Analysis of Intratumoral Crosstalk in Clonal Populations of OvarianTumor Cells

描述(由申请人提供)：控制癌症最重要的挑战之一是单个肿瘤肿瘤细胞的高度异质性。虽然肿瘤细胞的异质性已经在许多水平上得到了描述，对产生异质性的过程的了解也在不断涌现，但肿瘤细胞的不同亚群在其功能活动中的差异程度以及亚群之间的相互作用是否影响肿瘤的发生和发展还知之甚少。研究这些问题的主要障碍是缺乏方法学来维持人类肿瘤在培养中的异质性，以及从单个肿瘤中繁殖不同的克隆亚群。与我们的合作者一起，我们开发了克服这些障碍的方法，使分离、培养和鉴定卵巢肿瘤细胞克隆群体，然后跟踪从移植克隆的混合物产生的异种肿瘤内的条形码、表位标记的克隆群体成为可能。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，这显著地 影响肿瘤进展-最有趣的是，我们发现，无法自行启动肿瘤形成的克隆性癌细胞群体，当与“启动肿瘤”的克隆混合时，可以强烈促进原发肿瘤的扩张和特定器官部位的转移。这支持了一个假设，即肿瘤内的克隆亚群相互合作，促进肿瘤的扩张和转移。在这项建议中，我们描述了在人类卵巢肿瘤中使用系统的方法来测试这一假说的计划，方法是：(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差异的程度，(2)比较单个克隆群体和这些群体的混合的活动，以调查肿瘤内串扰的存在和性质，(3)随着时间的推移跟踪克隆群体的肿瘤内定位，以及(4)阐明肿瘤内串扰产生表型的机制。

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.