Potential contribution of endothelial colony-forming cells (ECFCs) in physiological and pathological lymphangiogenesis

Background: ECFCs are endothelial progenitor cells endowed with the unique ability to sustain adult vasculogenesis and are therefore under intense investigation for use in regenerative medicine. Their capacity to sustain also lymphovasculogenesis/lymphoangiogenesis has not been yet investigated. Indeed, understanding whether ECFCs may act as lymphatic endothelial progenitor cells would be particularly relevant to the comprehension of the mechanisms underlying pathological lymphangiogenesis, as occurring in cancer microenvironment. Aims: To investigate whether ECFCs can present a lymphatic phenotype and may participate in cancer lymphangiogenesis. Methods: ECFCs were isolated from the blood of adult healthy donors and analyzed for cell morphology, immunophenotype, proliferation, in-vitro vasculogenesis, migration to chemotactic stimuli. Lymphatic phenotype was assesed based on the expression of the lymphatic markers PROX-1, Podoplanin (PDPN), LYVE-1 and VEGFR-3. Immunophenotypic and functional characterization of ECFCs were assessed in basal conditions and upon stimulation in pro-lymphangiogenic conditions, mimicking the tumor microenvironment. Results: In basal conditions, ECFCs expressed lymphatic markers at variable levels, with a particularly high variability in PDPN expression that allowed the identification of PDPNhi and PDPNdimECFCs. PDPNhiECFCs maintained high levels of PDPN throughout culture, whereas PDPNdimECFCs showed a progressive decrease of PDPN with increasing culture passages. PDPNdimECFCs treated with different lymphangiogenic stimuli did not acquire a PDPNhiphenotype. Unstimulated PDPNhi and PDPNdimECFCs showed similar proliferative and vasculogenic activity, and their functions under pro-lymphangiogenic stimulation are currently under investigation. Conclusions: ECFCs can act as lymphatic endothelial progenitor cells and may therefore represent a useful model for the in vitro and in vivo study of cancer lymphangiogenesis.