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

Massimiliano Cadamuro, Simone Brivio, Joachim Mertens, Marta Vismara, Anja Moncsek, Chiara Milani, Christian Fingas, Maria Cristina Malerba, Giorgia Nardo, Luigi Dall'Olmo, Eleonora Milani, Valeria Mariotti, Tommaso Stecca, Marco Massani, Carlo Spirli, Romina Fiorotto, Stefano Indraccolo, Mario Strazzabosco, Luca Fabris

Research output: Contribution to journalArticle

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 (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.

Original languageEnglish
JournalJournal of Hepatology
DOIs
Publication statusE-pub ahead of print - Dec 13 2018

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Lymphangiogenesis
Myofibroblasts
Cholangiocarcinoma
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor C
Endothelial Cells
Neoplasms
Vascular Endothelial Growth Factor A
Fibroblasts
factor D (liver)
Blood Vessels
Neoplasm Metastasis
Cancer-Associated Fibroblasts
Sclerosing Cholangitis
Lymphatic Vessels
Inbred F344 Rats
Biliary Tract
Conditioned Culture Medium
Electric Impedance

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Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma. / Cadamuro, Massimiliano; Brivio, Simone; Mertens, Joachim; Vismara, Marta; Moncsek, Anja; Milani, Chiara; Fingas, Christian; Cristina Malerba, Maria; Nardo, Giorgia; Dall'Olmo, Luigi; Milani, Eleonora; Mariotti, Valeria; Stecca, Tommaso; Massani, Marco; Spirli, Carlo; Fiorotto, Romina; Indraccolo, Stefano; Strazzabosco, Mario; Fabris, Luca.

In: Journal of Hepatology, 13.12.2018.

Research output: Contribution to journalArticle

Cadamuro, M, Brivio, S, Mertens, J, Vismara, M, Moncsek, A, Milani, C, Fingas, C, Cristina Malerba, M, Nardo, G, Dall'Olmo, L, Milani, E, Mariotti, V, Stecca, T, Massani, M, Spirli, C, Fiorotto, R, Indraccolo, S, Strazzabosco, M & Fabris, L 2018, 'Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma', Journal of Hepatology. https://doi.org/10.1016/j.jhep.2018.12.004
Cadamuro, Massimiliano ; Brivio, Simone ; Mertens, Joachim ; Vismara, Marta ; Moncsek, Anja ; Milani, Chiara ; Fingas, Christian ; Cristina Malerba, Maria ; Nardo, Giorgia ; Dall'Olmo, Luigi ; Milani, Eleonora ; Mariotti, Valeria ; Stecca, Tommaso ; Massani, Marco ; Spirli, Carlo ; Fiorotto, Romina ; Indraccolo, Stefano ; Strazzabosco, Mario ; Fabris, Luca. / Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma. In: Journal of Hepatology. 2018.
@article{d05d5b723ff24b5b8644a5f53a52aba9,
title = "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 (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.",
author = "Massimiliano Cadamuro and Simone Brivio and Joachim Mertens and Marta Vismara and Anja Moncsek and Chiara Milani and Christian Fingas and {Cristina Malerba}, Maria and Giorgia Nardo and Luigi Dall'Olmo and Eleonora Milani and Valeria Mariotti and Tommaso Stecca and Marco Massani and Carlo Spirli and Romina Fiorotto and Stefano Indraccolo and Mario Strazzabosco and Luca Fabris",
note = "Copyright {\circledC} 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.",
year = "2018",
month = "12",
day = "13",
doi = "10.1016/j.jhep.2018.12.004",
language = "English",
journal = "Journal of Hepatology",
issn = "0168-8278",
publisher = "Elsevier B.V.",

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TY - JOUR

T1 - Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma

AU - Cadamuro, Massimiliano

AU - Brivio, Simone

AU - Mertens, Joachim

AU - Vismara, Marta

AU - Moncsek, Anja

AU - Milani, Chiara

AU - Fingas, Christian

AU - Cristina Malerba, Maria

AU - Nardo, Giorgia

AU - Dall'Olmo, Luigi

AU - Milani, Eleonora

AU - Mariotti, Valeria

AU - Stecca, Tommaso

AU - Massani, Marco

AU - Spirli, Carlo

AU - Fiorotto, Romina

AU - Indraccolo, Stefano

AU - Strazzabosco, Mario

AU - Fabris, Luca

N1 - Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

PY - 2018/12/13

Y1 - 2018/12/13

N2 - 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.

AB - 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.

U2 - 10.1016/j.jhep.2018.12.004

DO - 10.1016/j.jhep.2018.12.004

M3 - Article

JO - Journal of Hepatology

JF - Journal of Hepatology

SN - 0168-8278

ER -