N6-甲基腺苷（m6A）在肿瘤免疫微环境（TIME）中的作用仍未得到充分研究。在此，我们阐明了YTH N6-甲基腺苷RNA结合蛋白1（YTHDF1）在结肠直肠癌（CRC） TIME中的功能和机制。通过组织微阵列（N = 408）和TCGA（N = 526）队列评估了YTHDF1的临床意义。在同种移植瘤，肠特异性Ythdf1基因敲入小鼠和人类化小鼠中确定了YTHDF1的功能。使用单细胞RNA测序（scRNA-seq）来描绘TIME。使用甲基化RNA免疫沉淀测序（MeRIP-seq），RNA测序（RNA-seq）和核糖体测序（Ribo-seq）来识别YTHDF1的直接靶点。在体内使用胞状纳米粒（VNPs）封装的YTHDF1-siRNA来沉默YTHDF1。YTHDF1的表达与TCGA-CRC中的干扰素-γ基因签名负相关。一致地，独立组织微阵列队列中YTHDF1蛋白质与CD8 + T细胞浸润呈负相关，暗示其在TIME中的作用。Ythdf1的遗传缺失增强了CT26（MSS-CRC）和MC38（MSI-H-CRC）同种移植瘤的抗肿瘤免疫力，而Ythdf1基因敲入则促进了免疫抑制性TIME，有利于Azoxymethane-dextran sulphate-sodium或ApcMin / +模型中的CRC。scRNA-seq确定了骨髓源性抑制细胞（MDSCs）的减少，同时与Ythdf1敲出瘤组织中细胞毒性T细胞的增加相伴。整合MeRIP-seq，RNA-seq和Ribo-seq揭示了p65 / Rela是YTHDF1的靶点。YTHDF1促进p65翻译以上调CXCL1，并通过CXCL1-CXCR2轴增加MDSC迁移。增加的MSDC反过来会拮抗TIME中的功能CD8 + T细胞。重要的是，通过CRISPR（聚集的正则间隔短回文重复）或VNPs-siYTHDF1靶向YTHDF1，提高了MSI-H CRC的抗PD1疗效，并克服了MSS CRC的抗PD1耐药性。YTHDF1通过m6A-p65-CXCL1 / CXCR2轴损害抗肿瘤免疫力以促进CRC，并作为免疫检查点阻滞疗法中的治疗靶点。©作者（或其雇主）（2023）。在CC BY-NC下允许重复使用。无商业再利用。由BMJ出版。

The role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME.Clinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. YTHDF1 function was determined in syngeneic tumours, intestine-specific Ythdf1 knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated YTHDF1-siRNA was used for YTHDF1 silencing in vivo.YTHDF1 expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8+ T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of Ythdf1 augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while Ythdf1 knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or ApcMin/+ models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in Ythdf1 knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8+ T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-siYTHDF1 boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC.YTHDF1 impairs antitumour immunity via an m6A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.