Structural and Functional Characterization of the Hemolymphangiogenic Microenvironment in Lung Cancer

Background: The hypothesis of the existence of a Lung Cancer Initiating Cell (LCIC) offers new pathogenetic and therapeutic options. CICs express vascular-related molecules in order to induce neoangiogenesis and establish an aberrant vascular niche. Conversely, tumor angiogenesis and formation of a cancer vascular niche contribute to the maintenance of CIC. The limited success of anti-angiogenic strategies in lung cancer imposes a better knowledge of the biology and architecture of the tumor vascular microenvironment. The aim of our study was to characterize the structural and functional changes of blood and lymphatic vasculature in human lung cancer. Methods: Fresh samples of the neoplastic(T) and spared distal (Dist) lung from 30 patients affected by NSCLC (21 Adeno and 9 Squamous) and 9 Neuroendocrine tumors were processed for immunohistochemical analysis and cell isolation. Control healthy lung (CTRL) was represented by 12 samples collected at autopsy from patients who died in the absence of respiratory diseases and 6 surgical specimens of pneumothorax. Immunofluorescence and confocal microscopy were employed using specific antibodies to detect blood (CD31, vWF, a-SMA) and lymphatic (Podoplanin Pdn, Lyve-1 and Prox-1) vessels. Moreover, the distribution of cells expressing stem/progenitor cell associated antigens (c-kit, CD133, CD34 and PDGFR) was assessed. Phenotypical and functional characterization on immunomagnetically sorted hematic (CD31pos) and lymphatic (CD31pos/Pdnpos) endothelial cells was performed on cells isolated from Dist and T lung samples. Results: Results indicated that, compared to CTRL capillary density increased by 77% and 74% in Dist and T portions of the lung, respectively. Conversely, the numerical incidence of venules did not show significant difference. These parameters were similarly represented in the three different tumor histotypes. A significant increase in arteriolar density was observed in all tumor types compared to CTRL. Moreover, the number of arterioles within T was increased by 3-fold compared to Dist portion. The quantitative analysis of lymphatic vessels showed similar values in all types of cancer specimens although rarefaction of these vascular structures was observed compared to CTRL. Moreover, lymphatic vessels density was 10-fold higher in Dist lung than in T. Immunofluorescence confocal analysis documented a positive gradient of c-kitpos, CD34pos and PDGFRpos progenitor cells from Dist towards T in all cancer samples. However, compared to CTRL, cells expressing c-kit and CD34 were more numerous both in the Dist and T portion of the lung, while the increase in PDGFRpos cells was present only in T. Blood (BEC) and lymphatic (LEC) endothelial cells isolated from Dist and T samples of the lung, showed different growth properties and variable expression of Tumor Endothelial Marker (TEM) and receptor tyrosine kinases as VEGFR 2 and 3, PDGFRbeta, EGFR, IGF-1R, and c-met. Functional assays indicated that T derived LEC possess higher tube forming ability on matrigel than Dist LEC while this phenomenon was not observed in BEC. Moreover, wound healing assay showed a VEGF-C independent reduced migratory capacity of LEC isolated from T samples compared to the Dist counterpart. Conclusion: Specific changes in the composition and function of the tumor hemolymphangiogenic environment occur in lung cancer providing innovative pathogenetic and therapeutic approaches.