Mechanisms of melanoma brain metastasis by CTCs isolated from patients' blood and CSF

黑色素瘤脑转移（MBM）预后不良，中位总生存期仅为4-6个月。 如果患者发生软脑膜疾病，总体生存率甚至更低。MBM的发病率一直是 据报道，在临床环境中高达43%，在尸检系列中高达75%。虽然流传的观念 肿瘤细胞（CTC）作为顽固性转移的“种子”已经建立，没有知识表征 MBM-定殖CTC。单细胞CTC转录谱分析也表明，从人结肠癌细胞中分离的CTC可以被用于治疗结肠癌。 患者与广泛用于药物发现的细胞系非常不同。这一点更加引人注目 考虑到CTC和相应的原发性和转移性CTC之间的生物标志物的显著差异， 已经观察到肿瘤。此外，尽管脑脊液（CSF）中CTC的存在仍然是一个潜在的问题。 作为金标准，在第一次CSF分析时，细胞学的灵敏度仅为50-56%。因此 开发有效的治疗方法-基于CTC的测试-可能会对治疗产生巨大的临床影响 MBM。我们假设神经营养因子受体p75 NTR和乙酰肝素酶（HPSE），两种与神经营养因子受体p75 NTR和乙酰肝素酶（HPSE）有关的标志物， 在MBM模型中，是预测临床MBM的新型CTC生物标志物和预防MBM的潜在治疗靶点。 MBM。本申请的目的是证明p75 NTR/HPSE轴在临床中具有诊断性。 MBM;以及p75 NTR和HPSE是对抗MBM的新型治疗性CTC靶标。在目标1中，我们将：a）分离 并表征来自血液和CSF的p75 NTR/HPSE CTC亚群（多参数流式细胞术和流式细胞仪）。 DEPArrayTM技术等），并比较CTC中p75 NTR/HPSE组合的表达 诊断为患有或不患有MBM的黑素瘤患者; B）直接关联患者分离的CTC亚群， 具有p75 NTR/HPSE表达和组合的细胞，用于临床MBM。在目标2中，我们将评估 利用小分子p75 NTR调节p75 NTR/HPSE CTC亚群对MBM发育的作用， 新的HPSE抑制剂沿着CTC异种移植物;并通过调节性p75 NTR/HPSE补充这些作用 基因表达（pINDUCER慢病毒工具包）。在目标3中，我们将：a）确定CTC表达的Merlin的作用， p75 NTR/HPSE途径改变CTC增殖与生长停滞的重要整合者; B）描述 HPSE诱导的、多配体介导的Merlin/Hippo信号传导调节，以影响驱动MBM的CTC性质。 揭示MBM CTC表型提供了通过直接扩展研究来修改治疗的机会， 人黑素瘤。该项目由一个跨学科和良好整合的团队领导，将研究和验证新的 和负责CTC诱导的MBM的特异性CTC生物标志物。它具有很高的治疗效果，是典范- 转移我们在进行这项研究方面处于独特的地位，不仅可以获得广泛的队列研究， 黑色素瘤患者的血液/CSF样本，以及询问整个CTC的广泛专业知识 与该国其他集团不同，该集团通过结合多种互补的CTC技术，开发了一个频谱。

Melanoma Brain Metastasis (MBM) carries a dismal prognosis with a median overall survival of only 4-6 months. If patients develop leptomeningeal disease, the overall survival is even lower. The incidence of MBM has been reported to be up to 43% in clinical settings and up to 75% in the autopsy series. Although notions that Circulating Tumor Cells (CTCs) act as “seeds” of intractable metastasis are established, there is no knowledge characterizing MBM-colonizing CTCs. Single-cell CTC transcriptional profiling has also demonstrated that CTCs isolated from patients are very distinct from cell lines that are widely used for drug discovery. This is even more compelling considering that significant discrepancies of biomarkers among CTCs and corresponding primary and metastatic tumors have been observed. Moreover, while the presence of CTCs in the cerebrospinal fluid (CSF) remains the gold standard, the sensitivity of cytology is only 50-56% at time of the first CSF analysis. Therefore, the development of effective therapy approaches - CTC-based tests - could have a tremendous clinical impact to treat MBM. We hypothesize that the neurotrophin receptor p75NTR and Heparanase (HPSE), two markers implicated in MBM models, are novel CTC biomarkers to predict clinical MBM and potential therapeutic targets to prevent MBM. The objective of this application is to demonstrate that the p75NTR/HPSE axis is diagnostic in clinical MBM; and that p75NTR and HPSE are novel therapeutic CTC targets to combat MBM. In aim 1, we will: a) isolate and characterize p75NTR/HPSE CTC subsets from blood and CSF (multiparametric flow cytometry and DEPArrayTM technologies among others), and compare the expression of p75NTR/HPSE combinations in CTCs of melanoma patients diagnosed either with or without MBM; b) directly link patient-isolated CTC subsets, possessing p75NTR/HPSE expression and combinations, to clinical MBM. In aim 2, we will assess effects of regulating functions of p75NTR/HPSE CTC subsets on MBM development by using small-molecule p75NTR and new HPSE inhibitors along with CTC xenografts; and complement these effects with regulatory p75NTR/HPSE gene expression (pINDUCER lentiviral toolkit). In aim 3, we will: a) determine roles of CTC-expressed Merlin as an important integrator of p75NTR/HPSE pathways altering CTC proliferation vs. growth arrest; b) delineate HPSE-induced, syndecan-mediated modulation of Merlin/Hippo signaling to affect CTC properties driving MBM. Uncovering MBM CTC phenotypes offers the opportunity to modify treatment by extending studies directly to human melanoma. This project lead by an inter-disciplinary and well-integrated team will study and validate new and specific CTC biomarkers responsible for CTC-induced MBM. It has high therapeutic impact and is paradigm- shifting. We are uniquely positioned to perform this study not only for having access to an extensive cohort of blood/CSF samples from melanoma patients but also for the extensive expertise interrogating the entire CTC spectrum by combining multiple and complementary CTC technologies unlike other groups in the country.