P3.01-050 Isolation and Characterization of Lymphatic Endothelial Cells from Neoplastic and Normal Human Lung: Topic: Functional Biology in Lung Cancer

Abstract

Cell culture models may be crucial to study lung microvascular endothelium and its role in cancer progression and treatment. In addition, a high heterogeneity among endothelial cells from various districts has been demonstrated, with greater difference on the lymphatic circulatory system. Organ-specific endothelial cells are essential to elucidate signaling pathways involved in the pathogenetic mechanisms of neoplastic lung diseases and to provide novel approaches to reach the goal of a true personalized therapy. The aim of the present study was to isolate and characterize lymphatic endothelial cells from neoplastic and healthy human lung. A simple and inexpensive method, requiring minimal equipment and accessories was utilized to harvest, isolate and expand lymphatic endothelial cells from human lung (Lu-LECs). Specifically, samples from lung cancer (T) and spared distal lung (D) of 46 patients undergoing lobectomy or pneumonectomy for NSCLC, were processed. To obtain a pure population of Lu-LEC, a two-step purification tool based on sorting with monoclonal antibody to CD31 and podoplanin coated paramagnetic beads was employed. Immunohistochemical analysis on harvested pulmonary tissues was performed to assess the presence or absence of neoplastic cells and to identify blood and lymphatic vasculature. The purity of cultured endothelial cells was ascertained by morphologic criteria including TEM analysis, immunocytochemistry, flow cytometry and functional assays. T and D Lu-LECs were positive for CD31. Moreover, to define the lymphatic phenotype, we examined the specific markers podoplanin, LYVE-1 and Prox-1. No significant immunophenotypic differences between D and T Lu-LEC were detected by FACS. Although T Lu-LEC were larger than D Lu-LEC, morphologic features as cytoplasmic microvescicles, Weibel-Palade Bodies and aggregate of parallel intermediate filaments were equally observed. Cells were characterized in vitro for the ability to express several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation and in the development and progression of cancer. Cultured lymphatic endothelial cells variably expressed VEGFR-2, VEGFR-3, PDGFR-beta, c-met, and IGF-1R according to D or T origin. Moreover, FGFR-1 and EGFR-1 were present in a large fraction of T and D Lu-LEC. Matrigel assay documented that T Lu-LEC more efficiently organized in tubular structures at early time point when compared to D counterpart. Conversely, wound healing assay revealed that D Lu-LEC had a superior migratory ability. Primary lines of LECs from the human lung have been consistently obtained and may represent an important tool to study NSLCL microenvironment, lymphoangiogenesis and anti-cancer therapy.