Three-dimensional, mechanically tunable scaffold systems to model osteosarcoma heterogeneity and therapeutic response

项目摘要 这项研究的基本原理是需要研究肿瘤异质性和 分化影响骨肉瘤细胞的分化、可塑性和对治疗的抗性机制。 目前，癌症研究领域缺乏能够准确概括 肿瘤微环境包括对于分化和肿瘤细胞的产生至关重要的机械信号， 正常细胞表型。因此，本研究的目的是利用三维静电纺丝支架 具有可变的机械性能以平衡基底硬度对骨肉瘤细胞表型的影响， 可塑性和对治疗的反应。这项工作的基本假设是，当细胞培养在 在具有可变机械特性的3D环境中，它们将显示出更高的差异化倾向， 主要致病途径的差异激活和对治疗的可变反应。 拟议的研究将通过两个具体目标来完成。在第一个具体目标中，我们将 扩展初步数据，表明可变表型和三维可塑性增加 通过确定力学环境对塑性的影响， 骨肉瘤细胞分化将通过扫描电子显微镜评估制造的支架， 压汞法和单轴拉伸试验。然后将骨肉瘤细胞在支架上培养 可变的机械性能，并将暴露于生化分化线索诱导 成骨、成脂、成软骨谱系定型。重要的生化信号通路 骨肉瘤的发病机制和分化标志物将通过蛋白质印迹、qRT-PCR和流式细胞术进行评估。 细胞仪这一特定目标的结果将揭示机械环境在决定肿瘤中的作用 细胞可塑性和表型。第二个具体目标将阐明机械之间的相互作用 环境和分化表型影响骨肉瘤细胞对阿霉素治疗的反应 以及最近临床试验的对象地磷莫司。前两项研究将有助于阐明 IGF-1/mTOR和Wnt通路的分化和基本信号传导的特征， 骨肉瘤群体对治疗耐药或敏感。第三项研究旨在验证我们的模型， 结合使用从患者获得的原发性骨肉瘤样品。原代骨肉瘤细胞 如在该目的的前两项研究中那样用治疗剂治疗; 将评价治疗反应。 我们的目标是利用可调的3D机械环境来研究异常分化 在骨肉瘤中存在的不同细胞群中，以及分化和表型如何与 抵抗治疗的机制。这项拟议中的研究有可能为癌症研究人员提供 研究肿瘤细胞的可变群体的工具，这是不可能用标准培养技术。

Project Summary The rationale for the proposed research is the need for strategies to study how tumor heterogeneity and differentiation affect osteosarcoma cell differentiation, plasticity, and mechanisms of resistance to therapy. Currently, the field of cancer research suffers from a dearth of pre-clinical models that accurately recapitulate the tumor microenvironment including mechanical cues which are essential for differentiation and generation of normal cell phenotype. Therefore, the objective of the proposed research is to utilize 3D electrospun scaffolds with variable mechanical properties to leverage the effect of substrate stiffness on osteosarcoma cell phenotype, plasticity, and response to therapy. The fundamental hypothesis of this work is that when cells are cultured in 3D environments with variable mechanical properties, they will display a higher propensity for differentiation, differential activation of essential pathogenic pathways, and variable response to therapy. The proposed research will be accomplished through two specific aims. In the first specific aim, we will expand on preliminary data that suggests variable phenotype and increased plasticity in three-dimensional mechanical environments by determining the effects of the mechanical environment on the plasticity of osteosarcoma cell differentiation. Fabricated scaffolds will be assessed through scanning electron microscopy, mercury porosimetry, and uniaxial tensile testing. Osteosarcoma cells will then be cultured on scaffolds of variable mechanical properties and will be exposed to biochemical differentiation cues for induction of osteogenic, adipogenic, chondrogenic lineage commitment. Essential biochemical signaling pathways for osteosarcoma pathogenesis and markers of differentiation will be assessed via Western blot, qRT-PCR and flow cytometry. The outcomes of this specific aim will reveal the role of the mechanical environment in dictating tumor cell plasticity and phenotype. The second specific aim will elucidate how the interplay between mechanical environment and differentiation phenotype affects osteosarcoma cell response to therapy with the doxorubicin and ridaforolimus, the subject of recent clinical trials. The first two studies will serve to elucidate the molecular signatures of differentiation and essential signaling of IGF-1/mTOR and Wnt pathways that identify osteosarcoma populations as resistant or susceptible to therapy. The third study aims to validate our model by incorporating the use of primary osteosarcoma samples obtained from patients. Primary osteosarcoma cells will be treated with therapeutics as in the first two studies of this aim; the identified molecular signatures of therapeutic response will be evaluated. We aim to leverage the effects of a tunable, 3D mechanical environment to study aberrant differentiation in divergent cell populations present in osteosarcoma and how differentiation and phenotype relate to mechanisms of resistance to therapy. The proposed research has the potential to offer cancer researchers with the tools to study variable populations of tumor cells which is impossible with standard culture techniques.