Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow encompasses iterative antibody staining of proteins, high-content imaging, and machine learning based classification of mitotic states. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, the basis used for model-based prediction of spatially resolved affinities of various mitotic proteins. As a proof-of-concept, we mapped twelve epitopes in 3D cultured epithelial breast spheroids and investigated the network effects of mitotic cancer drugs with known limited success in clinical trials. Our approach reveals novel insights into spindle fragility and global chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell cultured models combined with our automized high content assay will inspire future functional protein expression and drug assays.

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening