Three-dimensional, mechanically tunable scaffold systems to model osteosarcoma heterogeneity and therapeutic response
Three-dimensional, mechanically tunable scaffold systems to model osteosarcoma heterogeneity and therapeutic response
批准号:
9770812
负责人:
Eric Rodolfo Molina
金额:
$4.12万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-09-15 至 2020-09-14
关键词:
3-DimensionalAdherent CultureAdolescenceAffectArchitectureBiochemicalBiological AssayBiologyCaliberCancer BiologyCell Differentiation processCell LineCell ProliferationCellsClinical TrialsCommon NeoplasmCuesCulture TechniquesCultured CellsDataDifferentiation AntigensDimensionsDiseaseDoxorubicinDrug resistanceElectrospinningEnvironmentExcisionExposure toFRAP1 geneFiberFlow CytometryGelatinGenerationsGleanGrowthHeterogeneityHistologyHydrogelsIn VitroInsulin-Like Growth Factor IMalignant Bone NeoplasmMalignant NeoplasmsMechanicsMercuryMesenchymal Stem CellsModelingModernizationMolecular ProfilingNeedlesNeoplasm MetastasisNormal CellOperative Surgical ProceduresOutcomePathogenesisPathogenicityPathway interactionsPatientsPharmaceutical PreparationsPhenotypePlayPolymersPolystyrenesPopulationPorosityPre-Clinical ModelPropertyQuantitative Reverse Transcriptase PCRResearchResearch PersonnelResistanceRoleSamplingScanning Electron MicroscopySignal PathwaySignal TransductionStem cellsSurfaceSurface PropertiesSurvival RateSuspension CultureSystemTestingTherapeuticTreatment FailureTumor InitiatorsWestern BlottingWorkanticancer researchbeta catenincancer cellcancer stem cellcancer typechemotherapyclinically relevantds-DNAin vivo Modelindividual variationmTOR Inhibitormechanical propertiesmonolayerneoplastic cellnovelosteogenicosteosarcomapolycaprolactoneprimary bone cancerresistance mechanismresponsesarcomascaffoldstem cell differentiationtherapy resistanttooltreatment responsetumortumor heterogeneitytumor microenvironmenttumorigenicvoltage
中文摘要
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。
英文摘要
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.
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