Multiplex cytological profiling assay to measure diverse cellular states

Abstract

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

Figure 1. The cell-painting assay applied to U2OS cells.

(A) Cells labeled with Hoechst 33342 (nuclei, blue), concanavalin A (ER), SYTO 14 (nucleoli), phalloidin (actin), WGA (Golgi), MitoTracker Deep Red (mitochondria). Scale bars 50 µm. (B) Ten diverse phenotypes in compound-treated U2OS cells: toroid nuclei (amperozide); giant, multinucleated cells (fenbendazole); abundant ER (tetrandrine); redistribution of ER to one side of nucleus (NPPB); reduced nucleolar size (rapamycin); large, flat nucleoli (etoposide); bright, abundant Golgi staining (Ca-074-Me); actin breaks (latrunculin B); extensive mitochondrial fission (Beta-dihydrorotenone); and redistribution of mitochondria (berberine chloride). Scale bars 50 μm.

Figure 2. Hierarchical clustering of image-based profiles.

Details are shown for three of the clusters that were highly enriched for annotation terms. These enriched clusters contain compounds with similar mechanisms of action, some with similar and some with distinct chemical structure. The presence of these enriched clusters indicates that the assay can identify subtle, physiologically relevant effects of compounds on cultured cells. U2OS cells labeled for nuclei (blue), ER (green), nucleoli (grey), actin and Golgi (yellow), and mitochondria (red). Scale bars 50 µm.