基于三维培养模型的肿瘤细胞代谢组AFAI-MS分析新方法研究

The change of cell metabolism is one of the important characteristics of the tumor, and metabolic abnormalities plays an important role in tumorigenesis and development. The development of novel analytical method for accurate and comprehensive characterization of intracellular small molecular metabolites is very critical. In this project, a three-dimensional in vitro culture model will be constructed to simulate in vivo tumor tissue, thus avoiding the infidelity of cellular metabolic information caused by traditional two-dimensional culture mode. Furthermore, high-efficiency and high-precision analysis for cellular metabolome and dynamic metabolic flux will be explored based on self-developed ambient air flow-assisted ionization mass spectrometry (AFAI-MS). An AFAI-MS method for cellular metabolome analysis will be developed and established based on three-dimensional culture model, to realize high-throughput, real-time and rapid acquisition of tumor cellular whole metabolome and dynamic metabolic change under physiological and pathological conditions. Finally, the metabolic response of glioma stem cells under acidic microenvironment will be interrogated by using the established AFAI-MS method, and the upstream metabolism-related enzymes, mRNA and target gene will also be characterized and validated by molecular biology techniques to realize the precise docking and network integration between metabolic phenotype and metabolite function. The success of this project will provide a convenient and high-efficiency cellular metabolomics protocol, and accelerate the development of AFAI-MS toward the more challenging frontiers.

细胞代谢的改变是肿瘤的重要特征之一，细胞代谢异常与肿瘤发生发展密切相关，发展针对细胞内源性代谢物准确全面表征的新方法对了解肿瘤代谢调控的分子机制非常关键。本项目将模拟体内肿瘤组织构建体外肿瘤细胞三维培养模型，避免传统二维培养模式可能导致的细胞代谢信息失真；同时基于自主研发的敞开式空气动力辅助离子化质谱技术（AFAI-MS）探索肿瘤细胞静态代谢组与动态代谢流的高效精准分析，建立基于三维培养模型的肿瘤细胞代谢组AFAI-MS分析新方法，高通量、实时快速获取在生理病理条件下肿瘤细胞代谢组全景式信息及其动态变化规律。应用该方法开展胶质瘤干细胞对酸性微环境的代谢应答研究，并采用分子生物学技术将代谢物上游相关的代谢酶、mRNA及靶基因进行表征，实现代谢表型与代谢物功能的精准对接与网络化整合。本项目的实施将为细胞代谢组学研究提供高效便捷的分析方法，并推动AFAI-MS技术向更具挑战性的前沿领域发展。

细胞代谢的改变是肿瘤的重要特征之一，发展针对细胞代谢物结构与功能活性表征的新方法对于了解肿瘤代谢调控的分子机制非常关键。本项目模拟体内肿瘤组织构建体外肿瘤细胞三维培养模型，同时基于自主研发的敞开式空气动力辅助离子化质谱成像技术（AFADESI-MSI）探索肿瘤细胞静态代谢组与动态代谢流的高效精准分析方法。首先采用超低吸附表面微孔板法构建了重复性好、可连续培养、易于操控的多细胞肿瘤球（MCTS）模型，成功用于胶质瘤、食管癌、肝癌等多种肿瘤细胞MCTS构建，并针对MCTS微小体积样本（直径0.5-1mm）建立了冰冻切片制备操作流程。采用改进的AFADESI-MSI装置核心部件，即内径 20μm 的新型解吸电喷雾探针，提高了解吸离子化过程的稳定性并实现较高空间分辨能力的成像，实现了氨基酸类、有机胺类、胆碱类、有机酸类、核苷及核苷酸类、脂肪酸类、磷脂类等数十类约1100个内源性代谢物的高灵敏、高覆盖成像分析。进一步结合稳定同位素示踪技术，建立了13C6-葡萄糖标记的糖酵解、TCA循环与核苷酸从头合成的靶向代谢流分析方法，探究了乏氧微环境下胶质瘤MCTS的代谢组特征及变化规律。开展了胶质瘤干细胞对酸性微环境的代谢应答研究，整合转录组学数据与生物学验证结果，阐明了酸性微环境下胶质瘤干细胞可能的代谢重编程机制，发现H6PD有望成为改善胶质瘤患者预后的潜在临床靶点。开发了基于多细胞肿瘤球的空间分辨药物代谢组学新方法，应用于研究胺碘酮、紫杉醇药物在3D细胞球中的渗透、代谢和药物效应。此外，整合代谢组学、脂质组学的LC-MS/MS新方法，以及化学标记质谱成像新方法等，建立完善了细胞分析平台，实现微小体积MCTS中代谢物组的多维度、多指标量化表征，为生理病理机制及药物的药效/毒理作用机制研究提供新方法和新思路。项目实施期间，发表论文7篇，包括中科院1区Top期刊论文4篇， Anal Chem 1篇，Anal Chim Acta 1篇，Cell Death & Disease 2篇等；申请发明专利2项；培养博士研究生2名，硕士生6名，研究生获奖4人次。完成项目预期目标。