Three-dimensional Model of Human Ewing Sarcoma

描述（由申请人提供）：拟定研究的总体目标是应用骨肉瘤细胞的静态和流动灌注生物反应器培养，在可靠模拟骨肿瘤生态位关键特征的组织工程聚合物/细胞外基质（ECM）混合模型上生长，以促进我们对IGF-1 R/mTOR癌症相关途径及其临床相关耐药机制的理解。我们的实验室报告了体内IGF-1 R/mTOR表达模式，与在人类尤文肉瘤肿瘤（ES）中观察到的密切相关，当建立的ES细胞系生长在创新的生物惰性3D静电纺丝聚（ε-己内酯）（PCL）微纤维支架上时，而不是传统的塑料单层上。本提案旨在阐明细胞-细胞和细胞-ECM相互作用在这种工程化的3D骨肿瘤微环境中刺激激活的IGF-1 R/mTOR信号传导状态的确切机制，因为这些相互作用在启动和维持ES中至关重要。在确定静态细胞培养下这些参数的影响的同时，将调整3D支架和培养条件以更好地模拟天然骨微环境：（a）将使用不同的流动灌注速率来评估剪切应力对细胞保留的影响，同时促进尤文细胞在支架内的均匀分布，（B）PCL支架将掺入IGF-1以模拟当肿瘤侵入周围骨时天然释放的高浓度IGF-1，（c）成骨ECM对IGF-1 R/IGF-1受体的影响。mTOR信号级联将使用脱细胞支架来测定，在该支架上首先生长向成骨细胞谱系分化的异型间充质干细胞，和（d）将ES细胞与内皮细胞（EC）共培养，以确定异型细胞如何在制造的3D骨肿瘤模型内相互作用以引发存活的ES肿瘤。最后，为了确定尤文肉瘤逃避mTOR/IGF-1 R联合靶向治疗敏感性的机制，新鲜来源的肿瘤标本（获自在IRB批准的临床试验中用Medi- 573/依维莫司治疗的尤文肉瘤患者），将在3D支架内的原代培养物中生长，并通过IGF-1 R/PDTX的蛋白质组学分析与2D培养物和患者来源的肿瘤外植体（PDTX）进行比较。mTOR通路和推定的抗性机制。这种在骨微环境的离体临床前模型中研究尤文肉瘤的新方法为理解化疗疗效和确定生物靶向治疗的耐药机制提供了巨大的潜力。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to apply static and flow perfusion bioreactor culture of bone sarcoma cells, grown upon a tissue-engineered polymer/extracellular matrix (ECM) hybrid model that reliably mimics key features of the bone tumor niche, to advance our understanding of the IGF-1R/mTOR cancer- related pathway and its clinically-relevant resistance mechanisms. Our laboratory has reported in vivo-like IGF- 1R/mTOR expression patterns, closely related to those observed in human Ewing sarcoma tumors (ES), when established ES cell lines are grown upon innovative biologically inert 3D electrospun poly(�-caprolactone) (PCL) microfiber scaffolds rather than upon traditional plastic monolayers. The present proposal seeks to elucidate the precise mechanisms by which cell-cell and cell-ECM interactions stimulate an activated IGF- 1R/mTOR signaling state within this engineered 3D bone tumor microenvironment, as those interactions are critically important in initiating and maintaining ES. In parallel with determining the influence of those parameters under static cell culture, 3D scaffolds and culture conditions will be adapted to better emulate the native bone microenvironment: (a) varied flow perfusion rates will be used to assess the effect of shear stress upon cell retention while facilitating uniform distribution of Ewing cells within the scaffold, (b) PCL scaffolds will incorporate IGF-1 to mimic the high concentration of IGF-1 naturally released as tumors invade surrounding bone, (c) the effect of an osteogenic ECM upon the IGF-1R/mTOR signaling cascade will be determined using decellularized scaffolds upon which heterotypic mesenchymal stem cells, differentiated toward an osteoblastic lineage, are first grown, and (d) ES cells will be co-cultured with endothelial cells (EC) to determine how heterotypic cells interact within a fabricated 3D bone tumor model to elicit viable ES tumors. Finally, to determine the mechanism(s) by which Ewing sarcoma evades sensitivity to combined mTOR/IGF-1R targeted therapy, freshly-derived tumor specimens (obtained from Ewing sarcoma patients treated with Medi- 573/everolimus in an IRB-approved clinical trial), will be grown in primary culture within 3D scaffolds and compared to 2D culture and patient-derived tumor explants (PDTX) by proteomic analysis of the IGF- 1R/mTOR pathway and putative resistance mechanisms. This novel approach of studying Ewing sarcoma within an ex vivo preclinical model of the bone microenvironment presents tremendous potential for understanding chemotherapeutic efficacy and for determining resistance mechanisms to biologically targeted therapies.