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Ricerca sul cancro

使用微生理系统模拟人体乳房组织中的乳腺癌

Published: April 23, 2021
doi:
10.3791/62009
, , , , , ,
1Department of Surgery,Louisiana State University Health Sciences Center, 2Department of Bioinnovation,Tulane University, 3Tulane University School of Medicine, 4Department of Biological and Agricultural Engineering,Louisiana State University, 5Department of Medicine,Tulane University School of Medicine

Summary

该协议描述了使用原始人体乳房组织与现成材料研究乳腺癌的体外微生理系统的构建。

Abstract

乳腺癌（BC）仍然是妇女的主要死因。尽管每年在不列颠哥伦比亚省的研究上投资超过7亿美元，但97%的候选BC药物没有通过临床试验。因此，需要新的模型来改善我们对这种疾病的理解。NIH微生理系统（MPS）计划旨在改善基础科学发现和有前途的新治疗策略的临床翻译。在这里，我们介绍了一种产生乳腺癌 MPS 的方法 （BC-MPS）。该模型通过将WAT夹在脂肪衍生的干细胞片（ASC）之间，来适应先前描述的培养原人类白脂肪组织（WAT）的方法。我们的BC-MPS的新方面包括将BC细胞植入非疾病的人类乳房组织（HBT），其中含有原生细胞外基质、成熟的脂肪细胞、常驻成纤维细胞和免疫细胞：并将BC-HBT混合在HBT衍生的ASC表之间。由此产生的BC-MPS在文化前活力中稳定至少14天。此模型系统包含影响 BC 的微环境的多个元素，包括脂肪细胞、频闪细胞、免疫细胞和细胞外基质。因此，BC-MPS可用于研究BC与其微环境之间的相互作用。

我们通过研究两种已知影响癌症进展和转移的 BC-MPS 行为来展示我们的 BC-MPS 的优势：1） BC 运动和 2） BC-HBT 代谢相声。虽然 BC-MPS 以前曾使用静脉成像来证明其运动性，但允许使用荧光显微镜在几天内进行高分辨率延时成像。此外，虽然代谢相声以前是使用BC细胞和穆林前脂肪细胞分化成不成熟的脂肪细胞来证明的，但我们的BC-MPS模型是第一个在体外证明原人类乳腺脂肪细胞和BC细胞之间的相声的系统。

Introduction

每年，超过40，000名美国妇女死于乳腺癌（BC）1。尽管每年在不列颠哥伦比亚省的研究上投资超过7亿美元，但97%的候选BC药物在临床试验中失败2，3。需要新的模式来改善药物开发管道和我们对不列颠哥伦比亚省的理解。NIH微生理学（MPS）计划界定了突破模型所需的功能，以改进将基础科学转化为临床成功4。其中包括使用原生人体细胞或组织，稳定培养4周，并纳入原生组织结构和生理反应。

目前的体外BC模型，如BC细胞系的二维培养，膜插入共同培养，三维球体和器官，不符合NIH的MPS标准，因为这些都没有回顾本地乳房组织结构。当细胞外基质 （ECM） 添加到这些系统中时，不使用乳房 ECM：相反，使用胶原蛋白凝胶和地下室膜矩阵。

目前体内系统，如患者衍生的异种移植物（PDX），同样不符合NIH的MPS标准，因为乳腺组织与人类乳房有很大的不同。此外，免疫系统-BC相互作用越来越被公认为肿瘤发展的关键，但用于生成PDX肿瘤的免疫功能低下的穆林模型缺乏成熟的T细胞、B细胞和天然杀伤细胞。此外，虽然PDX允许维持和扩大原发性乳房肿瘤，但由此产生的PDX肿瘤被原发性穆林频闪细胞和ECM5渗透。

为了克服这些挑战，我们开发了符合NIH MPS标准的新型、前活体、三维人类乳房MPS。我们的乳房MPS的基础是通过将原发性人类乳房组织（HBT）夹在两片脂肪衍生干细胞（ASCs）之间，也从HBT（图1）中分离出来。将细胞板转移到夹心的柱子可以3D打印或由简单的丙烯酸塑料制成（图1H，I）。这项技术适应了我们以前描述的培养人类白脂肪组织6，7的方法。然后，乳房MPS可以通过不列颠哥伦比亚省的模型进行播种，从标准的BC细胞系到人类原始乳房肿瘤。在这里，我们显示，这些BC-MPS在文化中稳定了几个星期（图2）：包括HBT的原生元素，如乳腺脂肪细胞，ECM，内皮，免疫细胞（图3）：并回顾BC和HBT之间的生理相互作用，如代谢相声（图4）。最后，我们表明，BC-MPS允许研究整个HBT（图5）中BC细胞的动物体运动。

Protocol

所有人体组织都是按照经LSUHSC机构审查委员会办公室批准的议定书#9189收集的。 1. 为细胞片播种脂肪衍生干细胞（ASC） 从商业来源购买ASC或按照既定协议8，9从原人类乳房组织分离。种子人类乳房ASCs在70%密度（+80，000细胞/厘米2表面积）到6井标准组织培养板在杜尔贝科的修改鹰介质（DMEM）补充10%胎儿牛血清，和1%青…

Representative Results

文化稳定BC-MPS 是一个稳定的微生理系统，可以在体外培养至少 14 天。以 100 倍的放大倍数拍摄了 ASC 细胞片的明亮场图像，以显示共体表 （图 2A）的条纹图案。ASC 细胞片在培养中稳定至少 4 周。BC-MPS在6口井板的一口井中进行了14天的培养，用彩色照相机进行成像，表明浮力高清在14天后仍然由ASC细胞板稳定地固定在井底（<strong class="…

Discussion

需要新的系统来模拟人类乳腺癌，以更好地了解这种疾病。开发人类微生理系统来模拟疾病设置，包括原生 ECM 和频闪细胞，将提高临床前研究的预测能力。这里介绍的BC-MPS模型是一个新开发的系统，克服了以前模型的局限性，允许在其原生HBT环境中对BC进行评估。该系统可用于癌细胞系或肿瘤外植，稳定至少14天。荧光标记 BC 细胞允许实时可视化癌细胞的活力和细胞-细胞相互作用、迁移、生存?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们要感谢杜兰流细胞测量和细胞分拣核心以及杜兰组织学核心的技术支持。这项工作得到了东南整形和重建外科医生协会2019年研究补助金和国家科学基金会（EPSCoR轨道2 RII，OIA 1632854）的支持。

Materials

Accumax Innovative Cell Technologies 1333 Cell disassoication solution for separation of BC-MPS
Accutase Corning 25-058-CI Cell detachment solution for passaging of cells
BioStor Container 16oz National Scientific Supply Co MPCE-T016 For Transport of sterile tissue
Cell Culture 75 cm flasks Corning 430641U For culturing ASCs
Conical Tubes 15mL  ThermoScientific 339650
Curved Forceps ThermoScientific 1631T5 For maneuvering tissue while mincing 
DMEM low glucose, w/ Glutamax Gibco 10567-014 For culturing ASCs and BC-MPS
FBS Qualified Gibco 26140-079
Gelatin Sigma G9391
HBSS 10x Gibco 14185-052
NaOH Sigma 221465
Nunc UpCell 6 well plates ThermoScientific 174901 Top ASC cell sheet
PBS Gibco 10010-023
Pen/Strep 5,000U Gibco 15070-063
Petri Dish 150 cm FisherBrand FB0875714 For holding tissue while mincing 
Razor Blades VWR 55411-055 Single Edge for mincing tissue
Strainer 250um  ThermoScientific 87791 For separation of BC-MPS
Tissue Culture 6 well plates Corning 3506 Bottom ASC cell Sheet
Weights/Washers BCP Fasteners BCP672 For weighing plungers down 1/2" inner diameter
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Riferimenti

  1. DeSantis, C. E., et al. Breast cancer statistics, 2019. CA: A Cancer Journal for Clinicians. 69 (6), 438-451 (2019).
  2. . NIH Categorical Spending -NIH Research Portfolio Online Reporting Tools (RePORT) Available from: https://report.nih.gov/categorical_spending.aspx (2020)
  3. Wong, C. H., Siah, K. W., Lo, A. W. Estimation of clinical trial success rates and related parameters. Biostatistics. 20 (2), 273-286 (2019).
  4. Sutherland, M. L., Fabre, K. M., Tagle, D. A. The National Institutes of Health Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline. Stem Cell Research & Therapy. 4, 1 (2013).
  5. Wang, X., et al. Breast tumors educate the proteome of stromal tissue in an individualized but coordinated manner. Science Signaling. 10 (491), (2017).
  6. Lau, F. H., et al. Sandwiched white adipose tissue: A microphysiological system of primary human adipose tissue. Tissue Engineering. Part C, Methods. 24 (3), 135-145 (2018).
  7. Scahill, S. D., Hunt, M., Rogers, C. L., Lau, F. H. A microphysiologic platform for human fat: sandwiched white adipose tissue. Journal of Visualized Experiments: JoVE. (138), e57909 (2018).
  8. Yu, G., et al. Adipogenic differentiation of adipose-derived stem cells. Adipose-Derived Stem Cells. 702, 193-200 (2011).
  9. Bunnell, B., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. Adipose-derived stem cells: Isolation, expansion and differentiation. Methods. 45 (2), 115-120 (2008).
  10. Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. S. Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and ‘smart’ polymers. Radiation Physics and Chemistry. 76 (8-9), 1409-1413 (2007).
  11. Lin, J. B., et al. Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering. Colloids and Surfaces B: Biointerfaces. 99, 108-115 (2012).
  12. Turner, W. S., Sandhu, N., McCloskey, K. E. Tissue engineering: Construction of a multicellular 3D scaffold for the delivery of layered cell sheets. Journal of Visualized Experiments. (92), e51044 (2014).
  13. Tinkercad. Create 3D digital designs with online CAD. Tinkercad. , (2020).
  14. Halfter, K., Mayer, B. Bringing 3D tumor models to the clinic – predictive value for personalized medicine. Biotechnology Journal. 12 (2), (2017).
  15. Wang, Y. Y., et al. Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells. JCI insight. 2 (4), 87489 (2017).
  16. Qiu, B., Simon, M. BODIPY 493/503 staining of neutral lipid droplets for microscopy and quantification by flow cytometry. Bio-Protocols. 6 (17), 1912 (2016).
  17. Fotos, J. S., et al. Automated time-lapse microscopy, and high-resolution tracking of cell migration. Cytotechnology. 51 (1), 7-19 (2006).
  18. Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. Open source software for quantification of cell migration, protrusions, and fluorescence intensities. Journal of Cell Biology. 209 (1), 163-180 (2015).
  19. Akiyama, Y., Kikuchi, A., Yamato, M., Okano, T. Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control. Langmuir: the ACS Journal of Surfaces and Colloids. 20 (13), 5506-5511 (2004).
  20. Shingyochi, Y., Orbay, H., Mizuno, H. Adipose-derived stem cells for wound repair and regeneration. Expert Opinion on Biological Therapy. 15 (9), 1285-1292 (2015).
  21. Tokunaga, M., et al. Fat depot-specific gene signature and ECM remodeling of Sca1(high) adipose-derived stem cells. Matrix Biology: Journal of the International Society for Matrix Biology. 36, 28-38 (2014).
  22. Belgodere, J. A., et al. Engineering breast cancer microenvironments and 3D bioprinting. Frontiers in Bioengineering and Biotechnology. 6, 66 (2018).
  23. Cao, Y. Adipocyte and lipid metabolism in cancer drug resistance. The Journal of Clinical Investigation. 129 (8), 3006-3017 (2019).
  24. Dirat, B., et al. Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion. Ricerca sul cancro. 71 (7), 2455-2465 (2011).
  25. Druso, J. E., Fischbach, C. Biophysical properties of extracellular matrix: Linking obesity and cancer. Trends in Cancer. 4 (4), 271-273 (2018).
  26. Luo, H., Tu, G., Liu, Z., Liu, M. Cancer-associated fibroblasts: A multifaceted driver of breast cancer progression. Cancer Letters. 361 (2), 155-163 (2015).
  27. Chandler, E. M., et al. Implanted adipose progenitor cells as physicochemical regulators of breast cancer. Proceedings of the National Academy of Sciences. 109 (25), 9786-9791 (2012).
  28. Oskarsson, T. Extracellular matrix components in breast cancer progression and metastasis. The Breast. 22, 66-72 (2013).
  29. Liu, J., et al. Collagen 1A1 (COL1A1) promotes metastasis of breast cancer and is a potential therapeutic target. Discovery Medicine. 25 (139), 211-223 (2018).
  30. Ahfeldt, T., et al. Programming human pluripotent stem cells into white and brown adipocytes. Nature Cell Biology. 14 (2), 209-219 (2012).
  31. Volz, A. -. C., Omengo, B., Gehrke, S., Kluger, P. J. Comparing the use of differentiated adipose-derived stem cells and mature adipocytes to model adipose tissue in vitro. Differentiation; Research in Biological Diversity. 110, 19-28 (2019).
  32. Zimta, A. A., et al. Molecular links between central obesity and breast cancer. International Journal of Molecular Sciences. 20 (21), 5364 (2019).
  33. Bousquenaud, M., Fico, F., Solinas, G., Rüegg, C., Santamaria-Martínez, A. Obesity promotes the expansion of metastasis-initiating cells in breast cancer. Breast Cancer Research. 20 (1), 104 (2018).
  34. Robado de Lope, L., Alcíbar, O. L., Amor López, A., Hergueta-Redondo, M., Peinado, H. Tumour-adipose tissue crosstalk: fuelling tumour metastasis by extracellular vesicles. Philosophical Transactions of the Royal Society B: Biological Sciences. 373 (1737), 20160485 (2018).
  35. Insua-Rodríguez, J., Oskarsson, T. The extracellular matrix in breast cancer. Advanced Drug Delivery Reviews. 97, 41-55 (2016).
  36. Sabol, R. A., et al. Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models. Breast Cancer Research: BCR. 21 (1), 67 (2019).
  37. Cui, X. D., Gao, D. Y., Fink, B. F., Vasconez, H. C., Pu, L. L. Q. Cryopreservation of human adipose tissues. Cryobiology. 55 (3), 269-278 (2007).
  38. Skardal, A., Shupe, T., Atala, A. Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling. Drug Discovery Today. 21 (9), 1399-1411 (2016).
  39. Clark, A. M., Allbritton, N. L., Wells, A. Integrative microphysiological tissue systems of cancer metastasis to the liver. Seminars in Cancer Biology. , (2020).

Tags

Breast CancerMicrophysiological SystemEx VivoAdipocytesImmune CellsVascular StructuresDuctal StructuresExtracellular MatrixPersonalized MedicinePharmaceutical DevelopmentTumor InitiationFibrosisExtracellular Matrix RemodelingGelatinPNIPAAmASCsPBSRazor BladeCancer Cell LinesBC-MPS Media
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Brown, L. M., Hebert, K. L., Gurrala, R. R., Byrne, C. E., Burow, M., Martin, E. C., Lau, F. H. Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System. J. Vis. Exp. (170), e62009, doi:10.3791/62009 (2021).
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