Identification of Circulating Lymphendothelial Cells in the Blood of Patients with Solid Tumors.
Authors: Loges Sonja , Suhrbier Karin , Otten Jasmin , Riethdorf Sabine , Schuch Gunter , Pantel Klaus , Bokemeyer Carsten , Fiedler Walter 
Published: April 2020

Abstract
Angiogenesis and lymphangiogenesis promote tumor growth and metastasis. In addition to vessel sprouting circulating bone marrow derived endothelial progenitor (ECPs) and endothelial cells (CECs) contribute to neoangiogenesis in a process termed vasculogenesis. CECs can be detected in the blood of cancer patients and increased numbers are related to tumor progression. CECs and EPCs express endothelial cell surface markers as CD144 (VE-Cadherin) and VEGFR-2. Sprouting of new lymphatics from the preexisting lymphatic vessels has been described in solid tumors. Circulating lymphatic endothelial progenitor cells expressing CD133 and VEGFR3 were shown to differentiate into LYVE+ lymphatic endothelial cells (LECs) in vitro. Recently integration of circulating lymphatic endothelial progenitor cells in lymphatic neovessels was demonstrated in vivo. Therfore a distinct mechanism similar to vasculogenesis exists in lymphangiogenesis. Goal of our study was to identify and characterize circulating lymphatic endothelial cells in cancer patients. Presence of lymphatic endothelial cells in the peripheral blood was investigated in 26 patients with metastatic cancer and 10 healthy individuals. Additionally PBMNCs from G-CSF mobilised peripheral blood were analyzed (n=5). By developing a sensitive immunocytochemical approach we could identify a novel population of circulating lymphatic endothelial cells (CLECs) in patients with metastatic cancer. CLECs were LYVE+ and could be detected in 16 of 26 patients (62%) with metastatic cancer and in 4 of 10 healthy subjects (40%). The mean number of lymphendothelial cells was significantly higher in cancer patients (8.8 cells/1x106 PBMNCs [range 0–131] vs. 0.3 cells/1x106 PBMNCs [range 0–2]; p=0.03). We additionally investigated if CLECs were mobilised from bone marrow upon stimulation with G-CSF. 60% of analysed PBMNCs from leukapheresis products contained low numbers of circulating lymphatic endothelial cells comparable to normal peripheral blood (0.3 cells/1x106 PBMNCs [range 0–1]). Consequently CLECs unlike CEPs are not mobilised after cytokine stimulation from bone marrow. To better characterize CLECs, co-expression of VEGFR3 and LYVE was analyzed. We found that 90% of LYVE+ cells were positive for VEGFR3 which is consistent with a LEC phenotype previously described. Coexpression of vascular endothelial specific cell adhesion molecule CD144 (VE-Cadherin) was determined on LYVE+ cells by two-colour immunofluorescence. LYVE+ cells were negative for VE-Cadherin consistent with their lymphatic nature. We investigated whether LYVE+ cells represented a differentiated or a progenitor phenotype by double staining for LYVE and CD34 (n=3). All LYVE+ cells were negative for the progenitor marker CD34 indicating a differentiated lymphendothelial phenotype. Macrophages posess the capacity to transdifferentiate into lymphatic endothelial cells. We investigated whether circulating LYVE+ cells expressed CD11b and found a subfraction with a mean of 19% expressing LYVE and CD11b probably representing this macrophage subpopulation (range 13–25%, n=3). In summary our data show significantly higher levels of LYVE+ circulating lymphendothelial cells in patients with metastatic cancer compared to healthy subjects. Further studies are needed to clarify the origin of the circulating LECs and to analyse their potential as surrogate marker for lymphangiogenesis in cancer patients.