PROJECT SUMMARY/ABSTRACT Role of Th2 cytokines in progenitor-mediated formation of tumor lymphatics Lymph node metastasis, a common event in breast cancer, is a strong indicator of poor outcome due to spread of nodal metastatic cells to distant organs which leads to mortality of patients. Metastatic burden in lymph nodes directly correlates with the density of tumor lymphatic vessels. Outgrowth of these vessels is promoted by bone marrow (BM) derived progenitors that co-express specific markers of lymphatic endothelial cells and M2- type macrophages. This subset dubbed Myeloid-derived Lymphatic Endothelial Cells Progenitors (M-LECP) is present in mice and patients with metastatic breast tumors but not in cancer-free individuals. We previously showed in clinical breast cancers that density of lymphatic progenitors significantly correlates with tumor lymphatic formation and metastases in lymph nodes. These data underscore the clinical significance of M-LECP and the need to define their properties and the mechanisms that induce tumor lymphatics formation. We previously showed that M-LECP are M2-myeloid cells with lymphatic-specific markers whereas newly formed lymphatic vessels express macrophage-specific markers. This misalignment between the markers and their respective lymphatic/myeloid lineages is well-documented but unexplained. However, it can be explained considering the traits of other BM progenitors known to adopt the phenotype of lineages targeted for repair, and to employ fusion as the means to initiate the repair by inserting a trigger for cell proliferation. Fusion of BM progenitors with targeted cells results in transfer of genomic material encoding for M2 myeloid markers which explains their appearance in new structures. We reasoned that M-LECP, myeloid progenitors with partial lymphatic identity, might similarly use BM-enabled fusogenic properties to expand lymphatic vessels. Our preliminary data show that myeloid precursors treated with Th2 cytokines IL-4, IL-13 and IL-10 in vitro differentiate into cells with combined M2-myeloid, lymphatic endothelial, and fusogenic phenotypes. This led to the hypothesis that activation of Th2 pathways in M2-biased myeloid precursors co-regulates lymphatic lineage-specific and fusogenic properties that collectively promote the ability of mature progenitors to induce new lymphatic vessels. This hypothesis is supported by our data that demonstrate co-development of M2, lymphatic, and fusogenic properties in differentiated M-LECP as well as multiple evidence for fusion of these progenitors with lymphatic endothelial cells in vivo and in vitro. We will test this hypothesis through the following Specific Aims: (1) Determine in vivo the significance of Th2 pathways in M-LECP driven tumor lymphangiogenesis; and (2) Determine whether fusogenic properties induced by Th2 factors and its regulator TLR4 enable M-LECP to trigger tumor lymphatic formation. Significance: Th2 factors could play a paramount role i...