Identification and characterization of cancer cell states by novel computational and experimental technologies - Resubmission - 1

摘要 肿瘤是由遗传和转录异质细胞组成的复杂系统，而这 异质性已被认为是耐药和总死亡率的原因之一。了解Intra- 因此，肿瘤的异质性可能会产生广泛的影响，无论是基础还是临床。……的到来 单细胞RNA-Seq已导致检测到在转录上不同状态的癌细胞跨越 广泛的肿瘤类型和分期。然而，该领域缺乏强有力的计算和实验。 从功能上识别和表征这些癌细胞状态的技术。在这个项目中，我们提取了一种基因 以模块为中心的视图，以严格且广泛适用的方式定义单元状态。我们将验证 使用人类样本和斑马鱼黑色素瘤模型研究每种细胞状态的重要性，该模型具有特殊的 在肿瘤进展和转移过程中对细胞状态进行成像和扰动的能力。至 通过实验系统地表征这些细胞状态，我们提出了分析它们的方法 肿瘤内和微环境因素之间的相互依赖关系。我们的基因工程 斑马鱼将用荧光报告器和消融盒标记每种癌细胞状态，提供 灵活的实验平台，可以研究和扰动每一种细胞状态。这位记者将使我们能够对癌症进行分类 并单独研究它们，特别强调它们的可塑性。通过系统地 扰乱癌细胞状态，我们将进一步阐明它们在肿瘤启动中的个体贡献， 进展和转移。使用空间转录组学方法并与单细胞rna-seq整合 数据，我们将绘制癌细胞状态与其微环境的关系图，以筛选可能的相互作用。 最后，我们将直接测试特定癌细胞状态和免疫之间的预测相互作用。 隔间使用T细胞缺陷的鱼。在我们的三个目标中，我们采用了系统生物学工作流程 在我们的泛函模型的观察模式、扰动模式和精化模式中迭代循环 癌细胞状态在肿瘤进展中的作用。我们的建议共同整合了互补的 怀特和柳井实验室的专业知识，并着手显著提高我们对肿瘤内的理解 通过癌细胞状态的晶状体的异质性。

SUMMARY Tumors are complex systems composed of genetically and transcriptionally heterogeneous cells, and this heterogeneity has been implicated as a cause of drug resistance and overall mortality. Understanding intra- tumor heterogeneity is therefore likely to have widespread impact, both fundamental and clinical. The advent of single-cell RNA-Seq has led to the detection of transcriptionally distinct states among cancer cells across a wide range of tumor types and stages. However, the field lacks robust computational and experimental technologies to functionally identify and characterize these cancer cell states. In this project, we take a gene module-centric view to define cell states in a rigorous and widely applicable manner. We will validate the importance of each cell state using human samples and the zebrafish melanoma model, which has exceptional capabilities for imaging and perturbation of cell states throughout tumor progression and metastasis. To systematically characterize these cell states experimentally, we propose methods to assay their interdependencies within the tumor and with elements of the microenvironment. Our genetically engineered zebrafish will mark each cancer cell state with a fluorescent reporter and an ablation cassette, providing a flexible experimental platform to study and perturb each cell state. The reporter will enable us to sort cancer cell states and study them individually, with a particular emphasis on their plasticity. By systematically disrupting cancer cell states, we will further elucidate their individual contributions to tumor initiation, progression and metastasis. Using a spatial transcriptomics approach and integrating with single-cell RNA-seq data, we will map cancer cell states in relation to their microenvironment to screen for putative interactions. Finally, we will directly test predicted interactions between specific cancer cell states and the immune compartment using T cell-deficient fish. Throughout our three Aims, we adopt a systems biology workflow that iteratively cycles through modes of observations, perturbations, and refinement of our model of the functional role of cancer cell states during tumor progression. Our proposal collectively integrates the complementary expertise of the White and Yanai labs and sets out to significantly improve our understanding of intratumoral heterogeneity through the lens of cancer cell states.