Analysis of Intratumoral Crosstalk in Clonal Populations of OvarianTumor Cells

描述（由申请人提供）：控制癌症最重大的挑战之一是个体肿瘤的肿瘤细胞内的高度异质性。虽然肿瘤细胞的异质性已在许多层面上进行了描述，并正在形成的异质性的过程的理解，在何种程度上的异质性肿瘤细胞亚群在其功能活动的变化，以及是否亚群之间的相互作用影响肿瘤的发生和发展是知之甚少。研究这些问题的主要障碍是缺乏方法来维持人类肿瘤在培养中的异质性，并从个体肿瘤中繁殖不同的克隆亚群。与我们的合作者一起，我们已经开发出克服这些障碍的方法，并使分离，培养和表征卵巢肿瘤细胞的克隆群体成为可能，然后在由移植克隆的混合物产生的肿瘤异种移植物内追踪条形码化，表位标记的克隆群体。在一项初步研究中使用这些方法，我们获得了克隆间合作的证据， 影响肿瘤进展-最有趣的是，我们发现不能自行启动肿瘤形成的癌细胞克隆群，当与“肿瘤启动”克隆混合时，可以强烈促进原发性肿瘤的扩张和特定器官部位的转移。这支持了肿瘤内的克隆亚群彼此合作以促进肿瘤扩张和转移的假设。在该提案中，我们描述了使用系统方法在人类卵巢肿瘤中测试该假设的计划，通过（1）分离和表征来自卵巢肿瘤的大量克隆群体之间的遗传和表型变异的程度，（2）比较单个克隆群体与此类群体的混合物的活性，以研究肿瘤内串扰的存在和性质，（3）随时间追踪克隆群体的肿瘤内定位，以及（4）阐明负责由肿瘤内串扰产生的表型的机制。

DESCRIPTION (provided by applicant): One of the most significant challenges in controlling cancer is the high degree of heterogeneity within the neoplastic cells of individual tumors. While tumor cell heterogeneity has been described at many levels and an understanding of the processes that create heterogeneity is emerging, the extent to which the heterogeneous subpopulations of tumor cells vary in their functional activities and whether interactions between subpopulation influence tumor initiation and progression is poorly understood. Major barriers to investigating these questions have been the lack of methodologies to maintain the heterogeneity of human tumors in culture and to propagate distinct clonal subpopulations from individual tumors. Together with our collaborators, we have developed methodologies that overcome these barriers and make it feasible to isolate, culture, and characterize clonal populations of ovarian tumor cells and then track barcoded, epitope-tagged clonal populations within tumor xenografts generated from mixtures of transplanted clones. Using these approaches in a pilot study, we obtained evidence for interclonal collaborations that significantly affect tumor progression - most interestingly, we found that clonal populations of cancer cells which are unable to initiate tumor formation on their own, can strongly promote the expansion of primary tumors and metastasis at specific organ sites when mixed with 'tumor-initiating' clones. This supports the hypothesis that clonal subpopulations within a tumor cooperate with one another to promote tumor expansion and metastasis. In this proposal, we describe plans to use a systematic approach to test this hypothesis in human ovarian tumors by (1) isolating and characterizing the extent of genetic and phenotypic variation among a large set of clonal populations derived from ovarian tumors, (2) comparing the activities of single clonal populations to mixtures of such populations in order to investigate the existence and nature of intratumoral cross talk, (3) tracking intratumoral localization of clonal populations over time, an (4) elucidating the mechanisms responsible for phenotypes generated by intratumoral crosstalk.