Deciphering the heterogeneity and cellular interactions of cancer-associated fibroblasts in extrahepatic cholangiocarcinoma by spatial transcriptomics.

4237 Background: Cholangiocarcinoma (CCA) is a rare and lethal malignancy characterized by a desmoplastic stroma. containing cancer-associated fibroblasts (CAFs). Although CAFs have been widely studied in intrahepatic CCA, their impact on TME remodeling and chemo-immunotherapy (CIT) efficacy in extrahepatic (e) CCA is not fully elucidated. A compressive characterization of the spatial orchestration of the eCCA TME and of the interaction between CAFs and adjacent tumor cells is needed for the identification of therapies able to improve CIT efficacy, that, so far, is limited in these patients. Methods: We employed spatial transcriptomics on tumor specimens from 8 patients with advanced distal (d) or perihilar (p) CCA treated with durvalumab + cisplatin/gemcitabine. 126 microregions were analyzed with the GeoMx Digital Spatial Profiler and classified according to pan-CK and ACTA2/αSMA straining, as follow: CK + αSMA - tumor, CK - αSMA + distant and peritumor CAF-enriched stroma, or CK - αSMA - peritumor stroma. Results: αSMA staining showed a high degree of stromal heterogeneity in all eCCA, with some tumor nests surrounded by a dense CAF-enriched stroma and others by αSMA - cells within the same sample. Based on CD45 staining all samples were classified as immune-desert or -excluded, as no immune cells were found intratumorally. Tumor-adjacent CAFs exhibited a distinct extracellular matrix (ECM) remodeling signature marked by the expression of COL11A1 , MMP11 and TGFB family members. GSEA showed a positive enrichment of epithelial-mesenchymal transition (EMT) transcripts linked to the downregulation of signatures of immune activation and IFN response, in both peritumoral CAF-enriched regions and adjacent tumor cells. Confirming this data, receptor-ligand interaction modeling performed using the CellChat tool confirmed that CAF-enriched regions exhibited strong interactions with adjacent tumor cells, throughout the secretion of several factors involved in ECM-remodeling and immunosuppression. Intriguingly, a higher expression of EMT-related genes linked to a higher downregulation of pathways of immune activation was observed in dCCA vs pCCA samples, with dCCA patients experiencing a shorter progression-free survival (PFS) from CIT compared to pCCA patients (PFS ranging from 7-12 and 10-24 months, respectively). Conclusions: Our findings suggest that COL11A1 + MMP11 + CAFs might limit CIT efficacy in eCCA by promoting eCCA EMT and, at the same time, by forming a collagen-dense physical barrier that hampers the intratumoral recruitment of immune cells, thus offering novel insights into potential therapeutic targets for interventions. A deeper characterization of CAF features and CAF-tumor interactions is ongoing.