Zebrafish Tumor Model Research Articles

Endothelial Cords Promote Tumor Initial Growth prior to Vascular Function through a Paracrine Mechanism.

The angiogenic switch is an important oncogenic step that determines whether microtumors remain dormant or progresses further. It has been generally perceived that the primary function of this tumorgenic event is to supply oxygen and nutrients through blood circulation. Using in vivo imaging of zebrafish and mouse tumor models, we showed that endothelial cords aggressively penetrated into microtumors and remained non-circulatory for several days before undergoing vascular blood perfusion. Unexpectedly, we found that initial tumor growth in both models was significantly reduced if endothelial cords were removed by blocking VEGF-VEGFR2 signaling or using a vascular deficient zebrafish mutant. It was further shown that soluble factors including IL-8, secreted by endothelial cells (ECs) were responsible for stimulating tumor cells proliferation. These findings establish that tumor angiogenesis play a much earlier and broader role in promoting tumor growth, which is independent of vascular circulation. Understanding this novel mechanism of angiogenic tumor progression offers new entry points for cancer therapeutics.

Non-metastatic 2 (NME2)-mediated suppression of lung cancer metastasis involves transcriptional regulation of key cell adhesion factor vinculin.

Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n = 382), survival data (n = 530) and lymph node metastases (n = 100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2, did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i-mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.

Xmrk, Kras and Myc Transgenic Zebrafish Liver Cancer Models Share Molecular Signatures with Subsets of Human Hepatocellular Carcinoma

Previously three oncogene transgenic zebrafish lines with inducible expression of xmrk, kras or Myc in the liver have been generated and these transgenic lines develop oncogene-addicted liver tumors upon chemical induction. In the current study, comparative transcriptomic approaches were used to examine the correlation of the three induced transgenic liver cancers with human liver cancers. RNA profiles from the three zebrafish tumors indicated relatively small overlaps of significantly deregulated genes and biological pathways. Nevertheless, the three transgenic tumor signatures all showed significant correlation with advanced or very advanced human hepatocellular carcinoma (HCC). Interestingly, molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples (24–29%) and there were conserved up-regulated pathways between the zebrafish and correlated human HCC subgroup. The three zebrafish liver cancer models together represented nearly half (47.2%) of human HCCs while some human HCCs showed significant correlation with more than one signature defined from the three oncogene-addicted zebrafish tumors. In contrast, commonly deregulated genes (21 up and 16 down) in the three zebrafish tumor models generally showed accordant deregulation in the majority of human HCCs, suggesting that these genes might be more consistently deregulated in a broad range of human HCCs with different molecular mechanisms and thus serve as common diagnosis markers and therapeutic targets. Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup.

Abstract B90: Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model

Abstract Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization. Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGFinduced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy. Citation Format: Pegah Rouhi, Samantha Lin Lee, Lasse Dahl Jensen, Danfang Zhang, Hong Ji, Giselbert Hauptmann, Philip Ingham, Yihai Cao. Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B90.

November 2012: This Month in JoVE

In this issue, Oestreicher et al. show us how to isolate magnetotactic bacteria from freshwater samples, and concentrate the bacteria at one end of a glass capillary. The magnetotactic bacteria can then be visualized by light and transmission electron microscopy, and used for various other assays. Also in the November 2012 issue, Boland et al. demonstrate how to reprogram fibroblasts into induced pluripotent stem cells (iPSCs), and how to isolate iPSC lines for injecting into tetraploid blastocysts, as demonstrated previously in JoVE. While mouth pipetting is generally not recommended because it can have adverse effects, this protocol requires the technique, executed carefully, to manipulate the embryos. This is one of the rare occasions when it's actually OK to mouth pipet in the lab. If the induced cells are fully pluripotent, they can result in full-term mice derived completely from iPSCs. In JoVE Neuroscience, Heermann and Krieglstein demonstrate a method for visualizing Schwann cell development along growing axons. To do this, our authors culture cervical ganglia explants onto collagen matrices, and treat the explants with nerve growth factor or other substances. The collagen gels can then be visualized using time-lapse imaging with fluorescence or bright-field microscopy, migrating along axons towards the periphery. Also in JoVE Neuroscience, Hoffmann et al. put tiny headphones on songbirds to study how they use auditory feedback to adjust their singing. The authors demonstrate how to construct the headphones and attach them to the bird's head; then, by adjusting the acoustic signal, they can study the computational and neurophysiological basis of vocal learning in birds. In JoVE Clinical & Translational Medicine, Iyengar et al. use a zebrafish tumor model to study genes that can modify the pathogenesis of melanoma. This is done by first creating transgenic zebrafish that express a gene of interest. Various assays can then be performed to study how different genes affect melanoma, including onset, invasion, and transplantability. In JoVE Bioengineering, Martin et al. demonstrate a gliding assay to measure the flexural rigidity of biopolymers (such as microtubules). By attaching motor proteins to a microscope slide, and adding fluorescently labeled microtubules, our authors can analyze the dynamics of cytoskeletal polymers. This preview summarizes just a few notable video-articles in the November 2012 issue of JoVE. Stop by throughout the month of November to check out the full length versions of these articles and many more.

One step forward: The use of transgenic zebrafish tumor model in drug screens

The zebrafish (Danio rerio) has been an experimental model in the developmental biology and toxicology since the 1950s. In recent years, with the aid of transgenic technology, it has also gained an increasing popularity to model human diseases, including various cancers. As a feasible vertebrate model for large-scale chemical screens, the zebrafish has also given us a new option for the search of potential anticancer drugs. It is hopeful that in the near future with automation and analytical tools, drug development processes will be significantly shortened for quick and effective identification of candidate drugs.

In VivoMagnetic Resonance Imaging to Detect Malignant Melanoma in Adult Zebrafish

Zebrafish cancer models are fast gaining ground in cancer research. Most tumors in zebrafish develop late in life, when fish are no longer transparent, limiting in vivo optical imaging methods. Thus, noninvasive imaging to track tumor in adult zebrafish remains challenging. In this study, we applied magnetic resonance microimaging (microMRI) to track spontaneous melanomas in stable transgenic zebrafish models expressing an RAS oncoprotein and lacking P53 (mitf:Ras::mitf:GFP X p53(-/-)). Tumors in live adult zebrafish were observed at various locations using a T(2)-weighted fast spin echo sequence at 9.4 T. Further, live imaging of tumors at ultrahigh field (17.6 T) revealed significant tumor heterogeneity. This heterogeneity was also confirmed by the significant differences in transverse relaxation time, T(2) measured in various regions of tumor. To our knowledge, this is the first report demonstrating the application of microMRI to detect the locations, invasion status, and characteristics of internal melanomas in zebrafish and suggesting that noninvasive microMRI can be applied for longitudinal studies to track tumor development and real-time assessment of therapeutic effects in zebrafish tumor models.

Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model

Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization. Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGF-induced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy.

02-P009 In vivo, live imaging of immune surveillance in a V12HRAS induced zebrafish tumor model

multifunctional regulatory nuclear compartment that, besides hosting ribosome biogenesis, regulates cellular growth, cell death, and the cell cycle. The Myc transcription factor stimulates tissue growth, in part by activating the expression of genes required for ribosome biogenesis. By qRT-PCR we show that Myc is necessary and sufficient for transcriptional activation of vito expression. Our results raise the hypothesis that the integration of signalling and nutritional cues that control cell growth in Drosophila requires Vito to regulate nucleolar function and architecture.