Testing the Role of Chromosomal Instability in Chemotherapy Resistance in High Grade Serous Ovarian Cancer

染色体改变（整个或部分染色体的增加或减少）在癌症中几乎无处不在，并且被认为为肿瘤细胞提供了迅速进化出对治疗的抗性的能力。然而，由于操纵肿瘤染色体不稳定性水平的困难，这从未在实验中直接测试过。卵巢癌尤其被认为依赖于染色体的改变，因为其他类型的突变在这种癌症中比较罕见。在过去的几年里，我们已经描述了细胞机制驱动染色体不稳定性（CIN）在结直肠癌（参考文献1）和卵巢癌（参考文献2）。这使我们能够开发实验方法来操纵CIN的比率，使我们能够第一次测试CIN在产生化疗耐药性中的直接作用。目的1：跟踪一组先前表征的高级别浆液性卵巢癌（HGSC）细胞系化疗耐药性的发展。我们将通过对单个癌细胞进行“条形码”来进行谱系追踪，然后通过对产生的细胞群进行深度测序，跟踪它们的克隆生长动态。我们将比较“天然CIN”HGSC细胞系和我们使用Tamura等人，BioRXiv 2019中描述的方法降低CIN的HGSC细胞系的化疗耐药动态。目标2：发现CIN机制在最近从HGSC患者身上提取的“离体”HGSC培养物中起作用（与曼彻斯特大学Taylor教授合作-见参考文献4）。这将涉及细胞生物学分析，以确定导致持续染色体复制和分离错误的细胞失调的比率和类型。目的3：在化疗耐药发展过程中，用体外培养重复谱系追踪实验。这将使我们能够测试CIN在已建立的细胞系和离体培养中产生化疗耐药性的重要性，这更准确地代表了患者的疾病。

Chromosomal changes (gains or losses of whole, or parts of chromosomes) are almost ubiquitous in cancer, and are thought to provide tumour cells with the ability to rapidly evolve resistance to therapy. However this has never been directly tested experimentally due to difficulties in manipulating tumour chromosomal instability levels. Ovarian cancer particularly is thought to rely on chromosomal changes because other types of mutations are rarer in this cancer. Over the last years we have characterized the cellular mechanisms driving chromosome instability (CIN) in colorectal (ref 1) and ovarian (ref 2) cancer. This has allowed us to develop experimental methods to manipulate the rates of CIN placing us in a position to test the direct role of CIN in developing chemotherapy resistance for the first time.Aim 1: Track the development of chemotherapy resistance in a panel of previously characterised high grade serous ovarian cancer (HGSC) cell lines. We will use lineage tracing by 'barcoding' individual cancer cells then following the dynamics of their clonal growth over time, by deep sequencing resulting cell populations. We will compare the dynamics of chemotherapy resistance in 'naturally CIN' HGSC cell lines, and also HGSC cell lines where we have reduced CIN using methods described in Tamura et al, BioRXiv 2019.Aim 2: Discover the CIN mechanisms operating in a panel of 'ex-vivo' HGSC cultures that have been recently derived from HGSC patients (in collaboration with Prof. Taylor, University of Manchester - see Ref 4). This will involve cell biological assays to determine the rates and types of cellular dysregulation that leads to ongoing chromosome replication and segregation errors.Aim 3: Repeat lineage tracing experiments during chemotherapy resistance development, with ex-vivo cultures.This will allow us to test the importance of CIN in developing chemotherapy resistance in both established cell lines, and also ex-vivo cultures, which more accurately represent disease in patients.