Tumor-derived Exosome Induction of Dendritic Cell Tolerization

项目总结/摘要 树突状细胞（Dendritic cells，DC）是专职抗原呈递细胞，其通过免疫调节作用连接先天性免疫和适应性免疫。 抗原的交叉呈递。DC在癌症免疫中起着不可或缺的作用， 细胞毒性T细胞清除肿瘤细胞。尽管如此，癌症已经开发出了使DC活化的方法， 以避免在称为DC耐受化的过程中的免疫清除。为了与其他细胞类型交流， 癌细胞利用称为外泌体的30-150 nm纳米囊泡。从肿瘤释放的外泌体转运到 远端部位，包括淋巴结，在那里它们促进肿瘤支持性肿瘤的形成。 通过递送特定的RNA和蛋白质，转移前微环境被称为“转移前生态位”。由于能够 外泌体促进长距离转移以及DC对肿瘤进展的重要性， 外泌体可能在DC耐受化中起作用。我们的实验室已经证明了DC脂肪酸 氧化（FAO）代谢程序是诱导耐受的主要决定因素。粮农组织诱导直流电 耐受化的特征在于促进免疫抑制细胞类型，如调节性T细胞（T细胞）， 以及DC呈递抗原的能力降低。初步数据显示，肿瘤来源的外泌体是 在局部和远处淋巴结中，这些外泌体被DC显著吸收，但这些外泌体的作用 仍然不清楚。我们假设肿瘤来源的外泌体驱动DC代谢朝向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 分离自BRAFV 600 E和PTEN-/-转基因小鼠，所述转基因小鼠经工程改造以表达CD 63-RFP融合物 荧光标记黑色素瘤外泌体的蛋白质。在肿瘤植入模型中，外泌体的生物分布与肿瘤细胞的生长密切相关。 将评估淋巴结和原发性肿瘤中的DC和由此产生的DC表型变化 使用流式细胞术和RNA-seq.接下来，我们将利用细胞代谢分析和高分辨率质量 测定肿瘤来源的外泌体对代谢物通量变化的影响 通过关键的代谢途径。我们预计，黑色素瘤外来体导致FAO增加， 随后的DC耐受性。最后，利用溶瘤性疱疹病毒载体的治疗应用 将研究oHSV 1在树突状细胞中杀死肿瘤细胞和抑制FAO的作用。oHSV 1将用于 驱动靶向CPT 1a的miR-33或shRNA的黑色素瘤特异性表达， 基序（x-motif）将这些小RNA靶向外泌体。这两种RNA都靶向CPT 1a，CPT 1a是一种重要的介导物， 粮农组织。因此，oHSV 1可用于用含有mir 33/shCPT 1a的外泌体靶向DC，减少 FAO和促进抗肿瘤免疫反应，同时杀死肿瘤细胞。最终，这些 提出的研究将产生一个更好的理解肿瘤衍生的因素，驱动树突状细胞， 使细胞进入免疫耐受状态。

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.