乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型
Summary
乳腺上皮细胞和内皮细胞之间的串扰对乳腺癌进展、肿瘤生长和转移有重要贡献。在这项研究中，球状体是由乳腺癌细胞与血管和/或淋巴内皮细胞一起制成的，并证明了它们作为乳腺癌研究 的体外 系统的适用性。
Abstract
乳腺癌是女性死亡的主要原因。乳腺癌细胞的生长及其随后的转移是其进展的关键因素。尽管使用MCF-7细胞等乳腺癌细胞的单一培养物对促进乳腺癌生长的机制进行了深入研究，但尚未深入研究其他细胞类型（例如密切参与肿瘤生长的血管和淋巴内皮细胞）的贡献。细胞 – 细胞相互作用在肿瘤生长和进展中起着关键作用。新血管生成或血管的发育对于肿瘤生长至关重要，而淋巴系统则是癌细胞迁移和随后转移的门户。最近的研究提供了证据表明，血管和淋巴内皮细胞可以显着影响癌细胞的生长。这些观察结果意味着需要开发体 外 模型，以更真实地反映 体内乳腺癌的生长过程 。此外，动物研究的限制要求开发 离体 模型，以更好地阐明所涉及的机制。
本文描述了由乳腺癌细胞（雌激素受体阳性MCF-7细胞）和血管和/或淋巴内皮细胞组成的乳腺癌球体的发展。该协议描述了使用两种不同的方法创建双细胞球体的详细分步方法，即悬挂式下降（黄金标准和便宜）和96孔U底板（昂贵）。提供了有关如何通过显微镜定量和使用死细胞和活细胞染色评估活力来精细拾取形成的球体以监测生长的深入说明。此外，还描述了用生长特异性抗体固定球体以进行切片和染色以区分球体中生长模式的程序。此外，还提供了用转染细胞制备球体的详细信息以及提取RNA进行分子分析的方法。总之，本文为制备用于乳腺癌研究的多细胞球体提供了深入的说明。
Introduction
使用动物进行实验有局限性。动物研究无法准确模拟人类的疾病进展，动物和人类对病原体的反应也不尽相同。此外，由于担心动物的痛苦和道德问题，动物实验受到限制1,2 越来越限制研究项目。 In vitro 系统已被广泛开发，以规避动物的使用;而且，利用人体细胞已经使 in vitro 与病理生理学和治疗学研究更相关的模型。传统的单层（2D）细胞培养物被广泛使用，因为它们在某种程度上模仿人体组织。然而，2D单一培养无法模仿人体器官，2D单一培养无法模拟原始器官的复杂微环境并模仿 in vivo 情况3,4,5,6.此外，在单层细胞培养物中，药物治疗很容易破坏/损伤所有细胞。重要的是，可以通过切换到多层三维（3D）细胞培养物来克服其中一些限制。7,8.事实上，3D培养模型已被证明可以更好地反映原代组织中细胞的结构，布局和功能。这些3D培养物可以使用各种细胞系形成，类似于功能器官。事实上，有两种3D文化模型。一种模型产生聚集细胞的球体，这些细胞形成簇并将它们重组为球体（无支架模型）。第二种产生类器官，其具有更复杂的结构，由多个器官特异性细胞的组合组成，这些细胞被认为是器官的微型版本。9,10.因此，3D培养系统代表了一种具有许多生物学和临床应用的创新技术。因此，球状体和类器官在疾病建模和与再生医学，药物筛选和毒理学研究相关的研究中具有许多应用。6,11,12,13,14,15.源自3D技术的致癌球体，重建相关细胞类型的形态和表型，模仿 in vivo 肿瘤微环境，并对肿瘤发展过程中可操作的细胞通信和信号通路进行建模16,17,18.此外，为了提高对癌症生物学的理解，肿瘤球状体/类器官还可用于识别潜在的患者特异性抗癌疗法（个性化）并评估其疗效，毒性和长期影响。19,20,21,22.球状体为研究病理生理学，疾病建模和药物筛选提供了突出的机会，因为它们能够保存细胞和三维组织结构，能够模仿 in vivo 情况，以及细胞 – 细胞相互作用。然而，人们还必须意识到该系统的局限性，例如缺乏血管/全身成分，功能性免疫或神经系统，并且与动物模型相比，该系统代表了还原论的方法。事实上，与动物模型相比，3D结构仅提供人体内生物学的近似值。了解3D方法的局限性可能有助于研究人员设计更精细和有效的过程，以产生在更大尺度上更好地代表器官的球体。23,24,25.
癌症是全世界死亡的主要原因，而乳腺癌是女性中最常见的癌症26、27、28。为了模拟乳腺癌的复杂微环境，应使用在乳腺肿瘤中起突出作用的细胞（即上皮细胞，内皮细胞，成纤维细胞和/或免疫细胞）培养乳腺癌球状体。此外，对于代表乳腺癌的球状体，还应考虑女性激素受体（雌激素/孕激素受体）的表达，保存患者肿瘤组织学状态的能力以及模仿治疗反应的能力。研究表明，3D共培养系统具有与体内原代组织相似的细胞组织，具有对刺激实时反应的能力，并具有功能性雄激素受体29、30、31、32。因此，类似的方法可能有助于在体外模拟乳腺肿瘤 。当前方案的目的是建立一种产生乳腺癌球体的新方法。该方法利用雌激素受体阳性MCF-7细胞（上皮细胞的永生化人细胞系）和血管内皮细胞（HUVECs）或淋巴内皮细胞（HMVEC-DNeo）来创建模拟或密切反映肿瘤内这些细胞之间相互作用的模型。虽然MCF-7（雌激素反应性）和内皮细胞在本研究中已被用于发展球状体，但其他细胞，如占乳腺肿瘤质量约80%的成纤维细胞，也可以在未来结合，以更好地代表和模仿乳腺肿瘤。
形成球体的方法有几种，例如：1）采用重力33，34的悬挂液滴法;2）利用磁性纳米颗粒暴露于外部磁铁的磁悬浮法35，以及3）通过在低附着板上接种细胞来执行的球状微孔板法36，37。在仅使用一种细胞类型的现有方法的基础上，对本方案已利用上皮细胞和内皮细胞进行了优化，以更好地模拟乳腺癌肿瘤在体内38、39、40、41的生长条件。这种方法可以在实验室中以低成本和最低的设备要求轻松实现。根据实验室的需求/目标，使用不同的方法来形成旋转体并从这些旋转体中获得相关的细胞材料。在这种情况下，对于DNA，RNA或蛋白质分析，3D球体是通过与悬挂滴法共培养内皮和上皮细胞来产生的。然而，对于功能研究，例如，为了监测短干扰（siRNA）转染和/或激素处理后的细胞生长，使用U底板生成球状体。
该技术方案的目的是为1）形成乳腺癌多细胞型球体，2）制备用于组织学染色的样品以及3）收集用于提取RNA，DNA和蛋白质的细胞提供详细的分步描述。廉价的悬挂式下降方法和更昂贵的U底板都用于形成球体。在这里，提供了制备（固定）球体的方案，用于切片和随后的免疫染色，并用标记物评估细胞增殖，凋亡以及球体内上皮和内皮细胞的分布。此外，该协议显示了使用ImageJ软件对组织学数据的完整分步分析。生物数据的解释因实验类型和所用抗体而异。固定球体的切片和随后的切片染色由常规病理学实验室（Sophistolab：info@sophistolab.ch）进行
Protocol
Representative Results
Discussion
与2D细胞培养相比，革命性的3D球体培养技术是重建器官微环境，细胞间相互作用和 体外药物反应的更好，更强大的工具。这是描述用于乳腺癌研究的多细胞（上皮和内皮）细胞系中球状体形成的第一个方案。该协议可确保球体的球体3D生长长达5天，并且可以在石蜡包埋，切片和组织学染色后检查球体。有趣的是，球体内的细胞元件仍然表达促进细胞生长的受体，并对增殖和凋亡刺激有反?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了癌症研究基金会/瑞士癌症联盟资助KFS-4125-02-2017给RKD和国家卫生研究院资助DK079307给EKJ的支持。
Materials
| 100 mm × 20 mm tissue-culture treated culture dishes | Corning | CLS430167 | |
| 149MULTI0C1 | |||
| 35 x 10 mm Tissue Culture Dish | Falcon | 353001 | |
| 5 mL Serological Pipet, Polystyrene, Individually Packed, Sterile | Falcon | 357543 | |
| Adobe Photoshop | Version: 13.0.1 (64-bit) | ||
| Antibiotic Antimycotic Solution (100×) | Sigma-Aldrich | A5955 | |
| Calcein-AM | Sigma-Aldrich | 17783 | |
| CD31 (cluster of differentiation 31) | Cell Marque | 131M-95 | monoclonal mouse ab clone JC70 |
| CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) | Invitrogen | C7025 | |
| CK8/18 (cytokeratins 8 and 18) | DBS | Mob189-05 | monoclonal mouse ab, clone 5D3 |
| CKX41 Inverted Microscope | Olympus Life Science | Olympus DP27 digital camera | |
| Cleaved Caspase 3 | Cell Signaling | 9661L | polyclonal rabbit ab |
| Collagen (rat tail) | Roche | 11 179 179 001 | |
| Coulter ISOTON II Diluent | Beckman Coulter | 844 80 11 | Diluent II |
| Coulter Z1, Cell Counter | Coulter Electronics, Luton, UK | ||
| Dehydrated Culture Media: Noble Agar | BD Difco | BD 214220 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
| Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham | Sigma-Aldrich | D6434 | |
| EBM-2 Endothelial Cell Growth Basal Medium-2 | Lonza | 190860 | |
| Ethidium homodimer | Sigma-Aldrich | 46043 | |
| Fetal Calf Serum (FCS) | Thermo Fisher Scientific | SH30070 | |
| Fetal Calf Serum Charcoal Stripped (FCS sf) | Thermo Fisher Scientific | SH3006803 | |
| Fluorescence stereo microscopes Leica M205 FA | Leica Microsystems | ||
| GlutaMAX Supplement (100x) | Gibco | 35050038 | |
| HBSS, no calcium, no magnesium, no phenol red | Gibco | 14175053 | |
| Hoechst 33342 | Life Technologies | H3570 | |
| HUVEC – Human Umbilical Vein Endothelial Cells | Lonza | CC-2517 | |
| ImageJ | National Institute of Health, USA | Wayne Rasband Version: 1.52a (64-bit) |
|
| Ki67 | Cell Marque | 275R-16 | monoclonal rabbit ab, clone SP6 |
| Leica Histocore Multicut Rotary Microtome | 149MULTI0C1 | ||
| Low Serum Growth Supplement Kit (LSGS Kit) | Gibco | S003K | |
| MCF-7 cells – human breast adenocarcinoma cell line | Clinic for Gynecology, University Hospital Zurich | Provived from Dr Andrè Fedier obtained from ATCC | |
| Nunclon Sphera 96U-well plate | Thermo Fisher Scientific | 174925 | |
| Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | |
| Phosphate-buffered saline (PBS) 1x | Gibco | 10010015 | |
| Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
| Protein Lysis Buffer | Cell Signaling, Danvers, USA | 9803 | |
| Quick-RNA Miniprep Kit | Zymo Research | R1055 | |
| RNA Lysis Buffer | Zymo Research | R1060-1-100 | Contents in Quick-RNA Miniprep Kit |
| Rotina 46R Centrifuge | Hettich | ||
| Round-Bottom Polystyrene Tubes, 5 mL | Falcon | 352003 | |
| Sonicator | Bandelin electronics, Berlin, DE | ||
| Tecan Infinite series M200 microplate reader | Tecan, Salzburg, AU | ||
| Tissue Culture Flasks 75 | TPP | 90076 | |
| Trypsin-EDTA solution | Sigma-Aldrich | T3924 |
Riferimenti
- Sakamoto, K. The pathology of Mycobacterium tuberculosis infection. Veterinary Pathology. 49 (3), 423-439 (2012).
- Morrisey, E. E., Hogan, B. L. Preparing for the first breath: genetic and cellular mechanisms in lung development. Developmental Cell. 18 (1), 8-23 (2010).
- Goodman, T. T., Ng, C. P., Pun, S. H. 3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. Bioconjugate Chemistry. 19 (10), 1951-1959 (2008).
- Yang, L., et al. Tumor organoids: From inception to future in cancer research. Cancer Letters. 454, 120-133 (2019).
- Velasco-Velázquez, M. A., Popov, V. M., Lisanti, M. P., Pestell, R. G. The role of breast cancer stem cells in metastasis and therapeutic implications. The American Journal of Pathology. 179 (1), 2-11 (2011).
- Imamura, Y., et al. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncology Reports. 33 (4), 1837-1843 (2015).
- Jensen, C., Teng, Y. Is it time to start transitioning from 2D to 3D cell culture. Frontiers in Molecular Biosciences. 7, 33 (2020).
- Russell, S., Wojtkowiak, J., Neilson, A., Gillies, R. J. Metabolic profiling of healthy and cancerous tissues in 2D and 3D. Scientific Reports. 7 (1), 15285 (2017).
- Sakalem, M. E., De Sibio, M. T., da Costa, F., de Oliveira, M. Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine. Biotechnology Journal. 16 (5), (2021).
- Zanoni, M., et al. Modeling neoplastic disease with spheroids and organoids. Journal of Hematology & Oncology. 13 (1), 97 (2020).
- Corrò, C., Novellasdemunt, L., Li, V. S. W. A brief history of organoids. American Journal of Physiology. Cell Physiology. 319 (1), 151-165 (2020).
- Ding, Y., et al. Three-dimensional tissue culture model of human breast cancer for the evaluation of multidrug resistance. Journal of Tissue Engineering and Regenerative Medicine. 12 (9), 1959-1971 (2018).
- Little, M. H. Organoids: a special issue. Development. 144 (6), 935-937 (2017).
- Rimann, M., Graf-Hausner, U. Synthetic 3D multicellular systems for drug development. Current Opinion in Biotechnology. 23 (5), 803-809 (2012).
- Breslin, S., O’Driscoll, L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today. 18 (5-6), 240-249 (2013).
- Hanahan, D., Coussens, L. M. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 21 (3), 309-322 (2012).
- Yamada, K. M., Cukierman, E. Modeling tissue morphogenesis and cancer in 3D. Cell. 130 (4), 601-610 (2007).
- Weigelt, B., Ghajar, C. M., Bissell, M. J. The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer. Advanced Drug Delivery Reviews. 69-70, 42-51 (2014).
- Lin, R. Z., Chang, H. Y. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnology Journal. 3 (9-10), 1172-1184 (2008).
- Fong, E. L., et al. Modeling Ewing sarcoma tumors in vitro with 3D scaffolds. Proceedings of the National Academy of Sciences of the United States of America. 110 (16), 6500-6505 (2013).
- Hauptmann, S., et al. Integrin expression on colorectal tumor cells growing as monolayers, as multicellular tumor spheroids, or in nude mice. International Journal of Cancer. 61 (6), 819-825 (1995).
- Vlachogiannis, G., et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science. 359 (6378), 920-926 (2018).
- Ashok, A., Choudhury, D., Fang, Y., Hunziker, W. Towards manufacturing of human organoids. Biotechnology Advances. 39, 107460 (2020).
- Lehmann, R., et al. Human organoids: a new dimension in cell biology. Molecular Biology of the Cell. 30 (10), 1129-1137 (2019).
- Verjans, E. T., Doijen, J., Luyten, W., Landuyt, B., Schoofs, L. Three-dimensional cell culture models for anticancer drug screening: Worth the effort. Journal of Cellular Physiology. 233 (4), 2993-3003 (2018).
- Bombonati, A., Sgroi, D. C. The molecular pathology of breast cancer progression. The Journal of Pathology. 223 (2), 307-317 (2011).
- DeSantis, C., Ma, J., Bryan, L., Jemal, A. Breast cancer statistics, 2013. CA: A Cancer Journal for Clinicians. 64 (1), 52-62 (2014).
- Solanki, M., Visscher, D. Pathology of breast cancer in the last half century. Human Pathology. 95, 137-148 (2020).
- Djomehri, S. I., Burman, B., Gonzalez, M. E., Takayama, S., Kleer, C. G. A reproducible scaffold-free 3D organoid model to study neoplastic progression in breast cancer. Journal of Cell Communication and Signaling. 13 (1), 129-143 (2019).
- Xu, H., et al. Organoid technology and applications in cancer research. Journal of Hematology & Oncology. 11 (1), 116 (2018).
- Yu, J., Huang, W. The progress and clinical application of breast cancer organoids. International Journal of Stem Cells. 13 (3), 295-304 (2020).
- Simian, M., Bissell, M. J. Organoids: A historical perspective of thinking in three dimensions. The Journal of Cell Biology. 216 (1), 31-40 (2017).
- Foty, R. A simple hanging drop cell culture protocol for generation of 3D spheroids. Journal of Visualized Experiments: JoVE. (51), e2720 (2011).
- Tung, Y. C., et al. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. The Analyst. 136 (3), 473-478 (2011).
- Türker, E., Demirçak, N., Arslan-Yildiz, A. Scaffold-free three-dimensional cell culturing using magnetic levitation. Biomaterials Science. 6 (7), 1745-1753 (2018).
- Froehlich, K., et al. Generation of multicellular breast cancer tumor spheroids: comparison of different protocols. Journal of Mammary Gland Biology and Neoplasia. 21 (3-4), 89-98 (2016).
- 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).
- Kaushik, G., Ponnusamy, M. P., Batra, S. K. Concise review: Current status of three-dimensional organoids as preclinical models. Stem Cells. 36 (9), 1329-1340 (2018).
- Marconi, A., Quadri, M., Saltari, A., Pincelli, C. Progress in melanoma modelling in vitro. Experimental Dermatology. 27 (5), 578-586 (2018).
- Saltari, A., et al. CD271 down-regulation promotes melanoma progression and invasion in three-dimensional models and in zebrafish. The Journal of Investigative Dermatology. 136 (10), 2049-2058 (2016).
- Yamauchi, M., et al. A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation. Journal of Radiation Research. 55 (2), 381-390 (2014).
- Jabs, J., et al. Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations. Molecular Systems Biology. 13 (11), 955 (2017).
- Arruabarrena-Aristorena, A., et al. FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer. Cancer Cell. 38 (4), 534-550 (2020).
- Carey, S. P., Starchenko, A., McGregor, A. L., Reinhart-King, C. A. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model. Clinical & Experimental Metastasis. 30 (5), 615-630 (2013).
- Park, S., et al. Differential functions of splicing factors in mammary transformation and breast cancer metastasis. Cell Reports. 29 (9), 2672-2688 (2019).
- Truong, D. D., et al. A human organotypic microfluidic tumor model permits investigation of the interplay between patient-derived fibroblasts and breast cancer cells. Ricerca sul cancro. 79 (12), 3139-3151 (2019).
- Zhang, J., et al. Energetic regulation of coordinated leader-follower dynamics during collective invasion of breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 116 (16), 7867-7872 (2019).
- Jeong, Y. H., et al. Vitrification for cryopreservation of 2D and 3D stem cells culture using high concentration of cryoprotective agents. BMC Biotechnology. 20 (1), 45 (2020).
