Reduced CH25H expression defines a high membrane fluidity, trogocytosis active state in colon cancer stem cells

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Introduction: Trogocytosis enables direct membrane exchange between contacting cells and has recently been implicated in tumor immune evasion. However, the cellular states and molecular mechanisms that regulate cancer cell trogocytosis remain largely unknown. Here, we investigated whether trogocytosis differs between cancer stem cells (CSCs) and non-stem cancer cells (non-CSCs) in colorectal cancer. Methods: Three-dimensional spheroid cultures of Caco-2, HCT116, and HT-29 colorectal cancer cells were used to enrich CSCs. Trogocytosis was quantified by co-culture with membrane-labeled Jurkat T cells via flow cytometry. Membrane fluidity, gene expression, STAT1 phosphorylation, and publicly available human colorectal cancer single-cell RNA-seq datasets were analyzed to investigate the underlying mechanisms. Results: 3D spheroid culture enriched CSC populations and markedly increased trogocytic membrane transfer from Jurkat T cells to tumor cells. Within spheroids, CSCs acquired significantly higher levels of T cell-derived membrane proteins and membrane components than non-CSCs, indicating preferential enhancement of trogocytosis in CSCs. Membrane order analysis revealed reduced membrane order and increased membrane fluidity in CSCs. Quantitative PCR analysis demonstrated reduced expression of cholesterol 25-hydroxylase (CH25H) and cholesterol efflux-related genes in CSCs Because CH25H is an interferon-responsive gene downstream of STAT1 signaling, we next examined STAT1 activity. CSCs exhibited reduced STAT1 phosphorylation, which was associated with enhanced trogocytic membrane acquisition. Furthermore, activation of STAT1 signaling by IFN-γ reduced the CSC population and suppressed trogocytosis. Analysis of human colorectal cancer single-cell RNA-sequencing datasets further supported an association between reduced STAT1 signaling and decreased CH25H expression. Discussion: These findings identify a CH25H-low CSC state characterized by increased membrane fluidity and enhanced trogocytosis. Reduced STAT1-CH25H signaling may promote membrane biophysical remodeling that facilitates trogocytosis in colon cancer stem cells and contributes to tumor progression and immune evasion.

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