Name: in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells
Uploaded: 2014-05-15T13:00:00
Description: Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

Dette arbejde blev støttet af finansiering fra Department of Defense-Prostata Cancer Research Program (W81XWH-12-1-0124), U54CA143876 fra National Cancer Institute, og Robert McCooey Urogenitale Oncology Research Fund. Vi vil gerne takke Dr. Annarita Lorenzo (Department of Pathology) for at levere VE-Cadherin antistoffer, og Dr. Marco Seandel (Department of Surgery) til at tilvejebringe HUVEC&#39;er.

1.. Dyrkning HUVEC&#39;er om Microslides for at observere CTC-endotel Interactions <ol><li> Under vævskultur hætte, først skylles mikroobjektglas, kanalbredde på 1 mm med PBS. Forsigtigt belægge mikroobjektglas med 200 pi 50 mg / ml fibronectin (opløst i PBS) under anvendelse af en 1 ml-Luer-lock sprøjte. </li><li> Dæk mikroobjektglas med låget og holde det inde i vævskultur hætte i 30 min. Langsom dispensering af væsken i mikroobjektglas forhindrer bobledannelse i kanalen. </li><li> Perfuse 200 pi varm (3...

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På grund af det lave antal CTCs blandt blodlegemer, er det vanskeligt at isolere CTCs som en ren population af celler. For at studere CTC / EF interaktioner, den sjældne og urene befolkning CTCs stiller to store udfordringer: a) identifikation af CTCs blandt blodlegemer; b) Observation af CTC / EF interaktioner. For at overvinde den første begrænsning identificere prostata CTCs blandt blodlegemer, tog vi fordel af det faktum, at stort set alle prostata tumorce...

Metastase er en kompleks flertrinsproces, der forbliver dårligt forstået. Den E-selectin/selectin ligand akse har vist sig at spille en vigtig rolle i tumormetastase ved at fremme primære adhæsive interaktioner mellem det vaskulære endotel celler og cancerceller 1,2. Endotel (E)-selectin er et transmembrant protein, der udtrykkes af aktiverede endotelceller, mens forskellige E-selectin ligand (r) udtrykkes af tumorceller 3. Talrige in vitro-fremgangsmåder med succes er blevet anvendt til at modellere E-selectin/selectin ligand interaktioner mellem tumorceller og endotelceller (EC&#39;er) 1.. At studere disse interaktioner er forskellige flowkammer systemer, der anvendes til at simulere blod kar-systemet. Blandt strømningskammeret samlinger er parallel-plade-strømningskammeret (PPFC), sammenholdt med EC&#39;er rutinemæssigt anvendes som en in vitro model, der simulerer in vivo shear stress. I dennemetode EC&#39;er dyrkes på en 35 mm skål og efter opnåelse et monolag, er EC&#39;er knyttet til PPFC og forskydningsspænding baserede eksperimenter udføres. Men PPFC og andre nuværende systemer præsentere mange begrænsninger til at studere klæbende interaktioner mellem cirkulerende tumorceller (CTCs) afledt fra patienter og ECS, primært fordi CTCs er en sjælden population af celler, skur fra den primære tumor, der cirkulerer blandt millioner af blodceller (1 CTC per 10 9 celler i blodet) 4.. Derfor, i modsætning til ubegrænset udbud af dyrkede cellelinier, lavt CTC tæller føre til meget få og sjældne CTC / EF interaktioner, der kræver ordentlig flow kanal bredde at registrere interaktioner for afspilning analyse. Derudover, da patientens afledte CTCs er et urent befolkning, derfor en identifikation markør er forpligtet til at spore CTCs i specifikke. For at løse dette problem, har vi udviklet en ny metode til at identificere prostatacancer (PSA) CTCs ved at udnytte det faktum, at næsten alle disse CTCs ekspres prostataspecifikt membran-antigen (PSMA) på deres celleoverflade 5,6. I denne rapport har vi brugt prostata cancer cellelinie, MDA PCa2b (MDA), at demonstrere potentialet nytte af vores nye system til at studere prostata CTC interaktioner med ECS sidst at forstå den mekanisme af metastaser. Vores metode kan anvendes til forskellige shear baserede eksperimenter, der simulerer in vivo vaskulære system 7-9. Udover behandlingen PCa CTC / EF interaktioner, kunne strømmen kammer system nemt tilpasses til analyse af perifert blod mononukleære celler eller tumorceller interaktioner med EC&#39;er. Den lette demontering og samling af flowet kammeret, en mikroobjektglas III (0,1) (herefter benævnt mikroobjektglas), giver mulighed for dyrkning EC&#39;er under perfusion og stimulerende EC&#39;er med forskellige cytokiner at inducere PRotein udtryk. Desuden dyrkes EC&#39;er rekombinante proteiner, såsom E-og P-selectin kan overtrækkes på mikroobjektglasset og interaktioner med tumorceller kan observeres under laminare strømningsbetingelser 10.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

Metastase er en proces, hvor tumorceller kaste fra den primære tumor intravasate blod vaskulære og lymfesystemet, derved at få adgang til ekstravasere og danne en sekundær niche. Ekstravasation af tumorceller fra blodet vaskulære system kan undersøges ved hjælp af endotelceller (EC&#39;er) og tumorceller opnået fra forskellige cellelinjer. Indledende undersøgelser blev udført ved hjælp af statiske forhold, men det er veldokumenteret, at ECS opfører sig anderledes under fysiologiske flow. Derfor er forskellige flow chamber samlinger i øjeblikket anvendes til at studere kræft celle interaktioner med EC&#39;er. Aktuelle flowkammer forsamlinger tilbyder reproducerbare resultater ved hjælp af enten forskellige cellelinjer eller væske ved forskellige shear stress. Men for at observere og studere interaktioner med sjældne celler såsom cirkulerende tumorceller (CTCs), skal gøres til den konventionelle flowkammer samling visse ændringer. CTCser en sjælden cellepopulation blandt millioner af blodlegemer. Derfor er det vanskeligt at opnå en ren population af CTCs. Forurening af CTCs med forskellige typer af celler, der normalt findes i omløb er uundgåelig hjælp til stede berigelse eller udtynding teknikker. I denne rapport beskriver vi en unik metode til fluorescens label cirkulerende prostatacancerceller og studere deres samspil med ECS en selvstændig samlet flow kammer system. Denne teknik kan anvendes yderligere til at observere interaktionen mellem prostata CTCs og helst protein af interesse.

Figur 1 viser en O / N-kulturen af et monolag af EC&#39;er på mikroobjektglas. Skalering på figur 1A viser, at 100% af mikroobjektglasset er synlig ved hjælp af 5X mål, mens 70% er synlig ved hjælp af et 10X objektiv (figur 1B). For E-selectin medierede interaktioner, celler rullende på kanterne ikke anset som gør mere end 70% af den mikroobjektglas tilgængelig for video-optagelse og afspilning analyse. Det er vores erfaring, i første omgang dette set-up samlin...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

In vitro metode til at observere E-selectin-medierede interaktioner mellem Prostata cirkulerende tumorceller afledt fra patienter og humane endotelceller

Vores rapport beskriver en unik metode til at visualisere og analysere CTC / EF interaktioner i prostatakræft under fysiologiske flow.

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

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Medicine

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

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Isolation, karakterisering og sammenlignende differentiering af humane Dental Pulp stamceller fra blivende tænder ved hjælp af to forskellige metoder

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

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Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Isolering af kræft stamceller fra humane Prostata Cancer Prøver

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

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Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

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Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Studere i bugspytkirtlen Kræft stamceller Karakteristik for udvikling af nye behandlingsstrategier

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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Isolation og formering af cirkulerende tumorceller fra en mus Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

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Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

Udtømning af Mouse Celler fra Humane tumorxenoplantater Markant Forbedrer Downstream Analyse af Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...