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 over TGF-β familieleden worden ondersteund door het Cancer Genomics Centre Nederland. Sijia Liu en Jiang Ren worden ondersteund door de China Scholarship Council voor 4 jaar van studie aan de Universiteit van Leiden. Wij danken Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA) voor de MCF10A cellijnen.

Al het onderzoek met behulp van de transgene fluorescerende zebravis Tg (Amst: EGFP) stam, die groen fluorescerend eiwit is verbeterd (EGFP) gemerkt vaatstelsel 20, met inbegrip van huisvesting en experimenten, werd uitgevoerd in overeenstemming met de internationale richtlijnen en werd goedgekeurd door de lokale Institutional Comité for Animal Welfare (Dier Ethische commissie (DEC) van het Leids Universitair medisch Centrum. Opmerking: Zoals weergegeven in figuur 1, wordt het proto...

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Hier beschreven we twee methoden om de invasief gedrag van borstkankercellen in Tg (fli1: EGFP) onderzoeken zebravis embryo&#39;s, met perivitelline ruimte en Doc injecties. Door het injecteren van kankercellen gelabeld met chemische kleurstof of fluorescent eiwit in transgene zebravis embryo, kan de dynamische en ruimtelijke eigenschappen van invasie en metastase duidelijk worden bijgehouden in real time bij de enkele-cel of cluster niveau onder een fluorescentiemicroscoop. In de meeste gevallen, de snelle pro...

Openlijke uitzaaiing in de kliniek omvat een reeks complexe en multi-step events zogenaamde &quot;metastatische cascade&quot;. De cascade is uitvoerig beoordeeld en kan worden ontleed in opeenvolgende stappen: de lokale invasie, intravasation, verspreiding, arrestatie, extravasatie, en kolonisatie 1, 2. Een beter begrip van de pathogenese van kanker metastase en de ontwikkeling van potentiële therapeutische strategieën in vivo vereisen robuuste gastheer modellen van kankercel te verspreiden. Knaagdiermodellen zijn goed ingeburgerd en worden veel gebruikt voor metastase 3 evalueren, maar deze benaderingen hebben lage efficiëntie en ethische beperkingen en zijn kostbaar als voorgrond model om te bepalen of een bepaalde manipulatie kunnen hebben voor de metastatische fenotype. Andere efficiënte, betrouwbare, goedkope modellen zijn nodig om snel toegang te krijgen tot de mogelijke effecten van (epi) genetische veranderingen of pharmacologsche verbindingen. Vanwege hun hoge genetische homologie voor mens en transparantie van de embryo zebravis (Danio rerio) naar voren gekomen als een belangrijke gewervelde model en worden steeds meer toegepast voor het onderzoek van ontwikkelingsprocessen, microbe-gastheer interacties, menselijke ziekten, screening van geneesmiddelen, enz . 4. De in de zebravis gevestigde uitzaaiing van kanker modellen kunnen een antwoord op de tekortkomingen van knaagdiermodellen 5, 6 te bieden. Het spontaan neoplasie nauwelijks is te zien in wild zebravis 7, zijn er verschillende langdurige technieken om de gewenste kanker in zebravis induceren. Carcinogeen-geïnduceerde genmutaties of signaleringsroute activering kan histologisch en moleculair model carcinogenese, nabootsen menselijke ziekte zebravis 7, 8, 9. door taking maken van diverse voorwaartse en omgekeerde genetische manipulaties van oncogenen of tumor suppressors, (transgene) zebravis konden ook potentieelstudies van vorming en instandhouding 6, 10 kanker. De geïnduceerde kankermodellen in zebravis bestrijken een breed spectrum, zoals spijsvertering, voortplanting, bloed, zenuwstelsel en epitheliale 6. Het gebruik van zebravis in kankeronderzoek is onlangs uitgebreid als gevolg van de oprichting van de menselijke tumorcel xenograft modellen in dit organisme. Dit werd voor het eerst gemeld met menselijke gemetastaseerd melanoom cellen die met succes werden geënt in zebravis embryo&#39;s in het blastula stadium in 2005 11. Verschillende onafhankelijke laboratoria hebben de haalbaarheid van dit pionierswerk gevalideerd door het inbrengen van een breed scala van zoogdierlijke kankercellen lijnen in zebravis op verschillende plaatsen en ontwikkelingsstadia 5 &lt;/ Sup&gt;. Bijvoorbeeld injecties bij de blastoschijf en blastocyst van het blastula stadium; injecties in de dooierzak, perivitelline ruimte, kanaal van Cuvier (Doc), en achterste hoofdader van 6-H- tot 5 dagen oude embryo&#39;s; en injecties in de buikholte van de 30-dagen oude immuungesuppresseerde larven zijn uitgevoerd 5, 12. Bovendien werden allogene transplantaties tumor ook vermeld in zebravis 12, 13. Een van de grote voordelen van het gebruik van xenotransplantaten is dat de geënte kankercellen kan gemakkelijk fluorescerend gemerkt en onderscheiden van normale cellen. Derhalve onderzoek naar het dynamische gedrag van microtumor formatie 14 cellen invasie en metastase 15, 16, 17, door tumor geïnduceerde angiogenese 15, 18, en de interacties tussen kankercellen en gastheerfactoren 17 duidelijk worden gevisualiseerd in levende vislichaam, vooral wanneer transgene zebravis lijnen toegepast 5. Geïnspireerd door het grote potentieel van zebravis xenograftmodellen metastase evalueren we aangetoond dat de transvasculaire extravasatie eigenschappen van verschillende borstkanker cellijnen in de staartvin gebied van Tg (Amst: EGFP) zebravis embryo&#39;s door middel van Doc injecties 16. De rol van transforming growth factor-β (TGF-β) 16 en bot morfogenetisch proteïne (BMP) 19 signaalwegen in pro- / anti-borstkankercellen invasie en metastase werden ook onderzocht in dit model. Bovendien hebben we recapituleerde ook de intravasation vermogen van verschillende borstkanker cellijnen in het verkeer met behulp van xenograft zebravismodellen met perivitelline ruimte injecties. <p class = &quot;jove_content&quot;&gt; Dit artikel presenteert gedetailleerde protocollen voor zebravis xenograft modellen gebaseerd op de injectie van humane borstkankercellen in de perivitelline ruimte of doe. Met behulp van hoge-resolutie fluorescentie beeldvorming, tonen we de representatieve werkwijze van intravasation in bloedvaten en het invasieve gedrag van andere menselijke borstkankercellen, die zich van de bloedvaten in de avasculaire zone staartvin.

<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 veel gevallen hebben kankerpatiënten niet sterven van een primaire tumor, maar eerder als gevolg van metastase. Hoewel tal van knaagdier modellen zijn beschikbaar voor het bestuderen van uitzaaiing van kanker in vivo, worden ook andere efficiënte, betrouwbare, goedkope modellen die nodig zijn om snel toegang te krijgen tot de mogelijke effecten van (epi) genetische veranderingen of farmacologische verbindingen. Als zodanig, we illustreren en uitleg over de haalbaarheid van het xenograft modellen met behulp van menselijke borstkankercellen geïnjecteerd in zebravis embryo&#39;s om dit doel te ondersteunen. Onder de microscoop zijn fluorescerende eiwitten of chemisch gemerkte menselijke borstkankercellen getransplanteerd in transgene zebravis embryo, Tg (fli: EGFP), de perivitelline ruimte of kanaal van Cuvier (doe) 48 uur na bevruchting. Kort daarna is de tijdruimtelijke proces van kankercel invasie, verspreiding, en metastase in het levende vis lichaam zichtbaar gemaakt onder een fluorescentiemicroscoop. De modellen met behulp van verschillende injectieplaatsen, dat wil zeggen, perivitelline ruimte of Doc complementair aan elkaar, als gevolg van de vroege fase (intravasation stap) en de late fase (stap extravasatie) van de meerstaps metastatische cascade van gebeurtenissen. Bovendien kan peritumorale en intratumorale angiogenese worden waargenomen met de injectie in de perivitelline ruimte. De gehele experimentele periode is niet meer dan 8 dagen. Deze twee modellen combineren cel labeling, micro-transplantatie, en fluorescentie beeldvormende technieken, waardoor de snelle evaluatie van kanker metastase in reactie op de genetische en farmacologische manipulaties.

In de embryonale xenograft zebravis model met een perivitelline ruimte injectie wordt de hematogene verspreiding van gemerkte kankercellen in de vis lichaam beschouwd als actieve migratie. Dit proces kan worden gedetecteerd en gekwantificeerd onder een fluorescentiemicroscoop, zoals beschreven in de werkwijzen hierboven. Deze xenograft model te illustreren, volgden we de verspreidingsproces verschillende borstkankercellijnen met bekende (of zonder) invasie / metastase potentieel volgens ...

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

We beschrijven hier xenograft zebravismodellen behulp van twee verschillende injectieplaatsen, dat wil zeggen, perivitelline ruimte en het kanaal van Cuvier, aan de invasief gedrag te onderzoeken en de intravasation en extravasatie potentieel van menselijke borstkankercellen respectievelijk beoordelen.

Invasief gedrag van menselijke borstkankercellen in embryonale zebravis

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 ...

The overall goal of this microinjection of human breast cancer cells into embryonic zebrafish is to investigate the invasive behavior and assess the metastatic potential of human breast cancer cells. This method can help to answer key questions regarding intravasation and extravasation to critical processes in cancer metastasis. The main advantage of this technique is that it provides an elegant, rapid, and cheap in vivo model to measure the ability of cancer cells to invade and metastasize.The implications of this technique extend towards the development of new pharmacological compounds with anti-metastatic activity. Though this measure can provide insight into cancer emission and the metastasis, it can also be applied to study some other phenomena, such as peritumoral angiogenesis and cancer cell dormancy. To begin, culture transduced human breast cancer MDA-MB-231 cells, breast epithelial MCF10A cells, and breast epithelial MCF10A-Ras cells in a T75 flask.Once the transduced cells reach 80%confluence, remove the medium from the T75 flask, wash the cells with phosphate-buffered saline, and detach the cells with 0.5%Trypsin-EDTA solution. Then neutralize the Trypsin with medium. Next, centrifuge the cells at 1, 000 RPM for five minutes to remove the medium, wash them with PBS, and centrifuge the sample one more time.After washing, resuspend the cells in approximately 200 microliters of PBS to obtain a suspension of no more than 1 x 10 to the eighth cells per milliliter. Store the cell suspension in four degrees Celsius for no longer than five hours. To prepare zebrafish embryos for implantation, incubate approximately 60 embryos in a petri dish filled with egg water, supplemented with 60 micrograms per milliliter of sea salts at 28 degrees Celsius.Then, using fine tweezers, remove the coriands from the embryos at 48 hours post-fertilization. To anesthetize the embryos for the implantation procedure, transfer them to a 40 microgram per milliliter solution of tricaine in egg water. Prior to the implantation, prepare injection needles.To do this, load a borosilicate microcapillary tube into a micro-pipette puller and pull the needle. Store the prepared needles in a needle-holder plate until needed. Aspirate 15 microliters of the prepared cell suspension into an injection needle.Next, attach the needle to a micromanipulator, and, using fine tweezers, break its tip to five to 10 microns. Adjust the pneumatic PICO pump and inject cells onto the petri dish pre-coated with sterile 1%agarose. Then, transfer approximately 10 anesthetized embryos at two to three days post-fertilization onto a flat injecting plate coated with 1%agarose.Arrange the position of the embryos with a hair loop tool so that they are oriented in the same direction. While injecting, manually adjust the injection plate position to place the embryos in a diagonal orientation, which is preferred for a precise needle insertion. Direct the needle tip at the injection site and gently insert it into the perivitelline space localized between the yolk sac and the paraderm of the zebrafish embryo.Lastly, inject approximately 400 tumor cells labeled with mCherry. Make sure not to rupture the yolk sac to avoid a misplaced implantation. To inject the cells into the duct of Cuvier, approach the vein from the dorsal side of the embryo with the needle kept at a 45-degree angle.Insert the needle at the starting point of the duct of Cuvier, localized dorsally to the point where the duct starts broadening over the yolk sac. Then, inject approximately 400 cells. Finally, transfer the injected zebrafish embryos to the egg water, and incubate them at 33 degrees Celsius until further analysis.To visualize metastasis, with a Pasteur pipette, transfer the injected zebrafish embryo to a glass-bottom polystyrene dish and remove excess egg water. Image the whole body of the embryo with a confocal microscope set to low magnification to obtain a general pattern of tumor cell dissemination. Use a 488 nanometer laser to visualize the zebrafish embryo vasculature, and a 543 nanometer laser to visualize implanted tumor cells labeled with red fluorescent marker.Next, scan the embryo in eight to 10 steps to acquire a high-quality image. Presented here are photographs of zebrafish embryos that were taken with a confocal microscope three days after perivitelline injection of MDA-MB-231 cells. MDA-MB-231 cells widely disseminated within the zebrafish body, manifesting highly invasive potential.MCF10A-Ras cells, when implanted to zebrafish embryos, revealed moderate invasive capacity with only a few cells spread to distant parts of the body. Upon transplantation, MCF10A cells remained non-invasive and concentrated within the injection site, confirming that all studied cells reveal different invasive potential. The highly invasive character of MDA-MB-231 cells was reflected in their enhanced migration capacity observed within the perivitelline space.To some extent, such migration was also observed for MCF10A-Ras cells. On the contrary, MCF10A cells did not migrate in the perivitelline space. Distinct metastatic behavior of MDA-MB-231 and MCF10A-Ras cells was also observed when both cell types were implanted into the embryos'duct of Cuvier.As shown, MDA-MB-231 cells migrated independently of one another, while MCF10A-Ras were prone to migrate in clusters. Following this procedure, other measures like immunofluorescence can be performed in order to answer additional questions, such as how cancer cells proliferate in the zebrafish, and how they affect the surrounding host of cells. After watching this video, you will have a good understanding of how to use zebrafish model to study an invasive behavior and assize intravasation and extravasation capacity of human breast cancer cells.

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