Fibroblast-derived thrombospondin-1 shapes macrophage polarization in advanced human co-culture models

Abstract Background Tumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. Methods We established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. Results The co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. Conclusion Our study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract