PROJECT SUMMARY/ABSTRACT Tumor cells produce and release large numbers of extracellular vesicles (EVs). A subset of small (30-150 nm) EVs derived from the endocytic compartment of the tumor cell is called TEX (Tumor-derived Exosomes). TEX carry various immunoregulatory molecules and relative to normal cell-derived exosomes, NTEX (Non-Tumor cell-derived Exosomes), are enriched in inhibitory proteins which suppress functions of immune effector cells. TEX have recently emerged as a major immunosuppressive system used by tumors to escape from the host immune system. Our results indicate that adenosine (ADO), now considered a major immune checkpoint in cancer, and the enzymes driving the adenosinergic pathway are carried and delivered to target immune cells by TEX. We hypothesize that TEX-mediated suppression of immune cells in cancer is due to ADO distributed by TEX, which thereby encourage tumor growth and metastasis. We will test this hypothesis first by comparing the adenosinergic pathway activity in TEX versus NTEX isolated from plasma of patients with melanoma by a unique immune capture method we have developed. In concert with newly introduced highly sensitive etheno-bridge chemistry and a state-of-the-art UPLC-MS/MS system, we now have means to measure the activity of the canonical and non-canonical ADO-producing biochemical pathways in paired TEX versus NTEX of each patient. We will define “adenosinergic drive” as the capacity of TEX to produce immunosuppressive ADO and will link it to in vitro functional assays performed with each patient’s immune cells. The discovery in Aim 1 of high adenosinergic drive in melanoma patients TEX relative to NTEX is expected to confirm the major role of TEX in tumor immune escape. Aim 1 will also provide an in vitro measure of adenosinergic drive in every melanoma patient for use in Aims 2 and 3 studies. In Aim 2, TEX with “high” versus “low” adenosinergic drive will be injected IV into mice to determine their in vivo effects on carcinogenesis, cancer growth, angiogenesis, immune dysfunction and metastasis formation. This Aim extends the role of TEX from immune suppression to tumor growth promoting activities. Various well-established mouse models will be used to measure adenosinergic activity of TEX in vivo. We will also examine whether in vivo blocking of the adenosine pathway reverses detrimental effects mediated by TEX. In Aim 3, we will assess the utility of adenosinergic drive in TEX as a biomarker of prognosis and response to immune checkpoint inhibitors. Utilizing TEX isolated from banked plasma of melanoma patients, we will correlate adenosinergic drive in the TEX with disease stage and clinicopathological endpoints as well as with prognosis and response to immunotherapy. By elucidating the role of TEX in ADO-mediated immune suppression as well as tumor progression in cancer, this study introduces new therapeutic targets and new cancer biomarkers that are expected to improve cancer therapy and patien...