Early tumorigenesis in vivo imaging, scRNA-seq to functional assays

项目摘要/摘要 肿瘤在细胞行为、细胞微环境和基因表达上的异质性已成为 肿瘤进展，并为理解癌症提供了一个重要的挑战。例如，采用 某些肿瘤细胞的侵袭表型是癌症从良性肿瘤状态发展的关键一步。 肿瘤细胞从表皮上皮层向真皮的迁移为肿瘤细胞提供了进入 血管系统，为远端转移提供了直接途径。了解某些细胞如何以及为什么能够 侵袭周围组织是了解肿瘤侵袭的关键，但目前体内侵袭的模型确实如此。 不容易让特定的侵袭细胞被回收和研究，以阐明肿瘤侵袭和 进步。 我们的目标是为后续的肿瘤发生创造一条新的管道，允许对肿瘤细胞的直接研究 多元化和侵略性。这一实验性管道旨在提供以下功能： (I)从肿瘤的最初起源到侵袭等关键事件，纵向追踪肿瘤的发生； (Ii)标记并选择性地提纯在这些时间进程研究中确定的肿瘤细胞亚群； (3)以公正的方式描述孤立人群中单个肿瘤细胞的特征。 这种方法将允许识别伴随着细胞事件和发育轨迹 肿瘤的生长和侵袭性。在肿瘤的关键亚组分中表征的分子相关性可以 与细胞亚组分的事件历史以及这些细胞和分子的重要性直接相关 事件可以通过扰动、烧蚀和功能阻断实验进行测试。这样一个功能齐全的 纵向实验流水线是创建更有针对性和更有信息量的药物筛选系统的关键。 我们已经开发了一套转基因斑马鱼品系和成像工具，可以重复、非侵入性地 成年动物内源性肿瘤发展的可视化研究。这使得肿瘤可以从他们的 在很长一段时间内，癌基因在皮肤上皮细胞表达后产生。我们的初步结果显示， 从肿瘤形成的最早阶段就存在异质性，因为只有一小部分细胞被诱导成 表达的癌基因继续形成侵袭性肿瘤；这样的结果为 我们对肿瘤异质性的产生和阐述的分析。 在这里，我们建议使用成像和标记选定的细胞群体，结合单细胞 RNA测序分析，研究侵袭性肿瘤的细胞行为和分化途径 发展。我们将通过调查建立一个实验性的管道：侵袭性特有的细胞类型 肿瘤(目标1)；导致侵袭性表型的关键分化途径(目标2)；以及利用这些 功能分析的洞察力(目标3)。这条管道将为侵袭性肿瘤的发展提供新的见解， 支持针对关键细胞亚群的更大规模的研究和抗肿瘤疗法的设计。

PROJECT SUMMARY/ABSTRACT Tumor heterogeneity, in cell behavior, cell microenvironment and gene expression, has become a hallmark of tumor progression, and provides an important challenge in understanding cancer. For example, the adoption of an invasive phenotype by some tumor cells is a key step as carcinomas advance from the benign tumor state. Migration of tumor cells from the epithelial layers of the epidermis to the dermis provides tumor cell access to the vasculature, offering a direct path for distal metastasis. Understanding how and why some cells are capable of invading surrounding tissues is key to understanding tumor invasion, but current in vivo models of invasion do not readily allow specific invasive cells to be recovered and studied to elucidate drivers of tumor invasion and progression. Our goal is to create a new pipeline for following tumorigenesis, permitting direct studies of tumor cell diversification and invasion. This experimental pipeline is designed to offer capabilities to: (i) track tumorigenesis longitudinally over time from the first origins of a tumor to key events such as invasion; (ii) label and selectively purify tumor cell subsets identified in these time-course studies; (iii) characterize individual tumor cells in isolated populations in unbiased way. This approach would allow identification of the cellular events and developmental trajectories that accompany tumor growth and invasiveness. Molecular correlates characterized in the key sub-fractions of the tumors can be directly related to the event history of the cell sub-fractions, and the importance of these cellular and molecular events can be tested through perturbation, ablation and function blocking experiments. Such a functional and longitudinal experimental pipeline is key to creating more targeted and informative drug screening systems. We have developed a set of transgenic zebrafish lines and imaging tools that allow repeated, non-invasive visualization of endogenous tumor development in adult animals. This permits tumors to be tracked from their origins, over long periods, after oncogene expression in skin epithelial cells. Our preliminary results show that heterogeneity is present from the earliest stages of tumor formation, as only a small fraction of cells induced to express oncogenes go on to form invasive tumors; such results provide the motivation for and the foundation for our analyses of the emergence and elaboration of tumor heterogeneities. Here, we propose to employ imaging and labeling of selected cell populations, in combination with single-cell RNA-sequencing analyses, to study the cell behaviors and differentiation routes key to invasive tumor development. We will establish an experimental pipeline by investigating: the cell-types specific to invasive tumors (Aim 1); the key differentiation pathways leading to an invasive phenotype (Aim 2); and leveraging these insights by functional assays (Aim 3). This pipeline will offer new insights into invasive tumor development, supporting larger studies and the design of anti-tumor therapeutics targeting key cell subsets.