Dobbelt virkningerne af melanom celle-afledte faktorer på knoglemarv adipocytter differentiering
Summary
Her præsenterer vi en pålidelig og enkel todimensionale (2D) coculture system til at studere samspillet mellem tumorceller og knoglemarv adipocytter, hvilket afslører en dobbelt effekt af melanom celle-afledte faktorer på knoglemarv adipocytter differentiering og udgør også en klassisk metode for mekanistiske studier af knogle metastaser.
Abstract
Krydstale mellem knoglemarv adipocytter og tumorceller kan spille en kritisk rolle i knogle metastaser. En række forskellige metoder er tilgængelige til at studere den betydelige krydstale; en to-dimensional transwell system for coculture er imidlertid fortsat en klassisk, pålidelig og let måde for denne krydstale undersøgelse. Vi præsenterer her, en detaljeret protokol, der viser coculture af knoglemarven adipocytter og melanom celler. Men sådan et coculture system kunne ikke kun bidrage til studie af celle signal transductions af kræftceller induceret af knoglemarven adipocytter, men også til fremtidens mekanistisk undersøgelse af knogle metastaser, der kan afsløre nye terapeutiske mål for knogle metastaser.
Introduction
Knoglemetastaser er udbredt blandt fremskreden kræftpatienter, men en kurativ behandling er stadig ikke tilgængelig. Ud over med speciale i lagre energi som fedt, kan adipocytter støtte tumorvækst og metastase i knoglemarven og andre organer1,2,3,4,5,6. Derudover spiller adipocytter en vigtig rolle i reguleringen af kræft celle biologi7,8,9,10 og metabolisme4,11,12 ,13,14,15,16, som godt som i knogle metastaser1,4,12. I knoglemarven niche, kan adipocytter også påvirke den biologiske opførsel af kræft celler4,6,17. Samspillet mellem knoglemarv adipocytter og kræftceller med osteotropism er væsentlig for forståelsen af knogle metastaser. Dog er lidt kendt.
Baseret på de aktuelle undersøgelser, der forskellige metoder anvendes på adipocytter, herunder to – eller tre – dimensionelle (2/3D) og ex vivo kulturer17,18,19,20,21. For nylig, Herroon et al. designet en ny 3D-kultur tilgang til at studere samspillet mellem knoglemarv adipocytter med kræft celler22. Selv om den 3D coculture er optimal for efterligne fysiologiske interaktioner mellem adipocytter og kræft celler, i vivo, det lider af dårlig reproducerbarhed22,23. Sammenlignet med en 2D coculture system, kan en 3D coculture system give forskellige cellulære fænotyper, såsom celle morfologi21,22,24,25,26. Desuden kan ex vivo kultur af isolerede spongiosa væv fragmenter føre til en robust udvækst af adipocytter fra kulturperler knoglemarv celler17.
I modsætning til disse tidligere modeller, men stadig 2D celle kultur model en klassisk, pålidelig og let teknik til hurtigt scanning kandidat molekyler og fænotyper ændres i enten adipocytter eller kræft celler in vitro-1, 4,6,12,15,27. For bedre at forstå krydstale mellem knoglemarv adipocytter og melanom celler, giver vi en detaljeret protokol for en 2D coculture system af knoglemarven adipocytter med melanom celler.
Protocol
Representative Results
Discussion
Cocultures med skær har været udbredt at studere celle-til-celle interaktioner. 2D coculture system er en effektiv måde at iagttage, hvordan de to dele krydstale in vitro-, som vi her viste to forskellige kræft celle-drevet effekter på knoglemarv adipocytter. Mange laboratorier har udnyttet denne metode til at undersøge krydstale mellem adipocytter og kræft celler6,12,27,39.
…Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker Dov Zipori (The Weizmann Institute of Science, Rehovot, Israel) venligst for at give os murine knoglemarv stromale celle linje 14F1.1. Denne undersøgelse blev støttet af tilskud fra den kinesiske National Natural Science Foundation (nr. 81771729) og det Yongchuan Hospital i Chongqing medicinske universitet (nr. YJQN201330; YJZQN201527).
Materials
| DMEM | Invitrogen Inc. | 11965092 | |
| Fetal Bovine Serum | Invitrogen Inc. | 16000–044 | |
| Phosphate Buffered Saline | Invitrogen Inc. | 14190-144 | |
| Insulin | Sigma-Aldrich | 91077C | |
| 3-isobutyl-1-methyl-xanthine | Sigma-Aldrich | I5879 | |
| Dexamethasone | Sigma-Aldrich | D4902 | |
| Oil Red o | Sigma-Aldrich | O0625 | |
| 24-well plate | Corning | CLS3527 | |
| Transwell insert | Millipore | MCHT24H48 | |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| isopropanol | Sigma-Aldrich | I9516 | |
| 0.25% trypsin | Thermo Scientific | 25200056 | |
| hemocytometer | Bio-Rad | 1450016 | |
| Culture incubator | Thermo Scientific | ||
| 50ml falcon | Corning | CLS430828 | |
| Clean Bench | Thermo Scientific | – | |
| Microscopy | Olympus | – | |
| 200 μL pipet tips | BeyoGold | FTIP620 | |
| 1000 mL pipet tips | BeyoGold | FTIP628 |
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