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

黑色素瘤脑转移(MBM)预后较差，中位生存期仅为4-6个月。 如果患者发展为软脑膜疾病，总体存活率甚至更低。多发性骨髓瘤的发病率一直是 据报道，在临床环境中高达43%，在尸检系列中高达75%。尽管流传着的观念 肿瘤细胞(CTCs)作为难治性转移的种子已建立，尚无知识表征 MBM--定植CTC。单细胞CTC转录图谱也表明，从 患者与广泛用于药物发现的细胞系非常不同。这一点更令人信服 考虑到CTC和相应的原发和转移瘤之间生物标记物的显著差异 已经观察到了肿瘤。此外，尽管脑脊液(CSF)中CTCs的存在仍然是 以细胞学为金标准，首次脑脊液分析时细胞学的敏感性仅为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。我们假设神经营养素受体p75NTR和乙酰肝素酶(HPSE)这两个标记物与 在MBM模型中，CTC是预测临床MBM和预防的潜在治疗靶点的新型CTC生物标志物 MBM。这项应用的目的是证明p75NTR/hPSE轴在临床上具有诊断价值。 P75NTR和HPSE是对抗MBM的新型治疗性CTC靶点。在目标1中，我们将：a)分离 并从血液和脑脊液中鉴定p75NTR/HPSE CTC亚群(多参数流式细胞术和 DEPArrayTM等技术)，并比较p75NTR/HPSE组合在CTCs中的表达 被诊断为有或没有MBM的黑色素瘤患者；b)直接链接患者分离的CTC亚群， P75NTR/HPSE的表达及其组合，对临床多发性骨髓瘤具有重要意义。在目标2中，我们将评估 P75NTR/HPSE CTC亚群在小分子p75NTR和HPSE调控MBM发育中的作用 新的HPSE抑制剂和CTC异种移植；并通过调节p75NTR/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的作用为 P75NTR/HPSE通路的重要整合者改变CTC增殖与生长停滞；b)描绘 HPSE诱导、Syndecan介导的Merlin/Hippo信号调节影响驱动MBM的CTC特性。 发现MBM CTC表型提供了修改治疗的机会，将研究直接扩展到 人类黑色素瘤。该项目由一个跨学科和集成良好的团队领导，将研究和验证新的 以及与CTC诱导的MBM有关的CTC生物标志物。它具有很高的治疗效果，是一种范例- 换档。我们处于独特的地位，可以进行这项研究，不仅是因为我们可以接触到广泛的 来自黑色素瘤患者的血液/脑脊液样本，以及询问整个CTC的广泛专业知识 与该国其他集团不同的是，该集团通过结合多种互补的CTC技术，实现了更高的频谱利用率。

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.