Department of Bioengineering, Rice University, Houston, TX, United States.
Department of Applied Physics, Rice University, Houston, TX, United States.
Acta Biomater. 2019 Dec;100:38-51. doi: 10.1016/j.actbio.2019.09.029. Epub 2019 Sep 19.
Current in vitro methods for assessing cancer biology and therapeutic response rely heavily on monolayer cell culture on hard, plastic surfaces that do not recapitulate essential elements of the tumor microenvironment. While a host of tumor models exist, most are not engineered to control the physical properties of the microenvironment and thus may not reflect the effects of mechanotransduction on tumor biology. Utilizing coaxial electrospinning, we developed three-dimensional (3D) tumor models with tunable mechanical properties in order to elucidate the effects of substrate stiffness and tissue architecture in osteosarcoma. Mechanical properties of coaxial electrospun meshes were characterized with a series of macroscale testing with uniaxial tensile testing and microscale testing utilizing atomic force microscopy on single fibers. Calculated moduli in our models ranged over three orders of magnitude in both macroscale and microscale testing. Osteosarcoma cells responded to decreasing substrate stiffness in 3D environments by increasing nuclear localization of Hippo pathway effectors, YAP and TAZ, while downregulating total YAP. Additionally, a downregulation of the IGF-1R/mTOR axis, the target of recent clinical trials in sarcoma, was observed in 3D models and heralded increased resistance to combination chemotherapy and IGF-1R/mTOR targeted agents compared to monolayer controls. In this study, we highlight the necessity of incorporating mechanical cues in cancer biology investigation and the complexity in mechanotransduction as a confluence of stiffness and culture architecture. Our models provide a versatile, mechanically variable substrate on which to study the effects of physical cues on the pathogenesis of tumors. STATEMENT OF SIGNIFICANCE: The tumor microenvironment plays a critical role in cancer pathogenesis. In this work, we engineered 3D, mechanically tunable, coaxial electrospun environments to determine the roles of the mechanical environment on osteosarcoma cell phenotype, morphology, and therapeutic response. We characterize the effects of varying macroscale and microscale stiffnesses in 3D environments on the localization and expression of the mechanoresponsive proteins, YAP and TAZ, and evaluate IGF-1R/mTOR pathway activation, a target of recent clinical trials in sarcoma. Increased nuclear YAP/TAZ was observed as stiffness in 3D was decreased. Downregulation of the IGF-1R/mTOR cascade in all 3D environments was observed. Our study highlights the complexity of mechanotransduction in 3D culture and represents a step towards controlling microenvironmental elements in in vitro cancer investigations.
目前，用于评估癌症生物学和治疗反应的体外方法在很大程度上依赖于在坚硬的塑料表面上进行单层细胞培养，而这些方法无法再现肿瘤微环境的基本要素。尽管存在许多肿瘤模型，但大多数模型都没有设计用于控制微环境的物理特性，因此可能无法反映机械转导对肿瘤生物学的影响。我们利用同轴静电纺丝技术开发了具有可调机械性能的三维（3D）肿瘤模型，以阐明基质刚度和组织架构对骨肉瘤的影响。通过一系列宏观测试（包括单轴拉伸测试）和利用原子力显微镜对单纤维进行的微观测试，对同轴静电纺丝网的机械性能进行了表征。在我们的模型中，宏观和微观测试的计算模量跨越了三个数量级。骨肉瘤细胞在 3D 环境中对基质刚度的降低做出反应，通过增加 Hippo 通路效应物 YAP 和 TAZ 的核定位，同时下调总 YAP。此外，在 3D 模型中观察到 IGF-1R/mTOR 轴的下调，该轴是最近肉瘤临床试验的靶点，与单层对照相比，预示着对联合化疗和 IGF-1R/mTOR 靶向药物的耐药性增加。在这项研究中，我们强调了在癌症生物学研究中纳入机械线索的必要性，以及机械转导的复杂性，即刚度和培养架构的融合。我们的模型提供了一个灵活的、可机械调节的基质，可用于研究物理线索对肿瘤发病机制的影响。
肿瘤微环境在癌症发病机制中起着关键作用。在这项工作中，我们设计了 3D 机械可调同轴静电纺丝环境，以确定机械环境对骨肉瘤细胞表型、形态和治疗反应的影响。我们在 3D 环境中表征了不同宏观和微观刚度对机械响应蛋白 YAP 和 TAZ 的定位和表达的影响，并评估了 IGF-1R/mTOR 通路的激活，这是最近肉瘤临床试验的靶点。当 3D 中的刚度降低时，观察到核 YAP/TAZ 的增加。在所有 3D 环境中都观察到 IGF-1R/mTOR 级联的下调。我们的研究强调了 3D 培养中机械转导的复杂性，并代表了朝着控制体外癌症研究中微环境因素迈出的一步。
