Augmented Drug Resistance of Osteosarcoma Cells within Decalcified Bone Matrix Scaffold: the role of Glutamine Metabolism

Due to the lack of a precise in vitro model that can mimic the nature microenvironment in osteosarcoma, the understanding of its resistance to chemical drugs remains limited. Here, we report a novel three-dimensional model of osteosarcoma constructed by seeding tumor cells (MG-63 and MNNG/HOS Cl #5) within in demineralized bone matrix scaffolds. Demineralized bone matrix scaffolds retain the original components of the natural bone matrix (hydroxyapatite and collagen type I), and possess good biocompatibility allowing osteosarcoma cells to proliferate and aggregate into clusters within the pores. Growing within the scaffold conferred elevated resistance to doxorubicin on MG-63 and MNNG/HOS Cl #5 cell lines as compared with two-dimensional cultures. Transcriptomic analysis showed an increased enrichment for drug resistance genes along with enhanced glutamine metabolism in osteosarcoma cells in demineralized bone matrix scaffolds. Inhibition of glutamine metabolism resulted a decrease in drug resistance of osteosarcoma, which could be restored by α-ketoglutarate supplementation. Overall, our study suggests that microenvironmental cues in demineralized bone matrix scaffolds can enhance osteosarcoma drug responses and that targeting glutamine metabolism may be a strategy for treating osteosarcoma drug resistance. To investigate the differences between OS cells cultured in 3D and 2D environment at whole transcriptional level, we performed transcriptome RNA sequencing (RNA-seq) of MING/HOS(HOS) cells at day7 after cell seeding.

由于缺乏能够模拟骨肉瘤天然微环境的精准体外模型，目前学界对其化疗药物耐药性的认知仍较为有限。本研究构建了一种新型骨肉瘤三维模型：将肿瘤细胞MG-63与MNNG/HOS Cl #5接种于脱钙骨基质支架（demineralized bone matrix scaffolds）中。该脱钙骨基质支架保留了天然骨基质的核心组分——羟基磷灰石（hydroxyapatite）与I型胶原（collagen type I），且具备优异的生物相容性，可支持骨肉瘤细胞在支架孔隙内增殖并聚集成团簇。与二维培养体系相比，在支架内生长的MG-63与MNNG/HOS Cl #5细胞株对多柔比星（doxorubicin）的耐药性显著升高。转录组分析（transcriptomic analysis）结果显示，接种于脱钙骨基质支架的骨肉瘤细胞中，耐药基因的富集程度显著上调，同时谷氨酰胺代谢（glutamine metabolism）活性增强。抑制谷氨酰胺代谢可降低骨肉瘤的耐药性，且该效应可通过补充α-酮戊二酸（α-ketoglutarate）得以逆转。综上，本研究表明脱钙骨基质支架所提供的微环境信号可增强骨肉瘤对化疗药物的响应，而靶向谷氨酰胺代谢或可成为治疗骨肉瘤耐药性的潜在策略。为在全转录组水平探究三维（3D）与二维（2D）培养环境下骨肉瘤细胞的基因表达差异，本研究对接种后第7天的MNNG/HOS细胞（简称HOS）进行了RNA测序（RNA-seq）。