High-resolution imaging of the dynamic tumor cell–vascular interface in transparent zebrafish

Abstract

Cell metastasis is a highly dynamic process that occurs in multiple steps. Understanding this process has been limited by the inability to visualize tumor cell behavior in real time by using animal models. Here, we employ translucent zebrafish and high-resolution confocal microscopy to study how human cancer cells invade in tissues, induce angiogenesis, and interact with newly formed vessels. We use this system to study how the human metastatic gene RhoC promotes the initial steps of metastasis. We find that RhoC expression induces a primitive amoeboid-like cell invasion characterized by the formation of dynamic membrane protrusions and blebs. Surprisingly, these structures penetrate the blood vessel wall exclusively at sites of vascular remodeling and not at regions of existing intact vessels. This process requires tumor cells to secrete VEGF, which induces vascular openings, which in turn, serve as portholes allowing access of RhoC-expressing cells to the blood system. Our results support a model in which the early steps in intravasation and metastasis require two independent events: (i) dynamic regulation of the actin/myosin cytoskeleton within the tumor cell to form protrusive structures and (ii) vascular permeablization and vessel remodeling. The integration of zebrafish transgenic technology with human cancer biology may aid in the development of cancer models that target specific organs, tissues, or cell types within the tumors. Zebrafish could also provide a cost-effective means for the rapid development of therapeutic agents directed at blocking human cancer progression and tumorinduced angiogenesis.angiogenesis ͉ cancer ͉ RhoC ͉ metastasis M etastasis is the major cause of death in cancer patients, and there are currently no therapeutic agents available to prevent this disease (1-2). Current models view metastasis as a highly dynamic process that occurs in multiple steps (1-3). The initial steps involve disruption of cell-cell adhesions, the migration of cells away from the primary tumor, and intravasation into the vasculature. The later steps involve tumor cells traveling to distant sites, where they must extravasate into permissible microenvironment to form secondary tumors. The individual steps in the metastatic cascade remain incompletely understood. This is especially true of the initial steps leading to intravasation, when small developing tumors and micrometastases are not easily detected. Most of our understanding of these early steps during the metastatic cascade has been derived from static images of large, advanced-stage tumors and their associated metastases. Consequently, fundamental questions remain as to how invasive cancer cells navigate through complex tissues, locate vessels, and intravasate. Thus, there is a crucial need to understand invasive mechanisms and angiogenic programs that facilitate metastasis so that therapeutic strategies can be developed to block disease progression.Progress has also been limited by the inability of existing animal models (mouse and chick) to facilitate...

Catch of the day: zebrafish as a human cancer model

“…Cancer cells derived from various species (mouse and human) and tissues (adenocarcinoma, fibrosarcoma, melanoma) have been introduced into both young embryos (2-5-days old post fertilization; Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007) and juvenile 30-day-old zebrafish (Stoletov et al, 2007). Both transplantation models have their specific benefits and limitations when it comes to understanding cancer cell behavior in a living organism (Table 1).…”

“…Cancer progression in these animals recapitulates many aspects of human disease and opens the door for studies to identify genetic and chemical modifiers of cancer (Amatruda et al, 2002;Smolowitz et al, 2002;Stern and Zon, 2003;Berghmans et al, 2005a;Goessling et al, 2007a;Kari et al, 2007). The attention has been further fueled by the development of xenograft models that allow the propagation and visualization of human cancer cells engrafted in optically transparent zebrafish (Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007;Stoletov et al, 2007). The integration of zebrafish genetics with the large tool chest of reagents available to study human cancer cells provides a powerful new vertebrate model to visualize and dissect the mechanisms that drive cancer formation, angiogenesis and metastasis.…”

“…A pair of adult fish produces several hundred fertilized eggs a week. The embryos develop externally and are transparent up to 1 month of age (Spitsbergen, 2007;Stoletov et al, 2007). Adult, casper mutant (roy À/À ;nacre À/À ) animals are also available that remain transparent throughout life and are amenable to in vivo transplantation studies (Figure 1) (White et al, 2008).…”

“…Adult, casper mutant (roy À/À ;nacre À/À ) animals are also available that remain transparent throughout life and are amenable to in vivo transplantation studies (Figure 1) (White et al, 2008). The remarkable transparency of zebrafish tissues allows direct imaging of cancer progression including cell invasion, intravasation, extravasation and angiogenesis (Stoletov et al, 2007;White et al, 2008). A complete sequence of the zebrafish genome is available and many human cancer genes are conserved structurally and functionally in zebrafish (http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish).…”

A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver

“…28 Additionally, zebrafish are a good animal model for studying human diseases due to their ability to produce pathological phenotypes comparable to those in humans. 29,30 For instance, a zebrafish carcinogen-induced liver tumor displays a transcriptome profile comparable to that of human liver cancer.…”