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).

Etik Statement: zebrafisk embryoner blev genereret efter en godkendt IACUC protokol. Disse forsøg blev udført i overensstemmelse med anbefalinger fra Georgetown University Animal Care og brug Udvalg. 1. Organiser Embryoner til injektion og Opret Stock Solutions <ol><li> Generere nødvendige zebrafisk larver at vurdere kræftcelle vaskulær invasion. <ol><li> Opsæt parvis eller gruppe i-cross parring med Tg (kdrl: grcfp) Zn1, mitfa b692; ednrb1 ...

<ol>
	<li>Cummings, M. C., 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=Metastatic+progression+of+breast+cancer:+insights+from+50+years+of+autopsies.">Metastatic progression of breast cancer: insights from 50 years of autopsies.</a> Am J Path. 232, 23-31 (2014).</li><li>Nguyen, D. X., Bos, P. D., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Reymond, N., d'&#193;gua, B. B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+endothelial+barrier+during+metastasis.">Crossing the endothelial barrier during metastasis.</a> Nat Rev Cancer. 13 (12), 858-870 (2013).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Method Enzymol. 409, 625-642 (2006).</li><li>Rahim, S., &#220;ren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+electrical+impedance+based+technique+to+measure+invasion+of+endothelial+cell+monolayer+by+cancer+cells.">A real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells.</a> J Vis Exp. (50), (2011).</li><li>Jeon, J. S., 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=Human+3D+vascularized+organotypic+microfluidic+assays+to+study+breast+cancer+cell+extravasation.">Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation.</a> Proc Natl Acad Sci USA. 112 (1), 214-219 (2015).</li><li>Shin, Y., 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=Microfluidic+assay+for+simultaneous+culture+of+multiple+cell+types+on+surfaces+or+within+hydrogels.">Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.</a> Nat Protoc. 7 (7), 1247-1259 (2012).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Mohanty, S., Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+metastasis+assay.">Experimental metastasis assay.</a> J Vis Exp. (42), (2010).</li><li>Minn, A. J., 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=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Nature. 436 (7050), 518-524 (2005).</li><li>Campbell, J. P., Merkel, A. R., Masood-Campbell, S. K., Elefteriou, F., Sterling, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+bone+metastasis.">Models of bone metastasis.</a> J Vis Exp. (64), e4260 (2012).</li><li>Arguello, F., Baggs, R. B., Frantz, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+experimental+metastasis+to+bone+and+bone+marrow.">A murine model of experimental metastasis to bone and bone marrow.</a> Cancer Res. 48 (23), 6876-6881 (1988).</li><li>Minn, A. J., 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=Distinct+organ-specific+metastatic+potential+of+individual+breast+cancer+cells+and+primary+tumors.">Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors.</a> J Clin Invest. 115 (1), 44-55 (2005).</li><li>Bos, P. D., 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=Genes+that+mediate+breast+cancer+metastasis+to+the+brain.">Genes that mediate breast cancer metastasis to the brain.</a> Nature. 459 (7249), 1005-1009 (2009).</li><li>Soares, K. C., 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=A+preclinical+murine+model+of+hepatic+metastases.">A preclinical murine model of hepatic metastases.</a> J Vis Exp. (91), e51677 (2014).</li><li>Kienast, Y., 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=Real-time+imaging+reveals+the+single+steps+of+brain+metastasis+formation.">Real-time imaging reveals the single steps of brain metastasis formation.</a> Nature Med. 16 (1), 116-122 (2010).</li><li>Hasegawa, H., Ushio, Y., Hayakawa, T., Yamada, K., Mogami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+the+blood-brain+barrier+in+experimental+metastatic+brain+tumors.">Changes of the blood-brain barrier in experimental metastatic brain tumors.</a> J Neurosurg. 59 (2), 304-310 (1983).</li><li>Amatruda, J. F., Shepard, J. L., Stern, H. M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+cancer+model+system.">Zebrafish as a cancer model system.</a> Cancer Cell. 1 (3), 229-231 (2002).</li><li>Feitsma, H., Cuppen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+cancer+model.">Zebrafish as a cancer model.</a> Molecular Cancer Res. 6 (5), 685-694 (2008).</li><li>Stoletov, K., Klemke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+of+the+day:+zebrafish+as+a+human+cancer+model.">Catch of the day: zebrafish as a human cancer model.</a> Oncogene. 27 (33), 4509-4520 (2008).</li><li>Stoletov, K., 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=Visualizing+extravasation+dynamics+of+metastatic+tumor+cells.">Visualizing extravasation dynamics of metastatic tumor cells.</a> J Cell Sci. 123 (13), 2332-2341 (2010).</li><li>Kanada, M., Zhang, J., Yan, L., Sakurai, T., Terakawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-initiated+extravasation+of+cancer+cells+visualized+in+zebrafish.">Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.</a> PeerJ. , (2014).</li><li>Teng, Y., Xie, X., Walker, S., White, D. T., Mumm, J. S., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+human+cancer+cell+metastasis+in+zebrafish.">Evaluating human cancer cell metastasis in zebrafish.</a> BMC Cancer. 13 (453), (2013).</li><li>Cross, L. M., Cook, M. A., Lin, S., Chen, J. -. N., Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+angiogenesis+drugs+in+a+live+fluorescent+zebrafish+assay.">Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay.</a> Arterioscl Throm Vas. 23 (5), 911-912 (2003).</li><li>Sharif, G. M., 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=Cell+growth+density+modulates+cancer+cell+vascular+invasion+via+Hippo+pathway+activity+and+CXCR2+signaling.">Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.</a> Oncogene. 34, 5879-5889 (2015).</li><li>Novoa, B., Figueras, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+model+for+the+study+of+inflammation+and+the+innate+immune+response+to+infectious+diseases.">Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases.</a> Adv Exp Med Biol. 946, 253-275 (2012).</li><li>Kitamura, T., Qian, B. -. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+promotion+of+metastasis.">Immune cell promotion of metastasis.</a> Nature Rev Immunol. 15 (2), 73-86 (2015).</li><li>He, S., 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=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J Pathol. 227 (4), 431-445 (2012).</li><li>Tulotta, C., 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=Inhibition+of+signaling+between+human+CXCR4+and+zebrafish+ligands+by+the+small+molecule+IT1t+impairs+the+formation+of+triple-negative+breast+cancer+early+metastases+in+a+zebrafish+xenograft+model.">Inhibition of signaling between human CXCR4 and zebrafish ligands by the small molecule IT1t impairs the formation of triple-negative breast cancer early metastases in a zebrafish xenograft model.</a> Dis Model Mech. 9 (2), 141-153 (2016).</li><li>Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W., Whyte, M. K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li><li>Berens, E. 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=Keratin-associated+protein+5-5+controls+cytoskeletal+function+and+cancer+cell+vascular+invasion.">Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.</a> Oncogene. , (2016).</li></ol>

Denne teknik udnytter zebrafisk model til effektivt teste den vaskulære invasive evne af cancerceller (se figur 1). Her påføres vi teknikken til et panel af brystcancer-cellelinier for at tilvejebringe en basislinie hvorpå andre forskere så kan bygge deres egne undersøgelser (se tabel 1; Figures 2 - 3). Den iagttagelse, at MDA-MB-231-celler let invaderet i den kaudale område af zebrafisk embryoner ville gøre denne cellelinie ideel til afprøvning midler der vil ...

Metastatisk sygdom er en væsentlig årsag til kræft dødelighed og mange mekanismer, som sætter celle formidling kræft mangler at blive opdaget 1. For at en kræftcelle med succes metastaserer, skal det først invadere gennem stroma, der omgiver en primær tumor, indtaste (intravasate) i kredsløbssygdomme, overleve i transit, exit (ekstravasatet) fra kredsløbet, og endelig etablere en levedygtig koloni ved den fjerne orgel site 2. Intravasation og ekstravasation er således afgørende skridt i den metastatiske kaskade, men hver kræftcelle er ikke i sig selv dygtige til at forstyrre og migrerer gennem endotel vejkryds 3. I virkeligheden er der en række unikke selektionstryk, der omgiver cancercelle vaskulær invasion og fremgangsmåden kan yderligere påvirkes af en række forskellige endogene og exogene 4 faktorer. Af disse grunde, teknikker, sonde den aggressive adfærd fremskreden kræft fokuserer ofte på vascular invasiv evne som et middel til at forudsige metastatisk spredning. Forskellige modelsystemer eksisterer for at lette undersøgelsen af kræftcellen vaskulær invasion in vitro. Den mest anvendte in vitro assays involverer enten transwell systemer til vurdering kræft cellemigrering gennem en endothelial barriere 5 eller Electric Cell-Substrat Impedans Sensing (ECIS) teknologi til at overvåge realtid afbrydelse af et intakt endotel monolag af kræftceller 6. Disse assays typisk mangler de fluide dynamik og stromale faktorer, som ellers ville påvirke cancercelle fastgørelse til en endothelial væg. Dette spørgsmål er noget omgås ved perfusable vaskulære netværk, der opstår fra 3D-kultur af endotel med støtte stromale celler, og disse 3D mikrofluide systemer nu repræsenterer forkant med de nuværende in vitro optioner 7,8. Stadig, disse tilgange udelade den robuste mikromiljø af et funktionelt kredsløbssygdommeog derfor kun delvis erstatning for in vivo-modeller. Den mest udbredte in vivo-model af vaskulær invasion er musen, i hvilken eksperimentel metastase assays almindeligvis udføres, fordi de forekommer på relativt korte tidsskalaer og har normalt indikerer metastatisk evne 9. Disse analyser involverer direkte injektion af kræftceller i omløb, og derfor modellere de endelige stadier af metastaser, nemlig ekstravasation og kræft celle kolonisering af organer. De eksperimentelle metastase analyser varierer baseret på stedet af kræftcellen indsprøjtning og organer til sidst analyseret. I den første analysetype, er kræftceller injiceret i halevenen af mus og cancercelle såning i lungerne overvåges 10,11. Det andet assay omfatter handlinger intrakardielle injektioner til direkte metastatisk podning mod knoglen mikromiljø 12-14, men også hjernen 15. I den tredje assay, kræft calen injiceres i milten for at tillade kolonisering af leveren 16 mens den fjerde tilførselsvej i carotidarterien bærer cancerceller til hjernen 17,18. Uanset kræftcellen leveringsmetode, orgel kolonisering er den accepterede eksperimentelle endpoint og er generelt bestemmes via luminescens, histologi, eller PCR-baserede teknikker. På trods af de fysiologiske fordele ved at gennemføre eksperimentelle metastase analyser inden for en muse vært, disse eksperimenter kræver stadig uger til måneder at gennemføre og analysere. Zebrafisk (Danio rerio) model har for nylig vist sig som et nyt system til at studere udviklingen af kræft 19,20, og muliggør vurdering af cancercellen vaskulær invasion i et funktionelt kredsløbssygdomme over et meget kortere tidsrum sammenlignet med mus 21-24. Metoden anvender en gennemsigtig zebrafisk stamme, der har sin endotel mærket med en grøn reef koral fluospare- protein reporter drevet af kdrl promotoren, zebrafisk receptor for vaskulær endotel vækstfaktor 25. I assayet, er cancerceller mærket med en rød fluorescerende markør og injiceret i præcardielt sinus af 2 dage gamle embryoner. Overalt mellem 48 til 96 timer efter injektionen, kan cancerceller, der har invaderet ud af vaskulaturen og ind i kaudale område af embryoner blive scoret effektivt på et fluorescerende mikroskop. Her anvender vi en teknik til et panel af almindeligt anvendte humane brystcancer cellelinjer at påvise markante forskelle i deres vaskulære invasive evne. Endvidere viser vi, at ændre injektionsstedet til embryo blommesækken muliggør studiet af heterogene celle-interaktioner som kræft cellepopulationer kan differentielt mærket med fluorescerende farvestoffer og injiceres i zebrafisk embryoner mangler fluorescerende vaskulatur. I sidstnævnte assay, cancerceller, der har invaderet blommen og intravasated ind i vasculature er scoret i caudale region 24 til 48 timer efter injektion. På grund af den effektivitet og bekvemmelighed af denne model, er zebrafisk stigende grad anvendes til hurtigt at teste den vaskulære invasive evne af cancerceller under en fysiologisk indstilling.

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			<td height="21" style="height:21px;">5 3/4&#34; Disposable Pastur Pipets, borosilicate Glass</td>
			<td>Fisher Brand</td>
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		</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 testede vi den vaskulære invasive evne almindeligt anvendte brystcancer cellelinjer i en zebrafisk embryo model (figur 1). Strenge kriterier blev anvendt i scoring ekstravasation for disse forskellige cellelinjer, hvor positive begivenheder kun blev talt, hvis kræftcellerne havde tydeligvis ekstravaseret, dette gøres hovedsageligt at begrænse eventuelle falsk-positive, der kunne opstå fra at lave cellerester. <p class="jove_content" fo:keep-together.within-p...

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 fremgangsmåde anvender zebrafisk embryoner til effektivt teste den vaskulære invasive evne af cancerceller. Fluorescerende cancerceller injiceres i præcardielt sinus eller blommesæk af fostre. Cancercelle vaskulær invasion og ekstravasation vurderes via fluorescensmikroskopi af halen område 24 til 96 timer senere.

Test af Vascular Invasiv evne kræftceller i Zebrafisk (<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 af cirkulerende tumorceller i en ortotopisk musemodel af kolorektal cancer

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 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 ...

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

En Time-lapse, etiket-fri, kvantitative fase Imaging undersøgelse af passive og aktive menneskelige kræftceller

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

I Vivo Målretning af Xenografted humane kræftceller med funktionaliserede fluorescerende Silica nanopartikler i zebrafisk

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

Generation af zebrafisk Larval Xenografts og Tumor Behavior Analysis

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-billeddannelse i en ortopisk musemodel af hepatocellulært karcinom

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 billeddannelse og flowcytometribaseret påvisning af flere fluorescerende ældningsmarkører i terapiinducerede senescente kræftceller

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 af hjernemetastase ved intern halspulsåreinjektion af kræftceller

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

Zebrafisklarver som model til evaluering af potentielle 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

Ortopisk implantation af patientafledte kræftceller hos mus rekapitulerer avanceret kolorektal cancer

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