Regenerative Medicine and Cancer Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
PLoS One. 2012;7(9):e40217. doi: 10.1371/journal.pone.0040217. Epub 2012 Sep 5.
Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.
生物物理和生物化学特性的微环境调节细胞反应，如生长、分化、形态发生和迁移在正常和癌细胞。由于二维（2D）培养缺乏细胞微环境的基本特征，三维（3D）培养已经被开发出来，以更好地模拟天然细胞外基质。迄今为止，3D 培养系统主要依赖于胶原和基质胶®水凝胶，只能对基质硬度、蛋白水解降解性和配体密度进行有限的控制。相比之下，生物工程水凝胶使我们能够独立地调整和系统地研究这些参数对细胞生长和分化的影响。在这项研究中，聚乙二醇（PEG）水凝胶，用精氨酸-甘氨酸-天冬氨酸（RGD）基序功能化，是细胞外基质蛋白中的常见细胞结合基序，以及基质金属蛋白酶（MMP）切割位点，对其硬度、扩散性能和支持雄激素依赖性 LNCaP 前列腺癌细胞生长的能力进行了表征。我们发现，机械性能调节了 LNCaP 细胞在 PEG 水凝胶中的生长动力学。在 28 天的培养期内，LNCaP 细胞发生形态发生变化，在 3D 培养中形成肿瘤样结构，具有缺氧和凋亡核心。我们进一步比较了 3D 和 2D 培养中受合成雄激素 R1881 刺激后的蛋白质和基因表达水平。有趣的是，免疫荧光染色观察到 2D 和 3D 培养中 R1881 刺激雄激素受体（AR）核易位的动力学不同。此外，微阵列研究表明，R1881 处理后雄激素反应基因的表达水平变化在 2D 和 3D 培养中差异很大。总之，在可调节的 PEG 水凝胶中培养 LNCaP 细胞揭示了 2D 和 3D 环境中细胞对雄激素刺激的反应差异。因此，我们建议所提出的 3D 培养系统是一种强大的高通量前列腺癌药物测试工具，可重现肿瘤微环境。
