Name: invasive behavior of human breast cancer cells in embryonic zebrafish
Uploaded: 2017-04-25T15:00:00
Description: Watch this Scientific Journal Video about Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish at JoVE.com

Studies on TGF-&#946; family members are supported by the Cancer Genomics Centre Netherlands. Sijia Liu and Jiang Ren are supported by the China Scholarship Council for 4 years of study at the University of Leiden. We thank Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA) for the MCF10A cell lines.

All research using the transgenic fluorescent zebrafish Tg (fli:EGFP) strain, which has enhanced green fluorescent protein (EGFP)-labeled vasculature20, including housing and experiments, was carried out according to the international guidelines and was approved by the local Institutional Committee for Animal Welfare (Dier Ethische Commissie (DEC) of the Leiden University Medical Center.
NOTE: As summarized in Figure 1, the protocol is roughly broken down into four steps: embryo collec...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diethylnitrosamine-induced+hepatic+degeneration+and+neoplasia+in+the+aquarium+fish,+Brachydanio+rerio.">Diethylnitrosamine-induced hepatic degeneration and neoplasia in the aquarium fish, Brachydanio rerio.</a> J. Natl. Cancer Inst. 34 (1), 117-130 (1965).</li><li>Lam, S. H., Wu, Y. L., Vega, V. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+expression+signatures+between+zebrafish+and+human+liver+tumors+and+tumor+progression.">Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.</a> Nat. Biotechnol. 24 (1), 73-75 (2006).</li><li>Spitsbergen, J. M., Kent, M. 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Here, we described two methods to investigate the invasive behavior of breast cancer cells in Tg (fli1:EGFP) zebrafish embryos, with perivitelline space and Doc injections. By injecting cancer cells labeled with chemical dye or fluorescent protein into transgenic zebrafish embryos, the dynamic and spatial characteristics of invasion and metastasis can be clearly tracked in real-time at the single-cell or cluster level under a fluorescence microscope. In most cases, the rapid progression of metastasis in zebrafis...

Overt cancer metastasis in the clinic comprises a series of complex and multi-step events known as the &#34;metastatic cascade&#34;. The cascade has been extensively reviewed and can be dissected into successive steps: local invasion, intravasation, dissemination, arrest, extravasation, and colonization1,2. A better understanding of the pathogenesis of cancer metastasis and the development of potential treatment strategies in vivo require robust host models of cancer cell spread. Rodent models are well established and are widely used to evaluate metastasis3, but these approaches have low efficiency and ethical limitations and are costly as a forefront model to determine whether a particular manipulation could affect the metastatic phenotype. Other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. Due to their high genetic homology to humans and the transparency of their embryos zebrafish (Danio rerio) have emerged as an important vertebrate model and are being increasingly applied to the study of developmental processes, microbe-host interactions, human diseases, drug screening, etc.4. The cancer metastasis models established in zebrafish may provide an answer to the shortcomings of rodent models5,6.
Although spontaneous neoplasia is scarcely seen in wild zebrafish7, there are several longstanding techniques to induce the desired cancer in zebrafish. Carcinogen-induced gene mutations or signaling pathway activation can histologically and molecularly model carcinogenesis, mimicking human disease in zebrafish7,8,9. By taking advantage of diverse forward and reverse genetic manipulations of oncogenes or tumor suppressors, (transgenic) zebrafish have also enabled potential studies of cancer formation and maintenance6,10. The induced cancer models in zebrafish cover a broad spectrum, including digestive, reproductive, blood, nervous system, and epithelial6.
The utilization of zebrafish in cancer research has expanded recently due to the establishment of human tumor cell xenograft models in this organism. This was first reported with human metastatic melanoma cells that were successfully engrafted in zebrafish embryos at the blastula stage in 200511. Several independent laboratories have validated the feasibility of this pioneering work by introducing a diverse range of mammalian cancer cells lines into zebrafish at various sites and developmental stages5. For example, injections near the blastodisc and blastocyst of the blastula stage; injections into the yolk sac, perivitelline space, duct of Cuvier (Doc), and posterior cardinal vein of 6-h- to 5-day-old embryos; and injections into the peritoneal cavity of 30-day-old immunosuppressed larvae have been performed5,12. Additionally, allogeneic tumor transplantations were also reported in zebrafish12,13. One of the great advantages of using xenografts is that the engrafted cancer cells can be easily fluorescently labeled and distinguished from normal cells. Hence, investigations into the dynamic behaviors of microtumor formation14, cell invasion and metastasis15,16,17, tumor-induced angiogenesis15,18, and the interactions between cancer cells and host factors17 can be clearly visualized in the live fish body, especially when transgenic zebrafish lines are applied5.
Inspired by the high potential of zebrafish xenograft models to evaluate metastasis, we demonstrated the transvascular extravasation properties of different breast cancer cell lines in the tailfin area of Tg (fli:EGFP) zebrafish embryos through Doc injections16. The role of transforming growth factor-&#946; (TGF-&#946;)16 and bone morphogenetic protein (BMP)19 signaling pathways in pro-/anti-breast cancer cell invasion and metastasis were also investigated in this model. Moreover, we also recapitulated the intravasation ability of various breast cancer cell lines into circulation using xenograft zebrafish models with perivitelline space injections.
This article presents detailed protocols for zebrafish xenograft models based upon the injection of human breast cancer cells into the perivitelline space or Doc. Using high-resolution fluorescence imaging, we show the representative process of intravasation into blood vessels and the invasive behavior of different human breast cancer cells, which move from the blood vessels into the avascular tailfin area.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>MP Biomedicals</td>
			<td>AGAF0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Harvard Apparatus</td>
			<td>300038</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera enterotoxin&#160;</td>
			<td>Calbiochem</td>
			<td>227035</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP5 STED</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-high glucose media containing L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 media containing L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>21041025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Merck Millipore</td>
			<td>01-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereo microscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>SigmaAldrich</td>
			<td>227035</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>SigmaAldrich</td>
			<td>I-6634</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF10A (M1) cell line</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)&#160;</td>
		</tr>
		<tr>
			<td>MCF10Aras (M2) cell line</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA)&#160;</td>
		</tr>
		<tr>
			<td>MDA-MB-231 cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual micromanipulator&#160;</td>
			<td>World Precision Instruments</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-tip Pasteur pipette (0,5-20 ul)</td>
			<td>Eppendorf</td>
			<td>F276456I</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSVG plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PLV-mCherry plasmid</td>
			<td>Addgene</td>
			<td>36084</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg-RRE (gag/pol) plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Houben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Pneumatic picoPump</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>SigmaAldrich</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 4 software</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV plasmid</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Prof. Dr. Rob Hoeben (Leiden University Medical Center, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg (fli:EGFP) zebrafish strain</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Dr. Ewa Snaar-Jagalska (Institute of Biology, Leiden University, Leiden, The Netherlands)</td>
		</tr>
		<tr>
			<td>Tris-base&#160;</td>
			<td>SigmaAldrich</td>
			<td>11814273001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (3-aminobenzoic acid)</td>
			<td>SigmaAldrich</td>
			<td>A-5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%)</td>
			<td>ThermoFisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene (60 &#215; 15 mm)</td>
			<td>SigmaAldrich</td>
			<td>P5481-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene dish with glass bottom</td>
			<td>WillCo</td>
			<td>GWST-5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

invasive behavior of human breast cancer cells in embryonic zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

In the embryonic xenograft zebrafish model with a perivitelline space injection, the hematogenous dissemination of labeled cancer cells in the fish body is considered as active migration. This process can be detected and quantified under a fluorescent microscope, as described in the methods above. To illustrate this xenograft model, we followed the dissemination process of different breast cancer cell lines with known (or without) invasion/metastasis potential according to in vitro</e...

Watch this Scientific Journal Video about Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish at JoVE.com

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

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Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...