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

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 N⁶-methyladenosine (m⁶A) 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 m⁶A, 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 m⁶A "reader" YTH N⁶-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 m⁶A-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.

N6‐methyladenosine; exosomes; gastric cancer; peritoneal metastasis; pre‐metastatic niche.