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

染色体变化（整个或部分染色体的获得或丢失）几乎普遍存在于癌症中，并且被认为为肿瘤细胞提供了快速发展对治疗的抗性的能力。然而，由于在操纵肿瘤染色体不稳定性水平方面的困难，这从未被直接实验测试。卵巢癌特别被认为依赖于染色体变化，因为其他类型的突变在这种癌症中更罕见。在过去的几年里，我们已经描述了在结直肠癌（参考文献1）和卵巢癌（参考文献2）中驱动染色体不稳定性（CIN）的细胞机制。这使我们能够开发实验方法来操纵率CIN放置我们在一个位置，以测试CIN在发展中的化疗resistance的直接作用为first.Aim 1：跟踪化疗耐药性的发展在一个小组以前的特点高级别浆液性卵巢癌（HGSC）细胞系。我们将通过对单个癌细胞进行“条形码”来使用谱系追踪，然后通过对所得细胞群进行深度测序来跟踪它们随时间的克隆生长动态。我们将比较“天然CIN”HGSC细胞系以及我们使用Tamura et al，BioRXiv 2019.Aim 2中描述的方法减少CIN的HGSC细胞系中化疗耐药性的动态：发现最近从HGSC患者中衍生的一组“离体”HGSC培养物中的CIN机制（与曼彻斯特大学Taylor教授合作-参见参考文献4）。这将涉及细胞生物学测定，以确定导致持续染色体复制和分离错误的细胞失调的速率和类型。在化疗耐药性发展过程中，使用离体培养物重复谱系追踪实验。这将使我们能够在已建立的细胞系和离体培养物中测试CIN在发展化疗耐药性中的重要性，这更准确地反映了患者的疾病。

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.