Name: spheroid assay to measure tgf-&#946;-induced invasion
Uploaded: 2011-11-16T13:00:00
Description: Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

Vi är tacksamma för Ken Iwata (OSI Pharmaceuticals, New York, USA) för reagenser och Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) för cellinjer.

1. Beredning av methocel lösning (500 ml) <ol><li> Autoklav 6 g metylcellulosa i en 500 ml flaska med en magnetomrörare. </li><li> Lös metylcellulosa i 250 ml förvärmd DMEM/F12 utan serum (60 ° C) i 20 min. </li><li> Tillsätt 250 ml DMEM/F12 (RT) med 10% häst serum. Blanda natten (4 ° C). </li><li> Rensa lösningen genom centrifugering (5000g, 2h, 4 ° C). </li><li> Ta den klara mycket viskös lösning till ett nytt rör (ca 90-95% av stamlösning). </li><li> Förvaras vid 4 ° C fram till användning. </li><...

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Vi etablerade en sfäroid modell där MCF10A1 celler och maligna derivat invadera till en kollagenmatrisen i en TGF-β-beroende sätt. Först är spheroids bildas i 96-brunnar runda plattor botten fjädring i närvaro av metylcellulosa. Naturligtvis kan även U-formad poly-HEMA plåt användas för detta ändamål. Metylcellulosa är absolut krävs i detta steg, eftersom celler dör i avsaknad av metylcellulosa. TGF-β läggs direkt till kollagen-methocel blandning eftersom vi upptäckt att celler svarade sämre på TGF...

<table border="1"><tbody><tr><td> Reagensets namn </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer (tillval) </td></tr><tr><td> Metylcellulosa </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avancerad Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH-indikator band </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-bra fjädring plattor </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-bra vidhäftning TC plattor </td><td> Greiner Bio-One </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β har motsatta roller i bröstcancer progression genom att fungera som en tumörsuppressor i det inledande skedet, men stimulerande invasion och metastasering i ett senare skede 1,2. Dessutom är TGF-β ofta överuttryckt i bröstcancer och dess uttryck korrelerar med dålig prognos och metastasering 3,4. De mekanismer genom vilka TGF-β inducerar invasion inte är välkända. TGF-β framkallar sina cellulära svaren via TGF-β typ II (TβRII) och typ I (TβRI)-receptorer. Vid TGF-β-inducerad heteromeric komplexbildning, fosforylerar TβRII den TβRI. Den aktiverade TβRI inleder intracellulära kanoniska signalväg genom fosforylerande receptor Smads (R-Smads), dvs Smad2 och Smad3. Dessa aktiveras R-Smads formen heteromeric komplex med Smad4, som ackumuleras i cellkärnan och reglera transkription av mål gener 5. Förutom den tidigare described Smad väg, aktivering av receptorn resulterar i aktivering av flera andra icke-Smad signalvägar, till exempel mitogen aktiverat protein kinas (MAPK) vägar 6. För att studera betydelsen av TGF-β i olika stadier av bröstcancer, gjorde vi användning av MCF10A cellen systemet. Detta system består av spontant förevigat MCF10A1 (M1) bröst epitelceller 7, förvandlade H-RAS M1-derivata MCF10AneoT (M2), som producerar premaligna lesioner hos möss 8 och M2-derivata MCF10CA1a (M4), som bildades ur M2 implanterad och bildar hög kvalitet carcinom med förmågan att metastasera till lungorna 9. Detta MCF10A serien erbjuder möjligheten att studera de svar av celler med olika grader av malignitet som inte är påverkade av en annan genetisk bakgrund. För analys av TGF-β-inducerad invasionen genererade vi homotypic MCF10A sfäroid cellkulturer embedded i en 3D kollagenmatrisen in vitro (Fig. 1). Sådana modeller liknar mycket humana tumörer in vivo genom att etablera en gradient av syre och näringsämnen, vilket resulterar i ett aktivt och invasiva celler på utsidan och inaktiv eller nekrotiska celler i insidan av sfäroid 10. Sfäroid baserade analyser har också visat att bättre rekapitulera resistens än monolayer kulturer 11. Denna MCF10 3D-modell systemet tillät oss att undersöka effekten av TGF-β signalering på invasiva egenskaper bröst celler i olika stadier av malignitet.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-ÃƒÅ½Ã‚Â²-induced Invasion

En analys för att kvantitativt mäta Transforming Growth Factor (TGF)-β-inducerad invasionen i 3-dimensionella kollagen geler beskrivs. Denna analys drar fördel av MCF10A serie av cellinjer som representerar olika stadier av bröstcancer utveckling. Denna metod kan antas för att användas tillsammans med andra cellinjer och kan användas för att undersöka andra potentiella aktivatorer eller hämmare av invasion.

Sfäroid analys för att mäta TGF-β-inducerad Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

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Spheroid Assay to Measure TGF-&#946;-induced Invasion

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