Multiplexed protein profiling reveals spatial subcellular signaling networks
Shuangyi Cai,
Thomas Hu,
Mythreye Venkatesan,
Mayar Allam,
Frank Schneider,
Suresh S. Ramalingam,
Shi-Yong Sun,
Ahmet F. Coskun
Affiliations
Shuangyi Cai
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA
Thomas Hu
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
Mythreye Venkatesan
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; Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, GA 30332, USA
Mayar Allam
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA
Frank Schneider
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, GA 30322, USA; Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
Suresh S. Ramalingam
Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA; Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA
Shi-Yong Sun
Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA; Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA
Ahmet F. Coskun
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA; Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, GA 30332, USA; Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA; Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA; Corresponding author
Summary: 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.
