M2 Macrophage Polarization Mediated by Complement C3 from Hedgehog-Activated Fibroblasts Establishes an Immunosuppressive Niche in Gastric Cancer

<b>Introduction</b>: The Hedgehog (Hh) signaling pathway is aberrantly activated in various types of cancer and plays a critical regulatory role. However, its biological significance in gastric cancer remains unclear. In this study, the mechanism underlying the role of Hh in gastric cancer progression and prognosis was investigated through bioinformatics analysis as well as in vitro and in vivo experiments. <b>Methods</b>: In this study, a systematic analysis of scRNA-seq datasets and bulk RNA-seq datasets from gastric cancer patients derived from the GEO database and TCGA database was performed by us, which revealed the activation characteristics of Hh in different cell types within the gastric cancer tumor microenvironment (TME). Furthermore, through conducting multiplex immunofluorescence staining experiments on clinical gastric cancer samples, we clarified the association mechanism between fibroblasts with highly activated Hh and the gastric cancer tumor immunosuppressive microenvironment. Finally, by means of in vitro and in vivo experiments, we elucidated the key molecular mechanism by which fibroblasts with highly activated Hh remodel the gastric cancer tumor immunosuppressive microenvironment. <b>Results</b>: We identified a distinct subpopulation of fibroblasts, designated MMP1 + FIB, in the gastric cancer tumor microenvironment. Studies revealed that this subpopulation can significantly activate Hh, suggesting it may play a crucial role in the regulation of the TME. Subsequent mechanistic investigations further confirmed that MMP1 + FIB exhibits a significant correlation with the immunosuppressive state of the TME (R = 0.29, <i>p</i> = 2.5 × 10^-0.8). In terms of the specific functions, the complement system in this fibroblast subpopulation is significantly activated (<i>p</i> < 0.05); further studies demonstrated that MMP1 + FIB can induce the polarization of macrophages toward the M2 subtype (an immunosuppressive phenotype) by specifically secreting complement C3 protein. Collectively, these processes contribute to the establishment of an immunosuppressive TME and ultimately promote the proliferation and metastasis of gastric cancer cells. <b>Discussion</b>: Aberrant activation of the Hh signaling pathway promotes gastric cancer progression via the MMP1 + FIB-C3-macrophage axis, providing a potential therapeutic strategy for targeting the tumor microenvironment.