Pfneissl, J. | bioRxiv Science

bioRxiv2026

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

BackgroundTumor-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. MethodsWe 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. ResultsThe 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. ConclusionOur 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 O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/728363v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@11b8c9borg.highwire.dtl.DTLVardef@1ac3aa2org.highwire.dtl.DTLVardef@3da223org.highwire.dtl.DTLVardef@5fc3a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Aggressive KRAS mutations direct TGF-β response towards partial EMT in patient-derived colorectal cancer tumoroids

Transforming growth factor beta (TGF-{beta}) exhibits complex and context-dependent cellular responses. While it mostly induces tumor-suppressive effects in early stages of tumorigenesis, its tumor promoting properties are evident in advanced disease. This TGF-{beta} duality is still not fully understood, and whether TGF-{beta} supports invasion and metastasis by influencing cancer cells directly, or rather through the stromal tumor compartment remains a matter of debate. Here, we utilized a library of colorectal cancer (CRC) patient-derived tumoroids (PDTs), representing a spectrum of tumor stages, to study cancer cell-specific responses to TGF-{beta}. Using medium conditions allowing for the differentiation of PDTs, we observed TGF-{beta} induced tumor-suppressive effects in early-stage tumoroids. PDTs with TGF-{beta} pathway mutations or PDTs derived from metastatic tumors were insensitive to the treatment. Notably, one tumoroid line harboring an atypical KRASQ22K mutation underwent partial epithelial-to-mesenchymal transition (EMT), associated with morphological changes and increased invasiveness. On a molecular level, this was accompanied by elevated expression of mesenchymal genes, as well as deregulation of pathways associated with matrix remodeling and cell adhesion. Our results suggest that tumor cell intrinsic responses to TGF-{beta} are critical in determining its tumor-suppressive or -promoting effects.

cancer biology↗