Name: testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)
Uploaded: 2016-11-03T15:00:00
Description: Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

We thank Peter Johnson of the Georgetown University Microscopy Core for assistance with imaging the zebrafish embryos. The Microscopy &amp; Imaging Shared Resource and the Zebrafish Shared Resource are partially supported by NIH/NCI grant P30-CA051008. This work was also supported by NIH/NCI CA71508 (AW) and CA177466 (AW).

Etikk Uttalelse: sebrafisk embryoer ble generert i henhold til godkjent IACUC protokollen. Disse eksperimentene ble utført i samsvar med anbefalinger fra Georgetown University Animal Care og bruk komité. 1. Organiser Embryoet for injeksjon og Opprett Stock Solutions <ol><li> Generere nødvendige sebrafisk larver å vurdere kreftcelle vaskulær invasjon. <ol><li> Sett opp parvis eller gruppe i-cross parring med Tg (kdrl: grcfp) zn1, mitfa b692, ednrb1...

<ol>
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Denne teknikken utnytter sebrafisk modell for effektivt å teste den vaskulære invasiv evnen av kreftceller (se figur 1). Her påføres vi teknikken til et panel av brystkreftcellelinjer for å tilveiebringe en grunnlinje på hvilken andre forskere kan deretter bygge sine egne studier (se tabell 1; figurer 2 - 3). Den observasjon at MDA-MB-231-celler lett invaderte inn i den caudale området av sebrafisk embryoer ville gjøre denne cellelinjen ideell for testing av mid...

Metastatisk sykdom er en vesentlig årsak til kreft dødelighet og mange mekanismer som gjør kreftcelle formidling gjenstår å bli oppdaget en. For at en kreftcelle for å kunne spre, må det først invadere gjennom stroma som omgir en primærtumor, skriv (intravasate) i sirkulasjonssystemet, overleve i transitt, exit (ekstravasere) fra sirkulasjonen, og til slutt etablere en levedyktig koloni på den fjerne organ for 2.. Intravasation og bloduttredelse er dermed avgjørende skritt i metastatisk cascade, men hver kreftcelle er ikke iboende flinke til å forstyrre og migrere gjennom endothelial veikryss 3. Faktisk er det en rekke unike seleksjonstrykk som omgir kreftcelle vaskulær invasjon og fremgangsmåten kan videre påvirkes av en rekke endogene og eksogene fire faktorer. Av disse grunner teknikker som tester den aggressive oppførselen til avansert stadium kreften ofte fokus på vascular invasiv evne som et middel til å forutsi metastatisk spredning. Ulike modellsystemer eksisterer for å legge til rette studiet av kreft celle vaskulær invasjon in vitro. Den mest benyttede in vitro-analyser innebære enten Transwell-systemer for å vurdere kreftcellemigrering gjennom en endotelial barriere 5 eller elektrisk celle-substrat Impedans Sensing (ECIS) teknologi for å overvåke i sanntid avbrudd av en intakt endotel-monolag av kreftceller 6. Disse analyser mangler vanligvis den væskedynamikk og stromale faktorer som ellers ville påvirke kreftcellebinding til en endotelial vegg. Dette problemet er noe omgått ved perfusable vaskulære nettverk som oppstår fra 3D-kulturen i endothelia med støtte stromale celler, og disse 3D microfluidic systemer representerer nå i forkant av dagens in vitro alternativer 7,8. Likevel, disse metodene utelate robust mikromiljøet av en funksjonell sirkulasjonssystemetog derfor bare delvis erstatning for in vivo-modeller. Den mest brukte in vivo modell av vaskulær invasjon er musa, hvori eksperimentelle metastase analysene er vanligvis utføres fordi de forekommer i forholdsvis korte tidsrammer, og er generelt et tegn på metastatisk evne 9. Disse analyser innebære direkte injeksjon av kreftceller i omløp og derfor modellere slutten stadier av metastasering, nemlig ekstravasasjon og kreftcelle kolonisering av organer. De eksperimentelle metastase analyser variere basert på tuftene av kreftcelle injeksjon og organene til slutt analysert. I det første analysetype, blir kreftceller injisert inn i halevenen til mus og kreftcelle seeding i lungene overvåkes 10,11. Den andre analysen innebærer utførelse intrakardielle injeksjoner for å lede metastatisk seeding mot benet mikromiljøet 12-14, men også hjernen 15. I den tredje analysen, kreft calen blir injisert inn i milten for å tillate kolonisering av leveren 16, mens den fjerde leveringsruten inn i karotidarterien bærer kreftceller i hjernen 17,18. Uavhengig av kreftcellen leveringsmåte, er organ kolonisering akseptert eksperimentelle endepunktet og er generelt bestemmes via luminescens, histologi, eller PCR-baserte teknikker. Til tross for de fysiologiske fordelene ved å drive eksperimentell metastase analyser innenfor en muse vert, disse eksperimentene fortsatt kreve uker til måneder å fullføre og analysere. Sebrafisk (Danio rerio) modellen har nylig dukket opp som et nytt system for å studere kreft progresjon 19,20, og åpner for vurdering av kreftcellen vaskulær invasjon innenfor en funksjonell sirkulasjonssystemet over en mye kortere tidsskala sammenlignet med mus 21-24. Metoden benytter en gjennomsiktig sebrafisk stamme som har sin endothelia merket med en grønn rev korall fluorescent protein reporter drevet av kdrl promoter, sebrafisk reseptor for vaskulær endotelial vekstfaktor 25. I analysen blir kreftceller merket med et rødt fluorescerende markør og injisert i precardiac sinus av to dager gamle embryoer. Hvor som helst mellom 48 til 96 timer etter injeksjonen, kan kreftceller som har invadert ut av vaskulaturen og inn i den caudale området av embryoer scores effektivt på et fluorescerende mikroskop. Her kan vi bruke teknikken til et panel av vanlig anvendte humane brystcancercellelinjer for å demonstrere sterk forskjeller i deres vaskulære invasive egenskaper. Videre demonstrerer vi at ved å endre injeksjonsstedet til embryoet plommesekken tillater studiet av heterogene celleinteraksjoner, som cancercellepopulasjoner kan differensielt merket med fluorescerende fargestoffer og injisert i sebrafisk embryoer mangler fluorescerende vaskulatur. I denne sistnevnte analyse, har kreftceller som har invadert den eggeplomme og intravasated inn i vasculature blir scoret i hale regionen 24-48 timer etter injeksjon. På grunn av effektiviteten og bekvemmeligheten av denne modellen, er sebrafisk stadig ansatt for å raskt teste vascular invasiv evne til kreftceller under en fysiologisk innstilling.

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			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-G Potassium</td>
			<td>Fisher Biotech</td>
			<td>BP914-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Plates, 100mm x 15mm</td>
			<td>Fisher Brand</td>
			<td>&#160;FB0875713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer II</td>
			<td>General Valve Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin Sulfate</td>
			<td>Fisher Biotech</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiI</td>
			<td>ThermoFisher Scientific</td>
			<td>V22885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiO</td>
			<td>ThermoFisher Scientific</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zebrafish&#160;</td>
			<td>Georgetown Zebrafish Shared Resources</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and HCC18-6 cells, which were mantained in RPMI + 10% FBS.</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Her testet vi den vaskulære invasiv evne brukte brystkreft cellelinjer i en sebrafisk embryo modell (figur 1). Strenge kriterier var ansatt i scoring bloduttredelse for disse ulike cellelinjer, hvor positive hendelser ble bare telles hvis kreftcellene hadde klart ekstravasert, dette blir gjort først og fremst for å begrense eventuelle falske-positive som kan oppstå fra ledelsen mobilnettet rusk. Vår analy...

Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

Denne metoden benytter sebrafisk embryoer for effektivt å teste den vaskulære invasiv evne til kreftceller. Fluorescent kreftceller blir injisert i precardiac sinus eller plommesekken utviklings embryoer. Kreftcelle vaskulær invasjon og ekstravasering blir vurdert via fluorescens mikroskopi av halen området 24 til 96 timer senere.

Teste Vaskulær Invasive Evne av kreftceller i Sebrafisk (<em&gt; Danio rerio</em&gt;)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Research

JoVE Journal

Cancer Research

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Isolering av sirkulerende tumorceller i en ortotopisk musemodell av kolorektal kreft

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Invasiv Opptreden av humane brystcancerceller i Embryonic Sebrafisk

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 ...

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

En Time-lapse, etikett-fri, kvantitativ fase tenkelig studie av sovende og aktive menneskelige kreftceller

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

In Vivo Målretting av Xenografted Human Cancer Celler med funksjonalisert fluorescerende silika nanopartikler i sebrafisk

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Generasjon av sebrafisk Larval Xenografts og tumor atferdsanalyse

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Ikke-invasiv PET/MR-avbildning i en ortotopisk musemodell av hepatocellulært karsinom

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Samtidig avbildning og flowcytometribasert påvisning av flere fluorescerende senescensmarkører i terapiinduserte senescent kreftceller

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Modellering av hjernemetastase ved intern karotisarterieinjeksjon av kreftceller

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Sebrafisk larver som en modell for å evaluere potensielle radiosensibilisatorer eller beskyttere

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Ortotopisk implantasjon av pasientavledede kreftceller hos mus rekapitulerer avansert kolorektal kreft

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...