Advanced tools for HCMI model genetic perturbation and metastasis characterization

摘要 人类癌症模型倡议（HCMI）正在创建下一代癌症模型，将推动未来 癌症精准医疗研究的一部分历史上的癌细胞系已被选择为它们的快速增殖 在组织培养塑料，这使得他们适合高通量筛选，如全基因组 CRISPR/Cas9敲除筛选。然而，历史线在他们的代表性方面有很大的差距， 人类癌症的多样性，并且它们可能缺乏生理相关性，因为它们对快速癌症的最佳化具有重要意义。 增殖下一代HCMI模型解决了这些问题，但需要开发新的 方法，使之有用。具体而言，基因组编辑的标准方法（涉及首先创建Cas9- 稳定表达细胞系，然后引入向导RNA）对于通常生长缓慢的增殖细胞将不起作用 在3D中。因此，我们将开发一体化的基因组编辑载体系统，使其能够将基因组编辑技术引入人类。 基因组编辑对HCMI模型的影响。此外，这种“脱落”筛选的标准生存力读数 涉及细胞在许多群体中的生长。但对于缓慢扩散的HCMI模型，替代方案 将需要读出以进行有效筛选。因此，我们将开发短期单细胞RNA测序 因此，可以使用scRNAseq（scRNAseq）方法，其将用作长期存活力的替代读出。鉴于临床注释 与HCMI模型相关联，也有巨大的机会将这些模型的使用扩展到 更复杂的，生理相关的表型，如器官特异性转移， 潜力因此，我们将开发方法，使其有可能确定转移潜力的下一个 第二代癌症模型，我们将创建一个公共资源的转移地图（MetMap）至少50 HCMI模型。所有数据和方案将不受限制地公开提供，所有试剂将 可通过Addgene获得，并且所有修改的模型可提供给ATCC用于分发。重要的是，在整个 在该项目中，所有细胞模型都将受到严格监测，以寻找遗传和表观遗传漂变的证据。在 在拟议项目结束后，我们预计将产生一套工具和数据，这将有助于推动 基于下一代癌症模型的癌症精准医疗的未来。

ABSTRACT The Human Cancer Models Initiative (HCMI) is creating next generation cancer models that will drive the future of cancer precision medicine research. Historical cancer cell lines have been selected for their rapid proliferation on tissue culture plastic, which has made them amenable to high throughput screening such as genome-wide CRISPR/Cas9 knock-out screens. However, the historical lines have large gaps in their representation of the diversity of human cancer, and they may lack physiological relevance given their optimization for rapid proliferation. Next generation HCMI models address these concerns, but will require the development of new methods to make them useful. Specifically, standard approaches to genome editing (involving first creating Cas9- stably expressing lines and then introducing guide RNAs) will not work for slowly proliferating cells often growing in 3D. We will therefore develop all-in-one genome editing vector systems that will make it possible to bring the power of genome editing to HCMI models. In addition, standard viability read-outs of such “drop-out” screens involve the growth of cells over many population doublings. But for slowly proliferating HCMI models, alternative readouts will be required for efficient screening. We will therefore develop short-term single cell RNA sequencing (scRNAseq) methods that will serve as surrogate read-outs for long-term viability. Given the clinical annotation associated with HCMI models, there is also enormous opportunity to expand the use of these models beyond viability measures to more complex, physiologically relevant phenotypes such as organ-specific metastatic potential. We will therefore develop methods that make it possible to determine the metastatic potential for next generation cancer models, and we will create a public resource of the metastasis map (MetMap) for at least 50 HCMI models. All data and protocols will be made publicly available without restriction, all reagents will be made available via Addgene, and all modified models made available to ATCC for distribution. Importantly, throughout the project, all cell models will be rigorously monitored for evidence of genetic and epigenetic drift. At the conclusion of the proposed project, we expect to have generated a set of tools and data that will help propel the future of cancer precision medicine based on next generation cancer models.