Abstract 3854: Lymphatic endothelial progenitors promote tumor lymphatic formation through cell-cell fusion

Abstract Introduction: Bone marrow (BM) derived myeloid-lymphatic endothelial cell progenitors (M-LECP) promote formation of tumor lymphatics and metastasis to lymph nodes. Regenerative capacity of BM progenitors to many other lineages is mediated through cell-cell fusion, a process that efficiently inserts a pro-mitotic message directly into damaged or division-restricted cells. We aimed to determine whether M-LECP use a similar mechanism to induce division of tumor lymphatic endothelial cells (LEC) which is an essential step towards the formation of new sprouts. Methods: Fusogenic markers were determined in M-LECP differentiated by Th2 and TLR4 ligands as well as in RAW264.7 macrophages by qPCR, flow cytometry, and immunohistochemistry (IHC) applied to mouse and clinical breast cancers. Fusion of myeloid and lymphatic cells stably expressing GFP and mCherry tags, respectively, was quantified using flow cytometry and microscopy by determining the percent of yellow cells resulting from merging of green- and red-colored cell partners. Fusion of lymphatic progenitors in vivo was determined in female chimera mice bearing MDA-MB-231 or EMT6 breast tumors that have been grafted with male BM prior to tumor implantation. Staining for LEC-specific markers in combination with probes for Y-chromosome quantified tumor lymphatic vessels fused with BM progenitors. Results: 10% to 25% of myeloid cells fused with co-cultured LEC quantified by two independent methods. M-LECP and RAW264.7 macrophages expressed a variety of fusogenic markers such as SIRP-a, CD36, DAP12, Stabilin-1, and DC-Stamp. These proteins were also expressed in M-LECP residing in human and mouse breast tumors. In vivo models showed up to 48% of tumor lymphatic vessels containing nuclear Y-chromosome indicating fusion. Supporting the hypothesis, Y-chromosome in LEC nuclei correlated with mitotic markers, nuclei doubling, lymphatic formation, and metastasis to nodes. Both in vitro and in vivo assays showed dependency of fusion on Th2 and TLR4 pathways. Conclusion: We show for the first-time using in vivo and in vitro assays that bone marrow derived myeloid-lymphatic progenitors induced by Th2 or TLR4 ligands are able to fuse with LEC. This event correlated with LEC proliferation and ultimately, metastatic behavior. These findings suggest a fundamentally novel mechanism of generating new lymphatic sprouts triggered by fusion with lymphatic endothelial cell precursors. Citation Format: Shaswati Athaiya, Lisa Volk-Draper, Emma Cox, Natalya Zinkevich, Sophia Ran. Lymphatic endothelial progenitors promote tumor lymphatic formation through cell-cell fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3854.