肿瘤异质性细胞亚群的多重荧光标记新技术与治疗耐受分子机制初探

The development of distant metastases and selection of resistance clones during anti-cancer therapy are the major causes of death for patients with a cancer. From an evolutionary perspective, a neoplasm can be viewed as a large, genetically and epigenetically heterogeneous population of individual cells. Genetic and epigenetic alterations that are beneficial to a neoplastic clone, enabling it to expand, are generally deleterious to the host that might lead to tumor invasion, metastasis and therapeutic resistance. Recent systemic cancer genomics studies, including large-scale cancer genome sequencing projects, have revealed significant genetic inter-tumor heterogeneity for cancers of the same histological subtype and remarkable intra-tumor heterogeneity between geographic regions (spatial heterogeneity) in the same tumor, as well as between the primary tumor and a subsequent local or distant recurrence in a same patient (temporal heterogeneity). Therefore, inter- and intra-tumor heterogeneity pose a great challenge to anti-cancer therapy, because a single biopsy or surgical excision is unlikely to accurately capture the complete genomic landscape of a patient's tumor and current methods of DNA sequencing cannot accurately portray the many mutations in a developing cancer that are present in only a minority of tumor cells. Although characterization of the inter- and intra-tumor heterogeneity is of paramount clinical importance for overcoming therapeutic resistance, it has been difficult to map heterogeneity within tumors and correlate it to specific biological features. The lack of powerful model system to study tumor heterogeneity thus becomes a great hurdle for our understanding and tackling the resistance phenotype. Recent technological advances in modeling brain neuron connectomes using a Cre-loxP based "Brainbow system" has facilitated our studies on modeling of heterogeneous tumor subpopulations and investigating their responsiveness to therapy. Our preliminary studies demonstrate that application of the "Brainbow system" enabled us to differentially color-coding clonal cell subpopulations. When these multi-color coded cell populations grown in vitro, clones of various color-coded subpopulations expanded and occupied a spatially distinct region with diverse growth patterns. When these same populations were inoculated into nude mice, they generated solid tumors exhibiting spatially distinct, color-coded neighborhoods of clonally derived cells, and different proclivity in metastasis. Thus, we proposed to apply this powerful color-coding model system to further address: 1) What are the molecular mechanisms underlying therapeutic resistance? 2) What are the genetic and epigenetic mechanisms for metastatic proclivity and organotropism? 3) Will sub-clones from an original single parental cell exhibiting different properties in metastasis and responsiveness to therapies? What are the molecular mechanisms that confer the phenotypic plasticity.

治疗耐受，包括药物与放射治疗耐受和肿瘤转移，是导致肿瘤病人死亡的主要原因：而肿瘤细胞异质性的存在，则是产生治疗耐受的根源。随着新一代大规模测序技术的发展与人类肿瘤基因组计划的实施，肿瘤异质性的研究已成为当前肿瘤研究新的热点。尽管在肿瘤细胞异质性与治疗耐受方面既往已做了大量的研究，但这一问题仍然没有得到实质性的突破。一个根本的原因是目前还没有建立一套合适的、能够标记并追踪不同异质性细胞亚群的实验体系。因此，本项目通过引入先进的"脑虹"多重荧光标记新技术，在不对肿瘤细胞进行放/化疗的预处理的前提下，依据"脑虹"标记细胞不同的荧光光谱，分选出不同的细胞亚群（克隆），研究各标记肿瘤细胞亚群中不同细胞亚群对治疗敏感性的内在性差异，并通过现代"组学"技术，阐明肿瘤细胞异质性与治疗耐受的分子机制。分离、鉴定"脑虹"标记的不同组织器官转移向性细胞亚群并阐明其分子机制。

为了明确肿瘤转移与治疗耐受的产生机制，我们分别利用定量蛋白质.组学(iTRAQ) 与全外显子测序(WES)技术等，对有相同遗传学背景、.不同转移潜能人肝癌来源细胞的异质性与进化关系进行了系统分析。为了.筛选和鉴定不同转移潜能三株肝癌细胞株的差异蛋白表达，我们用(iTRAQ).方法对这三株肝癌细胞系进行检测并定出 1170 种蛋白质，其中 47 个蛋白.质在高、低转移系显示有差异表达，另外有 65 个蛋白表达在肺转移细胞.系中特异性表达。生物信息学分析表明，转移相关的差异表达蛋白主要参.与酶催化和细胞间粘附及细胞代谢的过程。并应用 RT-PCR 和 Western .blot 分别在 RNA和蛋白水平上验证了部分差异表达的基因和蛋白。同时，.通过外显子组测序(WES)详细研究了这高低转移细胞系的基因组变化并分.析了它们之间的进化关系，构建了系统发育树，阐明了这些肿瘤细胞的演.化历程 。

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