Lymphatic vessels (LVs) are important in maintaining homeostasis and regulating pathological conditions. Besides being a passive route for lymph and immune cell transport, many studies have shown that LVs can actively participate in multiple immune regulations such as cytokine expression, cell adhesion regulation, and antigen cross-presentation. In many types of cancer, lymphangiogenesis is usually associated with poor prognosis due to its contribution to tumor metastasis and immune suppression. However, in melanoma and colorectal cancer, LVs can also promote immune infiltration and facilitate immunotherapy.

Our RNA sequencing data showed that Ch25h, the gene for cholesterol-25-hydroxylase, was upregulated in tumor-associated (TA-) lymphatic endothelial cells (LECs) compared to LECs from lymph nodes (LNs) and steady-state skin. Ch25h is an enzyme that converts cholesterol into 25-hydroxycholesterol (25HC) and plays critical roles in regulating lipid metabolism, gene expression, and immune activation. Unlike the well-known contribution upon viral infection, the functions of Ch25h in the context of tumor development have not been thoroughly addressed. Studies showed both promoting and inhibitory effects on tumor cells in vitro, while in vivo, a limited number of papers reported an anti-tumor role of intracellular Ch25h when it is expressed in different immune cells. However, the function of Ch25h expression in TA-LECs is still unknown.

Here, we show that LECs represent the primary source of extracellular 25HC in tumors in lymphangiogenic murine melanoma. Genetic abolishment of the expression of Ch25h in LECs lowers extracellular 25HC levels in the tumor microenvironment (TME) and increases tumor growth. Using two immunotherapy approaches, CpG+OVA tumor vaccination, and tumor-specific OT-1 CD8⁺T cell adoptive transfer, we observed an enhanced effect of Ch25h expression by TA LECs in controlling tumor growth and promoting anti-tumor immune response. Mechanistically, LEC-derived 25HC inhibits PPAR-γ in TA-monocytes and TA-macrophages, suppressing their immunosuppressive functions and leading to their transition into proinflammatory phenotypes, which in turn promotes anti-tumor T cell responses. These findings demonstrate a novel function of LVs in supporting anti-tumor immunity, indicating promising therapeutic potential when combined with immunotherapy.