The Adenosinergic Pathway in Tumor-derived Exosomes

项目总结/摘要 肿瘤细胞产生并释放大量的细胞外囊泡（EV）。一个小的子集（30-150 nm） 来源于肿瘤细胞内吞区室的EV被称为TEX（肿瘤来源的外泌体）。Tex 携带各种免疫调节分子，并且相对于正常细胞来源的外泌体，NTEX（非肿瘤 细胞来源的外泌体）富含抑制免疫效应细胞功能的抑制蛋白。 TEX最近成为肿瘤逃避宿主的主要免疫抑制系统 免疫系统我们的研究结果表明，腺苷（ADO），现在被认为是一个主要的免疫检查点， 癌症，并且驱动腺苷能途径的酶被携带并递送到靶免疫细胞， Tex.我们推测，TEX介导的癌症免疫细胞抑制是由于ADO分布在 TEX，从而促进肿瘤生长和转移。我们将首先通过比较 通过一种独特的方法从黑色素瘤患者血浆中分离的TEX与NTEX的腺苷能途径活性 免疫捕获方法。与新引入的高灵敏度乙烯桥相配合 化学和最先进的UPLC-MS/MS系统，我们现在有办法测量活性的 在每名患者的成对TEX与NTEX中的典型和非典型ADO产生生化途径。 我们将把“腺苷能驱动”定义为TEX产生免疫抑制性ADO的能力，并将其与 涉及对每个患者的免疫细胞进行的体外功能测定。Aim 1中的发现 预期TEX相对于NTEX在黑色素瘤患者中的腺苷能驱动将证实TEX在黑色素瘤中的主要作用。 肿瘤免疫逃逸AIM 1还将提供一种在体外测量每种黑色素瘤中腺苷能驱动的方法 用于目标2和3研究的患者。在目标2中，具有“高”与“低”腺苷能驱动的TEX将被 静脉注射到小鼠体内，以确定它们对致癌作用、癌症生长、血管生成、免疫调节、肿瘤生长、 功能障碍和转移形成。这一目标将TEX的作用从免疫抑制扩展到肿瘤 促进增长的活动。各种完善的小鼠模型将用于测量腺苷能 体内TEX活性。我们还将研究是否在体内阻断腺苷途径逆转 TEX介导的有害影响。在目标3中，我们将评估腺苷能驱动在TEX中的效用， 预后和对免疫检查点抑制剂的反应的生物标志物。利用从库存中分离的TEX 我们将TEX中的腺苷能驱动与疾病阶段相关联， 临床病理学终点以及预后和对免疫治疗的反应。通过阐明 TEX在ADO介导的免疫抑制以及癌症的肿瘤进展中的作用，这项研究介绍了新的 治疗靶点和新的癌症生物标志物有望改善癌症治疗和患者生存。

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 patient survival.