Herroon Mackenzie Katheryn, Diedrich Jonathan Driscoll, Podgorski Izabela
Department of Pharmacology, Wayne State University School of Medicine , Detroit, MI , USA.
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA; Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA.
Front Endocrinol (Lausanne). 2016 Jul 6;7:84. doi: 10.3389/fendo.2016.00084. eCollection 2016.
Adipocytes are a major component of the bone marrow that can critically affect metastatic progression in bone. Understanding how the marrow fat cells influence growth, behavior, and survival of tumor cells requires utilization of in vitro cell systems that can closely mimic the physiological microenvironment. Herein, we present two new three-dimensional (3D) culture approaches to study adipocyte-tumor cell interactions in vitro. The first is a transwell-based system composed of the marrow-derived adipocytes in 3D collagen I gels and reconstituted basement membrane-overlayed prostate tumor cell spheroids. Tumor cells cultured under these 3D conditions are continuously exposed to adipocyte-derived factors, and their response can be evaluated by morphological and immunohistochemical analyses. We show via immunofluorescence analysis of metabolism-associated proteins that under 3D conditions tumor cells have significantly different metabolic response to adipocytes than tumor cells grown in 2D culture. We also demonstrate that this model allows for incorporation of other cell types, such as bone marrow macrophages, and utilization of dye-quenched collagen substrates for examination of proteolysis-driven responses to adipocyte- and macrophage-derived factors. Our second 3D culture system is designed to study tumor cell invasion toward the adipocytes and the consequent interaction between the two cell types. In this model, marrow adipocytes are separated from the fluorescently labeled tumor cells by a layer of collagen I. At designated time points, adipocytes are stained with BODIPY and confocal z-stacks are taken through the depth of the entire culture to determine the distance traveled between the two cell types over time. We demonstrate that this system can be utilized to study effects of candidate factors on tumor invasion toward the adipocytes. We also show that immunohistochemical analyses can be performed to evaluate the impact of direct interaction of prostate tumor cells with adipocytes. Our models underline the importance of using the appropriate culture conditions to mimic physiological interactions between marrow adipocytes and metastatic tumor cells. These systems have a potential to be utilized for analyses of various factors that may be regulated by the adipocytes in bone. Their application likely extends beyond metastatic prostate cancer to other tumors that colonize the bone marrow microenvironment.
脂肪细胞是骨髓的主要组成部分，对骨转移进展具有关键影响。了解骨髓脂肪细胞如何影响肿瘤细胞的生长、行为和存活，需要利用能够紧密模拟生理微环境的体外细胞系统。在此，我们提出两种新的三维（3D）培养方法，用于体外研究脂肪细胞与肿瘤细胞的相互作用。第一种是基于Transwell的系统，由三维胶原蛋白I凝胶中的骨髓来源脂肪细胞和重组基底膜覆盖的前列腺肿瘤细胞球体组成。在这些三维条件下培养的肿瘤细胞持续暴露于脂肪细胞衍生因子，其反应可通过形态学和免疫组织化学分析进行评估。我们通过对代谢相关蛋白的免疫荧光分析表明，在三维条件下，肿瘤细胞对脂肪细胞的代谢反应与二维培养的肿瘤细胞显著不同。我们还证明，该模型允许纳入其他细胞类型，如骨髓巨噬细胞，并利用染料淬灭的胶原蛋白底物来检测对脂肪细胞和巨噬细胞衍生因子的蛋白水解驱动反应。我们的第二个三维培养系统旨在研究肿瘤细胞向脂肪细胞的侵袭以及两种细胞类型之间的后续相互作用。在这个模型中，骨髓脂肪细胞通过一层胶原蛋白I与荧光标记的肿瘤细胞分离。在指定时间点，用BODIPY对脂肪细胞进行染色，并通过整个培养物的深度进行共聚焦z轴堆叠，以确定两种细胞类型之间随时间的移动距离。我们证明该系统可用于研究候选因子对肿瘤细胞向脂肪细胞侵袭的影响。我们还表明，可以进行免疫组织化学分析来评估前列腺肿瘤细胞与脂肪细胞直接相互作用的影响。我们的模型强调了使用适当培养条件来模拟骨髓脂肪细胞与转移性肿瘤细胞之间生理相互作用的重要性。这些系统有可能用于分析可能由骨中的脂肪细胞调节的各种因素。它们的应用可能不仅限于转移性前列腺癌，还可扩展到其他定殖于骨髓微环境的肿瘤。
