Vurdering cellelevedygtighed og død i 3D Spheroid kulturer af kræftceller
Summary
Her præsenterer vi flere enkle metoder til at evaluere levedygtighed og død i 3D cancer celle sfæroider, som efterligner de fysisk-kemiske gradienter af in vivo tumorer meget bedre end 2D-kulturen. Den sfæide model giver derfor mulighed for evaluering af kræft stof effektivitet med forbedret oversættelse til in vivo betingelser.
Abstract
Tre-dimensionelle sfæroider af kræftceller er vigtige redskaber til både kræft stof skærme og for at få mekanistisk indsigt i kræft cellebiologi. Kraften i denne forberedelse ligger i dens evne til at efterligne mange aspekter af in vivo betingelser for tumorer, mens de er hurtige, billige, og alsidig nok til at tillade relativt høj-gennemløb screening. De sfæide dyrkningsbetingelser kan rekapitulere de fysisk-kemiske gradienter i en tumor, herunder den stigende ekstracellulære surhedsgrad, øget lactat og faldende glukose-og ilttilgængelighed fra den sfæide periferi til dens kerne. Også, de mekaniske egenskaber og celle-celle interaktioner af in vivo tumorer er til dels efterlignes med denne model. De specifikke egenskaber og dermed de optimale vækstbetingelser, af 3D sfæroider, varierer meget mellem forskellige typer af kræftceller. Desuden kræver vurderingen af cellernes levedygtighed og død i 3D sfæroider metoder, der delvis adskiller sig fra dem, der anvendes til 2D-kulturer. Her beskriver vi flere protokoller til forberedelse af 3D sfæroider af kræftceller, og for at bruge sådanne kulturer til at vurdere cellernes levedygtighed og død i forbindelse med evaluering af effekten af anticancermedicin.
Introduction
Brugen af flercellede sfæide modeller i kræft biologi er flere årtier gamle1,2, men har fået betydelig momentum i de seneste år. I vid del, dette afspejler øget bevidsthed om, hvor stærkt fænotype kræftceller er afhængig af deres mikromiljø og specifikke vækstbetingelser. Mikromiljøet i solide tumorer er fundamentalt forskellig fra det i tilsvarende normale væv. Dette omfatter fysisk-kemiske forhold såsom pH, oxygen spændinger, samt interstitiel tryk, koncentrations gradienter af opløselige faktorer såsom næringsstoffer, affaldsprodukter, og udskilles signalering forbindelser (vækstfaktorer, cytokiner). Endvidere, det omfatter tilrettelæggelse af ekstracellulære matrix (ECM), celle-celle interaktioner og intercellulære signalering, og andre aspekter af den særlige tredimensionelle (3D) arkitektur af tumor3,4, 5,6. De specifikke mikromiljømæssige forhold, hvor kræftceller findes, påvirker i høj grad deres genekspression profil og funktionelle egenskaber, og det er klart, at sammenlignet med celler dyrket i 2D, Fænotypen af 3D sfæroider meget tættere efterligner af in vivo-tumorer7,8,9,10,11. 2D-modeller, selv om de beskæftiger hypoxi, sure pH, og høje lactat koncentrationer til at efterligne kendte aspekter af Tumorens mikromiljø, stadig undlader at indfange gradienter af fysisk-kemiske parametre, der opstår i tumorer, samt deres 3D tumor Arkitektur. På den anden side, dyremodeller er dyrt, langsom, og etisk problematisk, og generelt har også mangler i deres evne til at rekapitulere menneskelige tumor betingelser. Derfor er 3D sfæroider blevet anvendt som en mellemliggende kompleksitet model i undersøgelser af en bred vifte af egenskaber af de fleste solide kræftformer9,11,12,13, 14,15,16,17.
En almindeligt anvendt brug af 3D sfæroider er i screening analyser af anticancer terapi effekt9,18,19,20. Behandling reaktioner er særligt følsomme over for tumor mikromiljø, der afspejler både virkningen af den tortuøsitet, begrænset diffusion, høj interstitiel tryk, og sure miljømæssige pH på medicin levering, og virkningen af hypoxi og andre aspekter af mikromiljøet på celledød svar9,17. Fordi miljøet i 3D sfæroider i sagens natur udvikler alle disse egenskaber7,8,9,10,11, beskæftiger 3D cellekulturer kan væsentligt forbedre oversættelsen af resultater til in vivo-forhold, men giver mulighed for en effektiv og økonomisk overkommelig screening af nettovæksten i høj produktion. Men det store flertal af undersøgelser af lægemiddel respons af kræftceller er stadig udført under 2D betingelser. Dette afspejler sandsynligvis, at mens nogle analyser relativt let kan implementeres for 3D cellekulturer, mange, såsom levedygtighed analyser, Western blotting, og immunofluorescens analyse, er meget mere bekvemt gjort i 2D end i 3D.
Formålet med det nuværende arbejde er at give let genstand analyser og præcise protokoller for analyser af effekten af behandling med anti-cancer medicin på kræftcellernes levedygtighed og overlevelse i en 3D tumor efterligner indstilling. Konkret leverer og sammenligner vi tre forskellige metoder til sfæoide dannelse, efterfulgt af metoder til kvalitative og kvantitative analyser af vækst, levedygtighed og narkotika respons.
Protocol
Representative Results
Discussion
Brugen af 3D cancer celle sfæroider har vist sig at være et værdifuldt og alsidigt værktøj ikke kun for anticancer Drug screening, men også for at få mekanistisk indsigt i reguleringen af kræftcellernes død og levedygtighed under betingelser, der efterligner dem i tumoren mikromiljø. Dette er især afgørende, da tilgængelighed, cellulær optagelse, og intracellulære virkninger af kemoterapeutiske lægemidler er dybt påvirket af de fysisk-kemiske forhold i tumoren, herunder pH, oxygen spændinger, tortuøsit…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi er glade for Katrine Franklin Mark og Annette Bartels for fremragende teknisk assistance og for Asbjørn Nøhr-Nielsen for at udføre eksperimenterne i figur 1D. Dette arbejde blev finansieret af Einar Willumsen Foundation, Novo Nordisk Foundation, og Fondation Juchum (alle til SFP).
Materials
| 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) | Invitrogen | # C10595 | For staining nuclei |
| 5-Fluorouracil (5-FU) | Sigma-Aldrich | #F6627 | Component in chemotherapeutic treatment |
| 5-(N-ethyl-isopropyl) amiloride (EIPA) | Life Technologies | #E3111 | Inhibitor of NHE1 |
| Antibody against PARP and cPARP | Cell signaling | #9542 | Used in western blotting |
| Antibody against Ki-67 | Cell signaling | #9449 | Used for IHC |
| Antibody against p53 | Cell Signaling | #2524 | Used for IHC |
| Antibody against β-actin | Sigma | A5441 | Used in western blotting |
| Bactoagar | BD Bioscience | #214010 | Used for agarose gel preparation |
| Benchmark protein ladder | Invitrogen | #10747-012 | Used for SDS-PAGE |
| Bio-Rad DC Protein Assay kit | Bio-Rad Laboratories | #500-0113, #500-0114, #500-0115 | Used for protein determination from lysates |
| Bürker chamber | Marienfeld | 610311 | For cell counting |
| BX63 epifluoresence microscope | Olympus | Used for fluorescent imaging | |
| CellTiter-Glo 3D Cell Viability Assay | Promega | #G9681 | Used for the cell viability assay |
| Cisplatin | Sigma-Aldrich | #P4394 | Component in chemotherapeutic treatment |
| Corning Spheroid Microplate, 96 well, Black with clear round bottom, Ultra-low attachment, With lid, Sterile | Corning | #4520 | Used for growing spheroids with luminescence measurements as end point |
| Corning 96 well, clear round bottom, Ultra-low attachment microplate, With lid, Sterile | Corning | #7007 | Sufficient for spheroid growth without luminescence measurements as end point |
| Criterion TGX Precast Gels | Bio-Rad | 5671025 | Used for SDS-PAGE |
| Doxorubicin | Abcam | #120629 | Component in chemotherapeutic treatment |
| FLUOStar Optima Microplate reader | BMG Labtech | Used for recording luminescence | |
| Formaldehyde | VWR Chemicals | #9713.1000 | Used for cell fixation |
| Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix | Gibco | #A1413202 | Keep at 4 °C to prevent solidification. Referred to as rBM in the protocol. |
| Heat-inactivated FBS | Sigma | #F9665 | Serum for growth media |
| ImageJ | NIH | Scientific Image analysis | |
| Medim Uni-safe casette | Medim Histotechnologie | 10-0114 | Used for storage of embedded spheroids |
| Mini protease inhibitor cocktail tablets | Roche Diagnostics GmBH | # 11836153001 | Used for lysis buffer preparation |
| MZ16 microscope | Leica | Used for light microscopic images | |
| NuPAGE LDS 4x Sample Buffer | Invitrogen | #NP0007 | Used for western blotting |
| Pierce ECL Western blotting substrate | Thermo scientific | #32106 | Used for western blotting |
| Ponceau S | Sigma-Aldrich | #P7170-1L | Used for protein band staining |
| Prism 6.0 | Graphpad | Scientific graphing and statistical software | |
| Propidium iodide (1mg/ml solution in water) | Invitrogen | P3566 | Light sensitive |
| Sterile reservoirs, multichannel | SPL lifesciences | 21002 | Used for seeding cells for spheroid formation |
| Superfrost Ultra-Plus Adhesion slide | Menzel-Gläser | #J3800AMNZ | Microscope glass slide used for embedding |
| Tamoxifen | Sigma-Aldrich | #T5648 | Used as chemotherapeutic treatment |
| Trans-blot Turbo 0.2 µm nitrocellulose membranes | Bio-Rad | #170-4159 | Used for western blotting |
| Tris/Glycine/SDS running buffer | Bio-Rad | #161 0732 | Used for SDS-PAGE |
| Trypsin-EDTA solution | Sigma | #T4174 | Cell dissociation enzyme |
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