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机制理解大部分是通过在人工二维环境中观察细胞行为获得的。尽管使用这种方法取得了进展，但重要的新证据表明，使用重建的3d基质和组织外植体，二维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计划提供的新证据表明，侵袭性细胞可以利用间充质类型的细胞侵袭，包括形成细长的不适应体和纺锤形形态，也可以利用原始的变形虫运动，包括通过细胞外基质中的小孔形成膜泡。这些突破性的发现提出了一种假设，即细胞具有不同的侵入性程序，使它们能够穿越复杂的组织，并在体内的外来部位定居。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，我们要确定肿瘤细胞在体内扩散所利用的侵袭机制，以便设计合适的治疗药物，彻底根除癌症在患者体内的扩散。然而，肿瘤细胞侵袭是一个复杂的动态过程，涉及肿瘤细胞与血管和基质重塑之间复杂的相互作用。在体内理解这一过程是困难的，因为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，利用人类癌细胞在光学透明斑马鱼体内生长的基因工程，在所有血管中表达绿色荧光蛋白。通过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诱导细胞快速侵袭过程，通过VEGF分泌诱导的血管开口促进细胞内渗。相比之下，间充质细胞的侵袭包括形成细长的侵足和血管壁的膜整合，但不包括细胞内渗。我们的目标是了解控制变形虫和间充质侵袭的信号机制，以及血管孔是如何形成的，以响应VEGF的分泌。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入侵。我们还假设，在人类癌症中发现的PI3K含有激活突变，通过激活FAK-Src-CAS-Crk-Rac信号模块诱导间充质细胞侵袭，这促进了行动蛋白介导的侵入性突起。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 -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PI3K信号通路如何调节癌细胞的侵袭和内渗，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.