An epigenetic switch in vascular phenotype augments anti-tumor immunity [bulkRNA-seq]
The abnormal tumor vasculature can present a barrier to the infiltration of anti-tumor immune cells, which impairs immune surveillance and response to immunotherapy. Here, we show that targeting the epigenetic factor DNA  methyltransferase 1 in endothelial cells (ECs) results in reduced angiogenesis while imparting profound changes to the tumor immune microenvironment (TIME), including increased proportions of CD4+ memory T-cells and NK cells. Depleting CD4+ T-cells, or blocking lymphocyte egress from the lymph nodes with FTY720, rescues tumor growth in mice with conditional deletion of Dnmt1 in ECs (Dnmt1iECKO) and dramatically shortens overall survival, whereas NK cells are dispensable. Tumors implanted in Dnmt1iECKO mice show reduced vascular branching, elevated expression of Vcam1, increased vessel-associated T-cells, and a shift in vascular specification including increased proportions of immune-permissive post-capillary venules (PCVs) and interferon-stimulated ECs (IFN_x0002_ECs). Deleting Dnmt1 in EC cultures strikingly potentiates responses to combinations of IFNg and TNFa and, notably, up-regulates important T-cell co-stimulatory molecules for memory CD4+ T-cells, including Icosl, Cd40, and Tnfsf4. Finally, immune checkpoint blockade (ICB) administered to Dnmt1iECKO mice with experimental melanoma lung metastasis reduces tumor burden, with some mice showing tumor eradication. Our findings identify endothelial Dnmt1 as a key regulator of vascular-mediated anti-tumor immunity, providing a rationale for integrating epigenetic modulation of the vasculature with cancer immunotherapy regimens. Overall design: Bulk rnaSequencing: Mouse lung ECs were seeded at 1×105 cells/mL in 6-well plates and cultured overnight at 37 °C in a humidified 5% CO2 incubator. The next day, cells were transfected with either control siRNA or DNMT1-targeting siRNA (BLOCK-iT oligos; Invitrogen) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) in Opti-MEM I Reduced Serum Medium for 4 h, following the manufacturer's instructions. A BLOCK-iT Alexa Fluor Red fluorescent oligo (Invitrogen) was used in parallel wells to monitor transfection efficiency. After 4 hrs, the transfection medium was replaced with growth medium containing 20% serum, and the cells were cultured for 24 hrs at 37 °C, humidified 5% CO2. A second siRNA transfection was performed 24 h later to reinforce the knockdown. Cells were then treated with TNFa (10 ng/mL) or IFNg (1,000 U/mL) for 16 hrs. Total RNA was isolated using a column-based RNA extraction kit (Zymo Research) according to the manufacturer's instructions. Libraries were prepared and sequenced by Novogene for bulk RNA-seq analysis. Experiments were performed with n = 4 biological replicates per condition.