Role of microRNAs released by Tumor Associated Macrophages within Exosomes in the chemoresistance of Neuroblastoma

项目总结 儿科肿瘤是一种非常沉重的社会和经济负担，有深刻的情感牵连 不仅对直接受影响的儿童，而且对他们的家人和朋友。在儿科肿瘤中， 神经母细胞瘤(NB)特别相关，因为它是儿童中最常见的实体癌 颅骨，它仍然杀死了大约40%被诊断为最具侵袭性形式的患者。因此，有一个 显然需要改进对这种致命疾病的治疗。实现这一目标的战略之一是 克服癌细胞对化疗产生的耐药性。 已发表的数据显示，肿瘤相关巨噬细胞(TAMs)对NB细胞和 增加他们对化疗的抵抗力。协调这种支持和沟通的机制 人们对这些细胞类型之间的交通工具知之甚少。我的初步数据已经开始阐明这一点， 通过以下发现：1)NB细胞分泌含有microRNAs(MiRs)的外体；2)miRs是 被周围巨噬细胞摄取，并能与Toll样受体8(TLR8)结合，触发TLR8激活 巨噬细胞；3)TLR8刺激导致巨噬细胞分泌含有特异性MIR的外体；4) Nb细胞摄取衍生的外体miRs，使Nb细胞中的BRG1、RHOB和FBXW7沉默。这些 蛋白质是众所周知的MYC(又名c-MYC)和Nb中MYCN表达的抑制因子；5)MYC的水平 和MYCN在与人单核细胞共培养的NB细胞和原代NBS与 渗透率较高。NB是一种由MYC驱动的肿瘤。已知MYCN会增加多药耐药性 单位：NB。我们提供的证据表明，在体外和体内，MYC也促进了NB对化疗的耐药性。 我们还鉴定了TAMS在与NB细胞共培养时分泌的一组细胞因子，这可能 与人单核细胞共培养可上调NB细胞MYC和MYCN的表达。 因此，我们的假设是TAMs分泌可溶性因子(外体MIR和细胞因子)，诱导- 调节NB细胞MYC和MYCN，增加其对化疗的抵抗力。这项建议有三个方面 明确的目标。首先，我们将研究外体miRs诱导抗性的机制。 北卡罗来纳州治疗。然后，我们将评估靶向衍生的可溶性因子的治疗潜力 (外体miRs和细胞因子)以克服对NB的抗性。最后，我们将关联衍生的循环 血中外切体含量与原发肿瘤侵袭程度相关的传统临床 影像技术和临床结果。我召集了一支经验丰富的科学家团队 在癌症以及微创放射治疗和癌症治疗领域具有成熟的专业知识。这项研究将导致 寻找新的分子靶点，开发克服耐药的新策略 (可能还有其他类型的MYC驱动的癌症)，以及新的循环生物标记物的可能定义 确定适合抗治疗的NB患者亚群。

PROJECT SUMMARY Pediatric tumors represent a very heavy social and economic burden with profound emotional involvement not only for the directly affected children but also for their family and friends. Within pediatric tumors, neuroblastoma (NB) is of particular relevance, since it is the most common solid cancer in children outside of the skull and it still kills about 40% of patients diagnosed with the most aggressive forms. Therefore, there is a clear need to improve the treatment for this deadly disease. One of the strategies to achieve this goal is to overcome the emergence of resistance to chemotherapy by cancer cells. Published data show that Tumor-Associated Macrophages (TAMs) provide positive support to NB cells and increase their resistance to chemotherapy. The mechanisms that mediate this support and the communication vehicles between these cell types are poorly understood. My preliminary data have started to elucidate this, through the following findings: 1) NB cells secrete exosomes that contain microRNAs (miRs); 2) The miRs are taken up by surrounding macrophages and can bind to Toll-like receptor 8 (TLR8), triggering TLR8 activation in macrophages; 3) TLR8 stimulation causes macrophages to secrete exosomes that contain specific miRs; 4) the NB cells uptake TAM-derived exosomic miRs which silence BRG1, RHOB, and FBXW7 in NB cells. These proteins are well known inhibitors of MYC (aka as c-MYC) and MYCN expression in NB; 5) the levels of MYC and MYCN are significantly higher in NB cells co-cultured with human monocytes and in primary NBs with higher TAM infiltration. NB is a very MYC-driven tumor. It is known that MYCN increases multi-drug resistance in NB. We provide evidence that also MYC promotes NB resistance to chemotherapy both in vitro and in vivo. We also identified a panel of cytokines secreted by TAMs when co-cultured with NB cells, which could contribute to the up-regulation of MYC and MYCN in NB cells when co-cultured with human monocytes. Therefore, our hypothesis is that TAMs secrete soluble factors (exosomic miRs and cytokines) that induce up- regulation of MYC and MYCN in NB cells, increasing their resistance to chemotherapy. This proposal has three Specific Aims. In the first we will examine the mechanisms by which exosomic miRs induce resistance to therapy in NB. Then we will assess the therapeutic potential of targeting TAM-derived soluble factors (exosomic miRs and cytokines) to overcome NB resistance. Finally, we will correlate circulating TAM-derived exosomes in the blood of NB patients with the degree of TAM infiltration in the primary tumor, traditional clinical imaging techniques and clinical outcome. I have brought together a team of seasoned scientists with well- established expertise in NB and in the field of miRs and cancer therapeutics. This research will lead to the identification of new molecular targets, the development of a new strategy to overcome drug resistance in NB (and possibly other types of MYC-driven cancers), and the possible definition of new circulating biomarkers to identify subsets of NB patients suitable for an anti-TAM therapy.