器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响
Summary
This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.
Abstract
Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.
Introduction
乳腺癌是女性最常见的癌症和癌症相关死亡1的第二大原因。乳腺癌的死亡率高的主要原因是常规治疗，以减轻和消除转移性疾病的故障;癌症相关死亡的约90％是由于转移2。理解转移级联的分子机制是至关重要的，以有效地早期和晚期乳腺癌治疗剂的开发。
过去的研究已经帮助阐明乳腺癌转移的多步性质以及它是假设，既癌症进展和转移的结果在很大程度上取决于癌细胞和主机环境3之间的相互作用。临床观察表明，许多癌症显示器官趋向性， 即 ，该倾向优先转移到特定organs.In的CAS乳腺癌E，病人的疾病通常传播或转移至5个主要景点，包括骨，肺，淋巴结，肝，脑4-6。许多理论已经发展到解释这个过程，但只有少数经受住了时间的考验。尤文转移的理论，在20世纪20年代提出的，假设转移thatthe分布严格，由于机械因素;由此肿瘤细胞通过正常定义生理血流模式整个身体携带并在第一毛细管床简单地阻止他们遇到7。相比之下，斯蒂芬·佩吉特氏1889“种子和土壤”假说认为，额外的分子相互作用负责存活和转移生长，因此癌细胞（“种子”），只能建立自己和产生相应的分子因素proliferatein器官微环境（“土“）8。将近一个世纪之后，伦纳德在韦斯花了此前公布的尸检资料进行了荟萃分析，证实了尤文的预言，在尸检时发现许多转移性肿瘤的转移是否器官取向是由血流模式单独确定，将有望在预期的比例被发现了。然而，在manyinstances有在某些网站，然后会被尤文提出的力学因素9预期形成更少或更多的转移。这些帐户和理论认为特定器官的微环境起到传播模式和多种癌症，包括乳腺癌的随后的生长和存活的关键作用。
过去的研究工作主要集中于肿瘤细胞衍生因子及其在乳腺癌转移10-12观察到的器官向性的贡献，从器官微环境衍生但是很少有研究探讨因素，可能为建立提供了有利的利基乳腺癌转移。这主要归因于在体外研究器官微环境的组件的技术挑战。
当前文章描述了一种用于研究淋巴结，骨，肺和大脑对人乳腺癌细胞的转移行为的水溶性组分的影响的综合体外模型系统。通过证明不同乳腺癌细胞系显示响应于器官条件培养基器官特异性细胞系特异性和恶性行为对应于它们的体内转移潜能13以前的研究已经证实该模型的系统。该模型系统可用于识别和评估可在乳房和其他类型的癌细胞，包括生长，迁移影响的转移行为发挥作用器官特异性的可溶性因子，干样行为，以及基因表达，以及鉴定潜在的新的治疗靶点为癌症。这是可以用来详细研究器官特异性转移行为，并评价在驱动癌转移的方法的器官来源的可溶性因子的作用的第一个体外模型系统。
Protocol
Representative Results
Discussion
转移是一个复杂的过程，其中一系列的细胞事件最终都是用于组织浸润和远处肿瘤形成4,30,31负责。这里提出的离体模型系统可用于转移进展的两个重要方面研究：癌细胞归巢或迁移到特定器官（“到达那里”）和生长在该机构（“生长在那里”）。先前许多研究都集中在识别与癌细胞本身有助于转移过程相关的关键分子的特性。例如，通过琼Massagué的组所做的工作已经确定肺特异?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master’s program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.
Materials
| 50 ml conical tubes | Thermo Scientific (Nunc) | 339652 | Keep sterile |
| 1X Phosphate-buffered saline | ThermoFisher Scientific | 10010-023 | Keep sterile |
| Nude mice | Harlan Laboratories | Hsd:Athymic Nude-Foxn1nu | Use at 6-12 weeks of age |
| Polystyrene foam pad | N/A | N/A | The discarded lid (~4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol. |
| Forceps | Fine Science Tools | 11050-10 | Keep sterile |
| Scissors | Fine Science Tools | 14058-11 | Keep sterile |
| Gauze pads | Fisher Scientific | 22-246069 | Keep sterile |
| 60 mm2 glass petri dishes | Sigma-Aldrich | CLS7016560 | Keep sterile |
| Scalpel blades | Fisher Scientific | S95937A | Keep sterile |
| DMEM:F12 | Life Technologies | 21331-020 | Warm in 37 °C water bath before use, keep sterile |
| 1 x Mito+ Serum Extender | BD Biosciences | 355006 | Referred to as "concentrated mitogen supplement" in the manuscript. Keep sterile |
| Penicillin-Streptomycin (10,000 U/mL) | Life Technologies | 15140-122 | Keep sterile |
| Rosewell Park Memorial Institute 1640 (RPMI 1640) | Life Technologies | 11875-093 | Warm in 37 °C water bath before use, keep sterile |
| Fetal Bovine Serum | Sigma-Aldrich | F1051-500ML | Keep sterile |
| Trypsin/EDTA solution | ThermoFisher Scientific | R-001-100 | Warm in 37 °C water bath before use, keep sterile |
| 6-well tissue culture plates | Thermo Scientific (Nunc) | 140675 | Keep sterile |
| 0.22 μm syringe filters | Sigma-Aldrich | Z359904 | Keep sterile |
| T75 tissue culture flasks | Thermo Scientific (Nunc) | 178905 | Keep sterile |
| Transwells | Sigma-Aldrich | CLS3464 | Keep sterile, use for migration assays |
| Anti-mouse Sca-1 | R&D Systems | FAB1226P | use at 10 µl/106 cells |
| Anti-mouse CD105 | R&D Systems | FAB1320P | use at 10 µl/106 cells |
| Anti-mouse CD29 | R&D Systems | FAB2405P-025 | use at 10 µl/106 cells |
| Anti-mouse CD73 | R&D Systems | FAB4488P | use at 10 µl/106 cells |
| Anti-mouse CD44 | R&D Systems | MAB6127-SP | use at 0.25 µg/106 cells |
| Anti-mouse CD45 | eBioscience | 11-0451-81 | use at 5 µl/106 cells |
| Anti-mouse gp38 | eBioscience | 12-5381-80 | use at 10 µl/106 cells |
| β-mercaptoethanol | Sigma-Aldrich | M6250 | Keep sterile |
| Protein arrays | RayBiotech Inc. | AAM-BLM-1-2 | Use 1 array per media condition (including negative control), in triplicate |
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