Generering av Microtumors Bruke 3D Menneskelig Biogel Kultur System og pasient-avledet glioblastom celler for Kinomic Profilering and Drug Response Testing
Summary
Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.
Abstract
The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.
Introduction
De vanligste primære intrakraniale ondartede hjernesvulster er klasse III astrocytomas og klasse IV glioblastoma multiforme (glioblastom eller GBM). Disse svulstene har dårlige prognoser med median ett-års overlevelse mellom 12 – 15 måneder med dagens terapi for GBM i USA 1-3. Multimodalitet behandlinger inkluderer kirurgi, stråling og kjemoterapi, inkludert temozolomid (TMZ) og kinase-målrettede midler. Kinase signalering blir ofte dysregulerte i GBM, blant annet undersett av tumorer med forsterkning eller aktiverende mutasjoner i epidermal vekstfaktor reseptor (EGFR), en økning i blodplateavledet vekstfaktor reseptor (PDGFR) signalering, økt Phosphatidyl-inositol-3-kinase (PI3K) og tumorbærende angiogen signalering gjennom vaskulær endotel vekstfaktor reseptor (VEGFR) så vel som andre kinase drevet veier 4-6. Current in vitro- og in vivo-modeller ofte miste disse representative forandringer <sup> 7. I tillegg har genetisk profilering ikke tilbudt forventede fordeler som kan gjenspeile det faktum at genetiske og epigenetiske endringene ikke alltid forutsi endringer på nivået av protein aktivitet, der de fleste kinase målsøkende midler handle direkte, og hvor behandling med andre virkningsmekanismer kan handle indirekte.
Den tradisjonelle udødeliggjort cellelinje som kan passeres ad infinitum har lenge vært standard for narkotika testing på grunn av deres enkle vedlikehold og reproduserbarhet. Imidlertid lider denne modellen fra et høyt næringsinnhold (og kunstig) vekst miljø som selekterer for hurtigvoksende celler som avviker sterkt fra den opprinnelige tumor. Som sådan, har det vært betydelig interesse for å utvikle mer realistiske modellsystemer som reflekterer en mer kompleks svulst biologisk system som er til stede i pasienten. Tumorxenotransplantater utviklet direkte fra en primær svulst vokst i mus ( "xenoline," pasient-avledet xenograft eller PDX) provide en mer reflekterende modellsystem, spesielt i innstillingen av kreftbehandling, som de følte seg til mer pålitelig måte å forutsi klinisk suksess. 8 Til tross for den mer reflekterende biologi, disse modellene er kostbare, og er vanskelig å etablere og vedlikeholde. Dessuten er de ikke mottagelig for high-throughput-studier. Behovet for å bedre utvikle biologiske modeller som mer nøyaktig gjenspeiler molekylære endringer i de primære svulster, og for å profilere og teste disse modellene bruker direkte tiltak av kinase aktivitet, ikke surrogat genetiske markører, er klart.
Det er velkjent at i motsetning til to-dimensjonale (2D) monolagskulturer, kan 3D- eller flercellede analysemodeller gi mer fysiologisk relevante endepunkter 9-11. Felles 3D kultur tilnærminger involvere matrix-belagt mikro og celle spheroid formasjon. Tumor kuler kan genereres via mobilnettet aggregering bruker spinner kolbe, PHEMA plate og henger slipp teknikker. Begrensninger for these tilnærminger inkluderer: manglende evne for noen celler til å danne stabile kuler, variasjon i vekst og utfordringer med blandede celletyper. Alternativt mange syntetiske (hydrogel, polymer) og dyr-avledet Engelbreth-Holm-Swarm (EHS) matrise fra mus sarkomer, kollagen) matriser er utviklet for 3D kultur studerer 12-14. Mus EHS matrise blir brukt i stor utstrekning, men er kjent for å fremme cellevekst og differensiering in vitro og in vivo 15.
For å gjenskape 3D tumorbiologi, ble et menneske BioMatrix system utviklet av Dr. Raj Singh et al. 16. Den naturlige, vekstfaktor-frie menneske Biogel tillater 3D kultur stillaser (perler, plater), som støtter langsiktig dyrking av flere celletyper. En serie av 3D menneske Biogel kultur design er etablert for å studere tumorvekst, vedheft, angiogenese og invasjon egenskaper. Fordeler og egenskaper til human Biogel sammenlignet med vanligmus EHS geler er oppsummert i tabell 1 og tabell 2.
| Kilde: | Menneskelig Amnions (Felles vev) Patogen-fri, IRB-fritak / godkjent |
| ECM naturen: | Non-denaturert Biogel (GLP-produksjon) |
| Nøkkel komponenter: | Col-I (38%), Laminin (22%), Col-IV (20%), Col-III (7%), Entactin og HSPG (<3%) |
| GF-free: | Undetectable EGF, FGF, TGF, VEGF, PDGF (Non-angiogen, Giftfri) |
Tabell 1: Egenskaper for menneskelig Biogel i forhold til vanlige HMS-Gel.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-side = "always">Tabell 2: Fordeler med Menneskelig Biogel i forhold til vanlige HMS-Gel.
Protocol
Representative Results
Discussion
Kritiske trinnene i protokollen overveiende knyttet til microtumor generasjon, så vel som medikamentdosering og vedlikehold. Fordi microtumor perler er skjøre og lett revet, er ekstrem forsiktighet nødvendig i både utviklingsstadier av en analyse og vedlikehold. Hvis det oppstår en feil under en av disse prosessene kan eksperimentell tolkning bli kompromittert, forårsaker utvidelse eller unødvendig repetisjon av forsøkene eller eksklusjon av data.
Modifikasjoner og feilsøking, spesi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Støttet av NIH R21 stipend (PI: C. Willey, CA185712-01), hjernesvulst SPORE award (PD: GY Gillespie, P20CA 151129-03) og SBIR kontrakt (PI: R. Singh, N43CO-2013-00026).
Materials
| Collagenase-I | Sigma-Aldrich | CO130 | |
| Trypsin EDTA (10X) | Invitrogen | 15400-054 | |
| Neurobasal-A | Life Technologies | 10888-022 | |
| N-2 Supplement | Life Technologies | 17502-048 | 1x final concentration |
| B-27 Supplement w/o Vitamin A | Life Technologies | 12587-010 | 1x final concentration |
| Recombinant Human FGF-basic | Life Technologies | PHG0266 | 10 ng/mL final concentration |
| Recombinant Human EGF | Life Technologies | PGH0315 | 10 ng/mL final concentration |
| L-Glutamine | Corning Cellgro Mediatech | 25-005-CI | 2 mM final concentration |
| Fungizone | Omega Scientific | FG-70 | 2.5 ug/mL final concentration |
| Penicillin Streptomycin | Omega Scientific | PS-20 | 100 U/mL Penicillin G, 100 ug/mL Streptomycin final concentration |
| Gentamicin | Life Technologies | 15750-060 | 50 ng/mL final concentration |
| MTT | Life Technologies | M6494 | prepared to 5 mg/mL in PBS and sterile filtered, 1 mg/mL in well |
| SDS | Fisher | BP166 | for MTT lysis buffer, prepared to 10% in 0.01M HCL, 5% in well |
| HCl | Fisher | A144SI-212 | for MTT lysis buffer, prepared to 0.01M with SDS, 5 mM in well |
| Calcein AM | Life Technologies | C1430 | 1 mM in DMSO stock, 2 uM in PBS staining solution, 1 uM in well |
| Halt’s Protein Phosphatase Inhibitor cocktail | Pierce ThermoScientific | 78420 | 1:100 ratio in MPER |
| Halt's Protein Protease Inhibitor | Pierce ThermoScientific | 87786 | 1:100 ratio in MPER |
| Mammalian Protein Extraction Reagent (MPER) | Pierce ThermoScientific | PI78501 | |
| Trypan Blue | Pierce ThermoScientific | 15250-061 | |
| DMSO | Fisher | BP231 | for dissolution of calcein AM & compounds |
| Phosphate-Buffered Saline without Ca/Mg | Lonza | 17-517Q | diluted to 1X with MiliQ ultrapure water and sterile filtered (for cell culture) |
| Dulbecco's Phosphate-Buffered Saline with Ca/Mg | Corning Cellgro Mediatech | 20-030-CV | diluted to 1X with MiliQ ultrapure water (for pre-fixation wash) |
| 10% Neutral Buffered Formalin | Protocol | 032-060 | |
| Trypan Blue | Pierce ThermoScientific | 15250-061 | |
| High Density Hubiogel | Vivo Biosciences | HDHG-5 | |
| Halt's Protein Phosphatase Inhibitor | Pierce | 78420 | |
| Halt's Protein Protease Inhibitor | Pierce | 87786 | |
| Mammalian Protein Extraction Reagent (MPER) | Thermo Scientific | 78501 | |
| Protein Tyrosine Kinase (PTK) Array Profiling chip | PamGene | 86312 | |
| PTK kinase buffer | PamGene | 36000 | 300 µl 10X PK buffer stock in 2.7 ml dH20, catalog number for PTK reagent kit |
| ATP | PamGene | 36000 | catalog number for PTK reagent kit |
| PY20- FITC-conjugated antibody | PamGene | 36000 | catalog number for PTK reagent kit |
| PTK Additive | PamGene | 32114 | |
| PTK-MM1 tube (10X BSA) | PamGene | 36000 | catalog number for PTK reagent kit |
| Serine/Threonine Kinase (STK) Array Profiling chip | PamGene | 87102 | |
| STK kinase buffer | PamGene | 32205 | catalog number for STK reagent kit |
| STK Primary Antibody Mix (DMAB tube) | PamGene | 32205 | catalog number for STK reagent kit |
| FITC-conjugated Secondary Antibody | PamGene | 32203 | |
| STK-MM1 tube (100X BSA) | PamGene | 32205 | catalog number for STK reagent kit |
| STK Antibody Buffer | PamGene | 32205 | catalog number for STK reagent kit |
| Equipment | |||
| #11 Blades, sterile | Fisher | 3120030 | |
| #3 scalpel handles, sterile | Fisher | 08-913-5 | |
| 100mm glass Petri dishes | Fisher | 08-748D | |
| Semicurved forceps | Fisher | 12-460-318 | |
| Trypsinizing flask | Fisher | 10-042-12B | |
| Magnetic stirrer | Fisher | 14-490-200 | |
| 3/4" stir bar | Fisher | 14-512-125 | |
| B-D cell strainer | Fisher | #352340 | |
| B-D 50ml Centrifuge tube | Fisher | #352098 | |
| PamStation 12 | PamGene | ||
| BioNavigator 6.0 kinomic analysis software | PamGene | ||
| Evolve Kinase Assay Software | PamGene | ||
| UpKin App software (upstream kinase prediction) | PamGene | ||
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | |
| Rotary Cell Culture System (RCCS) | Synthecon | RCCS-D | with 10 mL disposable HARV |
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