乳腺辐照有机体的生长与表征
Summary
对小鼠乳腺发育的有机体进行辐照, 并对其进行评估, 以评估上皮特征和与免疫细胞的相互作用。辐照有机体可用于更好地评估细胞相互作用, 这可能导致肿瘤细胞在辐照正常组织中的招募。
Abstract
从消化组织中提取的有机体是多细胞三维 (3D) 结构, 比细胞单层在体内条件下更有能力进行重述。虽然它们不能在体内完全模拟复杂性, 但它们保留了原始器官的一些功能。在癌症模型中, 有机体常用于研究肿瘤细胞的侵袭。该方案旨在开发和表征正常和辐照小鼠乳腺组织中的有机体, 以评估正常组织中的辐射反应。这些有机体可用于未来的体外癌症研究, 以评估肿瘤细胞与辐照有机体的相互作用。乳腺被切除, 照射到20戈, 并在胶原酶 VIII 溶液中消化。通过离心分化分离上皮性有机硅, 并在96好低粘附微板中制备三维有机生物。有机体表达了细胞角蛋白14的特征上皮标记。在共培养实验中观察到巨噬细胞与有机体的相互作用。该模型可用于研究肿瘤间质相互作用、免疫细胞浸润和在辐照微环境中巨噬细胞极化的研究。
Introduction
大约60% 的三重阴性乳腺癌 (TNBC) 患者选择保乳疗法 (BCT) 作为一种治疗形式 1。在这种治疗方式中, 含有部分乳房组织的肿瘤被切除, 周围的正常组织暴露在电离辐射下, 杀死任何残留的肿瘤细胞。治疗可减少大部分乳腺癌患者的复发;然而, 大约13.5% 的 TNBC 患者经历局部复发 2.因此, 研究辐射如何招募循环肿瘤细胞 (ctc) 将导致对局部复发3,4的重要洞察。
先前的研究表明, 正常组织的辐射增加了不同类型细胞的招募5。在 TNBC 的临床前模型中, 正常组织的照射增加了巨噬细胞, 随后肿瘤细胞被招募到正常组织5。免疫状态影响肿瘤细胞招募到辐照部位, 在免疫功能低下的受试者中观察到肿瘤细胞迁移。利用乳腺产生的有机体对这些相互作用进行复制, 可以通过显微镜和活细胞成像实时观察细胞迁移和细胞间质相互作用, 以确定辐射损伤在改变中的作用肿瘤细胞的行为。
小鼠乳腺组织已帮助阐明在乳腺发育的关键步骤。乳腺有机体是一种多细胞的三维结构, 是一种孤立的乳腺上皮, 大于 50μm6,7,8, 9,10.使用原发上皮类固醇, Simian 等人评估了乳腺分枝的必要因素 7.Shamir 等人发现, 传播可以发生没有上皮间充质过渡, 提供了洞察转移级联8。从乳腺组织中生成和表征有机体的方法已确定 6,11,12,13。然而, 据我们所知, 从乳腺中生长辐照有机体的方法还没有报道。一项生长和描述辐照有机体的协议将是重述辐射诱导的免疫和肿瘤细胞招募的关键一步。
本文报道了一种在低粘附微板中生长和表征辐照乳腺上皮细胞的方法, 这些微板上涂有一种支持球体形成的亲水性聚合物。这些有机体与巨噬细胞共同培养, 以检查免疫细胞浸润动力学。这项工作可以扩展到包括共培养有机体与脂肪细胞, 以重述乳房特征, 乳腺癌细胞可视化肿瘤细胞的招募, CD8 + T 细胞, 以研究肿瘤免疫细胞的相互作用。以前建立的协议可用于评估辐照有机体。早期的模型共同培养乳腺有机体和免疫细胞已经揭示了转移和传播的机制。德纳多等人发现, CD4 + T 细胞对肿瘤相关巨噬细胞的调节增强了乳腺腺癌的转移表型14。共同文化模型也被用来阐明生物发展的机制。Plaks 等人阐明了 CD4 + T 细胞作为乳腺器官发生的下调节剂的作用15。然而, 我们的小组是第一个建立一个程序, 可视化正常的组织照射如何影响免疫细胞的行为。由于正常组织照射已被证明可以增强肿瘤细胞的吸收5, 这一方案可以进一步发展, 以分析肿瘤细胞行为如何被正常组织和细胞的照射改变, 从而使人们对肿瘤细胞的行为有了更大的了解。癌症复发。
Protocol
动物研究是根据范德比尔特大学动物护理和使用机构委员会批准的机构准则和议定书进行的。 1. 小鼠的制备和细胞采集 (改编自 Nguyen-ngkoc 等人) 牺牲胸腺 Nu/Nu 小鼠 (8-10 大) 使用 co2 窒息, 然后颈椎脱位。使用70% 的乙醇清洁皮肤。 使用预先消毒的剪刀和钳子切除小鼠的腹部和腹股沟乳腺。切除前切除淋巴结。在无菌1x 磷酸盐缓冲盐水中…
Representative Results
从小鼠乳腺成功地获得辐照上皮乳腺有机体, 并在低粘附板上进行处理和培养 (图 1)。在不同生长环境下通过播种测试了有机产量 (图 2A-G)。将细胞直接播种到经过10厘米的细胞板上, 导致成纤维细胞过度生长。在与有机体相同的病灶平面内或附近的相对照显微镜下, 对成纤维细胞进行了鉴定, 并在几天内迅速从镀金有机体中生长出来。在基底膜和胶原蛋白基质中播…
Discussion
在该协议中, 我们开发了一种用于辐照乳腺有机体的可重复生长和表征的方法 (图 1)。应用20戈的辐照剂量来反映以前肿瘤细胞招募5的体内模型。在有机体形成之前的体内乳腺照射允许分离辐射损伤效应, 而不会相应的免疫细胞浸润。体外辐照正常组织模型的开发使人们能够实时观察可能导致辐射诱导的 ctc 招募11,12</su…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢劳拉博士 l. Bronsart 提供了 GFP 和 dtomato 标记的 RAW 264.7 巨噬细胞。这项研究得到了国家卫生研究院 #R00CA201304 资助的财政支持。
Materials
| 10% Neutral Buffered Formalin | VWR | 16004-128 | |
| Anti-cytokeratin 14 | abcam | ab181595 | Lot: GR3200524-3 |
| Bovine Serum Albumin | Sigma | A1933-25G | |
| Collagen Type I | Corning | 354236 | |
| Collagenase from Clostridium Histolyticum, Type VIII | Sigma | C2139 | |
| Collagenase I | Gibco | 17018029 | |
| DMEM/F12 | Thermofisher | 11320-033 | |
| DNAse | Roche | 10104159001 | |
| DPBS | Fisher | 14190250 | |
| E-Cadherin | Cell Signaling | 24E10 | Lot: 13 |
| FBS | Sigma | F0926 | |
| Gentamicin | Gibco | 15750 | |
| Goat anti-rabbit secondary | abcam | ab150077 | green Lot: GR3203000-1 |
| Goat anti-rabbit secondary | abcam | ab150080 | red Lot: GR3192711-1 |
| Hoechst 33342 | Fisher | 62249 | Lot: TG2611041 |
| Insulin (10 mg/mL) | Sigma | I9278 | |
| Insulin-Transferrin-Selenium, 100x | Gibco | 51500-056 | |
| Matrigel Basement Membrane (basement membrane extracted from Engelbreth-Holm-Swarm mouse sarcoma) | Corning | 356237 | |
| Normal Goat Serum | Vector Laboratories | S-1000 | |
| Nuclon Sphera 96 well plates | Thermo | 174927 | |
| PBS | VWR | 10128-856 | |
| Pen/strep | Fisher | 15140122 | |
| Phalloidin | abcam | ab176757 | Lot: GR3214582-16 |
| Tight Junction Protein 1 | Novus | NBP1-85047 | Lot: C115428 |
| Triton X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol) | Sigma | X100-100ML | |
| Trypsin | Gibco | 27250-018 | |
| Tween-20 (Polyethylene glycol sorbitan monolaurate) | Sigma | P1379-100ML |
References
- Lautner, M., et al. Disparities in the Use of Breast-Conserving Therapy Among Patients With Early-Stage Breast Cancer. Journal of the American Medical Association Surgery. 150 (8), 778-786 (2015).
- Lowery, A., Kell, M., Glynn, R., Kerin, M., Sweeney, K. Locoregional recurrence after breast cancer surgery a systematic review by receptor phenotype. Breast Cancer Research and Treatment. 133, 831-841 (2012).
- Kim, M. Y., et al. Tumor Self-Seeding by Circulating Cancer Cells. Cell. 139 (7), 1315-1326 (2009).
- Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. Recruitment of Circulating Breast Cancer Cells Is Stimulated by Radiotherapy. Cell Reports. 8 (2), 402-409 (2014).
- Rafat, M., et al. Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence following Radiotherapy in Immunosuppressed Patients. Recherche en cancérologie. 78 (15), 4241-4252 (2018).
- Shamir, E. R., Ewald, A. J. Three-dimensional organotypic culture: Experimental models of mammalian biology and disease. Nature Reviews Molecular Cell Biology. 15 (10), 647-664 (2014).
- Simian, M., Hirai, Y., Navre, M., Werb, Z., Lochter, A., Bissell, M. J. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development. 128, 3117-3131 (2001).
- Shamir, E. R., et al. Twist1-induced dissemination preserves epithelial identity and requires E-cadherin. Journal of Cell Biology. 204 (5), 839-856 (2014).
- Ewald, A. J., Brenot, A., Duong, M., Chan, B. S., Werb, Z. Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis. Developmental Cell. 14, 570-581 (2008).
- Nguyen-Ngoc, K. -. V., et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proceedings of the National Academy of Sciences. 89 (19), E2595-E2604 (2012).
- Nguyen-Ngoc, K. -. V., Shamir, E. R., Huebner, R. J., Beck, J. N., Cheung, K. J., Ewald, A. J. 3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion. Tissue Morphogenesis: Methods and Protocols. 1189, 135-162 (2015).
- Ewald, A. J. Isolation of mouse mammary organoids for long-term time-lapse imaging. Cold Spring Harbor Protocols. 8 (2), 130-133 (2013).
- Drost, J., Clevers, H. Organoids in cancer research. Nature Reviews. , (2018).
- DeNardo, D. G., et al. CD4+T Cells Regulate Pulmonary Metastasis of Mammary Carcinomas by Enhancing Protumor Properties of Macrophages. Cancer Cell. 16 (2), 91-102 (2009).
- Plaks, V., et al. Adaptive Immune Regulation of Mammary Postnatal Organogenesis. Developmental Cell. 34 (5), 493-504 (2015).
- Mandl, I., McLennan, J. D., Howes, E. L. Isolation and Characterization of Proteinase and Collagenase Fromcl. Histolyticum. The Journal of Clinical Investigation. 32, 1323-1329 (1953).
- Mandl, I., Zaffuto, S. F. Serological Evidence for a Specific Clostridium histolyticum Geltinase. The Journal of General Microbiology. 18, 13-15 (1958).
- Bond, M. D., Van Wart, H. E. Characterization of the Individual Collagenases from Clostridium histolyticum. Biochimie. 23 (13), 3085-3091 (1984).
- Zhang, L., et al. Establishing estrogen-responsive mouse mammary organoids from single Lgr5+cells. Cellular Signalling. 29, 41-51 (2016).
- Sokol, E. S., Miller, D. H., Breggia, A., Spencer, K. C., Arendt, L. M., Gupta, P. B. Growth of human breast tissues from patient cells in 3D hydrogel scaffolds. Breast Cancer Research. 18 (1), 1-13 (2016).
- Richert, M. M., et al. An atlas of mouse mammary gland development. Journal of Mammary Gland Biology and Neoplasia. 5 (2), 227-241 (2000).
- Maier, P., Hartmann, L., Wenz, F., Herskind, C. Cellular pathways in response to ionizing radiation and their targetability for tumor radiosensitization. International Journal of Molecular Sciences. 17 (1), (2016).
- LaBarge, M. A., Garbe, J. C., Stampfer, M. R. Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture. Journal of Visualized Experiments. (71), (2013).
- Campbell, J. J., Botos, L. A., Sargeant, T. J., Davidenko, N., Cameron, R. E., Watson, C. J. A 3-D in vitro co-culture model of mammary gland involution. Integrative Biology (United Kingdom). 6, 618-626 (2014).
- Chanson, L., et al. Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells. Proceedings of the National Academy of Sciences. 14 (7), 2293-2306 (2011).
- Chua, A. C. L., Hodson, L. J., Moldenhauer, L. M., Robertson, S. A., Ingman, W. V. Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium. Development. 137, 4229-4238 (2010).
- Gregoire, F. M., Smas, C. M., Sul, H. S. Understanding Adipocyte Differentiation. Physiological Reviews. 78 (3), 783-809 (1998).
- Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. Current Methods of Adipogenic Differentiation of Mesenchymal Stem Cells. Stem Cells and Development. 20 (10), 1793-1804 (2011).
- Gabryś, D., Greco, O., Patel, G., Prise, K. M., Tozer, G. M., Kanthou, C. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability. International Journal of Radiation Oncology, Biology, Physics. 69 (5), 1553-1562 (2007).
- Ewald, A. J. Practical considerations for long-term time-lapse imaging of epithelial morphogenesis in three-dimensional organotypic cultures. Cold Spring Harbor Protocols. 8, 100-117 (2013).
- Zhang, M., et al. A high M1/M2 ratio of tumor-associated macrophages is associated with extended survival in ovarian cancer patients. Journal of Ovarian Research. 7 (1), 1-16 (2014).
- Ma, J., Liu, L., Che, G., Yu, N., Dai, F., You, Z. The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BioMed Central Cancer. 10, 112 (2010).
Citer Cet Article
Hacker, B. C., Gomez, J. D., Batista, C. A. S., Rafat, M. Growth and Characterization of Irradiated Organoids from Mammary Glands. J. Vis. Exp. (147), e59293, doi:10.3791/59293 (2019).