N6‐methyladenosine‐regulated exosome biogenesis orchestrates an immunosuppressive pre‐metastatic niche in gastric cancer peritoneal metastasis

Abstract Background Gastric cancer peritoneal metastasis is clinically challenging, given the limited treatment options and poor prognosis. The molecular mechanisms that precede gastric cancer peritoneal metastasis, known as the pre‐metastatic niche (PMN), and its relationship with N 6 ‐methyladenosine (m 6 A) modification remain unclear. Methods We used 87 resected gastric cancer tissues and 4 public datasets to explore the association between methyltransferase‐like 3 (METTL3) expression and gastric cancer peritoneal metastasis. Roles of m 6 A, exosomes, or macrophages in PMN formation were explored in immunocompetent mouse models through exosome treatments or macrophage modifications. Key genes and regulatory mechanisms were uncovered using mass spectrometry, RNA/miRNA sequencing, RNA‐immunoprecipitation, dual‐luciferase assays, and point mutations in the ras‐related protein Rab‐27A ( RAB27A ) in cells. Macrophage and T‐cell functions were assessed using enzyme‐linked immunosorbent assay, flow cytometry, and cytotoxicity assays. Results METTL3 overexpression in gastric cancer cells enhanced RAB27A translation by methylating its mRNA A502 base, facilitated by its m 6 A “reader” YTH N 6 ‐methyladenosine RNA binding protein F1 (YTHDF1), and led to increased exosome biogenesis. The miRNA‐17‐92 cluster was enriched in METTL3‐overexpressed cell‐derived exosomes and targeted SRC kinase signaling inhibitor 1 (SRCIN1) to activate SRC proto‐oncogene, non‐receptor tyrosine kinase (SRC) signaling in peritoneal macrophages. Macrophage activation skewed cytokine production towards an immunosuppressive profile in the peritoneum, elevating the levels of interleukin (IL)‐10 and tumor necrosis factor (TNF) and reducing the levels of IL‐1 and IL‐6. These cytokine shifts inhibited T cell proliferation and cytotoxic activities, which created an immunosuppressive PMN and led to peritoneal metastasis. The association between METTL3, macrophages, and peritoneal metastasis was verified in clinical samples. Conclusions Our study identified an intricate m 6 A‐regulated mechanism of peritoneal PMN development that is mediated by exosome‐promoted macrophages. These insights into gastric cancer peritoneal metastasis offer promising directions for translational research.