Imagining Human Cancer Progression in a Novel Zebrafish Model

在新型斑马鱼模型中想象人类癌症的进展

• 批准号: 7526232
• 负责人: Richard L. Klemke
• 金额: $ 32.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526232
• 关键词: Actins; Address; Adhesions; Animals; Blood Vessels; Bulla; Cell model; Cells; Color; Complex; Computer Assisted; Computer software; Confocal Microscopy; Cytoplasmic streaming; Disruption; Distant; Down-Regulation; Engineering; Environment; Event; Extracellular Matrix; Fishes; Genes; Goals; Green Fluorescent Proteins; Hand; Human; Image; Intercellular Junctions; Invaded; Invasive; Life; Locomotion; Malignant Neoplasms; Mediating; Membrane; Mesenchymal; Modeling; Molecular; Monitor; Morphology; Mutate; Mutation; Myosin ATPase; Myosin Type II; Neoplasm Metastasis; Oncogenic; Optics; Organ; Pathway interactions; Patients; Phosphorylation; Physiological; Prevention; Process; Proteolysis; Public Health; Receptor Activation; Reporter; Research; Research Proposals; Resolution; Rho-associated kinase; Role; Shapes; Signal Pathway; Signal Transduction; Site; Standards of Weights and Measures; Structure; System; Therapeutic; Therapeutic Agents; Tissues; Transgenic Model; Transgenic Organisms; Tumor Cell Invasion; Tyrosine; Upper arm; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Work; Xenograft Model; Zebrafish; ameboid movement; base; cadherin 5; cancer cell; cell behavior; cell motility; cell type; design; extracellular; in vivo; migration; mutant; neoplastic cell; novel; programs; reconstitution; response; src-Family Kinases; tumor progression

DESCRIPTION (provided by applicant): Most of our mechanistic understanding of how human cancer cells migrate and invade has been obtained by observing cell behavior in an artificial 2 D environment. Although progress has been made using this approach, important new evidence indicates that cell migration in 2 D systems does not completely recapitulate events associated with locomotion in a more physiological environment using reconstituted 3 D matrices and tissue explants. Work by others and novel evidence provided in this research proposal demonstrate invasive cells can utilize either a mesenchymal type of cell invasion that involves formation of an elongated invadapodia and a spindle shaped morphology or a primitive amoeboid movement that involves membrane blebbing though small holes in the extracellular matrix. These breakthrough findings prompted the hypothesis that cells are armed with different invasive programs that allow them to traverse complex tissues and colonize foreign sites in the body. Most importantly though these findings indicate that therapeutic prevention of this process in patients will require a multifaceted approach that targets both modes of cell invasion. It is crucial then that we identify invasive mechanisms utilized by disseminating tumor cells in vivo so that the appropriate therapeutic agent(s) can be designed to completely eradicate the spread of cancer in patients. However, tumor cell invasion is a complex and dynamic process that involves the intricate interplay between the tumor cells and the remodeling vasculature and stroma. Understanding this process in vivo has been difficult because it has not been possible to visualize this process in high resolution in live animals. To address this problem, we have developed a novel model of cancer progression that utilizes human cancer cells growing in optical clear zebrafish genetically engineered to express green fluorescent protein in all blood vessels. Using this model and dual color high resolution confocal microscopy, we discovered that the metastatic gene RhoC induces a rapid cell invasion process that facilitates cell intravasation through vascular openings induced by VEGF secretion. In contrast, mesenchymal cell invasion involves formation of elongated invadopodia and membrane integration into the vascular wall, but not cell intravasation. Our goal in the proposed work is to understand the signaling mechanism that control amoeboid and mesenchymal invasion as cells intravasate and how the vascular pores form in response to VEGF secretion. Based on our preliminary findings and the work of others, we hypothesize that the metastatic gene RhoC mediates amoeboid invasion through Rho kinase activity (ROCK) and myosin II-mediated contractility. We also hypothesize that PI3K harboring activating mutations found in human cancers induces mesenchymal cell invasion through activation of the FAK-Src-CAS-Crk-Rac signaling module, which facilitates actin-mediated invadopodial protrusion. We hypothesize that the vascular pores form through disruption of cell-cell junctions, which is regulated by src phosphorylation of VE-cadherin. Therefore, our overall goal is to examine in detail how RhoC and mutated PI3K signaling pathways regulate cancer cell invasion and intravasation and the molecular signaling mechanisms that control vascular pore formation. PUBLIC HEALTH RELEVANCE: Cancer cells spread throughout the body by invading into blood vessels where they are carried to distant organs and form secondary tumors. Work in this proposal will determine the mechanism of how cancer cells invade through the vessel wall utilizing high resolution confocal imaging of optically transparent zebrafish harboring metastatic human cancer cells.

描述（由申请人提供）：我们对人类癌细胞如何迁移和入侵的大部分机械理解是通过观察人工2 D环境中的细胞行为获得的。虽然使用这种方法已经取得了进展，但重要的新证据表明，在2D系统中的细胞迁移并不完全重现使用重建的3D基质和组织外植体在更生理环境中与运动相关的事件。其他人的工作和本研究提案中提供的新证据表明，侵入性细胞可以利用间充质类型的细胞侵入，包括形成细长的侵入足和纺锤形形态，或原始的变形虫运动，包括通过细胞外基质中的小孔产生膜泡。这些突破性的发现促使人们提出了一种假设，即细胞配备了不同的侵入性程序，使它们能够穿过复杂的组织并在体内的异物部位定居。最重要的是，尽管这些发现表明，在患者中治疗性预防这一过程将需要针对两种细胞入侵模式的多方面方法。因此，至关重要的是，我们确定了体内扩散肿瘤细胞所利用的侵入性机制，以便可以设计适当的治疗剂以完全根除患者中的癌症扩散。然而，肿瘤细胞侵袭是一个复杂的动态过程，涉及肿瘤细胞与重塑血管系统和基质之间错综复杂的相互作用。在体内理解这一过程一直很困难，因为它一直不可能在活体动物中以高分辨率可视化这一过程。为了解决这个问题，我们开发了一种新的癌症进展模型，该模型利用在光学透明斑马鱼中生长的人类癌细胞，该斑马鱼经基因工程改造在所有血管中表达绿色荧光蛋白。使用这个模型和双色高分辨率共聚焦显微镜，我们发现，转移基因RhoC诱导一个快速的细胞侵袭过程，促进细胞通过血管内皮生长因子分泌诱导的血管开口内渗。相比之下，间充质细胞侵袭涉及细长侵袭伪足的形成和膜整合到血管壁中，但不涉及细胞内渗。我们的目标是在拟议的工作是了解信号机制，控制变形虫和间充质入侵细胞intravasate和血管孔的形成，以响应VEGF分泌。基于我们的初步发现和其他人的工作，我们假设转移基因RhoC通过Rho激酶活性（ROCK）和肌球蛋白II介导的收缩性介导变形虫侵袭。我们还假设在人类癌症中发现的PI 3 K携带激活突变通过激活FAK-Src-CAS-Crk-Rac信号传导模块诱导间充质细胞侵袭，这促进肌动蛋白介导的侵袭性伪足突起。我们推测血管孔的形成是通过破坏细胞间连接，而细胞间连接是由VE-钙粘蛋白的src磷酸化调节的。因此，我们的总体目标是详细研究RhoC和突变的PI 3 K信号通路如何调节癌细胞侵袭和内渗以及控制血管孔形成的分子信号机制。公共卫生相关性：癌细胞通过侵入血管扩散到全身，在血管中它们被携带到远处器官并形成继发性肿瘤。这项提案中的工作将确定癌细胞如何通过血管壁侵入的机制，利用高分辨率共聚焦成像的光学透明斑马鱼窝藏转移性人类癌细胞。