Name: in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis
Uploaded: 2012-02-17T13:00:00
Description: Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

Vi vil gerne takke Nikon Imaging Center på Harvard Medical School, især Jennifer Waters, Lara Petrak og Wendy Laks, for uddannelse og brug af deres timelapse mikroskoper. Vi vil også gerne takke Rosa Ng og Achim Besser for værdifulde diskussioner. Dette arbejde blev støttet af NIH Grant 5695837 (til M. Iwanicki) og GM064346 til fælles kontrolinstans, af en bevilling fra Dr. Miriam og Sheldon G. Adelson Medical Research Foundation (til JSB).

1. Kræft i æggestokkene Cell klumpformet Dannelse <ol><li> RFP-udtrykkende ovariecancerceller dyrkes i 10% basemedium (en brugerdefineret celledyrkningsmedium indeholdende en 50:50 blanding af 199 og MCDB105, 10% inaktiveret føtalt bovint serum og 1% pen-strep). For at udtrykke RFP i umærkede ovariecancerceller, transficere cellerne med et plasmid indeholdende RFP og selektere for celler, der udtrykker RFP. Alternativt kan virale vektorer kan anvendes til transient udtrykker fluorescerende proteiner, eller cellern...

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Den &quot;mesothelial Clearance Assay&quot; præsenteres her bruger time-lapse mikroskopi til at overvåge samspillet mellem kræft i æggestokkene flercellede kugler og mesothelial cellemonolag, i stor rumlig og tidsmæssig detaljer. Tidligere havde flere grupper 8-14 bruges endpoint-analyser til at vise, at ovariecancerceller lægger til og invadere ind mesothelial cellemonolag. Dette assay er enestående ved, at den anvender fluorescensmærkede celler at skelne tumorceller fra mesotelceller, således at dy...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> OVCA433 ovariecancerceller </td><td></td><td></td><td> Gave fra Dr. Dennis Slamon </td></tr><tr><td> ZT mesotelceller </td><td></td><td></td><td> Gave fra Dr. Tan Ince </td></tr><tr><td> Medium 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Cell Applications Inc. </td><td> 117-500 </td><td></td></tr><tr><td> FBS-varmeinaktiveret </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> 96-brønds plader </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylat (Poly-HEMA) </td><td> Sigma Aldrich </td><td> 192.066-25G </td><td> For poly-HEMA-opløsning opløses 6mg poly-HEMA pulver i 1 ml 95% EtOH </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> Fortynd til 95% i dH2O 0 </td></tr><tr><td> Cellekultur hætte </td><td> Nuaire </td><td> NU-425 til 300 </td><td></td></tr><tr><td> Vævsdyrkningsinkubator </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> inkubator for poly-HEMA-plader </td><td> Labline Instrumenter </td><td> Imperial III 305 </td><td></td></tr><tr><td> Bordcentrifuge </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 og glasbund skål </td><td> Mattek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> Fibronektin </td><td> Sigma </td><td> F1141-1mg </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21 til 040-CV </td><td></td></tr><tr><td> Timelapse Microscope: </td><td></td><td></td><td></td></tr><tr><td> Mikroskop </td><td> Nikon </td><td></td><td> Ti-E Inverted Motoriseret Fluorescens time-lapse mikroskop med integreret Perfect Focus System </td></tr><tr><td> Lens </td><td> Nikon </td><td></td><td> 20X-0,75 numerisk apeture </td></tr><tr><td> Halogen transilluminatoren </td><td> Nikon </td><td></td><td> 0,52 NA lange afstand kondensator </td></tr><tr><td> Excitations-og emissionsfiltre </td><td> Chroma Single Pass filtre i Nikon bolig </td><td></td><td> GFP Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Overført og Epifluoresce lysvej </td><td> Sutter </td&gt; <td></td><td> Smart Skodder </td></tr><tr><td> Lineær kodet motoriseret objektbord </td><td> Nikon </td><td></td><td></td></tr><tr><td> Cooled Charge coupled device kamera </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Mikroskop inkubation kammer med temperatur og CO 2-kontrol </td><td> specialbyggede </td><td></td><td></td></tr><tr><td> Vibrationsisolering tabel </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements software </td><td> Nikon </td><td></td><td> Version 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

Kræft i æggestokkene er den femte hyppigste årsag til kræft dødsfald i USA 1. Trods en positiv indledende respons på behandlinger, udvikle 70 til 90 procent af kvinder med kræft i æggestokkene nye metastaser, og fornyet ofte er dødelig 2. Det er derfor nødvendigt at forstå, hvordan sekundære metastaser opstår for at udvikle bedre behandlinger for mellemprodukt og sen fase ovariecancer. Ovariecancer metastase opstår, når maligne celler løsnes fra det primære tumorsted og sprede hele den peritoneale kavitet. De formidlede celler kan danne multicellulære klynger eller sfæroider, som enten vil forblive separate eller implantat på organer i den peritoneale kavitet 3 (figur 1, film 1). Alle organerne i den peritoneale kavitet er foret med en enkelt, kontinuerlig lag af mesotelceller 4-6 (fig. 2). Imidlertid mesotelceller er fraværende nedefraperitoneale tumormasser, som afsløret ved elektronmikrografi undersøgelser af udskårne humane tumor vævssnit 3,5-7 (figur 2). Dette antyder, at mesotelceller er udelukket fra undersiden af ​​tumormassen af ​​en ukendt fremgangsmåde. Tidligere in vitro-forsøg viste, at primære ovariecancerceller vedhæfte mere effektivt til ekstracellulær matrix end mesotelceller 8, og nyere undersøgelser viste, at primære peritoneale mesotelceller faktisk tilvejebringe en barriere mod ovariecancer celleadhæsion og invasion (sammenlignet med adhæsion og invasion på substrater, der ikke var dækket med mesothelial celler) 9,10. Dette tyder på, at mesotelceller virke som en barriere mod ovariecancer metastase. De cellulære og molekylære mekanismer, som ovariecancerceller overtræde denne barriere, og udelukke mesothelium har indtil for nylig, forblev ukendt. Her beskriver vi the metode til et in vitro assay, der modellerer den interaktionen mellem ovariecancer celle sfæroider og mesoteliale celler in vivo (figur 3, film 2). Vores protokol blev tilpasset fra tidligere beskrevne fremgangsmåder til analyse af ovariale tumorer celleinteraktioner med mesoteliale monolag 8-16, og blev først beskrevet i en rapport, der viser, at ovarie tumorceller anvender et integrin-afhængig aktivering af myosin og trækkraft for at fremme udelukkelse af mesotelceller fra under en tumor sfæroid 17. Denne model udnytter tidsforløb fluorescensmikroskopi for at overvåge de to cellepopulationer i realtid, giver rumlig og tidsmæssig information om interaktionen. De ovariecancerceller udtrykker røde fluorescerende protein (RFP), medens de mesoteliale celler udtrykker grønt fluorescerende protein (GFP). RFP-udtrykke kræft i æggestokkene celle sphæroider tillægger GFP-udtrykkende mesothelial monolag. Den sfæroider spredning, invaderer ogtvinge mesotelceller side skaber et hul i monolag. Dette hul er visualiseret som den negative rum (sort) i GFP-billedet. Det område af hullet kan derefter måles kvantitativt at analysere forskelle i afstand aktivitet mellem kontrol-og forsøgsdyr populationer af ovariecancer og / eller mesotelceller. Dette assay kræver kun et lille antal af ovariecancerceller (100 celler pr kugleformede X 20-30 sfæroider pr tilstand), så det er muligt at udføre dette assay under anvendelse kostbare primær tumor celleprøver. Endvidere kan dette assay kan let tilpasses til high throughput screening.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Den mesothelial clearance analysen beskrevet her drager fordel af fluorescens mærkede celler og time-lapse video mikroskopi til at visualisere og kvantitativt at måle samspillet mellem kræft i æggestokkene flercellede kugler og mesothelial cellemonolag. Dette assay modellerer de tidlige trin af ovariecancer metastase.

In vitro mesothelial Clearance Assay at modeller de tidlige stadier af kræft i æggestokkene Metastase

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

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Heterotypisk tredimensionale<em&gt; In Vitro</em&gt; Modellering af Stromal-Epiteliale Interactions Under Kræft i æggestokkene Indledning og Progression

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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 ...

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In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

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High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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