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

iScience (Sep 2022)

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

Journal volume & issue: Vol. 25, no. 9
p. 104980

Abstract

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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.

Published in iScience

ISSN: 2589-0042 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science
Website: http://www.cell.com/iscience/home

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