FREQUENCY, ANGIOGENIC POTENTIAL, PHENOTYPE AND MOLECULAR SIGNATURE OF ENDOTHELIAL COLONY-FORMING CELLS ISOLATED FROM PATIENTS WITH CLASSIC KAPOSI¿S SARCOMA

Abstract Background Kaposi’s Sarcoma (KS) is a lymphangioproliferative disease whose causative agent is herpesvirus HHV-8. KS is characterized by hyperproliferation of HHV-8 infected spindle cells - cells of endothelial origin that represent the typical component of KS lesions. In previous studies we reported that circulating endothelial progenitor cells (EPCs), identified as CD45dim/CD34+/KDR+, are increased in patients with classic KS (cKS) and we also demonstrated that in cKS patients EPCs - isolated and cultured as Endothelial colony-forming cells (ECFCs) - are HHV-8 infected and can act as viral reservoir. Therefore, in this study we investigated whether ECFCs isolated from cKS patients are endowed with features typical of spindle cells in order to evaluate whether they may represent the precursors of spindle cells. Moreover, in preliminary studies we surprisingly observed that not only ECFCs isolated from cKS patients but also ECFCs isolated from healthy donors expressed LYVE-1 and podoplanin – lymphatic markers expressed by spindle cells in KS lesions. Since several studies supported the hypothesis that adult lymphangiogenesis could be promoted by the presence of bone marrow derived lymphatic endothelial progenitor cells (LEPCs) we also investigated the lymphangiogenic potential of ECFCs to evaluate whether they can act also as LEPCs. In particular, we evaluated the expression of lymphatic markers in basal condition and we investigated whether the lymphatic differentiation of ECFCs could be fostered by fibronectin a component of the tumor microenvironment whose presence correlates with tumor lymphangiogenesis and metastasis. In addition, we also evaluated whether migration stimulating factor (MSF), an oncofetal isoform of fibronectin released by cancer stromal cells and tumor associated macrophages, could promote lymphatic differentiation of ECFCs. Methods 83 cKS patients and 86 healthy HHV-8 seronegative donors were enrolled in the study. ECFCs were isolated using a protocol previously optimized in our lab. PBMCs were seeded in EGM-2 medium in culture plates coated with fibronectin and ECFC colonies were identified by microscopic visual inspection as colonies of cells with cobblestone-like morphology. Once isolated, ECFC colonies were expanded in culture plates coated with collagen. During the isolation phase, the time of appearance and the frequency of ECFC colonies were analyzed. During the following expansion phase, ECFC phenotype and the presence of HHV8-infection - assessed as expression of the viral latent nuclear antigen (LANA) - were analyzed by immunofluorescence. ECFC were functionally characterized by evaluating their cell viability, proliferative potential, vasculogenesis ability by Matrigel assay and cytokine production by ELISA. The molecular signature of ECFCs was also analyzed by gene array analysis. To investigate the lymphatic differentiative potential of ECFCs the expression of typical lymphatic markers (PROX-1, podoplanin, LYVE-1 and VEGFR-3) was analyzed by Real Time PCR and confocal microscopy. To evaluate the possible role of fibronectin in promoting lymphatic differentiation, ECFCs were cultured on either fibronectin or collagen and the effects of stimulation with MSF were also analyzed. Results In our study ECFC colonies appeared earlier (p<0.001) and with higher frequency (p<0.001) when isolated from cKS patients than healthy donors. Moreover, the frequency of ECFC colonies was higher in cKS patients with rapidly evolving disease than in cKS patients with slowly evolving disease (p<0.05). All screened ECFC colonies isolated from cKS patients contained HHV8-infected cells. During the expansion phase, ECFCs isolated from cKS patients were endowed with a higher proliferative potential (p<0.05), a higher vasculogenic ability in vitro (p<0.05) and a higher production of IL-6 (p<0.05) than ECFCs isolated from healthy donors. In addition, preliminary analysis of the gene expression profile revealed that patients and healthy controls segregated by clustering, thus confirming that gene expression profile differs between ECFCs isolated from cKS patients and healthy donors. A further relevant result of this study was the observation that ECFCs are endowed with the ability to express the typical lymphatic markers PROX-1, podoplanin, LYVE-1 and VEGFR-3. In particular, lymphatic markers were expressed by donor-derived ECFCs cultured on both fibronectin and collagen and they were upregulated by ECFC treatment with MSF (p<0.05). Conclusion In this study we demonstrated that ECFCs obtained from cKS patients are endowed with features that may be particularly relevant to KS pathogenesis, suggesting that ECFCs may act as putative precursors of the spindle cells and contribute to the development of KS lesions. Moreover, we demonstrated that ECFCs isolated from peripheral blood of adult healthy donors expressed markers typical of lymphatic endothelium, suggesting that ECFCs may act also as LEPCs thus participating in lymphangiogenic and lymphovasculogenic processes.