Unraveling mitotic protein networks by 3D multiplexed epitope drug screening

• Published in: Molecular systems biology 2018-08, Vol.14 (8), p.e8238-n/a
• Main Authors: Maier, Lorenz J, Kallenberger, Stefan M, Jechow, Katharina, Waschow, Marcel, Eils, Roland, Conrad, Christian
• Format: Article
• Language: English
• Subjects: Affinity; Artificial intelligence; Automation; Cancer; Cell culture; Cell Line, Tumor; cell profiling; Chromatin; Coordinates; Drug Evaluation, Preclinical - methods; Drug screening; Drug Screening Assays, Antitumor; Drugs; EMBO06; EMBO17; EMBO22; Epitopes - genetics; Epitopes - immunology; Fragility; Gene Expression Regulation - drug effects; High-Throughput Screening Assays; Humans; Immunofluorescence; Iterative methods; Learning algorithms; Localization; Machine learning; Mass spectrometry; Mass spectroscopy; Mathematical models; Method; Methods; Mitosis; Mitosis - genetics; mitosis modeling; multiplexed immunostaining; Multiplexing; Networks; Peptide mapping; Protein expression; Protein interaction; Proteins; Proteins - drug effects; Proteins - genetics; protein–protein interactions; Screening; Spherical coordinates; Spheroids; Staining; Technology; Three dimensional imaging; Three dimensional models; Workflow
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.20188238

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 comprises iterative antibody staining, high‐content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model‐based prediction of spatially resolved affinities of proteins. As a proof‐of‐concept, we mapped twelve epitopes in 3D‐cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and 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 culture combined with our automated high‐content assay will inspire future functional protein expression and drug assays. Synopsis 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening combines iterative antibody staining, high‐content 3D imaging, and classification by machine learning. As a proof of concept, it is applied to quantitatively study mitotic stages. 3D SPECS allows to query complex protein networks using microscopic localizations. Twelve epitopes are mapped and the network effects of 12 mitotic cancer drugs are analyzed in 3D cultured spheroids. Modelling affinities explores new mitotic protein‐protein interactions. Mitotic protein networks appear highly connected. Graphical Abstract 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening combines iterative antibody staining, high‐content 3D imaging, and classification by machine learning. As a proof of concept, it is applied to quantitatively study mitotic stages.