An epigenetic switch in vascular phenotype augments anti-tumor immunity [scRNA-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: Tumor Endothelial Cell (TEC) Isolation and Sorting YUMMER1.7 tumors (~1 cm³) from three control mice and three Dnmt1iECKO mice were harvested, and tumors within each sample were pooled to obtain sufficient EC numbers. Samples were collected in ice-cold low-glucose DMEM (LG-DMEM), washed thoroughly, and minced into fragments <5 mm under sterile conditions. Tissue fragments were transferred to GentleMACS C tubes containing a digestion mix of collagenase II (2 mg/mL, Worthington LS004176), dispase (2.5 U/mL, Worthington LS02104), and DNase I (1 mg/mL, Worthington LS002006). Tumors were digested at 37 °C for 3045 min with agitation (120 rpm) and processed on a GentleMACS Dissociator using program m_imptumor_02 to achieve single-cell suspensions. Digested samples were passed through 100µm strainers, washed with FACS buffer (PBS + 0.5% BSA + 2 mM EDTA), and centrifuged at 1200 rpm for 5 min. Red blood cell lysis was performed using 1× Pharm Lyse B. Cell suspensions were first incubated with Live-or-Dye viability dye (Biotium 32002), followed by Fc block (Miltenyi 130-092-575) for 10 min on ice. Cells were then stained with PE-conjugated CD31 (BD 553373) and APC_x0002_conjugated CD45 (BD 559864) for 30 min on ice.Live CD31?CD45? tumor endothelial cells (TECs) were sorted on an Influx cell sorter (Becton Dickinson) into LG-DMEM supplemented with 10% FBS, washed, and resuspended in PBS + 0.04% BSA. Final cell suspensions were adjusted to 500700 cells/µL with >85% viability. Sorted TECs were immediately used for 10x Genomics Chromium Single Cell 3' v3 library preparation according to the manufacturer's protocol. Single-cell RNA-seq Library Preparation and Sequencing Sorted cells were processed using the 10x Genomics Chromium Single Cell 3' Gene Expression kit (GEM-X v4) following the manufacturer's protocol. Libraries were prepared targeting ~10,000 cells per sample and sequenced on an Illumina NextSeq 2000 using the P2 XLEAP-SBS 100-cycle kit (paired-end) at a depth of 20,00025,000 reads per cell.