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

Undersøgelser af TGF-β familiemedlemmer understøttes af Cancer Genomics Centre Holland. Sijia Liu og Jiang Ren understøttes af Kina Scholarship Rådet for 4 års studier ved universitetet i Leiden. Vi takker Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA) for de MCF10A cellelinjer.

Al forskning under anvendelse af transgene fluorescerende zebrafisk Tg (Fli: EGFP) stamme, som har forstærket grønt fluorescerende protein (EGFP) -mærket vaskulatur 20, herunder bolig og forsøg blev udført i overensstemmelse med de internationale retningslinier og er godkendt af den lokale Institutional Udvalg for Animal Welfare (Dier Ethische Commissie (DEC) i Leiden University Medical center. BEMÆRK: Som opsummeret i figur 1, er protokollen groft opdeles i fire trin: embryoni...

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Her beskrev vi to metoder til at undersøge den invasive opførsel af brystcancerceller i Tg (FLI1: EGFP) zebrafiskembryoer, med perivitelline rum og dok injektioner. Ved at injicere cancerceller mærket med kemisk farvestof eller fluorescerende protein i transgene zebrafiskembryoer, kan de dynamiske og rumlige karakteristika for invasion og metastase tydeligt spores i realtid på enkelt-celle eller klyngeniveau under et fluorescensmikroskop. I de fleste tilfælde den hurtige progression af metastase i zebrafis...

Åbenlys cancermetastase i klinikken omfatter en række komplekse og flere trin begivenheder kendt som &quot;metastatisk kaskade&quot;. Den kaskade er blevet grundigt gennemgået og kan dissekeres i successive trin: lokal invasion, intravasation, udbredelse, arrest, ekstravasation og kolonisering 1, 2. En bedre forståelse af patogenesen af kræft metastaser og udvikling af potentielle behandlingsstrategier in vivo kræver robuste vært modeller af kræftcelle spredning. Gnavermodeller er veletablerede og er almindeligt anvendt til at evaluere metastaser 3, men disse metoder har lav effektivitet og etiske begrænsninger og er dyre som en forkant model til at afgøre, om en bestemt manipulation kan påvirke metastatisk fænotype. Andre effektive, pålidelige, billige modeller er behov for hurtigt at få adgang til de potentielle virkninger af (epi) genetiske ændringer eller pharmacologgiske forbindelser. Grund af deres høje genetisk homologi til mennesker og gennemsigtigheden af deres embryoner zebrafisk (Danio rerio) er dukket op som en vigtig hvirveldyr model og i stigende grad anvendes til studiet af udviklingsprocesser, mikrobe-værtssammenspil, humane sygdomme, lægemiddelscreening, etc . 4. De kræft metastase-modeller, der er etableret i zebrafisk kan give et svar på manglerne i gnavermodeller 5, 6. Selvom spontan neoplasi næppe ses i vild zebrafisk 7, er der flere langvarige teknikker til at fremkalde den ønskede kræft i zebrafisk. Carcinogen-induceret genmutationer eller signalvejen aktivering kan histologisk og molekylært model carcinogenese, efterligner human sygdom i zebrafisk 7, 8, 9. ved taking fordel af forskelligartede forward og reverse genetiske manipulationer af onkogener eller tumorsuppressorer, (transgene) zebrafisk har også gjort det muligt potentielle studier af dannelsen og vedligeholdelsen 6, 10 cancer. De inducerede kræftmodeller i zebrafisk dækker et bredt spektrum, herunder fordøjelsessystemet, reproduktive, blod, nervesystem, og epitelial 6. Udnyttelsen af ​​zebrafisk i kræftforskning har udvidet for nylig på grund af etableringen af ​​menneskelige tumor-xenograftmodeller i denne organisme. Dette blev først rapporteret med human metastatisk melanom celler, der lykkedes indpodede i zebrafisk embryoner på blastula scenen i 2005 11. Flere uafhængige laboratorier har valideret gennemførligheden af dette pionerarbejde ved at indføre en bred vifte af cancerceller hos pattedyr linjer i zebrafisk på forskellige steder og udviklingsstadier 5 &lt;/ Sup&gt;. For eksempel injektioner nær kimskiven og blastocyt i blastula-stadiet; injektioner i blommesækken, perivitelline rum, kanalen i Cuvier (dok), og posterior kardinalvene af 6-H- til 5 dage gamle embryoner; og injektioner i peritonealhulen af 30 dage gammel immunundertrykt larver er blevet udført 5, 12. Derudover blev også rapporteret allogen tumor transplantationer i zebrafisk 12, 13. En af de store fordele ved at bruge xenotransplantater er, at podede cancerceller let kan fluorescensmærket og skelnes fra normale celler. Derfor undersøgelser af dynamiske adfærd af microtumor formation 14, og -metastase 15, 16, 17, tumor-induceret angiogenese 15, 18, og samspillet mellem cancerceller og vært faktorer 17 klart kan visualiseres i levende fisk krop, især når transgene zebrafisk linjer påføres 5. Inspireret af det store potentiale af zebrafisk xenograftmodeller at evaluere metastase, demonstrerede vi de transvaskulær ekstravasation angår forskellige brystcancercellelinier i halefinne område i Tg (Fli: EGFP) zebrafiskembryoer gennem Doc injektioner 16. Rollen af transformerende vækstfaktor-β (TGF-β) 16 og knoglemorfogenetisk protein (BMP) 19 signalveje i pro / anti-brystcancer og -metastase blev også undersøgt i denne model. Desuden har vi også sammenfattet den intravasation evne forskellige brystcancercellelinier i omsætning med xenograft zebrafisk modeller med perivitelline space injektioner. <p class = &quot;jove_content&quot;&gt; Denne artikel præsenterer detaljerede protokoller for zebrafisk xenograftmodeller baseret på injektionen af ​​humane brystcancerceller i perivitelline rum eller dok. Ved hjælp af høj opløsning fluorescensimagografi, viser vi den repræsentative proces med intravasation i blodkarrene og den invasive opførsel af forskellige humane brystcancerceller, som flytter fra blodkarrene i den avaskulære halefinne område.

<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>
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invasive behavior of human breast cancer cells in embryonic zebrafish

I mange tilfælde behøver cancerpatienter ikke dø af en primær tumor, men snarere på grund af metastaser. Skønt talrige gnavermodeller er tilgængelige til undersøgelse cancermetastase in vivo, er behov for andre effektive, pålidelige, billige modeller til hurtigt at få adgang de potentielle virkninger af (epi) genetiske ændringer eller farmakologiske forbindelser. Som sådan, vi illustrere og forklare mulighederne for xenograftmodeller med humane brystkræftceller sprøjtes ind zebrafiskembryoer at støtte dette mål. Under mikroskopet er fluorescerende proteiner eller kemisk mærkede humane brystcancerceller transplanteret ind transgene zebrafiskembryoer, Tg (Fli: EGFP), ved perivitelline rum eller kanalen i Cuvier (dok) 48 timer efter befrugtningen. Kort efter er den tidsmæssige-rumlige proces med cancercelleinvasion, formidling, og metastase i den levende fisk krop visualiseres under et fluorescerende mikroskop. Modellerne anvender forskellige injektionssteder, dvs. privitelline rum eller Doc er komplementære til hinanden, hvilket afspejler den tidlige fase (intravasation trin) og sen fase (ekstravasation trin) af flertrins-metastatiske kaskade af hændelser. Desuden kan peritumoral og intratumoral angiogenese iagttages med injektion i perivitelline rum. Hele forsøgsperiode er ikke mere end 8 dage. Disse to modeller kombinerer celle mærkning, mikro-transplantation, og fluorescens billeddannelse teknikker, giver mulighed for hurtig evaluering af kræft metastaser som reaktion på genetiske og farmakologiske manipulationer.

I det embryoniske xenograft zebrafisk model med en perivitelline space injektion er hæmatogen spredning af mærkede cancerceller i fiskekroppen betragtes som aktiv migration. Denne proces kan detekteres og kvantificeres under et fluorescensmikroskop, som beskrevet i fremgangsmåderne ovenfor. For at illustrere dette xenograft model, fulgte vi formidlingsprocessen forskellige brystcancercellelinier med kendt (eller uden) invasion / metastase potentiale ifølge in vitro og in...

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

Her beskriver vi xenograft zebrafisk modeller ved hjælp af to forskellige injektionssteder, dvs. perivitelline plads og kanal- af Cuvier, for at undersøge den invasive adfærd og vurdere intravasation og ekstravasation potentiale af humane brystkræftceller, hhv.

Invasiv Opførsel af humane brystcancerceller i embryonale zebrafisk

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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