Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma

Abstract

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 (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, 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 marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo.

Results: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs 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 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRβ inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node 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 critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis.

Lay summary: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

Keywords: Cholangiocytes; Lymphatic endothelial cells; Tumor reactive stroma; VEGF-C; VEGFR3.

Fig.1:. In CCA, lymphatic microvascular density (LMVD) is much more preponderant than in HCC, at variance with blood microvascular density (BMVD).

(A-B) In human archival paraffin sections, LMVD was more extensively represented in CCA compared to HCC, as shown by IHC for podoplanin and Lyve-1 (lymphatic endothelial cell marker). (C) On the contrary, BMVD, evaluated as number of CD34⁺ (blood endothelial cell marker) cells, was increased in HCC samples. Right-side the plots, representative pictures of podoplanin⁺ (A), Lyve-1⁺ (B), and CD34⁺ (C) structures are shown for CCA and HCC; some faint expression of podoplanin is expressed also by CAF. n=6; *p<0.01, using two-tail t test. Original magnification: 200x.

Fig.2:. In human CCA specimens, lymphatic endothelial cells (LEC) closely align with CAF.

(A) Within the stroma of CCA, LEC were localized in close proximity to CAF, as shown by dual IHC for podoplanin (blue) and α-SMA (brown). (B, D) α-SMA⁺ CAF (green) expressed VEGF-A and VEGF-C (red), while (C, E) podoplanin⁺ LEC (green) reciprocally expressed VEGFR2 and VEGFR3 (red). (F) In malignant cholangiocytes (K19, green), expression of VEGF-A was weak and uneven (red), whilst (G) VEGF-C (red) was constantly not expressed. Original magnification: A-G, 200x; Insets: 400x.

Fig.3:. PDGF-D-stimulated secretion of VEGF-A and VEGF-C by human fibroblasts is dependent on ERK and JNK activation.

(A, B) While evaluating the intracellular signaling mediating VEGF-A and VEGF-C secretion by fibroblasts, we found that ERK and JNK inhibition abrogated the stimulatory effects of PDGF-D on both VEGF-A (A) and VEGF-C (B) secretory levels, similarly to the PDGFRβ inhibitor, imatinib mesylate (IM). n=5–7 experiments in duplicate; **p<0.01 vs Ctrl; ^p<0.05 vs PDGF-D; ^^p<0.01 vs PDGF-D, using two-tail t test.

Fig.4:. Upon PDGF-D stimulation, cultured human fibroblasts promote LEC recruitment, in vitro.

(A) Conditioned medium (CM) from fibroblasts exposed to PDGF-D ((PDGF-D) CM), potently stimulated LEC migration, an effect prevented by PDGFRβ antagonism with imatinib mesylate (IM) ((PDGF-D+IM) CM). This effect was reproduced by VEGF-A and VEGF-C, but not by the PDGF-D itself. (B) Representative pictures of Boyden Chamber inserts (M = 200x). n=4–11 experiments; **p<0.01 vs Ctrl; ^^p<0.01 vs (PDGF-D) CM, using two-tail t test.

Fig.5:. Upon PDGF-D stimulation, liver fibroblasts conversely from EGI-1 cells exert multiple lymphangiogenetic functions, inducing lumen formation, tubular branching and tubular lengthening of cultured LEC, in vitro.

(A) Representative micrographs of LEC tubulization and branching in a fibronectin/matrigel sandwich for the main treatment conditions. (B-D) CM from PDGF-D-treated fibroblasts ((PDGF-D) CM) induced 3-D cultured LEC to increase significantly the vessel area (B), the vessel length (C) and the number of junctions (D), with respect to controls. These effects were significantly attenuated by PDGFRβ antagonism in PDGF-D-treated fibroblasts ((PDGF-D + IM) CM), as well as by pre-treatment of LECs with inhibitors of VEGFR2 ((PDGF-D) CM + αVEGFR2) or VEGFR3 ((PDGF-D) CM + αVEGFR3). Similar effects to CM from PDGF-D-treated fibroblasts were obtained with VEGF-A and VEGF-C, but not with CM from EGI-1 cells, which induced only a slight increase in the vessel area (B). n=6–14 experiments; *p<0.05 vs Ctrl; **p<0.01 vs Ctrl; ^p<0.05 vs (PDGF-D) CM; ^^p<0.01 vs (PDGF-D) CM, using two-tail t test. Original magnification: 200x.

Fig.6:. Upon PDGF-D stimulation, liver fibroblasts reduce trans-endothelial resistance of LEC monolayers (TEER) and stimulate trans-endothelial migration (TrEM) of CCA cells (EGI-1-EGFP).

(A) CM from PDGF-D-treated fibroblasts ((PDGF-D) CM) dramatically impaired the integrity of the lymphatic endothelial barrier, more effectively than VEGF-C and VEGF-A. This effect was only partially counteracted by the concomitant treatment with αVEGFR2 ((PDGF-D) CM + αVEGFR2), but completely abrogated by the supplementation with αVEGFR3 ((PDGF-D) CM + αVEGFR3) or with bevacizumab ((PDGF-D) CM + Bev) or anti-VEGF-C ((PDGF-D) CM + αVEGF-C), as well as by PDGFRβ antagonism in PDGF-D-treated fibroblasts ((PDGF-D + IM) CM). (B) In TrEM experiments, CM from PDGF-D-treated fibroblasts ((PDGF-D) CM) enabled the CCA cell line EGI-1-EGFP to cross the LEC monolayer (similar to VEGF-A and VEGF-C), an effect blunted by the treatment of PDGF-D-stimulated fibroblasts with PDGFRβ antagonist ((PDGF-D + IM) CM), or with bevacizumab ((PDGF-D) CM + Bev) or anti-VEGF-C ((PDGF-D) CM + αVEGF-C), or by the treatment of LEC with αVEGFR3 ((PDGF-D) CM + αVEGFR3), but not with αVEGFR2 ((PDGF-D) CM + αVEGFR2). For TEER, n=4–15 experiments in duplicate. For TrEM, n=3–4; *p<0.05 vs Ctrl, **p<0.01 vs Ctrl, ^p<0.05 vs (PDGF-D) CM, ^^p<0.01 vs (PDGF-D) CM, using two-tail t test.

Fig.7:. In a syngeneic rat model of CCA, targeting CAF by navitoclax associates to a decreased lymphatic vascularization and lymph node metastasisation.

(A) In Fischer 344 male rats transplanted with BDE-neu rat CCA cells, selective depletion of CAF by navitoclax was accompanied by a significant decrease in Lyve-1⁺ LEC without affecting CD31⁺ blood endothelial cells compared to untreated rats. Up-sided, representative images of CCA sections, with dual immunofluorescence for CD31 (red) and Lyve-1 (green), show the stark differences in lymphatic and blood vessels between navitoclax and vehicle groups. (B) Concomitantly, navitoclax led to a reduction in the number of lymph node metastases that was significant at the peritoneal region (p<0.05), and close to significance at the paraortic region (p=0.068). (n=6 for each group). Original magnification: 100x. **p<0.01 vs Vehicle, using two-tail t test.