Name: evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1
Uploaded: 2020-12-05T13:30:00
Description: Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Dette arbejde blev støttet af et tilskud til videnskabelig forskning C (bevilling nr. 19K09834 til K.N.) fra Ministeriet for Uddannelse, Videnskab og Kultur, Japan.

Alle procedurer blev udført i henhold til den protokol, der blev godkendt af den etiske komité for menneskers velfærd. Alle patienter gav skriftligt informeret samtykke til forskningsbrug af deres prøver, og indsamling og brug af væv til denne undersøgelse blev godkendt af Human Genome, Gene Analysis Research Ethics Committee ved Teikyo University.
1. Isolering og dyrkning af stamceller fra æggestokkræft (OCSC'er) fra patienter med kræft i æggestokkene og ascites i et niveau 2 biosikke...

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G., Reya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+fate+in+cancer+growth,+progression+and+therapy+resistance.">Stem cell fate in cancer growth, progression and therapy resistance.</a> Nature Reviews Cancer. 18 (11), 669-680 (2018).</li><li>Dontu, G., 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes and Development. 17 (10), 1253-1270 (2003).</li><li>Lonardo, E., 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=Nodal/Activin+signaling+drives+selfrenewal+and+tumorigenicity+of+pancreatic+cancer+stem+cells+and+provides+a+target+for+combined+drug+therapy.">Nodal/Activin signaling drives selfrenewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy.</a> Cell Stem Cell. 9 (5), 433-446 (2011).</li><li>Ricci-Vitiani, L., 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=Identification+and+expansion+of+human+colon-cancer-initiating+cells.">Identification and expansion of human colon-cancer-initiating cells.</a> Nature. 445 (7123), 111-115 (2007).</li><li>Ohata, H., 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=Induction+of+the+stem-like+cell+regulator+CD44+by+Rho+kinase+inhibition+contributes+to+the+maintenance+of+colon+cancer-initiating+cells.">Induction of the stem-like cell regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating cells.</a> Cancer Research. 72 (19), 5101-5110 (2012).</li><li>Ishiguro, T., 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=Establishment+and+characterization+of+an+in+vitro+model+of+ovarian+cancer+stem-like+cells+with+an+enhanced+proliferative+capacity.">Establishment and characterization of an in vitro model of ovarian cancer stem-like cells with an enhanced proliferative capacity.</a> Cancer Research. 76 (1), 150-160 (2016).</li><li>Singh, S. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cells:+role+in+metastasis+and+opportunity+for+therapeutic+targeting.">Ovarian cancer stem cells: role in metastasis and opportunity for therapeutic targeting.</a> Cancers (Basel). 11 (7), 934 (2019).</li><li>Liz&#225;rraga-Verdugo, E., 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=Cancer+stem+cells+and+its+role+in+angiogenesis+and+vasculogenic+mimicry+in+gastrointestinal+cancers.">Cancer stem cells and its role in angiogenesis and vasculogenic mimicry in gastrointestinal cancers.</a> Frontiers in oncology. 10, 413 (2020).</li><li>Renaud, J., Martinoli, M. 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Den præsenterede protokol beskriver, hvordan man efterligner tumormikromiljøet af OCSC'er i en in vitro-indstilling. Den primære komponent i metoden udgør den meget reproducerbare kokulturmodel opnået ved hjælp af NICO-1-systemet, et indirekte Transwell-samkultursystem. Mange af de aktuelt tilgængelige kokulturmodeller undersøger virkningerne af direkte cellecellekontakt på kokulturerede cellepopulationer 12,13,14,15,16,17,18.<sup class="xref"...

Kræft i æggestokkene er den ottende mest almindelige malignitet hos kvinder over hele verden med ca. 300.000 nye diagnoser og anslået 180.000 dødsfald årligt1. Ved den første diagnose præsenterer kræft i æggestokkene ofte med alvorlige symptomer, hvor ca. 75% af patienterne allerede er i fase III-IV. Følgelig er den 5-årige overlevelsesrate <30%, og dødeligheden er den højeste blandt gynækologiske kræftformer2, hvor effektiviteten af behandlingen af kræft i æggestokkene er meget afhængig af kliniske faktorer såsom den vellykkede gennemførelse af debulking-kirurgi, modstand mod kemoterapi og gentagelse efter den indledende behandling.
Ovariecancervæv er hierarkisk organiseret, hvor ikke alle tumorkomponenter er lige i stand til at generere efterkommere. De eneste celler, der er i stand til at forny sig selv og producere en heterogen tumorcellepopulation, anses for at repræsentere kræftstamceller (CSC'er)3. CSC selvfornyelse og tumorinitiering ledsages af fremme af angiogenese for at ombygge deres tumormikromiljø med det formål at opretholde en støttende niche. Tidligere modeller kunne imidlertid ikke anvendes til in vitro-analyser på grund af den begrænsede reproducerbarhed af dyrkning af CSC'er afledt af kliniske prøver på grund af forstyrrelse af sfæroider efter flere passaging. For nylig er eksperimentelle metoder til dyrkning af CSC'er fra patienter blevet udviklet til flere applikationer 4,5,6,7. Især ved at udnytte karakteristikken ved CSC'er til at vokse ved at danne sfæroider i ultralave fastgørelsesplader med serumfrit medium, induceres de dyrkede CSC'er til at udtrykke en stamcelleoverflademarkør, der ikke udtrykkes i normale tumorceller med multilineagedifferentieringspotentiale 8,9.
Nylige data har vist, at persistensen af sovende ovarie (O) CSC'er visualiseret som formidling ved peritoneum er forbundet med deres regenerering som tilbagevendende tumorer10. Forståelse af de molekylære og biologiske egenskaber ved OCSC'er kan således muliggøre effektiv målretning og udryddelse af disse celler, hvilket resulterer i potentiel tumorremission. Især er der lidt kendt om de cellulære og molekylære mekanistiske træk ved CSC'ers roller i angiogenese11. Derfor brugte vi i den nuværende protokol patientafledte OCSC'er i en in vitro-indstilling til at undersøge den angiogene egenskab af endotelceller ved hjælp af co-kulturmodellen, som kan efterligne tumormikromiljøet af CSC'er og endotelceller på det metastatiske sted i den kliniske indstilling. I sidste ende, da neovaskularisering udgør en kritisk proces, der er nødvendig for at understøtte tumorvækst og metastase, vil en bedre forståelse af dens mekanisme muliggøre udviklingen af en ny målretningsterapi til OCSC'er på det metastatiske sted.
Her præsenterer vi en protokol til at visualisere neovaskulariseringstrinnet omkring CSC'erne på en tidsforløbsmåde. Fordelen ved protokollen inkluderer at tillade fuldt reproducerbare undersøgelser ved hjælp af 3D-samkultursystemet, NICO-1, hvilket muliggør observation af virkningerne på patienter af den OCSC-afledte tumorinitieringsevne under endotelcelleangiogenese.

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			<td>170356N</td>
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			<td>15 mL tube</td>
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			<td>339650</td>
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			<td>200 &#181;L Pipet tip</td>
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			<td>430290</td>
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			<td>AccuMAX</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
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			<td>BioCoatTM Collagen I 60mm Dish</td>
			<td>Corning</td>
			<td>356401</td>
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			<td>KUBOTA</td>
			<td>2800</td>
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			<td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
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			<td>Endothelial Cell Growth Medium 2&#160;</td>
			<td>PromoCell</td>
			<td>C-22011&#160;</td>
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			<td>D2158</td>
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			<td>HUEhT-1 cell</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB1458</td>
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			<td>ICCP Filter 0.6 &#181;m</td>
			<td>Ginrei Lab.</td>
			<td>2525-06</td>
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			<td>Insulin, human</td>
			<td>SIGMA (Roche)</td>
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			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356234</td>
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			<td>Yokogawa</td>
			<td>CQ-1</td>
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			<td>NICO-1</td>
			<td>Ginrei Lab.</td>
			<td>2501-02</td>
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			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005290</td>
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			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
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			<td>Gilson</td>
			<td>F123601</td>
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			<td>14190-144</td>
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			<td>StemPro hESC SFM</td>
			<td>Thermo (Gibco)</td>
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			<td>FALCON</td>
			<td>357575</td>
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			<td>Y-27632</td>
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			<td>253-00513</td>
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evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

Kræftstamceller (CSC'er) befinder sig i en støttende niche, der udgør et mikromiljø bestående af tilstødende stromale celler, kar og ekstracellulær matrix. CSC'ernes evne til at deltage i udviklingen af endotel udgør en vigtig egenskab, der direkte bidrager til den generelle forståelse af mekanismerne for tumorigenese og tumormetastase. Formålet med dette arbejde er at etablere en reproducerbar metode til at undersøge tumorinitieringsevnen hos ovariecancerstamceller (OCSC'er). Heri undersøgte vi neovaskulariseringsmekanismen mellem endotelceller og OCSC'er sammen med de morfologiske ændringer af endotelceller ved hjælp af in vitro-co-kulturmodellen NICO-1. Denne protokol tillader visualisering af neovaskulariseringstrinnet omkring OCSC'erne på en tidsforløbsmåde. Teknikken kan give indsigt i de angiogenetiske egenskaber af OCSC'er i tumormetastase.

Vi indsamlede ascitesvæsker opnået fra patienter med avanceret kræft i æggestokkene under operation eller paracentese med det formål at udføre en langsigtet stabil kultur for sfæroider. Her præsenterer vi tilfælde af en langsigtet sfæroidkultur af ovarie-CSC'er kaldet CSC1 og CSC2. Begge cellelinjer bærer den samme diagnose og histologiske profiler. De mekanistiske roller af OCSC'er, der ligger til grund for interaktionen med endotelceller, der er nødvendige for at inducere ne...

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Ovariecancer stamceller (OCSC) er ansvarlige for kræftinitiering, gentagelse, terapeutisk resistens og metastase. OCSC-vaskulær niche anses for at fremme selvfornyelse af OCSC'er, hvilket fører til kemoresistance. Denne protokol danner grundlag for etablering af en reproducerbar OCSC-vaskulær nichemodel in vitro.

Evaluering af de angiogenetiske egenskaber ved stamceller til æggestokkræft ved hjælp af det tredimensionelle samkultursystem, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

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FRET Imaging i Tre-dimensionelle Hydrogeler

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

Udvikling af en Insert Co-kultur System af to Cellular typer i fravær af celle-celle-Kontakt

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Tre-dimensionelle genopbygning af den vaskulære arkitektur i passiv klarhed-ryddet mus æggestokkene

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...