评估癌细胞3D球形培养中的细胞活力和死亡
Summary
在这里,我们提出了几种简单的方法来评估3D癌细胞球体的生存能力和死亡,它模仿体内肿瘤的物理化学梯度比2D培养要好得多。因此,球形模型允许评估癌症药物疗效,并改进对体内条件的翻译。
Abstract
癌细胞的三维球体是癌症药物屏幕和获得对癌细胞生物学的机械洞察力的重要工具。这种制剂的力量在于它能够模仿肿瘤体内状况的许多方面,同时快速、廉价和通用,足以允许相对较高的通量筛查。球体培养条件可以重述肿瘤中的物理化学梯度,包括细胞外酸度增加、乳酸增加、葡萄糖和氧气供应减少,从球体边缘到其核心。此外,该模型部分模拟了体内肿瘤的机械特性和细胞-细胞相互作用。3D球体的具体性质和最佳生长条件在不同类型的癌细胞之间差别很大。此外,评估3D球体的细胞活力和死亡需要部分不同于2D培养方法的方法。在这里,我们描述了几种协议,用于制备癌细胞的3D球体,并在评估抗癌药物疗效的背景下利用这些培养物评估细胞的生存能力和死亡。
Introduction
在癌症生物学中使用多细胞球体模型已有几十年的历史,但近年来却获得了巨大的发展势头。这在很大程度上反映了人们对于癌细胞表型对其微环境和特定生长条件的依赖程度的日益提高的认识。实体肿瘤的微观环境与相应的正常组织有着根本的不同。这包括物理化学条件,如pH、氧张力,以及间质压力、可溶性因子(如营养物质、废物产品和分泌信号化合物)的浓度梯度(生长因子、细胞因子)。此外,它包括组织细胞外基质(ECM),细胞-细胞相互作用和细胞间信令,以及肿瘤的特定的三维(3D)结构的其他方面3, 5,6.癌细胞存在的特定微环境条件深刻地影响其基因表达特征和功能特性,很明显,与在二维中生长的细胞相比,3D球体的表型更接近于模仿体内肿瘤7,8,9,10,11。2D模型,即使它们采用缺氧、酸性pH值和高乳酸浓度来模拟肿瘤微环境的已知方面,仍然无法捕获肿瘤内产生的物理化学参数及其3D肿瘤的梯度建筑。另一方面,动物模型是昂贵,缓慢,道德问题,一般来说,也有缺陷,在能力,以重新报告人类肿瘤条件。因此,3D球体已作为中间复杂性模型应用于大多数固体癌症的多种特性的研究9,11,12,13, 14,15,16,17.
一种广泛使用的3D球体是筛选癌症治疗疗效9,18,19,20的筛选方法。治疗反应对肿瘤微环境特别敏感,既反映了肿瘤的侵权性、扩散受限、间隙压力高、酸性环境pH值对药物输送的影响,也反映了缺氧等的影响。微环境方面对细胞死亡反应9,17。因为3D球形内的环境本身发展了所有这些属性7,8,9,10,11,使用3D细胞培养可以大幅度提高结果在体内条件的转化,同时允许对净增长进行高效且经济实惠的高通量筛选。然而,绝大多数关于癌细胞药物反应的研究仍在二维条件下进行。这可能反映了,虽然一些检测可以相对容易地用于 3D 细胞培养,但许多,如可行性测定,西方印迹和免疫荧光分析,在 2D 比在 3D 中更容易完成。
本工作的目的是提供易于接受的测定和精确的方案,用于分析抗癌药物治疗对3D肿瘤模拟环境中癌细胞活力和存活率的影响。具体来说,我们提供和比较三种不同的球形形成方法,然后是对生长、活力和药物反应进行定性和定量分析的方法。
Protocol
1. 飞球体的生成 为球形形成准备细胞悬浮液注:不同的细胞系具有非常不同的附着力,必须在每种情况下建立最合适的球形形成协议。我们发现MCF-7和BxPC-3细胞适合自发球体形成,而MDA-MB-231、SKBr-3、Panc-1和MiaPaCa需要添加重组的基体膜才能成功形成球体。只有MDA-MB-231和BxPC-3细胞用于挂降协议,但其他细胞系当然适用。 将细胞生长为单层,直到70-80%的汇合。 用磷酸盐?…
Representative Results
基于球形形成方案的球形生长测定图1A和图1B,作为分析3D肿瘤抗癌药物治疗效果的起点模仿设置。球形的形成容易是细胞系特定的,一些细胞系需要补充rBM才能形成相干球体22。添加的rBM浓度对球体的形态有深刻的影响。如图1C和图1D所示,在0?…
Discussion
使用3D癌细胞球体已被证明是一个有价值的和多功能的工具,不仅用于抗癌药物筛选,而且获得机械洞察力,在模拟肿瘤条件下的癌细胞死亡和生存能力的调节微。这一点尤其重要,因为化疗药物的可及性、细胞接受性和细胞内效应受到肿瘤物理化学条件(包括pH、氧张力、曲解性、物理和物理和化学细胞-细胞相互作用9,17。例如,在许多实体肿瘤25、26?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢卡特琳·富兰克林·马克和安妮特·巴特尔斯的出色技术援助,感谢阿斯比约恩·内尔-尼尔森在图1D中执行实验。这项工作由艾纳尔·维伦森基金会、诺和诺德基金会和Juchum基金会(全部为SFP)资助。
Materials
| 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) | Invitrogen | # C10595 | For staining nuclei |
| 5-Fluorouracil (5-FU) | Sigma-Aldrich | #F6627 | Component in chemotherapeutic treatment |
| 5-(N-ethyl-isopropyl) amiloride (EIPA) | Life Technologies | #E3111 | Inhibitor of NHE1 |
| Antibody against PARP and cPARP | Cell signaling | #9542 | Used in western blotting |
| Antibody against Ki-67 | Cell signaling | #9449 | Used for IHC |
| Antibody against p53 | Cell Signaling | #2524 | Used for IHC |
| Antibody against β-actin | Sigma | A5441 | Used in western blotting |
| Bactoagar | BD Bioscience | #214010 | Used for agarose gel preparation |
| Benchmark protein ladder | Invitrogen | #10747-012 | Used for SDS-PAGE |
| Bio-Rad DC Protein Assay kit | Bio-Rad Laboratories | #500-0113, #500-0114, #500-0115 | Used for protein determination from lysates |
| Bürker chamber | Marienfeld | 610311 | For cell counting |
| BX63 epifluoresence microscope | Olympus | Used for fluorescent imaging | |
| CellTiter-Glo 3D Cell Viability Assay | Promega | #G9681 | Used for the cell viability assay |
| Cisplatin | Sigma-Aldrich | #P4394 | Component in chemotherapeutic treatment |
| Corning Spheroid Microplate, 96 well, Black with clear round bottom, Ultra-low attachment, With lid, Sterile | Corning | #4520 | Used for growing spheroids with luminescence measurements as end point |
| Corning 96 well, clear round bottom, Ultra-low attachment microplate, With lid, Sterile | Corning | #7007 | Sufficient for spheroid growth without luminescence measurements as end point |
| Criterion TGX Precast Gels | Bio-Rad | 5671025 | Used for SDS-PAGE |
| Doxorubicin | Abcam | #120629 | Component in chemotherapeutic treatment |
| FLUOStar Optima Microplate reader | BMG Labtech | Used for recording luminescence | |
| Formaldehyde | VWR Chemicals | #9713.1000 | Used for cell fixation |
| Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix | Gibco | #A1413202 | Keep at 4 °C to prevent solidification. Referred to as rBM in the protocol. |
| Heat-inactivated FBS | Sigma | #F9665 | Serum for growth media |
| ImageJ | NIH | Scientific Image analysis | |
| Medim Uni-safe casette | Medim Histotechnologie | 10-0114 | Used for storage of embedded spheroids |
| Mini protease inhibitor cocktail tablets | Roche Diagnostics GmBH | # 11836153001 | Used for lysis buffer preparation |
| MZ16 microscope | Leica | Used for light microscopic images | |
| NuPAGE LDS 4x Sample Buffer | Invitrogen | #NP0007 | Used for western blotting |
| Pierce ECL Western blotting substrate | Thermo scientific | #32106 | Used for western blotting |
| Ponceau S | Sigma-Aldrich | #P7170-1L | Used for protein band staining |
| Prism 6.0 | Graphpad | Scientific graphing and statistical software | |
| Propidium iodide (1mg/ml solution in water) | Invitrogen | P3566 | Light sensitive |
| Sterile reservoirs, multichannel | SPL lifesciences | 21002 | Used for seeding cells for spheroid formation |
| Superfrost Ultra-Plus Adhesion slide | Menzel-Gläser | #J3800AMNZ | Microscope glass slide used for embedding |
| Tamoxifen | Sigma-Aldrich | #T5648 | Used as chemotherapeutic treatment |
| Trans-blot Turbo 0.2 µm nitrocellulose membranes | Bio-Rad | #170-4159 | Used for western blotting |
| Tris/Glycine/SDS running buffer | Bio-Rad | #161 0732 | Used for SDS-PAGE |
| Trypsin-EDTA solution | Sigma | #T4174 | Cell dissociation enzyme |
Referências
- Sutherland, R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 240 (4849), 177-184 (1988).
- Mueller-Klieser, W., Freyer, J. P., Sutherland, R. M. Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids. British Journal of Cancer. 53 (3), 345-353 (1986).
- Gaedtke, L., Thoenes, L., Culmsee, C., Mayer, B., Wagner, E. Proteomic analysis reveals differences in protein expression in spheroid versus monolayer cultures of low-passage colon carcinoma cells. Journal of Proteome Research. 6 (11), 4111-4118 (2007).
- Chen, J. L., et al. The genomic analysis of lactic acidosis and acidosis response in human cancers. PLoS Genetics. 4 (12), 1000293 (2008).
- Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science. 294 (5547), 1708-1712 (2001).
- Gudjonsson, T., Ronnov-Jessen, L., Villadsen, R., Bissell, M. J., Petersen, O. W. To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods. 30 (3), 247-255 (2003).
- Pampaloni, F., Reynaud, E. G., Stelzer, E. H. The third dimension bridges the gap between cell culture and live tissue. Nature Reviews in Molecular and Cell Biology. 8 (10), 839-845 (2007).
- Hirschhaeuser, F., et al. Multicellular tumor spheroids: an underestimated tool is catching up again. Journal of Biotechnology. 148 (1), 3-15 (2010).
- Jacobi, N., et al. Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling. Oncotarget. 8 (64), 107423-107440 (2017).
- Rodriguez-Enriquez, S., et al. Energy metabolism transition in multi-cellular human tumor spheroids. Journal of Cell Physiology. 216 (1), 189-197 (2008).
- Kunz-Schughart, L. A. Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor. Cell Biology International. 23 (3), 157-161 (1999).
- Andersen, A. P., et al. Roles of acid-extruding ion transporters in regulation of breast cancer cell growth in a 3-dimensional microenvironment. Molecular Cancer. 15 (1), 45 (2016).
- Swietach, P., Patiar, S., Supuran, C. T., Harris, A. L., Vaughan-Jones, R. D. The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths. Journal of Biological Chemistry. 284 (30), 20299-20310 (2009).
- Walenta, S., Doetsch, J., Mueller-Klieser, W., Kunz-Schughart, L. A. Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts. Journal of Histochemistry and Cytochemistry. 48 (4), 509-522 (2000).
- Kunz-Schughart, L. A., Groebe, K., Mueller-Klieser, W. Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation. International Journal of Cancer. 66 (4), 578-586 (1996).
- Feng, H., et al. Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer. Journal of Cancer Research and Clinical Oncology. 143 (9), 1771-1786 (2017).
- Santini, M. T., Rainaldi, G., Indovina, P. L. Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Critical Reviews in Oncology/Hematology. 36 (2-3), 75-87 (2000).
- Vinci, M., et al. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology. 10, 29 (2012).
- Pickl, M., Ries, C. H. Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene. 28 (3), 461-468 (2009).
- Wong, C., Vosburgh, E., Levine, A. J., Cong, L., Xu, E. Y. Human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy. Journal of Visual Experiments. (66), e4218 (2012).
- Friedrich, J., et al. A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay. Journal of Biomolecular Screening. 12 (7), 925-937 (2007).
- Ivascu, A., Kubbies, M. Diversity of cell-mediated adhesions in breast cancer spheroids. International Journal of Oncology. 31 (6), 1403-1413 (2007).
- Crouch, S. P., Kozlowski, R., Slater, K. J., Fletcher, J. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods. 160 (1), 81-88 (1993).
- Andersen, A. P., et al. The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms. International Journal of Cancer. , (2018).
- Vaupel, P. Tumor microenvironmental physiology and its implications for radiation oncology. Seminars in Radiation Oncology. 14 (3), 198-206 (2004).
- Vaupel, P. W., Frinak, S., Bicher, H. I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma. Pesquisa do Câncer. 41 (5), 2008-2013 (1981).
- Helmlinger, G., Yuan, F., Dellian, M., Jain, R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nature Medicine. 3 (2), 177-182 (1997).
- Zhang, X., Lin, Y., Gillies, R. J. Tumor pH and its measurement. Journal of Nuclear Medicine. 51 (8), 1167-1170 (2010).
- Gillies, R. J., Raghunand, N., Karczmar, G. S., Bhujwalla, Z. M. MRI of the tumor microenvironment. Journal of Magnetic Resonance Imaging. 16 (4), 430-450 (2002).
- Vukovic, V., Tannock, I. F. Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan. British Journal of Cancer. 75 (8), 1167-1172 (1997).
- Song, C. W., Griffin, R., Park, H. J., Teicher, B. A. . Cancer Drug Resistance. , 21-42 (2006).
- Lotz, C., et al. Role of the tumor microenvironment in the activity and expression of the p-glycoprotein in human colon carcinoma cells. Oncology Reports. 17 (1), 239-244 (2007).
- Sant, S., Johnston, P. A. The production of 3D tumor spheroids for cancer drug discovery. Drug Discovery Today: Technologies. 23, 27-36 (2017).
- Stratmann, A. T., et al. Establishment of a human 3D lung cancer model based on a biological tissue matrix combined with a Boolean in silico model. Molecular Oncology. 8 (2), 351-365 (2014).
- Kuen, J., Darowski, D., Kluge, T., Majety, M. Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model. PLoS One. 12 (7), 0182039 (2017).
- Bochet, L., et al. Adipocyte-derived fibroblasts promote tumor progression and contribute to the desmoplastic reaction in breast cancer. Pesquisa do Câncer. 73 (18), 5657-5668 (2013).
- Amann, A., et al. Development of a 3D angiogenesis model to study tumour – endothelial cell interactions and the effects of anti-angiogenic drugs. Scientific Reports. 7 (1), 2963 (2017).
- LaBonia, G. J., Ludwig, K. R., Mousseau, C. B., Hummon, A. B. iTRAQ Quantitative Proteomic Profiling and MALDI-MSI of Colon Cancer Spheroids Treated with Combination Chemotherapies in a 3D Printed Fluidic Device. Analytical Chemistry. 90 (2), 1423-1430 (2018).
- Hulikova, A., Vaughan-Jones, R. D., Swietach, P. Dual role of CO2/HCO3(-) formula buffer in the regulation of intracellular pH of three-dimensional tumor growths. Journal of Biological Chemistry. 286 (16), 13815-13826 (2011).
- Wallace, D. I., Guo, X. Properties of tumor spheroid growth exhibited by simple mathematical models. Frontiers in Oncology. 3, 51 (2013).
- Michel, T., et al. Mathematical modeling of the proliferation gradient in multicellular tumor spheroids. Journal of Theoretical Biology. 458, 133-147 (2018).
- Meijer, T. G., Naipal, K. A., Jager, A., van Gent, D. C. Ex vivo tumor culture systems for functional drug testing and therapy response prediction. Future Science OA. 3 (2), (2017).
Citar este artigo
Rolver, M. G., Elingaard-Larsen, L. O., Pedersen, S. F. Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells. J. Vis. Exp. (148), e59714, doi:10.3791/59714 (2019).