Genomic and molecular dissection of the collective invasion pack

 描述(由申请人提供)：基因组分析显示单个肿瘤内存在显著的分子异质性。这种异质性仍然是理解肿瘤进展的生物学驱动因素的主要障碍。为此，我们开发了一种新的基于成像的技术，通过精确地从他们的自然环境中选择和提取感兴趣的活细胞来获得任何细胞或群体的基因组图谱。这些提纯的和 然后，提取的细胞可以进行基因组分析，或者进行扩增和培养，用于分子研究。我们将这种图像引导的基因组学技术称为时空基因组分析(SAGA)。由于我们长期以来对肿瘤侵袭和转移的兴趣，我们使用SAGA通过集中肿瘤细胞侵袭来探讨癌症侵袭，这是小鼠模型和患者的主要转移方式。我们使用SAGA来：1)进行第一次基因组分析，直接比较集体侵袭包中纯化的高侵袭性先导细胞和流向先导细胞的跟随细胞的基因组表达谱。2)选择、扩增和保持第一批纯化的先导细胞系和跟随细胞系，这使我们现在可以获得几乎无限数量的以前罕见的侵袭细胞类型我们的基因组数据表明，与跟随细胞相比，先导细胞具有不同的表达谱，其关键的侵袭相关基因过表达。由于我们可以将这些罕见的细胞类型保留在培养中，并且它们随着时间的推移保持各自的表型，我们发现领导细胞侵袭但增殖能力较差，而跟随者表现出相反的情况，侵袭能力较弱，增殖速度较快。重要的是，这两种细胞类型的混合导致了一种协同作用，其中领导细胞重复它们的角色，并积极寻找跟随细胞来促进集体侵袭；跟随细胞反过来促进有效的领导细胞增殖。这些观察结果使我们认识到，由于细胞间的协同作用，集体侵袭包内的肿瘤细胞特化可增加转移成功率。假设如果是真的，这表明共同入侵的细胞代表了专门为促进侵袭而准备的专门劳动力。为了测试这一点，我们将通过转移到我们独特的细胞系的体内分析来确定侵袭单位内的细胞专门化是否决定了侵袭潜力、原发肿瘤形成和向次要部位的转移。然后，我们将利用我们目前的基因组数据集，探索领导细胞入侵行为的分子基础，并确定在集体细胞入侵过程中，哪些基因驱动领导细胞和跟随细胞的行为和交流。综上所述，这些方法提供了一个独特的机会来理解罕见细胞类型的机制基础以及集体侵袭和增殖中的异质性。

 DESCRIPTION (provided by applicant): Genomic analysis has revealed significant molecular heterogeneity within single tumors. This heterogeneity remains a major obstacle to understanding the biological drivers of tumor progression. To that end, we have developed a new imaging-based technique to obtain genomic profiles of any cell or population by precisely selecting and extracting living cells of interest from their native environment. These purified and extracted cells can then be subjected to genomic analysis, or amplified and cultured for molecular studies. We have termed this image-guided genomics technique, spatiotemporal genomic analysis (SAGA). Due to our long-standing interest in cancer invasion and metastasis, we have used SAGA to probe cancer invasion by focusing on collective tumor cell invasion, which is major mode of metastasis in murine models and patients. We have used SAGA to: 1) perform the first genomic analysis directly comparing the genomic expression profile of purified highly invasive leader cells of the collective invasion pack, to follower cells, which stream behin the leader cells. 2) select, amplify, and maintain the first purified leader cell lines and followe cell lines, which now give us virtually unlimited quantities of previously rare invading cell types Our genomics data show that leader cells have distinct expression profiles with overexpression of key invasion-related genes compared to follower cells. Since we can keep these rare cell types in culture, and they maintain their respective phenotype over time, we show that leader cells invade aggressively yet proliferate poorly, whereas followers show the converse by invading poorly and proliferating rapidly. Importantly, mixing these two cell types leads to a synergy where leader cells reprise their roles and actively seek out follower cells to promote collective invasion; follower cells, in turn, promote effective leader cell proliferation. These observations lead us to tumor cell specialization within the collective invasion pack results in increased metastatic success due to cell-cell cooperativity. hypothesize that If true, it suggests that collectively invading cells represent a specialized labor force that are specifically primed t promote invasion To test this, we will determine if cell specialization within the invasion unit dictates invasive potential, primary tumor formation, and metastasis to a secondary site by moving to in vivo assays with our unique cell lines. Then we will probe the molecular basis of leader cell invasive behavior by leveraging our current genomic data set and determine which genes drive leader and follower cell behavior and communication during collective cell invasion. Taken together, these approaches provide a unique opportunity to understand the mechanistic underpinnings of rare cell types and heterogeneity within the collective invasion pack and proliferation.