Abstract 400: A new model for macrophage transdifferentiation into lymphatic endothelial cell progenitors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to tumor and inflammatory lymphangiogenesis, but the mechanisms regulating their recruitment, differentiation, and function in vivo are largely unknown. The main challenge to better understanding of M-LECP biology is their low (2-5%) frequency in vivo and lack of models allowing in-depth analyses in vitro. Our goal was two-fold: 1) to establish a cell-based model that recapitulates macrophage-LECP transdifferentiation; and 2) to characterize this model under controlled conditions. Methodology/Results: M-LECPs have been identified in an inflammatory peritonitis model in situ by dual expression of myeloid (CD11b and Ly6C) and lymphatic-specific (VEGFR-3, LYVE-1, and podoplanin) markers. The model was induced by i.p. injections of lipopolysaccharide (LPS) that mobilized CD11b+ macrophages and induced their incorporation into diaphragm lymphatic vessels. To reconstruct the emergence of this sub-population in vitro, a mouse macrophage cell line, RAW264.7, was treated by LPS following by analyses of expression profile and functional assays. RT-qPCR showed significant overlap (>67%) in the LPS-induced expression of lymphatic-specific genes in endogenous CD11b+/VEGFR-3+ cells from LPS-treated mice and RAW264.7 macrophages in vitro. LPS treatment of RAW264.7 cells coincidently increased VEGFR-3 and VEGF-C expression by respectively 20- and 40-folds. This activated an autocrine loop shown by a time-dependent tyrosine phosphorylation of VEGFR-3 that was abolished by a VEGF-C-blocking agent. To monitor the functional behavior in vivo, GFP-tagged RAW264.7 cells (RAW-GFP) were injected into either saline- or LPS-treated mice. Diaphragms were harvested seven days later and stained for GFP, myeloid and lymphatic markers. The results showed that RAW-GFP cells were massively recruited to the diaphragms in LPS- but not in saline-treated mice. The lymphangiogenic potential of GFP-labeled RAW264.7 macrophages was indicated not only by their widespread incorporation into pre-existing lymphatic vessels but also by their ability to form new LYVE-1+ structures both inside and outside of the diaphragm tissues. Conclusions/Significance: We found that LPS-treated RAW264.7 macrophages can serve a model of macrophage transdifferentiation into LECPs. Using this novel model, we established for the time, that this inflammation-induced process requires transient expression of VEGFR-3 followed by VEGF-C-mediated autocrine signaling and de novo expression of a broad panel of lymphatic-specific genes. In contrast to endogenous M-LECPs, RAW264.7 cells comprise homogeneous population, are available in unlimited amount, and amendable to genetic manipulations. We, therefore, believe that the “RAW model” can serve as a great vehicle for detailed molecular studies of LECP biology at inflammatory and tumor sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 400. doi:1538-7445.AM2012-400