ABSTRACT Endothelial cells (ECs) form an essential part of the vasculature and are strategically located between blood and tissues, functioning as a fundamental barrier between the tissue and the immune system. As such, ECs can be viewed as an essential and active component regulating immune responses. We propose that EC's angiogenic vs. immune-modulatory function can be linked through the same genetic mechanism. We recently reported that Myct1 (MYC target 1), encoding a plasma membrane protein, is a novel regulator of angiogenesis. Myct1 expression is mainly restricted to ECs and tumor ECs. Global and EC-specific (Cdh5-Cre; Myct1f/f) Myct1 KO mice display decreased tumor angiogenesis and reduced tumor growth. Unexpectedly, defective tumor angiogenesis leads to an anti-tumor immune microenvironment. Particularly, tumors from Myct1 KO mice contain more CD8+ cytotoxic T lymphocytes (CTLs) than tumors from littermate control mice. While Myct1 deficient ECs display defects in EC motility, they support more robust CD8+ T cell trans-endothelial migration (TEM). Importantly, analysis of human cancers has also identified MYCT1 as a modulator of cancer patients' angiogenic and immune outcomes. Inhibition of Myct1 through knockout, siRNA treatment, or blocking monoclonal antibodies, in combination with anti-PD1 antibody, significantly improved complete tumor regression, suggesting that the better control of cancer depends on reduced angiogenesis and enhanced recruitment of CD8+ CTLs. We identified that Myct1 could control the outcome of angiogenesis vs. CTL recruitment to tumors through Rhoa vs. Rac1 Rho GTPase pathways. Moreover, we identified ZO1, also known as tight junction protein 1, to associate with MYCT1. Deeper mechanistic investigations on the Myct1-Rho GTPases and MYCT1-ZO1 interaction will help better understand how ECs control angiogenesis vs. immunity. As Myct1 function is conserved between mice and humans, findings from the proposed studies have high translational potential and can be applied to human anti-cancer therapies. The overall goal is to uncover novel molecular mechanisms and functions of MYCT1 in ECs and anti-tumor immunity. Particularly, we will test the hypothesis that Myct1 can modulate EC angiogenesis vs. immune regulatory outcome. Aim 1 determines how Myct1-Rho GTPase regulates tumor angiogenesis vs. tumor immunity. Aim 2 investigates the functional significance of MYCT1-ZO in EC permeability and vessel integrity. Aim 3 is to further assess the efficacy of anti-MYCT1 antibodies for targeting MYCT1 in tumor angiogenesis and growth. By completing the proposed studies, we will gain a deeper mechanistic understanding of how Myct1 regulates angiogenesis and the consequence of immune output played by ECs. The outcome will significantly impact the basic EC biology and therapeutic modality for cancer treatment.