Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma

BACKGROUND: /Aims. In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAF) and lymphatic endothelial cells (LEC). Cholangiocarcinoma cells recruit and activate CAF by secreting PDGF-D. Here we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion (ELISA) was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants stimulated by PDGF-D, with/without the PDGFRβ inhibitor imatinib. Using human LEC incubated with conditioned medium from PDGF-D-stimulated fibroblasts (with/without imatinib), we assessed migration (Boyden chambers), 3-D vascular assembly (AngioTool), trans-endothelial electric resistance and trans-endothelial migration of cholangiocarcinoma cells (EGI-1). In Fischer-344 rats transplanted with a syngeneic cholangiocarcinoma cell line, we studied effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymphnode metastases. RESULTS: In cholangiocarcinoma specimens, CAF and LEC were closely adjacent. CAF expressed VEGF-A and VEGF-C, while LEC expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3-D assembly, increased LEC monolayer permeability, and promoted trans-endothelial EGI-1 migration. These effects were all suppressed by imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion markedly reduced lymphatic vascularization and lymphnode metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This process critical for the early metastatization of cholangiocarcinoma, may be blocked by inducing CAF apoptosis or by inhibiting PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma (CCA) is a highly malignant cancer affecting the biliary tree. In CCA, a rich stromal reaction densely populated by cancer-associated fibroblasts promotes early metastatic spread. Here we show that CCA-derived PDGF-D recruiting fibroblasts, also stimulates them to secrete VEGF-A and VEGF-C. These vascular growth factors kindle both lymphatic vascularization and tumour cell vascular invasion. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce CCA invasiveness.