Guido Carpino1, Vincenzo Cardinale2, Alessandra Di Giamberardino3, Diletta Overi4, Samantha Donsante3, Tania Colasanti5, Gaia Amato6, Gianluca Mennini7, Matteo Franchitto8, Fabrizio Conti5, Massimo Rossi7, Mara Riminucci3, Eugenio Gaudio4, Domenico Alvaro6, Carmine Mancone9. 1. Division of Health Sciences, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy. 2. Department of Medico-Surgical Sciences and Biotechnologies, Polo Pontino, Sapienza University of Rome, Rome, Italy. 3. Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. 4. Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy. 5. Rheumatology Unit, Department of Clinical Internal, Anesthetic and Cardiovascular Sciences, Sapienza University of Rome, Rome, Italy. 6. Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy. 7. General Surgery and Organ Transplantation Unit, Department of General Surgery and Surgical Specialties P. Stefanini, Sapienza University of Rome, Rome, Italy. 8. Department of Medical-Surgical Sciences and Translation Medicine, Sant'Andrea Hospital, Sapienza University of Rome, Rome, Italy. 9. Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy. Electronic address: carmine.mancone@uniroma1.it.
Abstract
BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.
BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.
Authors: Jan C Kamp; Lavinia Neubert; Maximilian Ackermann; Helge Stark; Christopher Werlein; Jan Fuge; Axel Haverich; Alexandar Tzankov; Konrad Steinestel; Johannes Friemann; Peter Boor; Klaus Junker; Marius M Hoeper; Tobias Welte; Florian Laenger; Mark P Kuehnel; Danny D Jonigk Journal: Int J Mol Sci Date: 2022-01-29 Impact factor: 5.923