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

doi:10.1016/j.isci.2022.104980

Multiplexed protein profiling reveals spatial subcellular signaling networks

iScience. 2022 Aug 18;25(9):104980. doi: 10.1016/j.isci.2022.104980. eCollection 2022 Sep 16.

Authors

Shuangyi Cai¹, Thomas Hu^{1

2}, Mythreye Venkatesan^{1

2

3}, Mayar Allam¹, Frank Schneider^{4

5}, Suresh S Ramalingam^{5

6}, Shi-Yong Sun^{5

6}, Ahmet F Coskun^{1

3

5

7}

Affiliations

¹ Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
² School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
³ Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, GA 30332, USA.
⁴ Department of Pathology and Laboratory Medicine, Emory University School of Medicine, GA 30322, USA.
⁵ Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA.
⁶ Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA.
⁷ Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA.

Abstract

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.

Keywords: Biological sciences; Biological sciences research methodologies; Biology experimental methods; Biotechnology.

Grants and funding

P50 CA217691/CA/NCI NIH HHS/United States