Name: three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation
Uploaded: 2016-08-18T15:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

MERK: Ingen etikk vurdering siden animalske og menneskelige kreftceller ble kjøpt eller ber gitt til oss. 1. Making Kollagen Plugger (Pseudo-primær tumor) <ol><li> Forbered en kollagen spredning. Type I kollagen fra rottehale sener (RGT) kan enten trekkes ut og sterilisert som tidligere rapportert 17, eller kjøpes. Spre frysetørret RTT kollagen (3,25-3,50 mg / ml i 0,02 N eddiksyre) ved hjelp av en blender (high-speed setting; fem 2 min løper) for en jevn blanding. </li><li> Harvests (trypsin-E...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+influence+of+angiogenesis+in+in+vitro+cancer+model+systems.">Studying the influence of angiogenesis in in vitro cancer model systems.</a> Adv Drug Deliv Rev. , (2016).</li><li>Bill, R., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+EMT+in+breast+cancer+metastasis:+Correlation+or+causality.">The relevance of EMT in breast cancer metastasis: Correlation or causality.</a> FEBS Lett. 589 (14), 1577-1587 (2015).</li><li>Kimlin, L. C., Casagrande, G., Virador, V. 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I., 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+Novel+Hanging+Spherical+Drop+System+for+the+Generation+of+Cellular+Spheroids+and+High+Throughput+Combinatorial+Drug+Screening.">A Novel Hanging Spherical Drop System for the Generation of Cellular Spheroids and High Throughput Combinatorial Drug Screening.</a> Biomater Sci. 3 (4), 581-585 (2015).</li><li>Janvier, R., Sourla, A., Koutsilieris, M., Doillon, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Fibroblasts+are+Required+for+PC-3+Human+Prostate+Cancer+Cells+to+Produce+Capillary-like+Formation+of+Endothelial+Cells+in+a+Three-dimensional+Co-culture+System.">Stromal Fibroblasts are Required for PC-3 Human Prostate Cancer Cells to Produce Capillary-like Formation of Endothelial Cells in a Three-dimensional Co-culture System.</a> Anticancer Res. 17 (3A), 1551-1557 (1997).</li><li>Doillon, C. J., Gagnon, E., Paradis, R., Koutsilieris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Culture+System+as+a+Model+for+Studying+Cancer+Cell+Invasion+Capacity+and+Anticancer+Drug+Sensitivity.">Three-dimensional Culture System as a Model for Studying Cancer Cell Invasion Capacity and Anticancer Drug Sensitivity.</a> Anticancer Res. 24 (4), 2169-2177 (2004).</li><li>Gobeil, S., Zhu, X., Doillon, C. J., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+shRNA+Screen+Identifies+GAS1+as+a+Novel+Melanoma+Metastasis+Suppressor+Gene.">A Genome-Wide shRNA Screen Identifies GAS1 as a Novel Melanoma Metastasis Suppressor Gene.</a> Genes Dev. 22 (21), 2932-2940 (2008).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+Of+Ready-To-Use,+Storable+And+Reconstituted+Type+I+Collagen+From+Rat+Tail+Tendon+For+Tissue+Engineering+Applications.">Preparation Of Ready-To-Use, Storable And Reconstituted Type I Collagen From Rat Tail Tendon For Tissue Engineering Applications.</a> Nat Protoc. 1 (6), 2753-2758 (2007).</li><li>Horie, 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=Characterization+of+Human+Lung+Cancer-associated+Fibroblasts+in+Three-dimensional+In+Vitro+Co-culture+Model.">Characterization of Human Lung Cancer-associated Fibroblasts in Three-dimensional In Vitro Co-culture Model.</a> Biochem Biophys Res Commun. 423 (1), 158-163 (2012).</li><li>Banyard, 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=Identification+of+Genes+Regulating+Migration+and+Invasion+Using+a+New+Model+of+Metastatic+Prostate+Cancer.">Identification of Genes Regulating Migration and Invasion Using a New Model of Metastatic Prostate Cancer.</a> BMC Cancer. 30 (14), 387 (2014).</li><li>Palumbo, J. S., Degen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+Tumor+Cell+Metastasis.">Fibrinogen and Tumor Cell Metastasis.</a> Haemostasis. 31, 11-15 (2001).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Stroma,+Tumor+Blood+Vessels,+and+Antiangiogenesis+Therapy.">Tumor Stroma, Tumor Blood Vessels, and Antiangiogenesis Therapy.</a> Cancer J. 21 (4), 237-243 (2015).</li><li>Luoto, K. R., Kumareswaran, R., Bristow, R. 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Som et viktig teknisk fotnote, er det avgjørende at ikke i noe gap er til stede på grenseflaten mellom det sentrale og det perifere geler. Ellers kan det redusere kapasiteten av cellene for å migrere / invaderer fibringel. Et mellomrom mellom kollagen og fibrin-geler kan dannes i løpet av den første 24-timers kultur hvis trombin ikke er passende fortynnet. Det er også mulig at cellelinjen testet kunne føre kollagen gelen for å trekke seg sammen i løpet av kulturen, for derved å forårsake en forholdsvis stor p...

Gransker de grunnleggende og biomedisinske egenskaper ved kreftcelle invasjon / migrasjon og påfølgende metastase etablering er gjenstand for en intens forskning 1,2. Metastase er den ultimate stadium av kreft og dens kliniske behandlingen fortsatt ukjent. En bedre forståelse av metastaser på cellulære og molekylære nivåer vil muliggjøre utvikling av mer effektive behandlingsformer 3. Flere egenskaper av metastatiske celler kan utforskes in vitro 4 inkludert deres stemness og potensial til å skaffe seg en overgang tilstand (f.eks epithelioid-mesenchymale overgang) for å migrere og invadere i og fra den primære svulsten 5. Imidlertid har den in vitro vurderingen av invasjons / metastaseprosesser vært en utfordring, siden det praktisk talt utelukker bidraget av blod / lymfatiske sirkulasjon. Organotypiske kulturer som embed tumorfragmenter i kollagen gels har previouslu blitt brukt til å overvåke kreft aggressivitet. Selv om kompleksiteten av tumorer er bevart (f.eks tilstedeværelsen av ikke-kreftceller), tumorfragmenter blir utsatt for begrenset diffusjon medium, for prøvetaking variasjon, og til en overvekst av stromale celler 6. En alternativ metode består i voksende kreftceller innenfor komponenter i den ekstracellulære matriks (ECM), som etterligner den tredimensjonale (3D) cellemiljøet. Spredning av brystkreftcellelinjer i en kollagen-gel og / eller en basalmembran-avledet matriks er blant de best karakteriserte eksempler på 3D-cellekultur. Ved å bruke spesielle 3D cellekultur miljøer, kan uorganisert montering observert for brystkreft celler dyrket under standardbetingelser reverseres i den spontane dannelsen av melke acini og rørformede strukturer 7-10. Videre er dannelsen av flercellede tumor sfæroider avledet fra adenokarsinom-cancerceller deltar ved hjelp av ulike teknikker (f.eks hengende dråper, flytende sfæroider, agar embedment) nå utgjør den mest brukte 3D-cellekultur-analyse 11-13. Imidlertid er denne analysen begrenset av den begrensede sett av kreftcellelinjer som kan danne sfæroider og ved den korte perioden tilgjengelig for å studere celler i disse tilstandene. I denne visualisert teknikken, vi heri innføre en sofistikert 3D-cellekultur-analyse hvor kreftcellene av interesse er innleiret i en kollagen-gel for å tillate in vitro dannelse av en pseudo-primærtumor som kan alternativt belegges med en basalmembran-avledet matrise. En gang er dannet, blir den pseudo-primær tumor deretter klemt i en acellulær matrise (fibringel i det foreliggende tilfelle), noe som gjør at kreftceller til å krysse grenseflaten mellom de to kamrene matriks (se figur 1). Interessant, sekundær tumor-lignende strukturer som stammer fra den pseudo-primærtumor sammen med aggressive kreftceller vises ifibringel. Et slikt 3D kultursystem gir den fleksibilitet som kreves for å undersøke, for eksempel anticancer legemidler, genekspresjon og celle-celle og / eller celle-interaksjoner ECM 14-16. <img alt="Figur 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> Figur 1:.. Oversikt over Method Skjematisk oppsummering av metode for å generere 3D cellekultur system som modell for kreftstudier <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freeze-dried collagen</td>
			<td>Sigma-Aldrich</td>
			<td>C7661</td>
			<td>from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Micro-spoons</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td>Fisherbrand Handi-Hold Microspatula</td>
		</tr>
		<tr>
			<td>96 well plate, round base</td>
			<td>Sarstedt</td>
			<td>3925500</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sarstedt</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s Medium</td>
			<td>Sigma Chemical, Co.</td>
			<td>D5546</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>CAA15-701</td>
			<td>FBS, Canadian origin.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Chemical, Co.</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>Sigma Chemical, Co.</td>
			<td>H8264</td>
			<td>HBSS</td>
		</tr>
	</tbody>
</table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

Pattedyrcellekultur i monolag er mye brukt til å studere forskjellige fysiologiske og molekylære prosesser. Men denne tilnærmingen til å studere voksende celler genererer ofte uønskede gjenstander. Derfor cellekultur i et tre-dimensjonalt (3D) miljø, ofte ved hjelp av ekstracellulære matriks-komponenter, dukket opp som et interessant alternativ på grunn av sin nære likhet med det native in vivo vev eller organ. Vi har utviklet en 3D-cellekultursystem med to avdelinger, nemlig (i) en sentral del som inneholder kreftceller innleiret i en kollagen-gel som virker som et pseudo-primær macrospherical tumor og (ii) en perifer cellefri kammer laget av et fibrin gel, det vil si et ekstracellulært matrisekomponent forskjellig fra den som anvendes i midten, hvor kreftceller kan migrere (invasjon foran) og / eller dannelse av mikrosfærisk tumorer som representerer sekundære eller satellitt tumorer. Dannelsen av satellitt tumorer i det perifere kammer erbemerkelsesverdig korrelert til den kjente aggressivitet eller metastatisk opprinnelse av de innfødte kreftceller, noe som gjør denne 3D kultur systemet unikt. Denne cellekultur tilnærming kan anses for å vurdere kreftcelle invasivitet og motilitet, celle-ekstracellulær matriks interaksjoner, og som en metode for å evaluere anti-kreft legemidler egenskaper.

Som tidligere nevnt, en interessant trekk ved denne 3D-cellekultur-analyse er at kreftceller ikke bare kan migrere fra kollagen pluggen til den tilstøtende fibrin-gelen, men også etablere sekundære tumorer (for eksempel satellitt-tumor-lignende strukturer). Dette kan observeres direkte med et invertert fasekontrastmikroskop ved lave og høye forstørrelser gjennom gelen tykkelse, spesielt med en lang arbeidsavstand kondensator (figur 2). Ved hjelp av denne 3D...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Tredimensjonal Kultur analysen til Explore Cancer Cell Invasivitet og satellitt tumordannelse

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

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En Tredimensjonal Tissue Culture modell for å studere Primær Menneskelig Bone Marrow og dets Malignancies

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A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

En mus Tumor Model of Surgical Stress å Utforsk Mekanismer for Postoperativ Immunsuppresjon og evaluere Novel Perioperativ immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Tredimensjonal kulturer: Et verktøy for å studere Normal acinar Arkitektur vs malign transformasjon av brystkreft celler

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

Kvantifisering av Breast Cancer Cell Invasivitet Ved hjelp av en tredimensjonal (3D) modell

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Tredimensjonal Co-kultur Modell for Tumor-stromal Interaksjon

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Mammosphere Formation analysen fra Menneskelig Breast Cancer Vev og cellelinjer

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...