Abstract
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.
Highlights
Using 3D SPECS, we first studied sub-cellular differences in protein localizations in the tumorigenic and nontumorigenic 3D cell lines distinguishing between the cell cycle states metaphase and segregation
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
Iterative antibody labeling overcomes the spectral limit of total number of fluorescent antibodies that can be applied simultaneously to individual cells[1,3]. We successfully extend this technique of chemically bleached fluorescently labeled antibodies, to 3D cell cultured spheroids in Matrigel[9] combined with drug treatment
Summary
Using 3D SPECS, we first studied sub-cellular differences in protein localizations in the tumorigenic and nontumorigenic 3D cell lines distinguishing between the cell cycle states metaphase and segregation. We analyzed the effects of twelve targeted inhibitors on mitotic proteins of dividing MCF10A and MCF10CA cells (Fig. 3a,b), on protein concentrations and preferred localizations.
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