Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成
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
最常见的原发性脑恶性脑肿瘤是III级星形细胞瘤和Ⅳ级胶质母细胞瘤 （成胶质细胞瘤或GBM）。这些肿瘤提供与12之间平均一年的生存预后差- 15个月，在美国的1-3电流疗法GBM。多模态治疗包括手术，放疗和化疗，包括替莫唑胺（TMZ）和激酶靶向剂。激酶信号经常失调在GBM，包括在表皮生长因子受体（EGFR），增加血小板源生长因子受体（PDGFR）信令，增加的磷脂肌醇-3-激酶（PI3K），并与扩增的肿瘤的子集或激活突变肿瘤通过血管内皮生长因子受体（VEGFR），以及其他激酶驱动路径4-6支承血管生成信令。当前在体外和体内模型中经常失去这些代表性的改变<SUP> 7。此外，遗传分析没有提供可能反映出遗传和表观遗传变化并不总是在蛋白活性的水平，其中大多数激酶靶向剂直接作用预测的变化，并且其中与其它作用机制的治疗可充当预期的效益间接的影响。
可以传代无限传统的永生化细胞系一直是药物测试标准，由于其易于维护和重现性。然而，该模型从选择用于快速增长，从原来的肿瘤有很大的不同的细胞的高营养（和人工的）的生长环境受到影响。因此，出现了在显影反映一个更复杂的肿瘤生物学系统如存在于病人更现实的模型系统相当大的兴趣。肿瘤异种移植物直接从小鼠生长的原发肿瘤（“xenoline，”以病人为衍生异种移植或PDX）provi开发德一多个反射模型系统，特别是在癌症治疗的设置，因为它们被认为更可靠地预测临床成功。8尽管多个反射生物学，这些模型是昂贵的，并且难以建立和保持。此外，它们不适合于高通量研究。有必要更好地发展生物模型，更准确地反映原发肿瘤的分子改变，以及个人资料，并使用激酶活性的直接措施，检验这些模型，而不是代孕遗传标记，是显而易见的。
它众所周知，不同于二维（2D）单层培养，三维或多细胞测定模型可以提供更多的生理学相关端点9-11。常见的三维培养方法涉及涂基质微载体和细胞球体的形成。肿瘤球状体可以通过蜂窝聚合使用旋转器烧瓶中，的pHEMA板与悬滴技术产生。限制在tHESE方法包括：不能对一些细胞形成稳定的球状体，变异成长和挑战，混合细胞型。可替代地，许多合成的（水凝胶，聚合物）和动物衍生Engelbreth-Holm的-群基质已用于3D培养开发从小鼠肉瘤（EHS）矩阵，牛胶原）研究12-14。鼠标EHS矩阵被广泛使用，但已知促进细胞生长和分化的体外 和体内 15。
为了复制3D肿瘤生物学，人类生物基质系统是由拉吉辛格博士等人的 16开发的。 自然，生长自由因子人biogel允许三维培养支架（珠片），它支持多种类型的细胞长期培养。一系列的三维人体biogel文化设计都建立了研究肿瘤的生长，黏附，血管生成和侵袭特性。优势和人力biogel的性能比普通鼠标EHS凝胶总结在表1和表2。
| 资源： | 人类羊膜 （池组织） 无病原体，IRB豁免/批准 |
| ECM性质： | 非变性Biogel（GLP-生产） |
| 键 组件： | COL-I（38％），层粘连蛋白（22％），COL-IV（20％），COL-III（7％），巢蛋白和HSPG（<3％） |
| GF-免费： | 检测不到 EGF，FGF，TGF，VEGF，PDGF（非血管，无毒） |
表1：相比于常见的EHS凝胶人力Biogel的性质。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within页="“总是”">表2：相比于常见的EHS凝胶人力Biogel的优势。
Protocol
Representative Results
Discussion
在协议中的关键步骤主要涉及microtumor代，以及药物剂量和维护。因为microtumor珠粒是脆弱的，容易撕裂，需要在检测和维修都发育阶段特别小心。如果在这些过程之一中出现错误，实验解释可能会受到损害，从而导致延长或实验的不必要的重复，甚至排除的数据。
修改和排查，尤其是microtumor发展过程中，包括为使用使得microtumor珠的自定义工具疏水性的设计和生产。这个工具?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
，脑肿瘤SPORE奖（PD：GY吉莱斯皮，P20CA 151129-03）和SBIR合同（PI：R.辛格，N43CO-2013-00026）：由美国国立卫生研究院资助R21（C.威利，CA185712-01 PI）的支持。
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).
