Realtidsovervågning af menneskelige Glioma celle migration på dorsale root ganglion Axon-Oligodendrocyte Co-kulturer
Summary
Her præsenterer vi et ex-vivo blandet enkeltlags kultur system til studiet af human gliom Cell (HGC) migration i realtid. Denne model giver mulighed for at observere interaktioner mellem hGCs og både myelinerede og ikke-myelinerede axoner i et opdelt kammer.
Abstract
Glioblastoma er en af de mest aggressive menneskelige kræftformer på grund af omfattende cellulære heterogenitet og migrations egenskaber af hGCs. For bedre at forstå de molekylære mekanismer underliggende gliom celle migration, en evne til at studere samspillet mellem hgcs og axoner i tumor mikromiljø er afgørende. For at modellere denne cellulære interaktion, udviklede vi et blandet kultur system bestående af hgcs og dorsale root basale ganglier (DRG) Axon-oligodendrocyte Co-kulturer. DRG-kulturerne blev udvalgt, fordi de kan isoleres effektivt og kan danne de lange, omfattende fremskrivninger, der er ideelle til migrationsundersøgelser af denne art. Renset rotte oligodendrocytter blev derefter tilsat på renset rotte DRG axoner og induceret til myelinat. Efter at have bekræftet dannelsen af kompakt myelin, blev hgcs endelig føjet til co-kulturen og deres interaktioner med DRG axoner og oligodendrocytter blev overvåget i realtid ved hjælp af tidsforskudt mikroskopi. Under disse betingelser, hgcs danner tumor-lignende aggregat strukturer, der udtrykker GFAP og Ki67, migrere langs både myelinerede og ikke-myelerede axonal spor og interagere med disse axoner gennem dannelsen af pseudopodia. Vores ex vivo Co-Culture system kan bruges til at identificere nye cellulære og molekylære mekanismer af hGC migration og kunne potentielt anvendes til in vitro Drug effektivitet test.
Introduction
Glioblastoma er en af de mest aggressive og dødbringende tumorer i den menneskelige hjerne. Den nuværende standard for pleje omfatter kirurgisk resektion af tumoren efterfulgt af stråling1 plus samtidig og adjuverende administration af temozolomid2. Selv med denne multi-terapeutiske tilgang, tumor gentagelse er uundgåelig3. Dette skyldes til dels den omfattende migrerende karakter af tumorceller, som invaderer hjernen parenkym skabe flere finger-lignende fremskrivninger i hjernen4 , der gør fuldstændig resektion usandsynligt.
I de seneste år, er det blevet klart, at aggressivitet af glioblastoma skyldes, til dels, tilstedeværelsen af en population af kræft stamceller i tumormassen5,6, som udviser højt migrerende potentiale7,8, resistens over for kemoterapi og stråling9,10 og evnen til at danne sekundære tumorer11. GSCs er i stand til at rekapitulere oprindelige polyklonale tumorer, når xenografted til Nude mus5.
På trods af den rigdom af viden om Glioblastomas genetiske baggrund, er undersøgelser af gliom Cell (GC) migration i øjeblikket hæmmet af mangel på effektive in vitro-eller in vivo-migrations modeller. Især mens gliom celle-axonal interaktioner moduleret af cellulære og miljømæssige faktorer er en kernekomponent i gliom invasion, til vores viden er der i øjeblikket ingen eksperimentel system med evnen til at modellere disse interaktioner12,13,14. For at afhjælpe denne mangel udviklede vi et ex vivo-kultur system af primære Hgc’er, der er co-dyrket med rensede DRG Axon-oligodendrocytter, som resulterer i forhøjet ekspression af differentierede tumor markører samt omfattende migration og interaktion af hGCs med myelinerede og ikke-myelerede fibre. Denne ex vivo platform, på grund af sin opdelte layout, er egnet til at teste virkningerne af nye Therapeutics på hGC migration mønstre.,
Protocol
Representative Results
Discussion
Migrationsundersøgelser for hGCs kan udføres ved hjælp af BoyDen kammer systemer eller ridse assays. Men, mens disse eksperimenter undlader at give nogen oplysninger om samspillet mellem tumorceller med andre omgivende væv, det nuværende system kan rekapitulere GC interaktioner med myelinerede og ikke-myelerede fibre. Endvidere, at studere tumordannelse og slutpunkt migration, organotypiske skive kulturer af gnaver hjernen eller in vivo implantation af gliom celler i gnaver hjernen eller flanke har tidligere været …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af interne fonde af Department of neurosurgery, Brown University til N.T.
Materials
| 100 mm Suspension Culture Dish | Corning | 430591 | |
| 2.5S NGF | ENVIGO | B.5025 | |
| 60 mm Suspension Culture Dish | Corning | 430589 | |
| ACK Lysing Buffer | Thermo Fisher | A1049201 | |
| Ammonium Hydroxide Solution | Fisher Scientific | A669-500 | Concentrated |
| Animal-Free Recombinant Human EGF | Peprotech | AF-100-15 | |
| Animal-Free Recombinant Human FGF-basic (154 a.a.) | Peprotech | AF-100-18B | |
| Anti-A2B5 MicroBeads, human, mouse, rat | Miltenyi Biotec | 130-093-392 | |
| Antibiotic-Antimycotic (100X) | Thermo Fisher | 15240062 | |
| AutoMACS Rinsing Solution (PBS, pH 7.2) | Miltenyi Biotec | 130-091-222 | |
| B27 Supplement | Thermo Fisher | 17504044 | |
| B27 Supplement, minus vitamin A | Thermo Fisher | 12587001 | |
| Bacteriological Plate | BD Falcon | 351029 | |
| Biotin | Sigma | B4639 | |
| BSA | Sigma | A9418 | |
| Campenot Chamber | Tyler Research | CAMP-10 | |
| Cell Culture Dish | Corning | 430165 | 35mm X 10mm |
| Cell Strainer | BD Falcon | 352350 | 70 uM, Nylon |
| Cell Strainer | BD Falcon | 352340 | 30 uM, Nylon |
| Collagenase/Dispase | Roche | 11097113001 | |
| Cultrex Rat Collagen I | Trevigen | 3440-100-01 | |
| D-Glucose | Sigma | G5146 | |
| DMEM | Thermo Fisher | 10313021 | |
| DNase I | Sigma | D7291 | |
| Dow Corning High-Vacuum Grease | Fisher Scientific | 14-635-5D | |
| Dumont #5 Forceps | Roboz | RS-5045 | |
| E16 Timed Pregnant Sprague Dawley Rat | |||
| EBSS | Sigma | E7510 | |
| EGTA | Sigma | E3889 | |
| FBS | Hyclone | SH30070.02 | |
| FUDR | Sigma | F0503 | |
| GlutaMAX Supplement | Thermo Fisher | 35050061 | |
| Ham's F-12 Nutrient Mix | Thermo Fisher | 11765054 | |
| HBSS | Thermo Fisher | 14175095 | |
| Hemostatic Forceps | Roboz | RS-7035 | |
| Heparin Sodium Salt, 0.2% in PBS | Stem Cell Technologies | 07980 | |
| Hypodermic Needle, 18G | BD | 511097 | |
| Insulin-Transferrin-Selenium G | Thermo Fisher | 41400045 | |
| L-Cysteine | Sigma | C7477 | |
| L-Glutamine | Thermo Fisher | 25030081 | |
| Leibovitz's L-15 Medium | Thermo Fisher | 11415064 | |
| MACS BSA Stock Solution | Miltenyi Biotec | 130-091-376 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| MEM | Thermo Fisher | 1190081 | |
| Mg2SO4 | Sigma | M2643 | |
| MiniMACS Separator | Miltenyi Biotec | 130-042-102 | |
| MS Columns plus tubes | Miltenyi Biotec | 130-041-301 | |
| NAC | Sigma | A8199 | |
| NaHCO3 | Sigma | S5761 | |
| Neurobasal Medium | Thermo Fisher | 21103049 | |
| Neurobasal-A Medium | Thermo Fisher | 10888022 | |
| Ordinary forceps | |||
| P2 Sprague Dawley Rat Pups | |||
| Papain | Worthington | LS003126 | |
| Penicillin-Streptomycin | Thermo Fisher | 15140148 | |
| Pin Rake | Tyler Research | CAMP-PR | |
| Progesterone | Sigma | P8783 | |
| StemPro Accutase Cell Dissociation Reagent | Thermo Fisher | A1110501 | |
| Syrine Grease Applicator | Tyler Research | CAMP-GLSS | |
| Transferrin | Sigma | T2036 | |
| Uridine | Sigma | U3003 |
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