2A), PDGF–D (Fig. 2D), and selleck screening library for PDGFRα (Fig. 2E), but only weakly positive (<30%) for PDGF-B (Fig. 2B), and negative for PDGF-C (Fig. 2C) and PDGFRβ (Fig. 2F). CAFs, identified as α-SMA-positive cells localized
in close vicinity to neoplastic ducts outside of vascular structures, were instead extensively positive for PDGFRβ (Fig. 2F), whereas their expression of PDGFRα was patchy (Fig. 2E). In extratumoral liver samples, bile ducts were consistently negative for PDGF ligands and receptors (not shown). This reciprocal expression of the members of the PDGF family between neoplastic bile ducts and CAFs suggests a role for PDGF-mediated cross-talk in CAF recruitment. In addition to CCA cells, IF studies showed that PDGF-D was also expressed by a fraction of CD45-positive inflammatory cells, scattered within the tumor reactive stroma, whereas it
was negative in ECs (Supporting Fig. 2A,B). This finding indicates learn more that inflammatory cells populating the stromal microenvironment behave as an additional paracrine source of PDGF-D (Table 1; Fig. 2 and Supporting Fig. 2). The immunophenotype of cultured CCA cells assessed by ICC reproduced the expression pattern of the neoplastic bile ducts observed in CCA histological sections (Supporting Table 2; Supporting Figs. 3 and 4). Expression of PDGFRs was confirmed by western blotting. ELISA was used to assess the secretory functions of the different PDGF isoforms. PDGF-B secretion was undetectable in both CCA cells and controls; secretion of PDGF-A was similar between CCA cells and controls, whereas PDGF-D was variably secreted only by CCA cells (from 65.88 to 420.52 pg/mL), and was undetectable in controls (Table 2). In CCA cells with high PDGF-D secretion (EGI-1, TFK-1, and CCA1), PDGF-D secretion was also measured in conditions of chemical hypoxia after treatment with DMOG. In all three CCA cell lines, DMOG induced
a further and significant increase in PDGF-D secretion (Supporting Fig. PDK4 5A), greater than 3 times, and was associated with a significant up-regulation of hypoxia-inducible factor (HIF)−1α of the same degree (Supporting Fig. 5B). Specimens from xenotransplanted CCA were analyzed by dual and triple IF to assess expression of PDGF ligands and receptors. PDGF-A (Supporting Fig. 6A) and -D (Supporting Fig. 6B) were expressed by EGI-1 cells, together with PDGFRα (Supporting Fig. 6C), but not PDGFRβ (Supporting Fig. 6D). Conversely, CAFs localized in close vicinity to EGFP-positive cells were diffusely and intensely decorated by the anti-PDGFRβ antibody (Supporting Fig. 6D), but unevenly by anti-PDGFRα. These findings confirmed that the reciprocal expression of ligands and receptors between cholangiocytes and CAFs observed in native CCA was maintained in our experimental model of CCA.