To effekter av melanom Cell-derived faktorer på benmarg adipocytter differensiering
Summary
Her presenterer vi en pålitelig og enkel todimensjonal (2D) coculture system for å studere samspillet mellom kreftceller og benmarg adipocytter, som avslører en dobbel effekt av melanom celle-avledet faktorer på benmarg adipocytter differensiering og utgjør også en klassisk metode for mekanistisk studier av bein metastasering.
Abstract
Crosstalk mellom benmarg adipocytter og kreftceller kan spille en avgjørende rolle i prosessen med bein metastasering. En rekke metoder er tilgjengelige for å studere den betydelige crosstalk; imidlertid fortsatt en todimensjonal transwell system for coculture en klassisk, pålitelig, og enkel måte for denne crosstalk studien. Her presenterer vi en detaljert protokoll som viser coculture av benmarg adipocytter og melanom celler. Likevel kunne slikt coculture system ikke bare bidra til studiet av cellen signal transductions av kreftceller av benmarg adipocytter, men også i fremtiden mekanistisk studie av bein metastasering som kan avsløre nye terapeutiske mål for bein metastasering.
Introduction
Benmetastaser er utbredt blant avansert kreftpasienter, men en helbredende behandling er fremdeles utilgjengelig. Utover spesialiserer seg på lagring av energi som fett, kan adipocytter støtte tumor vekst og metastasering i benmargen og andre organer1,2,3,4,5,6. Videre spiller adipocytter en viktig rolle i å regulere kreft cellen biologi7,8,9,10 og metabolisme4,11,12 ,13,14,15,16, så vel som i bein metastasering1,4,12. I benmargen nisje, kan adipocytter også påvirke biologisk atferd av kreft celler4,6,17. Samspillet mellom benmarg adipocytter og kreftceller med osteotropism er viktig for en forståelse av bein metastasering. Men er lite kjent.
Basert på dagens studier, brukes ulike metoder på adipocytter, inkludert to – eller tre – dimensjonale (2/3D) og ex vivo kulturer17,18,19,20,21. Nylig utviklet Herroon et al. en ny 3D-kultur tilnærming til studere interaksjoner av benmarg adipocytter med kreft celler22. Selv om det 3D coculture er optimal for mimicking fysiologiske interaksjoner mellom adipocytter og kreft celler i vivo, den lider av dårlig reproduserbarhet22,23. I forhold til en 2D coculture system, kan en 3D coculture-system gi mobilnettet Norge, for eksempel cellen morfologi21,22,24,25,26. Videre kan ex vivo kulturen av isolerte cancellous bein vev føre til en robust utvekst av adipocytter kulturperler bein margtransplantasjon celler17.
I motsetning til disse tidligere modeller imidlertid 2D celle kultur modellen en klassisk, pålitelig og enkel teknikk for raskt skanne kandidat molekyler og av fenotyper i enten adipocytter eller kreft celler i vitro1, 4,6,12,15,27. For bedre å forstå crosstalk mellom benmarg adipocytter og melanom celler, gir vi en detaljert protokoll for et 2D coculture system av benmarg adipocytter med melanom celler.
Protocol
Representative Results
Discussion
Cocultures med innlegg har vært mye brukt til å studere celle til celle interaksjoner. 2D coculture systemet er en effektiv måte å observere hvordan de to delene crosstalk i vitro, som vi her viste to forskjellige kreft celle-drevet effekter på benmarg adipocytter. Mange labs har brukt denne metoden for å undersøke crosstalk mellom adipocytter og kreft celler6,12,27,39.
<p c…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Vi takker Dov Zipori (The Weizmann-Instituttet for vitenskap, Rehovot, Israel) ber for å gi oss murine benmarg stromal cellen linje 14F1.1. Denne studien ble støttet av tilskudd fra kinesiske National Natural Science Foundation (nr. 81771729) og Yongchuan Hospital i Chongqing medisinske universitet (Nos. 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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