方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居
Summary
协议描述为研究乳腺癌细胞的迁移，增殖和定植于人体骨组织外植体模型系统。
Abstract
骨是乳腺癌转移的最常见的部位。虽然它已被广泛接受的微环境影响癌细胞行为，知之甚少人体骨组织的天然微环境中的乳腺癌细胞的特性和行为。我们已开发的方法来跟踪，量化和的微环境中调节人乳腺癌细胞培养的人骨组织碎片从以下全髋关节置换手术丢弃股骨头隔离。使用设计用于萤光素酶和增强型绿色荧光蛋白（EGFP）表达的乳腺癌细胞中，我们能够后可重复地定量迁移和增殖型态使用生物发光成像（BLI），轨道细胞相互作用使用荧光显微镜的骨碎片内，并评估乳腺细胞定植流式细胞仪。这种模式的主要优势包括：1）一个人，建筑完好无损组织microenvironment包括有关人体的细胞类型，以及2）直接进入微环境，这有利于快速定量和定性监测和乳腺癌和骨细胞的特性，行为和交互作用扰动。一个主要的限制，目前，是组织片段，该组合限制住研究短期培养的窗口的有限的可行性。该模型系统的用途包括研究乳腺癌和其他骨寻求恶性肿瘤的基本生物学的转移性利基内，并且开发治疗策略，以有效地针对乳腺癌细胞在骨组织中。
Introduction
肿瘤微环境是目前公认的癌细胞行为乳腺癌进展和转移过程中的关键决定因素传播1-3。这里提出的方法的目标是促进人体骨组织，乳腺癌转移4-6的最频繁的部位的微环境中的乳腺癌细胞的研究。骨是由矿化和骨髓室7-9，这两个怀有细胞和分泌的因子牵连转移进展10,11。尽管广泛使用在体内小鼠模型促进转移过程12-15系统性研究，骨架内细胞的相互作用是不进行观察和直接扰动容易获得。模型系统研究和培养小鼠骨髓组织和小鼠骨髓细胞提供更好的访问，并已经取得了对串扰许多见解和机制基本乳腺癌细胞米etastasis小鼠骨骼16-22。然而，研究表明，有可能是osteotropism的物种特异性的模式为骨组织23,24。这些模式可以反映固有物种特异性在骨组织性质的差异，从改变的骨髓群体中的免疫缺陷小鼠11产生的差异，和/或小鼠的相对年轻的年龄在实验中使用的，所有这些都在骨的可能的决定因素微环境。
体外方法用于人骨共培养研究乳腺癌细胞已经典型地集中于特定骨的细胞类型，如骨髓来源的成骨细胞或基质细胞作为单层培养的或工程化的三维模型系统25-35内。虽然2维细胞培养模型已形成的在体外的支柱接近为癌症研究，人们早就认识到，细胞的行为是从根本上改变在米onolayer培养系统18,36,37。这导致了设计的微环境，模仿的3-维活组织的复杂性，包括矩阵可与天然材料，如胶原蛋白构成的支架的基于模型的开发，或合成的聚合物接种有特定的细胞类型，以创建组织样的微环境36-41。设计方案还包括使用生物反应器平台的控制和研究微18,19,41-43体内的荷尔蒙环境。虽然仿生模型和生物反应器提供了一个复杂的组织微环境中的受控环境的许多元素，并且已成功地应用到推进乳腺癌转移到骨18,19,35,42,43的研究中，工程化的模型系统一般不掺入天然骨环境中存在的细胞类型和细胞外基质组分的全谱。
直到最近，乳腺癌细胞尚未研究天然的，完整的，三维人体骨组织的微环境中。我们最近报道使用人体骨组织碎片从全髋关节置换分离（THR）的手术标本44的共培养模型的发展。这些标本既怀有必要研究潜在的微转移在短期内培养机制的矿化和骨髓车厢。在以前的工作中，我们建立了验证性原则，使用生物发光成像（BLI）监测荧光素酶表达乳腺癌细胞（MDA-MB-231-fLuc）共培养骨碎片44的扩散。在这里，我们提出详细的实验方案为研究使用人骨组织的外植体的天然微环境的上下文中乳腺细胞癌的增殖，定植，和迁移。
首先，我们提出了一个协议相邻骨骼碎片来衡量乳房共培养乳腺癌细胞使用BLI细胞增殖。在这一节中，乳腺癌细胞悬液接种作为细胞的斑点在邻近骨头碎片，这是由骨蜡件固定6孔板中。对照孔含有骨蜡，但是没有骨片段。一旦细胞斑附着，培养基，并将该板是24小时，在这之后的生物发光信号的强度（与细胞数目相关联）的测量是用BLI培养。在接下来的步骤中，我们提出直接接种到骨碎片研究定居和增殖共培养的乳腺癌细胞的方法。这里的乳房细胞悬浮液被直接吸移到所述骨组织碎片，其通过BLI和荧光显微镜在培养监测随着时间的推移追踪定植和细胞数。在该方法中，骨髓隔室可以从骨碎片被刷新为在定植乳腺癌细胞通过流式细胞分析的任何时间点术，或骨髓存活测定。
此外，我们取消划线两种不同的方法，用于测量乳腺癌细胞迁移。在第一种方法的步骤进行了概述，用于测量朝向骨组织培养上清液迁移。在这个协议中的乳腺癌细胞接种到Transwell小室的内，上表面插入膜具有8微米的孔径（ 如图1A）。插入件，然后放入一个接收机板用含有骨组织上清液或对照培养基的孔中。超过20小时的温育期的过程中，少量的乳腺癌细胞通过插入膜迁移入下面的组织培养孔中，其中它们附着于检测通过BLI。在第二迁移方法的乳腺癌细胞接种到Transwell小室的插入件的膜的下部，外表面。骨组织碎片放入插入杯子和乳腺癌细胞向上迁移通过膜拓殖骨碎片（ 见图1B），其然后用成像BLI。
Protocol
股骨头从择期THR手术矫形外科系在斯坦福大学医学院的患者收集。所有的组织收集的去鉴定标本按照斯坦福大学的研究合规办公室法规。 1。选择乳腺癌细胞系（S） 获得或转染所需的乳腺癌细胞系（S）与萤光素酶-GFP报道构建。下面实验使用的MDA-MB-231和MCF-7乳腺癌细胞系工程改造为萤火虫荧光素酶的稳定表达（fLuc）和增强型绿色荧光蛋白（EGFP）的睡美人转座子的质粒PKT2 / LuBi…
Representative Results
从股骨头分离组织碎片股骨头从男性和女性患者（44-90岁）在接受矫形外科系选修全髋关节置换手术在斯坦福大学医学院的数量相等收集。每个丢弃股骨头含有股骨上端，其中窝藏小梁骨组织的矿化骨针和骨髓组成的一小部分。此组织是可透过轴（图2A）的使用外科咬骨钳提取小片段的外科手术暴露的横截面。所提取的片的组织学示于图2B中 ，揭示了?…
Discussion
梅拉等人以前描述了死后收集和骨标本分析转移性前列腺癌患者收获在尸检时，露出高质量组织和验证的使用人骨样品来研究转移过程47。在这里，我们已经描述了协议，用于收集，处理和使用从下面的THR手术在短期共培养系统来研究乳腺癌细胞的迁移，定居和增殖丢弃股骨头获得的人骨组织碎片。大约300,000髋关节置换手术是在美国（矫形外科医生的美国学院; Orthoinfo.aaos.org）每年…
Declarações
The authors have nothing to disclose.
Acknowledgements
这些研究提供资金，部分通过从替代研究与发展基金会（107588），美国国立卫生研究院（1U54CA136465-04S1），和加州乳腺癌研究发展计划（201B-0141）资助。我们感谢博士。安德鲁·威尔伯和R.斯科特·麦基弗的慷慨捐赠自己转座子和PK / hUbiC-SB11转座质粒。我们非常感谢约翰Tamaresis博士对统计方法的建议，蒂莫西·布朗的表演术流，乔其纱亨里奇上迁移试验的意见，和南希Bellagamba为促进THR标本采集。
Materials
| DMEM (1X) + GlutaMAX-l | Life Technologies | 10569-010 | Supplement all DMEM with 10% fetal bovine serum and 1% Pen/Strep |
| McCoy's 5A Medium (1X) | Life Technologies | 16600-082 | Supplement all McCoy's with 10% fetal bovine serum and 1% Pen/Strep |
| Fetal bovine serum | Life Technologies | 16000-044 | |
| Penicillen Streptomycin | Life Technologies | 15140-122 | |
| Blastocidin (10 mgl/ml) | InvivoGen | ant-bl | Supplement media used for transfected cell lines. |
| Falcon® Cell Culture Inserts for 24 Well Plate with Transparent PET Membrane (8.0 μm pore size) | Corning Incorporated | 353097 | |
| Falcon® 24 Well TC-Treated Polystyrene Cell Culture Insert Companion Plate | Corning Incorporated | 353504 | |
| LIVE/DEAD Viability/Cytotoxicity Kit *for mammalian cells* | Invitrogen Detection Technologies | L-3224 | |
| FluoroBrite DMEM | Life Technologies | A18967-01 | |
| D-luciferin firefly, potassium salt 5 grams (30 mg/mL) | Life Technologies | L-8220 | |
| Sterile Cell Strainer 70 μm | Fisher Scientific | 22363548 | |
| 6 Well TC-Treated Cell Culture Cluster | Corning Incorporated | 3516 | |
| 12 Well TC-Treated Cell Culture Cluster | Corning Incorporated | 3513 | |
| 24 Well TC-Treated Cell Culture Cluster | Corning Incorporated | 3526 | |
| Friedman-Pearson Rongeur | Fine Science Tools | 16021-14 | |
| Bone wax | Surgical Specialties Corporation | 903 | |
| IVIS 50 Imaging Platform | Caliper Life Sciences/PerkinElmer | ||
| Living Image Software Program | Caliper Life Sciences/PerkinElmer | 133026 | |
| EVOS FL Imaging System | Life Technologies | Version 4.2 | |
| OsteoSense 680 EX | PerkinElmer | NEV10020EX | |
| MCF-7 breast cancer cells | ATCC | HTB-22 | |
| MDA-MB-231 breast cancer cells | ATCC | HTB-26 | |
| Monoclonal mouse anti-human cytokeratin antibody | DAKO Cytomation | Clone (AE1/AE3) |
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Templeton, Z. S., Bachmann, M. H., Alluri, R. V., Maloney, W. J., Contag, C. H., King, B. L. Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche. J. Vis. Exp. (97), e52656, doi:10.3791/52656 (2015).