Tumor-derived Exosome Induction of Dendritic Cell Tolerization

肿瘤源性外泌体诱导树突状细胞耐受

• 批准号: 10524740
• 负责人: Michael Paul Plebanek
• 金额: $ 7.43万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524740
• 关键词: Affect; Antigen Presentation; Antigen-Presenting Cells; Automobile Driving; Biodistribution; Cell Line; Cell physiology; Cells; Cellular Metabolic Process; Chimeric Proteins; Communication; Complex; Cross Presentation; Cytotoxic T-Lymphocytes; Data; Dendritic Cells; Development; Distant; Engineering; Flow Cytometry; Generations; Glycolysis; Immune; Immune checkpoint inhibitor; Immune response; Immunity; Immunosuppression; Immunotherapeutic agent; Impairment; Implant; In Vitro; Label; Laboratories; Lymph Node Tissue; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Mediator; Melanoma Cell; Metabolic; Metabolic Pathway; Metabolism; Methods; MicroRNAs; Modeling; Natural Immunity; Neoplasm Metastasis; Nucleotides; Oncology; Oncolytic; Oncolytic viruses; Oxidative Phosphorylation; Oxygen Consumption; PTEN gene; Patient-Focused Outcomes; Patients; Phenotype; Play; Primary Neoplasm; Process; Proteins; RNA; Regulation; Regulatory T-Lymphocyte; Resolution; Rest; Role; Signal Pathway; Site; Small RNA; Testing; Therapeutic; Transgenic Mice; Transgenic Organisms; Tumor Immunity; Tumor-Derived; Viral Vector; Work; adaptive immunity; anti-tumor immune response; cancer cell; cell type; delivery vehicle; draining lymph node; exosome; fatty acid oxidation; immune clearance; immunoregulation; improved; in vivo; innovation; lymph nodes; melanoma; nanovesicle; neoplastic cell; novel; novel therapeutics; paracrine; programs; red fluorescent protein; small hairpin RNA; success; transcriptome sequencing; tumor; tumor eradication; tumor immunology; tumor microenvironment; tumor progression; uptake; vector

Project Summary/Abstract Dendritic cells (DCs) are professional antigen presenting cells that bridge innate and adaptive immunity via the cross-presentation of antigens. DCs play an indispensable role in cancer immunity where they activate cytotoxic T cells to clear tumor cells. Despite this, cancers have developed methods to inactivate DCs in order to avoid immune clearance in a process called DC tolerization. In order to communicate with other cell types, cancer cells utilize 30-150 nm nanovesicles called exosomes. Exosomes released from the tumor transit to distant sites, including lymph nodes, where they promote the formation of a tumor supportive microenvironment called the ‘pre-metastatic niche’ by delivering specific RNAs and proteins. Due to the ability of exosomes to promote metastasis over long distances and the importance of DCs to tumor progression, it is likely that exosomes play a role in DC tolerization. Our laboratory has demonstrated that a DC fatty acid oxidation (FAO) metabolic program is a major determining factor in inducing tolerization. FAO-induced DC tolerization is characterized by the promotion of immunosuppressive cell types like regulatory T cells (Tregs) and by a reduced ability of DCs to present antigen. Preliminary data shows that tumor-derived exosomes are significantly taken up by DCs, both locally and in distant lymph nodes, but the effects of these exosomes remain unclear. We hypothesize that tumor-derived exosomes drive DC metabolism towards a FAO state resulting in an immunotolerant phenotype and cancer progression. First, we will use melanoma cell lines isolated from BRAFV600E and PTEN-/- transgenic mice that are engineered to express a CD63-RFP fusion protein to fluorescently tag melanoma exosomes. In tumor implant models, the biodistribution of exosomes to DCs and the resulting phenotypic changes of DCs in the lymph nodes, and primary tumor will be assessed using flow cytometry and RNA-seq. Next, we will utilize cellular metabolism analysis and high-resolution mass spectrometry to determine the impact of tumor-derived exosomes on changes in the flux of metabolites through key metabolic pathways. We anticipate that melanoma exosomes cause increased FAO leading to subsequent DC tolerization. Finally, the therapeutic application of utilizing oncolytic herpes viral vectors (oHSV1) to both kill tumor cells and suppress FAO in dendritic cells will be investigated. oHSV1 will be used to drive melanoma specific expression of either miR-33 or shRNA targeted to CPT1a preceded by a nucleotide motif (x-motif) targeting these small RNAs to exosomes. Both these RNAs target CPT1a an important mediator of FAO. Therefore, oHSV1 can be utilized to target DCs with mir33/shCPT1a containing exosomes, reduce FAO and promote an anti-tumor immune response while simultaneously killing tumor cells. Ultimately, these proposed studies will generate an improved understanding of the tumor-derived factors that drive dendritic cells towards an immunotolerant state.

项目摘要/摘要 树突状细胞(DC)是一种专职的抗原提呈细胞，通过其自身的功能，将先天免疫和获得性免疫联系起来。 抗原的交叉呈递。树突状细胞在癌症免疫中发挥着不可或缺的作用 清除肿瘤细胞的细胞毒性T细胞。尽管如此，癌症已经发展出了灭活树突状细胞的方法 在称为DC耐受的过程中避免免疫清除。为了与其他类型的小区通信， 癌细胞利用被称为外体的30-150 nm纳米囊泡。从肿瘤转移释放的外体到 远处的部位，包括淋巴结，在那里它们促进肿瘤的形成 微环境通过提供特定的RNA和蛋白质而被称为“转移前的生态位”。由于有能力 外周小体促进远距离转移以及树突状细胞对肿瘤进展的重要性 可能外切体在DC耐受中起作用。我们的实验室已经证明了一种DC脂肪酸 氧化(FAO)代谢程序是诱导耐受性的主要决定因素。粮农组织诱导的DC 耐受性的特征是促进免疫抑制细胞类型，如调节性T细胞(Tregs) 树突状细胞呈递抗原的能力降低。初步数据显示，肿瘤来源的外切体是 树突状细胞在局部和远处的淋巴结中都有显著的摄取，但这些外切体的作用 目前仍不清楚。我们假设肿瘤来源的外切体驱动DC代谢进入FAO状态 导致免疫耐受表型和癌症进展。首先，我们将使用黑色素瘤细胞系 从表达CD63-RFP融合基因的BRAFV600E和PTEN-/-转基因小鼠中分离出来 荧光标记黑色素瘤外切体的蛋白质。在肿瘤移植模型中，外切体的生物分布 DCs和由此导致的淋巴结和原发肿瘤中的DC的表型变化将被评估 采用流式细胞术和RNA-seq。接下来，我们将利用细胞代谢分析和高分辨率质量 测定肿瘤来源的外切体对代谢产物流量变化的影响的光谱分析 通过关键的新陈代谢途径。我们预计黑色素瘤外切体导致粮农组织增加，从而导致 随后的DC容差。最后，利用溶瘤疱疹病毒载体的治疗应用 (OHSV1)同时杀灭肿瘤细胞和抑制树突状细胞中FAO的作用将被研究。OHSV1将用于 驱动针对CPT1a的miR-33或shRNA的黑色素瘤特异性表达 基序(x-基序)将这些小RNA定向到外切体。这两种RNA都以CPT1A为靶标，CPT1A是一种重要的调节因子 粮农组织的。因此，oHSV1可以被用来靶向带有Mir33/shCPT1a的DC，其中Mir33/shCPT1a包含外体，Reduced 粮农组织和促进抗肿瘤免疫反应，同时杀死肿瘤细胞。最终，这些 拟议中的研究将促进对驱动树突状细胞的肿瘤衍生因素的理解 细胞进入免疫耐受状态。