Spatial transcriptomic profiling of cancer-associated fibroblast niches in extrahepatic cholangiocarcinoma

Background. Cholangiocarcinoma (CCA) is a rare and highly lethal malignancy characterized by a dense desmoplastic stroma containing cancer-associated fibroblasts (CAF). While CAF have been extensively studied in intrahepatic CCA, their impact on TME remodeling and chemo-immunotherapy (CIT) efficacy in extrahepatic (e) CCA remains largely unexplored. A comprehensive characterization of CAF in eCCA is needed to identify therapeutic strategies able to improve the CIT efficacy, that, so far, is limited in these patients. Methods. Spatial transcriptomics was performed on treatment-naive tumor specimens from 8 patients with advanced eCCA treated with durvalumab + cisplatin/gemcitabine. A total of 126 microregions were profiled with the GeoMx Digital Spatial Profiler and classified based on pan-CK and ACTA2/alphaSMA straining, as follow: tumor (CK + alphaSMA - ), distant and peritumor CAF-enriched stroma (CK - alphaSMA + ) or peritumor alphaSMA-negative stroma (CK - alphaSMA - ). Results. alphaSMA staining revealed marked stromal heterogeneity across all eCCA, with some tumor nests surrounded by a dense CAF-enriched stroma and others by alphaSMA - cells within the same sample. Based on CD45 staining all samples were classified as immune-desert or -excluded, with no intratumoral immune infiltration. GSEA showed a positive enrichment of epithelial-mesenchymal transition (EMT) signatures, coupled with the downregulation of immune activation and IFN response pathways, in both CAF-enriched regions and tumor cells surrounded by alphaSMA + cells. Interestingly, genes encoding matrix metalloproteinase 11 (MMP11) and secreted phosphoprotein 1 (SPP1) were found overrepresented in CAF-enriched regions adjacent to tumor nests, as compared to distant CAF regions, suggesting CAF as major source of stromal MMP11 and SPP1. Finally, receptor-ligand interaction modeling performed using the CellChat tool showed that CAF-enriched regions exhibited significant interactions with adjacent tumor cells, throughout the secretion of factors involved in EMT-remodeling, tumor progression and immunosuppression (i.e., POSTN, HGF, WNT5A, TGFB1, INHBA, SPP1). Conclusions. Our findings suggest that MMP11 + SPP1 + CAF may promote desmoplastic barrier formation in eCCA, thereby hindering intratumoral recruitment of immune cells and limiting CIT efficacy. Ongoing analyses aim to further characterize CAF states and CAF-tumor crosstalk to support the development of strategies to enhance CIT responsiveness in eCCA.