Tumor-Restricted CXCR4 and MIF Signaling Axis is Associated with T Cell-Mediated Macrophage Exclusion in Gastrointestinal Stromal Tumors.

Purpose: Gastrointestinal stromal tumors (GISTs), driven by KIT/PDGFRA mutations and treated with tyrosine kinase inhibitors (TKIs), remain challenged by drug resistance, suggesting that poorly understood mechanisms sustain an immunosuppressive tumor microenvironment (TME). This study aimed to investigate the cellular and molecular basis of the immune architecture in GISTs through integrated multi-omics analysis. Patients and Methods: We integrated bulk RNA-sequencing data from 20 GISTs and 20 matched normal tissue samples (GSE225819) with single-cell RNA-sequencing data from 2 GISTs and 2 normal tissue samples (GSE162115) to dissect the cellular and molecular basis of TME. Weighted gene co-expression network analysis (WGCNA), immune deconvolution (CIBERSORTx), cell-cell communication inference (CellChat), and pseudotime trajectory analysis (Monocle 3) were employed. In vitro functional validation was performed using the GIST-T1 cell line, and clinical specimens were analyzed by RT-qPCR and flow cytometry. Results: Transcriptomic profiling of GISTs revealed an inverse correlation between activated T-cell signatures and macrophage abundance, alongside significant transcriptional remodeling (1071 upregulated and 1211 downregulated genes). scRNA-seq further identified a tumor cell subpopulation with high C-X-C Motif Chemokine Receptor 4 (CXCR4) expression that, together with activated CD4⁺ memory T cells, produced macrophage migration inhibitory factor (MIF). Ligand-receptor analysis identified the MIF pathway as the dominant intercellular signaling network, primarily targeting macrophages, thereby offering a mechanistic basis for their reduced presence in the TME. In vitro, CXCR4 knockdown suppressed MIF mRNA expression and cell proliferation. In clinical specimens, tumor tissue CXCR4 and MIF transcripts were elevated and positively correlated with T-cell-derived MIF protein, which inversely correlated with macrophage infiltration. Conclusion: Our study identifies a CXCR4⁺ GISTs tumor subpopulation associated with CD4⁺ T cell activation and MIF signaling amplification, which correlates with reduced macrophage infiltration. These findings nominate the CXCR4/MIF axis as a candidate therapeutic target, although further functional validation is warranted.