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 ønsker å takke Nikon Imaging Center ved Harvard Medical School, spesielt Jennifer Waters, Lara Petrak og Wendy Laks, for opplæring og bruk av deres timelapse mikroskoper. Vi ønsker også å takke Rosa Ng og Achim Besser for verdifulle diskusjoner. Dette arbeidet ble støttet av NIH Grant 5695837 (til M. Iwanicki) og GM064346 til JSB, av en bevilgning fra Dr. Miriam og Sheldon Adelson G. medisinske forskningsstiftelse (til JSB).

1. Ovarian Cancer Cell Spheroid Formation <ol><li> RFP-uttrykke eggstokkreft celler dyrkes i 10% Base Medium (et egendefinert celle kultur medium med en 50:50 blanding av 199 og MCDB105, 10% inaktivert fosterutvikling bovint serum og 1% penn-strep). For å uttrykke RFP i umerkede eggstokkreft celler, transfektere cellene med et plasmid som inneholder RFP og velg for celler som uttrykker RFP. Alternativt kan virale vektorer brukes til midlertidig uttrykke fluorescerende proteiner, eller cellene kan være pre-inkuber me...

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Den &quot;mesothelial Clearance Assay&quot; presenteres her bruker time-lapse mikroskop for å overvåke interaksjoner av eggstokkreft flercellede spheroids og mesothelial celle monolayers, i stor romlig og tidsmessig detalj. Tidligere hadde flere grupper 8-14 brukes endepunkt analyser å vise at eggstokkreft celler feste til og invadere inn mesothelial celle monolayers. Denne analysen er unik i at den bruker fluorescensmerkede celler å skille kreftceller fra mesothelial celler, slik at dynamikken i disse to...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> OVCA433 eggstokkreft celler </td><td></td><td></td><td> Gave fra Dr. Dennis Slamon </td></tr><tr><td> ZT mesothelial celler </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 Søknader Inc. </td><td> 117-500 </td><td></td></tr><tr><td> FBS-varme inaktivert </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 og plater </td><td> Corning CoStar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylate (poly-HEMA) </td><td> Sigma Aldrich </td><td> 192 066-25G </td><td> For poly-HEMA løsning oppløse 6mg poly-HEMA pulver i 1 ml 95% EtOH </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> Fortynn til 95% i dH 2 0 </td></tr><tr><td> Cellekultur Hood </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> Tissue kulturinkubatoren </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> inkubator for poly-Hema plater </td><td> Labline Instrumenter </td><td> Imperial III 305 </td><td></td></tr><tr><td> Tabletop sentrifuge </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 godt glass-bunn parabolen </td><td> Mattek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> Fibronectin </td><td> Sigma </td><td> F1141-1 mg </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Timelapse mikroskop: </td><td></td><td></td><td></td></tr><tr><td> Microscope </td><td> Nikon </td><td></td><td> Ti-E Inverted Motorisert Fluorescence time-lapse mikroskop med integrert 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 transilluminator </td><td> Nikon </td><td></td><td> 0,52 NA lang arbeidsavstand kondensator </td></tr><tr><td> Magnetisering og utslipp filtre </td><td> Kroma single pass filter 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> Overføres og Epifluoresce lysbanen </td><td> Sutter </td&gt; <td></td><td> Smarte Shutters </td></tr><tr><td> Lineær-kodet motorisert scenen </td><td> Nikon </td><td></td><td></td></tr><tr><td> Avkjølt ladet-kombinert enhet kamera </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Microscope inkubasjon kammer med temperatur og CO 2 kontroll </td><td> spesialbygd </td><td></td><td></td></tr><tr><td> Vibrasjonsisolering bord </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements programvare </td><td> Nikon </td><td></td><td> Versjon 3 </td></tr></tbody></table>

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

Ovarian kreft er den femte største årsaken til kreft dødsfall i USA 1. Til tross for en positiv initiell respons på behandling, 70 til 90 prosent av kvinner med eggstokkreft utvikle nye metastaser, og tilbakefall er ofte dødelig to. Det er derfor nødvendig å forstå hvordan sekundære metastaser oppstår for å utvikle bedre behandlingsmetoder for middels og sent stadium eggstokkreft. Eggstokkreft metastasering oppstår når ondartede celler løsner fra den primære svulsten området og spre hele bukhulen. De spres cellene kan danne flercellede klynger, eller spheroids, som enten vil forbli fristilt, eller implantat på organer innenfor bukhulen 3 (Figur 1, Movie 1). Alle organene i bukhulen er foret med en enkelt, sammenhengende, lag mesothelial celler 4-6 (figur 2). Men mesothelial celler er fraværende fra undersidenperitoneal tumor masser som åpenbart av elektron micrograph studier av excised menneskelige svulstvev seksjoner 3,5-7 (figur 2). Dette tyder på at mesothelial celler er ekskludert fra under tumormasse av en ukjent prosess. Forrige in vitro eksperimenter viste at primære eggstokkreft celler feste mer effektivt å ekstracellulær matrix enn å mesothelial celler 8, og nyere studier viser at primær peritoneal mesothelial celler faktisk gir en barriere mot eggstokkreft celle adhesjon og invasjon (i forhold til heft og invasjon på underlag som ikke var dekket med mesothelial celler) 9,10. Dette ville foreslå at mesothelial celler fungere som en barriere mot eggstokkreft metastasering. De cellulære og molekylære mekanismer som eggstokkreft celler bryter denne barrieren, og utelukke mesothelium har inntil nylig, forble ukjent. Her beskriver vi the metodikk for en in vitro metode som modeller samspillet mellom eggstokkreft celle spheroids og mesothelial celler in vivo (figur 3, Movie 2). Vår protokollen ble tilpasset fra tidligere beskrevet metoder for å analysere ovarian tumorcellelinjer interaksjoner med mesothelial monolayers 8-16, og ble først beskrevet i en rapport som viser at eggstokkene tumorceller utnytte en integrin-avhengig aktivering av myosin og trekkraft for å fremme utelukkelse av mesothelial celler fra under en svulst sfæriske legemet 17. Denne modellen tar fordel av time-lapse fluorescensmikroskopi å overvåke de to celle populasjoner i sanntid, noe som gir romlig og tidsmessig informasjon om interaksjon. De eggstokkreft celler uttrykker rød fluorescerende protein (RFP), mens de mesothelial celler uttrykker grønt fluorescerende protein (GFP). RFP-uttrykke eggstokkreft celle spheroids feste til GFP-uttrykke mesothelial monolayer. Den spheroids spredning, invadere, ogtvinge mesothelial cellene til side skaper et hull i monolayer. Dette hullet er visualisert som den negative plass (svart) i GFP bildet. Arealet av hullet kan da måles til kvantitativt analysere forskjeller i clearance aktivitet mellom kontroll og eksperimentelle populasjoner av eggstokkreft og / eller mesothelial celler. Denne analysen krever bare et lite antall eggstokkreft celler (100 celler per Spheroid X 20-30 spheroids per tilstand), så det er mulig å utføre denne analysen bruker dyrebare primære tumor celleprøver. Videre kan denne analysen enkelt tilpasses for høy gjennomstrømming 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 beskrives her utnytter fluorescensmerkede celler og time-lapse video mikroskopi for å visualisere og kvantitativt måle interaksjoner av eggstokkreft flercellede spheroids og mesothelial celle monolayers. Denne analysen modellerer de tidlige trinnene på eggstokkreft metastasering.

In vitro mesothelial Clearance Assay at modeller av de tidlige trinnene på eggstokkreft 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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

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Heterotypic Tredimensjonal<em&gt; In Vitro</em&gt; Modellering av stromal-Epiteliale interaksjoner Ovarian Cancer initiering og progresjon

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

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Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro berikelse for eggstokkreft Tumor-initiere Cells

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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

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

I Vivo og Ex Vivo tilnærminger til Study Ovarian Cancer Metastatisk Kolonisering av Milky Spot Structures i Peritoneal adipose

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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Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...