基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验
Summary
我们描述了一个基于球体的三维体外模型的进化, 它使我们能够测试细胞系头颈部鳞状细胞癌实验治疗方案的现行标准, 旨在评估治疗未来人体标本对原代细胞的敏感性和耐药性。
Abstract
目前的治疗方案的晚期和复发头颈部鳞状细胞癌 (HNSCC) 包围辐射和化疗的方法与或没有手术。虽然铂基化疗方案目前代表的是金标准的功效, 并在绝大多数情况下, 新的化疗方案, 即免疫疗法正在出现。然而, 无论是化疗方案的反应率和治疗耐药性机制都难以预测, 而且仍未得到充分了解。目前已知的化疗和耐辐射机制的广泛变化。本研究介绍了一种标准化的高通量体外检测方法, 以评估 HNSCC 细胞系对各种治疗方案的反应, 希望能将个体患者的原细胞作为个性化肿瘤的未来工具。治疗。本试验的目的是将 HNSCC 患者的质量控制标准算法纳入我们的三级护理中心;然而, 这将是未来研究的主题。从实际患者的肿瘤活检中, 技术可行性看起来很有希望。标本然后转移到实验室。活检是机械分离后, 酶消化。细胞, 然后培养在超低黏附细胞培养瓶, 促进可再生, 标准化和自发形成三维球形细胞企业集团。球体然后准备在必要时暴露在化学辐射协议和免疫治疗协议中。最终细胞生存能力和球体大小是治疗敏感性的指标, 因此可以考虑在未来评估患者的可能的治疗反应。这一模式可能是一个宝贵的, 经济高效的工具, 以个性化治疗头颈癌。
Introduction
头颈鳞状细胞癌 (HNSCC) 是世界上第六最常见的癌症, 其发病率上升, 粘膜人乳突病毒 (HPV) 感染相关的发病机制, 在大多数情况下, 过量的尼古丁和酒精引起消耗量1,2。虽然较小的肿瘤和前侵入阶段通常是很好的治疗与手术切除, 通常结合宫颈淋巴结清扫, 晚期和复发 HNSCC 治疗仍然是挑战, 由于侵袭性肿瘤侵袭转移扩散和抗辐射和化疗协议3,4,5,6,7,8。最近的研究表明细胞表型的变异很高, 循环和弥散肿瘤细胞的亚型特征刚刚开始9,10。早期对固体、均匀肿瘤质量的信念必须根据最近几年的研究进行修订:11,12,13,14。目前的肿瘤鉴定和关键突变鉴定方法可以识别出一些与治疗耐药性相关的基因, 但仍然是一种成本密集型的方法。而且, 基因型的知识并不一定能对表型及其治疗反应作出可靠的预测。
在改善晚期和复发性疾病的总体和无疾病生存方面, 进展甚少。对于尼古丁-以及与病毒相关的癌症, 目前的治疗方案除了手术外, 还附上了积极的辐射和铂基化学疗法。对 HPV 阴性和阳性癌的反应速率有不同的影响;然而, 这还没有导致一般治疗指南的改变。抗辐射和化疗是在所有肿瘤阶段普遍存在的现象, 并存在于铂基化疗以及靶向治疗 (抗 EGFR;表皮生长因子受体)和最近出现的检查点抑制15。无效的放疗和化疗在吞咽困难、粘膜炎、干口和肾脏或心脏功能下降的风险等方面有很高的病人发病率。预测治疗反应之前, 对每个病人的一般治疗概念的决定似乎是关键的目标, 防止不必要的治疗概念, 副作用和成本。
我们试图建立一个模型, 以测试病人的治疗敏感性目前标准的化疗-辐射, 可纳入常规和质量控制的肿瘤治疗算法从一个技术的站点。远的目标是使用该模型, 而不使用严重改变和老化细胞系, 因为他们不太代表实际的人肿瘤细胞没有他们的变异性和异质性, 因为我们现在知道, 而建立的协议是在不同的细胞系进行。为了独立于商业上可用的细胞线, 我们最近成功地从人类肿瘤标本的原始 HNSCC 细胞中生成了一种称为 “异食癖” 的中间细胞线, 其表面和有限通道上有保守的细胞标记16. 这个异食细胞系应作为一个准备, 以发展的道路上, 然后以后的试验与新鲜的人类癌细胞从肿瘤活检。已经表明, 三维细胞培养细胞在体内的反应不同, 并且比在单分子膜17,18,19,20 中生长的肿瘤药物的治疗更像是癌症药品的管理.21, 主要是由于某些单元格子集22 、23、24的迁移和分异属性的保护。在这里, 我们描述了从中间细胞线和原发性人鳞状细胞癌细胞的球体为基础的三维模型的协议, 以及如何将这种模型整合到头颈外科医生和肿瘤学家的癌症治疗中 (图 1)。
Protocol
Representative Results
Discussion
我们能够建立一个协议, 以产生可再生的球体从细胞悬浮, 对细胞系和在初步实验中, 原发性人类肿瘤细胞。我们首先评估了两种以前描述的方法, 并确定了 ULA 方法, 这种方法是使用超低粘附表面的培养板, 是一个更安全, 更可靠的三维球体生成。通过结合两种不同的方法来测定读出 (大小/面积和细胞的生存能力), 这种多模式检测是足够敏感, 以确定小的差异组之间。这是迈向技术可行性证明的第一?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项目由慕尼黑大学 (FöFoLe 项目-no: 789-781) 拨款资助。
Materials
| Dulbeccos modified Eagles medium (DMEM) | Biochrom, Berlin, Germany | F 0425 | |
| Fetal bovine serum | Gibco Life Technologies, Paisley, UK | 10500-064 | |
| penicillin/streptomycin | Biochrom, Berlin, Germany | A2212 | |
| sodium pyruvate | Biochrom, Berlin, Germany | L0473 | |
| non-essential amino acids | Biochrom, Berlin, Germany | K0293 | |
| L-Glutamine | Biochrom, Berlin, Germany | K0293 | |
| Liberase | Roche Life Sciences, Basel, Switzerland | 5401127001 | |
| GravityPLUS 3D Culture and Assay Platform | InSphero, Schlieren, Switzerland | PB-CS 06-001 | |
| GravityTRAP plate | InSphero, Schlieren, Switzerland | PB-CS-01-001 | |
| Ultra-low attachment (ULA) culture plates | Corning, Corning, NY, USA | 4520 | |
| airway epithelial cell growth medium | Promocell, Heidelberg, Germany | C-21060 | |
| amphotericin B | Biochrom, Berlin, Germany | A 2612 | |
| airway epithelial cell growth medium supplement mix | Promocell, Heidelberg, Germany | C39165 | |
| WST-8 test | Promocell, Heidelberg, Germany | PC PK-CA705-CK04 | |
| Keratinocyte SFMedium + L-Glutamine 500mL | Invitrogen | #17005-034 | |
| Bovine Pituitary Extract (BPE), 25mg | Invitrogen | #37000015 | |
| Recombinant human Epithelial Growth Factor 2.5 µg | Invitrogen | #37000015 | |
| DMEM High Glucose | Invitrogen | #21068-028 | |
| Penicillin Streptomycin 10000U/mL Penicillin/ 10000µg/mL Streptomycin | Invitrogen | #15140-122 | |
| F12 Nutrient Mix | Invitrogen | #21765-029 | |
| Glutamax (200 mM L-Alanyl-L-Glutamin-Dipeptide in NaCl) | Invitrogen | #35050087 | |
| HBSS (Ca, Mg) | Life Technologies | #14025-092 | (no phenol red) |
| 1x TrypLE Expres Enzyme | Invitrogen | #12604-013 | (no phenol red) |
| Accutase (enzymatic cell detachment solution) | Innovative cell technologies | Cat# AT104 | |
| 70 µm Falcon cell strainer | BD Biosciences, USA | #352350 |
Referências
- Hunter, K. D., Parkinson, E. K., Harrison, P. R. Profiling early head and neck cancer. Nat Rev Cancer. 5 (2), 127-135 (2005).
- Jerjes, W., et al. The effect of tobacco and alcohol and their reduction/cessation on mortality in oral cancer patients: short communication. Head Neck Oncol. 4, 6 (2012).
- Chen, H. H. W., Kuo, M. T. Improving radiotherapy in cancer treatment: Promises and challenges. Oncotarget. 8 (37), 62742-62758 (2017).
- Boeckx, C., et al. Anti-epidermal growth factor receptor therapy in head and neck squamous cell carcinoma: focus on potential molecular mechanisms of drug resistance. Oncologist. 18 (7), 850-864 (2013).
- Brand, T. M., Iida, M., Wheeler, D. L. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Cancer Biol Ther. 11 (9), 777-792 (2011).
- Seidl, D., Schild, S. E., Wollenberg, B., Hakim, S. G., Rades, D. Prognostic Factors in Patients Irradiated for Recurrent Head-and-Neck Cancer. Anticancer Res. 36 (12), 6547-6550 (2016).
- Shirai, K., et al. Clinical Outcomes of Definitive and Postoperative Radiotherapy for Stage I-IVB Hypopharyngeal Cancer. Anticancer Res. 36 (12), 6571-6578 (2016).
- Theile, D., et al. Evaluation of drug transporters’ significance for multidrug resistance in head and neck squamous cell carcinoma. Head Neck. 33 (7), 959-968 (2011).
- Slade, M. J., et al. Comparison of bone marrow, disseminated tumour cells and blood-circulating tumour cells in breast cancer patients after primary treatment. Brit J Cancer. 100 (1), 160-166 (2009).
- Mockelmann, N., Laban, S., Pantel, K., Knecht, R. Circulating tumor cells in head and neck cancer: clinical impact in diagnosis and follow-up. Eur Arch Otorhinolaryngol. 271 (1), 15-21 (2014).
- Gerlinger, M., et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 366 (10), 883-892 (2012).
- Ledgerwood, L. G., et al. The degree of intratumor mutational heterogeneity varies by primary tumor sub-site. Oncotarget. 7 (19), 27185-27198 (2016).
- Loyo, M., et al. Lessons learned from next-generation sequencing in head and neck cancer. Head Neck. 35 (3), 454-463 (2013).
- Morris, L. G., et al. The Molecular Landscape of Recurrent and Metastatic Head and Neck Cancers: Insights From a Precision Oncology Sequencing Platform. JAMA Oncol. , (2016).
- Bauml, J. M., Cohen, R. B., Aggarwal, C. Immunotherapy for head and neck cancer: latest developments and clinical potential. Ther Adv Med Oncol. 8 (3), 168-175 (2016).
- Mack, B., et al. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens. PLoS One. 8 (1), e55540 (2013).
- Ham, S. L., Joshi, R., Thakuri, P. S., Tavana, H. Liquid-based three-dimensional tumor models for cancer research and drug discovery. Exp Biol Med (Maywood). 241 (9), 939-954 (2016).
- Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng. 83 (2), 173-180 (2003).
- Kunz-Schughart, L. A., Freyer, J. P., Hofstaedter, F., Ebner, R. The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen. 9 (4), 273-285 (2004).
- Lin, R. Z., Chang, H. Y. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J. 3 (9-10), 1172-1184 (2008).
- Weiswald, L. B., Bellet, D., Dangles-Marie, V. Spherical cancer models in tumor biology. Neoplasia. 17 (1), 1-15 (2015).
- Cancer Genome Atlas, N. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 517 (7536), 576-582 (2015).
- Duarte, S., et al. Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect. Oncol Rep. 28 (3), 1057-1062 (2012).
- Reid, P. A., Wilson, P., Li, Y., Marcu, L. G., Bezak, E. Current understanding of cancer stem cells: Review of their radiobiology and role in head and neck cancers. Head Neck. 39 (9), 1920-1932 (2017).
- Cossu, F., et al. Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic. PLoS ONE. 7 (11), e49527 (2012).
- Hagemann, J., et al. Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer. Anticancer Res. 37 (5), 2201-2210 (2017).
- Worp, H. B., et al. Can animal models of disease reliably inform human studies?. PLoS Med. 7 (3), e1000245 (2010).
- Wilding, J. L., Bodmer, W. F. Cancer cell lines for drug discovery and development. Cancer Res. 74 (9), 2377-2384 (2014).
- Chitcholtan, K., Asselin, E., Parent, S., Sykes, P. H., Evans, J. J. Differences in growth properties of endometrial cancer in three dimensional (3D) culture and 2D cell monolayer. Exp Cell Res. 319 (1), 75-87 (2013).
- Longati, P., et al. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer. 13, 95 (2013).
