Generation af Microtumors Brug 3D Menneskelig Biogel Kultur System og Patient-afledte glioblastomaceller for Kinomic Profilering and Drug respons 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 mest almindelige primære intrakranielle maligne hjernetumorer er klasse III astrocytomer og grad IV glioblastoma multiforme (glioblastom eller GBM). Disse tumorer tilbyde dårlige prognoser med median et-års overlevelse mellem 12 – 15 måneder med nuværende behandlinger for GBM i USA 1-3. Multimodalitet terapier omfatter kirurgi, stråling, og kemoterapi herunder temozolomid (TMZ) og kinase-målrettede midler. Kinase signalering ofte dysreguleret i GBM, herunder delmængder af tumorer med forstærkning eller aktiverende mutationer i epidermal vækstfaktor receptor (EGFR), stigninger i blodpladeafledt vækstfaktorreceptor (PDGFR) signalering, øget phosphatidylinositol-3-kinase (PI3K) og tumor understøtter angiogen signalering gennem karendotelvækstfaktor Receptor (VEGFR) samt andre kinase drevet veje 4-6. Nuværende in vitro og in vivo modeller ofte mister disse repræsentative ændringer <sup> 7. Derudover har genetisk profilering ikke tilbudt de forventede fordele, der kan afspejle, at genetiske og epigenetiske ændringer ikke altid forudsige ændringer i niveauet af protein-aktivitet, hvor de fleste kinase målrettet midler virker direkte, og hvor terapier med andre virkningsmekanismer kan handle indirekte.
Den traditionelle immortaliseret cellelinie, som kan passeres ad infinitum har længe været standard til prøvning lægemidlet på grund af deres lette vedligeholdelse og reproducerbarhed. Men denne model lider af en høj næringsstof (og kunstige) vækstmiljø, der selekterer for hurtigt voksende celler, der afviger meget fra den oprindelige tumor. Som sådan har der været en betydelig interesse i at udvikle mere realistiske modelsystemer, der afspejler en mere kompleks tumor biologisk system som er til stede i patienten. Tumorxenoplantater udviklet direkte fra en primær tumor dyrket i mus ( "xenoline," patient-afledt xenograft eller PDX) bestemde en mere reflekterende modelsystem, især i fastsættelsen af kræftmedicin, som de er følt til mere pålideligt forudsige klinisk succes. 8. På trods af den mere reflekterende biologi, disse modeller er dyre og er vanskelige at etablere og vedligeholde. Desuden er de ikke egnede til high-throughput undersøgelser. Behovet for bedre at udvikle biologiske modeller, der mere præcist afspejler molekylære ændringer i de primære tumorer, og at profilere og teste disse modeller bruger direkte målinger af kinase aktivitet, ikke surrogat genetiske markører, er klar.
Det er velkendt, at i modsætning til to-dimensionel (2D) monolagskulturer, kan 3D eller flercellede assay modeller giver mere fysiologisk relevante endpoints 9-11. Fælles 3D kultur tilgange involverer matrix-coatede mikrobærere og celle klumpformet formation. Tumor sfæroider kan genereres via cellulære sammenlægning hjælp spinner kolbe pHEMA plade og hængende drop teknikker. Begrænsninger for tisse tilgange omfatter: manglende evne til nogle celler til at danne stabile sfæroider, variation i væksten og udfordringer med blandede celletyper. Alternativt mange syntetiske (hydrogel, polymer) og animalsk afledte Engelbreth-Holm-Swarm (EHS) matrix fra muse sarkomer, bovint kollagen) matrixer er blevet udviklet til 3D kulturundersøgelser 12-14. Muse EHS matrix benyttes flittigt men kendt for at fremme cellevækst og differentiering in vitro og in vivo 15.
For at replikere 3D tumorbiologi, blev en human biomatrix system udviklet af Dr. Raj Singh et al. 16. Den naturlige, vækstfaktor-fri human Biogel tillader 3D kultur stilladser (perler, skiver), der støtter langsigtet dyrkning af flere celletyper. En serie af 3D human Biogel kultur designs er etableret til undersøgelse tumorvækst, adhæsion, angiogenese og invasion egenskaber. Fordele og egenskaber af humant Biogel sammenlignet med fællesmuse EHS-geler er sammenfattet i tabel 1 og tabel 2.
| Kilde: | Menneskelig Amnions (Pooled væv) Patogenfri, IRB-fritaget / godkendt |
| ECM natur: | Ikke-denatureret Biogel (GLP-produktion) |
| Nøgle komponenter: | Col-I (38%), laminin (22%), col-IV (20%), col-III (7%), entactin & HSPG (<3%) |
| GF-fri: | Målbart EGF, FGF, TGF, VEGF, PDGF (Non-angiogene, Ikke-giftige) |
Tabel 1: Egenskaber af menneskelige Biogel Sammenlignet med Common EHS Gels.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page = "altid">Tabel 2: Fordele ved menneskelige Biogel Sammenlignet med Common EHS Gels.
Protocol
Representative Results
Discussion
Kritiske skridt i protokollen overvejende vedrører microtumor generation, samt dosering narkotika og vedligeholdelse. Fordi microtumor perler er skrøbelig og let revet, er der behov ekstrem omhu i begge udviklingsstadier i et assay og vedligeholdelse. Hvis der opstår en fejl under en af disse processer, kan eksperimentel fortolkning blive kompromitteret, forårsager udvidelse eller unødvendig gentagelse af forsøgene eller endda udelukkelse af data.
Ændringer og fejlfinding, især…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Støttet af NIH R21 tilskud (PI: C. Willey, CA185712-01), Brain Tumor SPORE award (PD: GY Gillespie, P20CA 151.129-03) og SBIR kontrakt (PI: R. Singh, N43CO-2013-00.026).
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 |
References
- Ohgaki, H., Kleihues, P. Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol. 64 (6), 479-489 (2005).
- Wen, P. Y., Kesari, S. Malignant gliomas in adults. N Engl J Med. 359 (5), 492-507 (2008).
- Thumma, S. R., et al. Effect of pretreatment clinical factors on overall survival in glioblastoma multiforme: a Surveillance Epidemiology and End Results (SEER) population analysis. World J Surg Oncol. 10 (75), (2012).
- Furnari, F. B., Cloughesy, T. F., Cavenee, W. K., Mischel, P. S. Heterogeneity of epidermal growth factor receptor signalling networks in glioblastoma. Nat Rev Cancer. 15 (5), 302-310 (2015).
- Mischel, P. S., Cloughesy, T. F., Nelson, S. F. DNA-microarray analysis of brain cancer: molecular classification for therapy. Nat Rev Neurosci. 5 (10), 782-792 (2004).
- Verhaak, R. G., et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 17 (1), 98-110 (2010).
- De Witt Hamer, P. C., et al. The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids. Oncogene. 27 (14), 2091-2096 (2008).
- Shankavaram, U. T., et al. Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma. J Cell Mol Med. 16 (3), 545-554 (2012).
- Abbott, A. Cell culture: biology’s new dimension. Nature. 424 (6951), 870-872 (2003).
- Rao, S. S., Lannutti, J. J., Viapiano, M. S., Sarkar, A., Winter, J. O. Toward 3D biomimetic models to understand the behavior of glioblastoma multiforme cells. Tissue Eng Part B Rev. 20 (4), 314-327 (2014).
- Pampaloni, F., Reynaud, E. G., Stelzer, E. H. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 8 (10), 839-845 (2007).
- Hollister, S. J. Porous scaffold design for tissue engineering. Nat Mater. 4 (7), 518-524 (2005).
- Rowley, J. A., Madlambayan, G., Mooney, D. J. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials. 20 (1), 45-53 (1999).
- Hughes, C. S., Postovit, L. M., Lajoie, G. A. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics. 10 (9), 1886-1890 (2010).
- Yoshino, J. E., et al. Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane. Glia. 3 (5), 315-321 (1990).
- Siegal, G. P., Singh, R., Foundation, T. U. R. Biologically active native biomatrix composition. US patent. , (2010).
- Sarkaria, J. N., et al. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin Cancer Res. 12 (7), 2264-2271 (2006).
- Strober, W. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 111, 1-3 (2015).
- Anderson, J. C., et al. Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines. Radiother Oncol. 111 (3), 468-474 (2014).
- Anderson, J. C., et al. Kinomic profiling of electromagnetic navigational bronchoscopy specimens: a new approach for personalized medicine. PLoS One. 9 (12), 116388 (2014).
- Jarboe, J. S., et al. Kinomic profiling approach identifies Trk as a novel radiation modulator. Radiother Oncol. 103 (3), 380-387 (2012).
- Anderson, J. C., et al. High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes. PLoS One. 10 (9), 0139267 (2015).
- Anderson, J. C., et al. Kinomic Alterations in Atypical Meningioma. Medical Research Archives. 3, (2015).
- Hothi, P., et al. High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells. Oncotarget. 3 (10), 1124-1136 (2012).
- Quartararo, C. E., Reznik, E., deCarvalho, A. C., Mikkelsen, T., Stockwell, B. R. High-Throughput Screening of Patient-Derived Cultures Reveals Potential for Precision Medicine in Glioblastoma. ACS Med Chem Lett. 6 (8), 948-952 (2015).
- Ma, L., et al. Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system. Biomaterials. 33 (17), 4353-4361 (2012).
- Pedron, S., Becka, E., Harley, B. A. Regulation of glioma cell phenotype in 3D matrices by hyaluronic acid. Biomaterials. 34 (30), 7408-7417 (2013).
- Rape, A., Ananthanarayanan, B., Kumar, S. Engineering strategies to mimic the glioblastoma microenvironment. Adv Drug Deliv Rev. 79-90, 172-183 (2014).
- Willey, C. D., Gilbert, A. N., Anderson, J. C., Gillespie, G. Y. Patient-Derived Xenografts as a Model System for Radiation Research. Semin Radiat Oncol. 25 (4), 273-280 (2015).
