Cai Shuangyi, Hu Thomas, Venkatesan Mythreye, Allam Mayar, Schneider Frank, Ramalingam Suresh S, Sun Shi-Yong, Coskun Ahmet F
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
iScience. 2022 Aug 18;25(9):104980. doi: 10.1016/j.isci.2022.104980. eCollection 2022 Sep 16.
Protein-protein interaction networks are altered in multi-gene dysregulations in many disorders. Image-based protein multiplexing sheds light on signaling pathways to dissect cell-to-cell heterogeneity, previously masked by the bulk assays. Herein, we present a rapid multiplexed immunofluorescence (RapMIF) method measuring up to 25-plex spatial protein maps from cultures and tissues at subcellular resolution, providing combinatorial 272 pairwise and 1,360 tri-protein signaling states across 33 multiplexed pixel-level clusters. The RapMIF pipeline automated staining, bleaching, and imaging of the biospecimens in a single platform. RapMIF showed that WNT/β-catenin signaling upregulated upon the inhibition of the AKT/mTOR pathway. Subcellular protein images demonstrated translocation patterns, spatial receptor discontinuity, and subcellular signaling clusters in single cells. Signaling networks exhibited spatial redistribution of signaling proteins in drug-responsive cultures. Machine learning analysis predicted the phosphorylated β-catenin expression from interconnected signaling protein images. RapMIF is an ideal signaling discovery approach for precision therapy design.
蛋白质-蛋白质相互作用网络在许多疾病的多基因失调中会发生改变。基于图像的蛋白质多重分析揭示了信号通路，以剖析细胞间的异质性，而这种异质性在之前的整体分析中被掩盖了。在此，我们提出了一种快速多重免疫荧光（RapMIF）方法，可在亚细胞分辨率下从培养物和组织中测量多达25重的空间蛋白质图谱，在33个多重像素级簇中提供组合的272种两两组合和1360种三蛋白信号状态。RapMIF流程在单个平台上实现了生物样本的自动染色、漂白和成像。RapMIF显示，在抑制AKT/mTOR通路后，WNT/β-连环蛋白信号上调。亚细胞蛋白质图像展示了单细胞中的易位模式、空间受体不连续性和亚细胞信号簇。信号网络在药物反应性培养物中呈现出信号蛋白的空间重新分布。机器学习分析从相互连接的信号蛋白图像中预测了磷酸化β-连环蛋白的表达。RapMIF是一种用于精准治疗设计的理想信号发现方法。
