TNF-α-Mediated Tumor Promotion Is Characterized by Enhanced Vasculogenesis and Generation of Myeloid/Endothelial Vascular Leukoctyes.

Whereas in many contexts myeloid cells are cytotoxic, it is well-established that as yet unknown microenvironment cues instruct the infiltrating tumor associated myeloid cells (TAMs) to drive malignant progression and dissemination. Recently, we and others have characterized a significant subpopulation of tumor associated myeloid cells that co-express endothelial and myeloid markers designated “vascular leukocytes”. Studies suggest that vascular leukocytes play an important role in tumor progression and also demonstrate modest contribution to functional vessels, i.e. vasculogenesis, suggesting that they represent a critical tumor-promoting TAM subpopulation. We have identified TNFα as a key regulator of the vascular transdifferentiation of myeloid progenitors in vitro and within the tumor milieu. TNFα at 40ng/ml significantly increased the numbers of flk-1/VE-cadherin dual positive, early outgrowth EPCs from human CD14+ cells by day 7 (about five fold of the control), starting with increased spindle-shaped population appeared as early as day 3. Consistent with this, we observed increased flk-1 expression by ∼9-fold (p<0.05) in cells treated with 40ng/ml TNFα by real time RT-PCR. Transcripts for VE-cadherin and tie2, both endothelial-enriched, were detected by day 3 in cells exposed to 40ng/ml TNFa but not in its absence (control). TNFα-directed upregulation of endothelial markers in mouse monocytes in vitro was dependent on TNFα receptors as monocytes isolated from mice lacking both TNF receptors displayed significantly delayed endothelial marker upregulation. These data suggested that TNF was a component of the molecular pathway that accelerated, but was not required for, endothelial transdifferentiation of murine and human myeloid cells. Enhanced TNFα expression in both B16 murine melanoma and PyV-mT tumor showed local TNFα significantly promoted tumor growth versus control (>5-fold increase for B16 tumor, p=0.04; >8-fold increase for PyV-mT tumor, p<0.01). Both tumor models indicated that overexpressing TNFα caused higher vascular density over control, while tumor necrosis was significantly reduced. Additionally, we observed increased bone marrow-derived vessels (vasculogenesis) in mouse TNFα-overexpressing tumors, which can be specifically inhibited by an anti-TNFα blocking antibody. A significant increase in association of vascular leukoctyes was detected in tumors overexpressing TNFα by FACs, which was abrogated in the mice lacking TNF receptors. Interestingly, TNF-overexpressing tumors did not recruit greater overall numbers of tumor-associated (myeloid or lymphoid) leukocytes, suggesting a specific role in myeloid to endothelial transdifferentiation in vivo. Our studies suggest that TNFα constitutes part of the microenvironment repertoire that biases recruited myeloid cells towards a proangiogenic/provasculogenic phenotype.