analysis, and genetic manipulations, we show that, while anti-PDL1/CD40 agonist immunotherapy can reprogram TANs to gain strong anti-tumor activities in squamous cell carcinomas (SCCs), a subset of neutrophils residing at the tumor-stroma interface can preserve their immune-suppressive state. Importantly, we identified a group of Sox2Hi tumor-initiating stem cells (tSCs) at the tumor-stroma interface that can activate Fatty Acid Desaturase 1 (Fads1) and produce arachidonic acid (AA) to induce autocrine prostaglandin E2 signaling which can disrupt the interferon responses in neutrophils. Thus, through this interaction, tSCs fine-tunes the cell states of neutrophils, shapes neutrophil heterogeneity, and sculpts a protective micro-niche to drive cancer relapse following immunotherapy. Overall design: To define how immunotherapy treatments impact TANs, we sought to employ a highly immunogenetic mouse cancer model that is induced by exposing the mouse skin epithelium to chemical mutagen 7,12-dimethylbenz[a]anthracene (DMBA) followed by repeated applications of phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). With this ideal model, we next treated the mice bearing spontaneous skin SCCs with PDL1 blocking and CD40 agonist antibodies which were recently demonstrated to induce anti-tumor activities in neutrophil. Immediately after the treatment (e.g. day two after the last dose), we collected these tumors, isolated total CD45+ immune cells, and subjected them, together with immune cells isolated from untreated naïve tumors, to single cell RNA-sequencing.