The role of extracellular vesicles in regulating response to immunotherapy

摘要 据估计，美国有12,390例胶质母细胞瘤(GBM)患者的中位存活率 尽管在手术、化疗和放射治疗方面取得了进展，但只有14.6个月。微环境 这些肿瘤中常有免疫抑制细胞高度渗透，包括肿瘤相关的巨噬细胞。 (TAMS)。检查点阻断疗法依赖于具有细胞毒性T细胞的肿瘤的渗透才能有效，并且 因此，一些GBM患者可能对这些药物反应不佳。因此，GBM一直是一个有吸引力的目标 嵌合抗原受体(CAR)T细胞疗法克服免疫逃避在 通过产生对GBM肿瘤新抗原表皮生长因子受体反应的T细胞来治疗GBM患者 (EGFR)变体III(EGVRvIII)。尽管在反应方面取得了一些进展，但微环境可以发挥重要的作用 在调节肿瘤对免疫调节治疗的反应中的作用。无反应的GBM患者 去关卡封锁治疗往往有较高水平的渗透，这可能会限制CAR的疗效 T疗法。先前的工作表明，肿瘤来源的细胞外小泡(EVS)可以调节 微环境向着有利于肿瘤、免疫抑制的环境发展。具体地说，它们可以由 巨噬细胞将它们推向亲肿瘤、免疫抑制的表型。许多研究都有 概述了外切体直接结合T细胞和阻断抗肿瘤细胞毒活性的能力。一种基于血液的 “液体活检”是确定哪些患者对免疫治疗有反应而不需要侵入性治疗的理想方法。 活组织检查。因此，我假设肿瘤来源的电动汽车将货物运送到免疫系统进行调节。 对免疫治疗的治疗反应和这批货物可以作为预后的生物标志物。 在第一个目标中，我将确定肿瘤EV分泌对免疫渗透和免疫治疗的影响 通过使用Rab27a KO减少EV分泌的疗效。这些模型将在人源化的小鼠身上进行测试， 以及同基因模型测试EV分泌对CAR T细胞疗效和关卡阻断的影响 治疗(分别)。在第二个目标中，我将使用新型微流体来捕获肿瘤EV，测量mRNAs， 测试这些差异表达的mRNAs中是否有任何一个是预测或预测的生物标记物。我会再加一次 使用多光谱成像将这些标记物与同一患者中的免疫渗透进行比较。 我的建议集中在一个主要问题上：EVS如何调节免疫系统和肿瘤反应 去接受免疫治疗。对EV mRNA货物的洞察，将既识别对免疫治疗有反应的患者 以及帮助确定克服免疫抑制环境的替代治疗方法。

Summary There are an estimated 12,390 cases of glioblastoma (GBM) in the United States with a median survival rate of only 14.6 months despite advances in surgery, chemotherapy, and radiation therapy. The microenvironment of these tumors is often highly infiltrated with immunosuppressive cells, including tumor-associated macrophages (TAMs). Checkpoint blockade therapies rely on the infiltration of tumors with cytotoxic T cells to be effective, and therefore some GBM patients may not respond well to them. Therefore, GBM has been an attractive target for treatment with chimeric antigen receptor (CAR) T cell therapy to overcome immune evasion often observed in GBM patients by producing T cells that respond to the GBM tumor neo-antigen epidermal growth factor receptor (EGFR) variant III (EGVRvIII). Despite some advances in response, the microenvironment can play an important role in modulating the response of tumors to immune modulatory therapies. GBM patients that do not respond to checkpoint blockade therapy often have high levels of TAM infiltration, which might limit the efficacy of CAR T therapy. Previous work has shown that tumor derived extracellular vesicles (EVs) can modulate the microenvironment toward a pro-tumor, immune inhibitory environment. Specifically, they can be taken up by macrophages to drive them toward a pro-tumor, immune-suppressive phenotype. Numerous studies have outlined the ability of exosomes to directly bind T cells and block cytotoxic activity against tumors. A blood-based ‘liquid biopsy’ is ideal to determine which patients will respond to immunotherapy without the need for invasive biopsies. I therefore hypothesize that tumor derived EVs transmit cargo to the immune system regulating therapeutic response to immunotherapy and this cargo can serve as prognostic biomarkers. In the first aim, I will determine the impact of tumor EV secretion on immune infiltrate and immunotherapy efficacy by reducing EV secretion using Rab27a KO. These models will be tested in both humanized mice as well as syngeneic models to test the impact of EV secretion of CAR T cell efficacy and checkpoint blockade therapy (respectively). In the second aim I will use novel microfluidics to capture tumor EVs, measure mRNAs, test if any of these differentially expressed mRNAs are prognostic or predictive biomarkers. I will additionally compare these markers to immune infiltrate in the same patients using multispectral imaging. My proposal focuses on one major question: How EVs regulate the immune system and the tumor response to immunotherapy. Insights into EV mRNA cargo, will both identify patients that will respond to immunotherapy as well as help identify alternative treatments to overcome an immunosuppressive environment.