Imagining Human Cancer Progression in a Novel Zebrafish Model

描述(由申请人提供)：我们对人类癌细胞如何迁移和侵袭的大部分机械理解是通过观察细胞在人工2D环境中的行为而获得的。尽管使用这种方法已经取得了进展，但重要的新证据表明，使用重组的3D基质和组织外植体，2D系统中的细胞迁移并不能完全概括与更生理环境中的运动相关的事件。其他人的工作和本研究提案中提供的新证据表明，侵袭细胞可以利用间叶型细胞侵袭，包括形成细长的内皮膜和纺锤形形态，或者利用原始阿米巴运动，涉及通过细胞外基质上的小孔进行膜气泡。这些突破性的发现引发了一种假设，即细胞配备了不同的侵入性程序，使它们能够穿越复杂的组织并在体内的外来部位定居。然而，最重要的是，这些发现表明，对患者这一过程的治疗性预防将需要一种针对两种细胞侵袭模式的多方面方法。因此，至关重要的是，我们要确定肿瘤细胞在体内扩散的侵袭机制，以便设计出合适的治疗剂(S)来完全根除患者体内的癌症扩散。然而，肿瘤细胞的侵袭是一个复杂的动态过程，涉及到肿瘤细胞与重建的血管和间质之间错综复杂的相互作用。在活体中理解这一过程一直很困难，因为还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，该模型利用人类癌细胞在光学透明斑马鱼中生长，并通过基因工程在所有血管中表达绿色荧光蛋白。利用该模型和双色高分辨率共聚焦显微镜，我们发现转移基因RhoC诱导了一个快速的细胞侵袭过程，该过程通过分泌血管内皮生长因子诱导的血管开放促进了细胞内的生长。相反，间充质细胞的侵袭包括形成细长的内陷和膜整合到血管壁中，而不是细胞内。我们在拟议工作中的目标是了解控制阿米巴和间充质细胞侵入的信号机制，以及血管孔是如何响应血管内皮生长因子的分泌而形成的。根据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介导的收缩作用介导阿米巴侵袭。我们还假设，在人类癌症中发现的含有激活突变的PI3K通过激活FAK-Src-CAS-Crk-Rac信号模块诱导间充质细胞侵袭，该信号模块促进肌动蛋白介导的腹内突起。我们假设血管孔是通过破坏细胞-细胞连接形成的，这是由VE-cadherin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PI3K信号通路如何调控癌细胞的侵袭和血管内渗，以及控制血管孔形成的分子信号机制。

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.