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

早期肿瘤发生体内成像、scRNA-seq 到功能测定

• 批准号: 10355131
• 负责人: SCOTT E FRASER
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-13 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10355131
• 关键词: Ablation; Address; Adopted; Adoption; Adult; Automobile Driving; Basement membrane; Behavior; Benign; Biological Assay; Carcinoma; Cell Differentiation process; Cell Fraction; Cell Proliferation; Cell Separation; Cell physiology; Cells; Complex; Data; Dermis; Development; Distal; Drug Screening; Epidermis; Epithelial Cells; Event; Excision; Exhibits; Foundations; Gene Expression; Gene Expression Profile; Genes; Goals; Growth; Harvest; Heterogeneity; Image; Imaging Device; Individual; Inequality; Invaded; Label; Lasers; Link; Maintenance; Malignant - descriptor; Malignant Neoplasms; Molecular; Motivation; Natural History; Neoplasm Metastasis; Oncogenes; Organ; Outcome; Pathway interactions; Phase; Phenotype; Play; Population; Positioning Attribute; Process; Recording of previous events; Reporter; Resolution; Role; Route; Skin; Skin Tissue; System; Testing; Time; Tissues; Transgenic Organisms; Tumor Cell Invasion; Tumor Cell Migration; Tumor stage; Visualization; Zebrafish; base; cancer imaging; cell behavior; cell type; design; experimental study; imaging study; in vivo; in vivo Model; in vivo imaging; insight; mature animal; migration; neoplastic cell; non-invasive imaging; novel marker; prevent; single-cell RNA sequencing; spatiotemporal; therapeutic target; transcriptome; transcriptomics; tumor; tumor growth; tumor heterogeneity; tumor initiation; tumor microenvironment; tumor progression; tumorigenesis

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.

项目摘要/摘要 肿瘤异质性，细胞行为，细胞微环境和基因表达，已成为 肿瘤进展，并在理解癌症方面带来了重要的挑战。例如，采用 某些肿瘤细胞的侵入性表型是癌从良性肿瘤状态前进的关键步骤。 肿瘤细胞从表皮上皮层迁移到真皮可提供肿瘤细胞的访问 脉管系统，为远端转移提供了直接路径。了解某些细胞如何以及为什么能够 入侵周围组织是了解肿瘤侵袭的关键，但是当前的体内侵袭模型DO 不容易使特定的侵入性细胞被恢复并研究以阐明肿瘤侵袭的驱动因素和 进展。 我们的目标是创建一条新的管道，用于遵循肿瘤发生，允许直接研究肿瘤细胞 多样化和入侵。该实验管道旨在提供以下功能： （i）随着时间的流逝，从肿瘤的第一个起源到诸如入侵之类的关键事件，纵向延伸肿瘤发生； （ii）在这些时间表研究中鉴定出的标记和选择性净化肿瘤细胞亚群； （iii）以公正的方式以孤立群体中的单个肿瘤细胞来表征。 这种方法将允许识别伴随的细胞事件和发育轨迹 肿瘤的生长和侵入性。在肿瘤的关键亚差异中特征的分子相关性可以 与细胞亚裂缝的事件历史直接相关，这些细胞和分子的重要性 可以通过扰动，消融和功能阻塞实验来测试事件。这样的功能和 纵向实验管道是创建更多有针对性和信息丰富的药物筛查系统的关键。 我们已经开发了一套转基因斑马鱼线和成像工具，这些工具允许重复，无创的 成年动物内源性肿瘤发展的可视化。这允许从他们的 长期以来，起源于皮肤上皮细胞中的癌基因表达后。我们的初步结果表明 异质性是从肿瘤形成的最早阶段存在的，因为只有一小部分被诱导的细胞 表达致癌基因继续形成侵袭性肿瘤。这样的结果为动机和基础提供了 我们对肿瘤异质性的出现和阐述的分析。 在这里，我们建议采用选定细胞群体的成像和标记，并结合单细胞 RNA测序分析，研究细胞行为和分化途径的侵入性肿瘤的关键 发展。我们将通过研究：特定于侵入性的细胞类型来建立实验管道 肿瘤（AIM 1）；导致侵入性表型的关键分化途径（AIM 2）；并利用这些 通过功能分析的见解（AIM 3）。该管道将​​为侵入性肿瘤发展提供新的见解， 支持更大的研究和针对关键细胞子集的抗肿瘤疗法的设计。