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

Studier av TGF-β familjemedlemmar stöds av Cancer Genomics Center Nederländerna. Sijia Liu och Jiang Ren stöds av Kina stipendium rådet för 4 års studier vid universitetet i Leiden. Vi tackar Dr Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA) för cellinjerna MCF10A.

All forskning med den transgena fluorescerande zebrafisk Tg (fli: EGFP) stam, som har förbättrat grönt fluorescerande protein (EGFP) -märkt vaskulatur 20, inklusive bostäder och experiment genomfördes i enlighet med de internationella riktlinjer och godkändes av den lokala Institutional kommittén for Animal Welfare (Dier Ethische Commissie (DEC) i Leiden University Medical Center. OBS: Som sammanfattas i figur 1, är det protokoll som grovt delas upp i fyra steg: embryosamlin...

<ol>
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Här, beskrev vi två metoder för att undersöka det invasiva beteendet hos bröstcancerceller i Tg (FLI1: EGFP) zebrafisk embryon, med perivitellina utrymmes- och Doc injektioner. Genom att injicera cancerceller märkta med kemisk färgämne eller fluorescerande protein i transgena zebrafisk embryon, kan de dynamiska och rumsliga egenskaper invasion och metastas tydligt spåras i realtid vid encelliga eller klusternivå under ett fluorescensmikroskop. I de flesta fall, säkerställer den snabba utvecklingen a...

Overt cancermetastas i kliniken innefattar en serie av komplexa och flera steg händelser känd som &quot;metastaserande kaskad&quot;. Kaskaden har i stor utsträckning granskats och kan dissekeras in successiva steg: lokal invasion, intravasering, spridning, gripande, extravasering och kolonisering 1, 2. En bättre förståelse av patogenesen av cancermetastaser och utveckling av potentiella behandlingsstrategier in vivo kräver robusta värd modeller av cancer cell spridning. Gnagarmodeller är väletablerade och används i stor utsträckning för att utvärdera metastas 3, men dessa tillvägagångssätt har låg verkningsgrad och etiska begränsningar och är kostsamma som en framkant modell för att bestämma huruvida en speciell manipulation kan påverka den metastatiska fenotypen. Det behövs andra effektiva, tillförlitliga, billiga modeller för att snabbt komma åt de potentiella effekterna av (EPI) genetiska förändringar eller Pharmacological föreningar. På grund av deras höga genetiska homologi till människor och insyn i deras embryon zebrafisk (Danio rerio) har dykt upp som en viktig ryggradsdjur modell och tillämpas i allt högre grad till studiet av utvecklingsprocesser, mikrob-värd-interaktioner, mänskliga sjukdomar, läkemedelsscreening, etc . 4. De cancermetastas modeller är etablerade i zebrafisk kan ge ett svar på bristerna i gnagarmodeller 5, 6. Även om spontan neoplasi knappast ses i vildzebrafisk 7, finns det flera långvariga tekniker för att framkalla den önskade cancer hos zebrafisk. Karcinogen-inducerade genmutationer eller signaleringsvägen aktivering kan histologiskt och molekylärt modell karcinogenes, imitera human sjukdom i zebrafisk 7, 8, 9. Med taking fördel av olika framåt- och bakåt genetiska manipulationer av onkogener eller tumörsuppressorer, (transgena) zebrafisk har också gjort det möjligt potentiella studier av cancerbildning och underhåll 6, 10. De inducerade cancermodeller i zebrafisk täcka ett brett spektrum, inklusive matsmältnings, reproduktiva, blod, nervsystemet, och epitelial 6. Utnyttjandet av zebrafisk inom cancerforskningen har expanderat nyligen på grund av etableringen av humana tumörcells xenograft-modeller i denna organism. Detta rapporterades först med humana metastaserande melanomceller som framgångsrikt inympade i zebrafisk embryon vid blastula skede 2005 11. Flera oberoende laboratorier har validerat genomförbarheten av detta banbrytande arbete genom att införa en mängd olika däggdjurscancercellinjer i zebrafisk vid olika platser och utvecklingsstadier 5 &lt;/ Sup&gt;. Till exempel injektioner nära blastocysten och blastocyst av blastula skede; injektioner i gulesäcken, perivitellina utrymme, kanalen hos Cuvier (Doc), och posterior kardinal åder av 6-h- till 5 dygn gamla embryon; och injektioner i bukhålan av 30 dagar gamla immunsupprimerade larver har utförts 5, 12. Dessutom var allogen tumörtransplantationer rapporterades också i zebrafisk 12, 13. En av de stora fördelarna med att använda xenotransplantat är att transplanterade cancerceller kan enkelt fluorescensmärkt och särskiljas från normala celler. Hence, undersökningar av de dynamiska beteenden av microtumor bildning 14, cellinvasion och metastas 15, 16, 17, tumörinducerad angiogenes 15, en8, och interaktionerna mellan cancerceller och värdfaktorer 17 kan tydligt visualiseras i levande fisk kroppen, speciellt när transgena zebrafisklinjer appliceras 5. Inspirerad av den höga potentialen för zebrafisk xenograft-modeller för att utvärdera metastas, vi visat de transvaskulär extravasation egenskaper hos olika bröstcancercellinjer i stjärtfenan området för Tg (fli: EGFP) zebrafisk embryon genom Doc injektioner 16. Rollen av transformerande tillväxtfaktor-β (TGF-β) 16 och benmorfogenetiskt protein (BMP) 19 signalvägar i pro- / anti-bröstcancercellinvasion och metastas undersöktes också i denna modell. Dessutom rekapituleras vi också intravasering förmågan hos olika bröstcancercellinjer i omlopp med hjälp av xenograft zebrafisk modeller med perivitellina utrymme injektioner. <p class = &quot;jove_content&quot;&gt; Den här artikeln presenterar detaljerade protokoll för zebrafisk xenograft modeller baserade på injektion av humana bröstcancerceller i perivitellina utrymme eller Doc. Med användning av högupplösande fluorescensavbildning, visar vi det representativa förfarandet enligt intravasering i blodkärl och det invasiva beteendet hos olika humana bröstcancerceller, som rör sig från blodkärlen in i den avaskulära tailfin området.

<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

I många fall gör cancerpatienter inte dö av en primärtumör, utan snarare på grund av metastaser. Trots att många gnagare modeller finns för att studera cancermetastaser in vivo behövs andra effektiva, tillförlitliga, modeller låg kostnad för att snabbt få tillgång till de potentiella effekterna av (EPI) genetiska förändringar eller farmakologiska föreningar. Som sådan, vi belysa och förklara möjligheten att xenograft modeller med hjälp av humana bröstcancerceller injiceras i zebrafisk embryon för att stödja detta mål. Under mikroskop, är fluorescerande proteiner eller kemiskt märkta humana bröstcancerceller transplanteras in i transgena zebrafiskembryon, Tg (fli: EGFP), vid perivitellina utrymme eller kanal av Cuvier (Doc) 48 h efter befruktning. Kort därefter, den temporala-spatial process av cancer cellinvasion, spridning och metastaser i den levande fiskkroppen visualiseras under ett fluorescerande mikroskop. Modellerna använder olika injektionsställen, dvs perivitelline utrymme eller Dok är komplementära till varandra, vilket återspeglar den tidigt stadium (intravasering steg) och sent stadium (extravasering steg) av flerstegs metastaserad kaskad av händelser. Vidare kan observeras peritumoral och intratumoral angiogenes med injektion i perivitellina rymden. Hela försöksperioden är inte mer än 8 dagar. Dessa två modeller kombinera cellmärkning, mikro-transplantation, och fluorescens avbildningstekniker, som möjliggör snabb utvärdering av cancermetastaser som svar på genetiska och farmakologiska manipulationer.

I den embryonala xenograft zebrafisk modell med en perivitellina utrymme injektion, är hematogen spridning av märkta cancerceller i fiskkroppen betraktas som aktiv migration. Denna process kan detekteras och kvantifieras under ett fluorescerande mikroskop, såsom beskrivits i metoderna ovan. För att illustrera denna xenotransplantatmodell följde vi spridningsprocessen av olika bröstcancercellinjer med känd (eller utan) invasion / metastas potential enligt in vitro och ...

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

Här beskriver vi xenograft zebrafisk modeller med hjälp av två olika injektionsställen, dvs perivitellina utrymme och kanal Cuvier, för att undersöka invasiva beteende och för att bedöma intravasering och extravasering potential av mänskliga bröstcancerceller, respektive.

Invasiv Beteende av humana bröstcancerceller i embryonal 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.

Research

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