PROJECT SUMMARY/ABSTRACT Blood vascular endothelial cells (BEC) control tumor growth through angiogenesis and differentiation of support vessels, and direct the host immune response to cancer by regulating immune cell recruitment from the blood. Recent studies emphasize the importance of specialized subsets of BEC in tumor biology, including high endothelial venules (HEV, vessels specialized for lymphocyte recruitment) which help direct effective tumor immunity. In spite of their central role in tumor biology, little is known about the BEC at the molecular level, mechanisms that regulate tumor angiogenesis are poorly understood, and the precursors that give rise to angiogenic tumor EC and to tumor-associated leukocyte-recruiting vessels remain to be determined. Under Aim 1 we will apply state-of-the-art single cell high dimensional mass label (CyTOF) flow cytometry and single cell RNAseq analyses to uncover the diversity of EC subsets in tumors and their environment, define the kinetics and subset-specificity of proliferative responses during tumor angiogenesis, and monitor the emergence and maturation of functional HEV and other post capillary venules (PCV) for immune cell traffic. Trajectory analyses will reveal developmental relationships of identified subsets including candidate progenitors, and immunofluorescence histology and confocal tissue imaging will define their location within the tumor and associated vasculature. Single cell BEC signatures will be mapped to the tumor vasculature using quantitative tissue immunohistology. Under Aim 2, innovative fate mapping approaches will elucidate precursor-product relationships among BEC subsets and will define clonal contributions of precursors to angiogenic tumor EC, to specialized EC of support vessels, and to high endothelium. Aim 3 will mine transcriptional profiles of induced EC subsets and apply pan-EC and EC subset-specific inducible gene targeting systems to define pathways and mechanisms that control progenitor cell and amplifying tumor EC activation, and that direct the differentiation of tumor-associated HEV. Novel bioinformatics tools will be applied to uncover transcriptional programs and pathways that induce recruiting vessels in settings of immunotherapy. Generation of a comprehensive atlas of blood endothelial cell subsets, molecular phenotypes and responses to tumorigenesis will open up new areas of investigation in cancer biology and immunology. Elucidation of the mechanisms of endothelial cell specialization and homeostasis, including mechanisms regulating endothelial cells that control lymphocyte traffic into tumors, may lead to novel targets and approaches to enhance cancer immunotherapies.