Targeting the RNA m6A reader YTHDF2 in tumor-associated macrophages shapes tumor immunity and immunotherapy [scRNA-seq]

The presence of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) affects cancer progression and immunotherapy response. The RNA m6A methylation, an epigenetic modification, has recently been found to play a pivotal role in shaping the TME. However, the role and underlying mechanisms by which RNA m6A methylation regulates TAMs function and anti-tumor immunity remain elusive. Here we show that depletion of YTHDF2, a well-known m6A reader, in TAMs suppresses tumor growth and metastasis. Myeloid YTHDF2 deficiency reprograms TAMs to anti-tumorigenic type and increases their cross-presentation ability, thereby enhancing CD8+T cell-mediated anti-tumor immunity. Transcriptome-wide screening identifies YTHDF2 deficiency facilitates anti-tumorigenic TAMs reprogramming through targeting IFN-?-STAT1 signaling. Selectively targeting YTHDF2 in TAMs using toll-like receptor 9 agonist - conjugated small interfering RNA against YTHDF2 effectively promotes anti-tumor immunity, restrains tumor growth, and enhances the efficacy of anti-PD-L1 therapy. Together, our findings suggest that YTHDF2 in TAMs might be a promising therapeutic target for cancer immunotherapy. Overall design: Tumors from WT or cKO mice were extracted and digested with collagenase type IV (1 mg/ml) and DNase type I (30?U/ml) for 30?min at 37?°C. The cells were filtered through the 70?µm cell strainers, washed with phosphate-buffered saline (PBS), lysed in red blood cell buffer, and resuspended in PBS. Tumor-infiltrating immune cells (CD45+ cells) were sorted by FACSAria Fusion Flow Cytometer (BD Biosciences). The cell suspension (300-600 living cells per microliter) was loaded onto the Chromium single cell controller (10 x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Libraries were subsequently prepared sing Single Cell 5'Library and Gel Bead Kit and sequenced on an Illumina NovaSeq 6000 instrument with pair-end 100?bp (PE100) reading strategy.