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Ringers C, Holmberg D, Larsson A, Jung B, Jarvius M, Luchniak A, Rosén D, Johansson M, Georgiev P, Seashore-Ludlow B, Visnes T, Flobak Å, Carreras-Puigvert J, Spjuth O
Nat Commun 17 (1) - [2026-10-21; online 2026-10-21]
Cell Painting is a popular high-content imaging assay that captures the shape and internal organization of 2D cell cultures at scale. While 3D cell cultures are more physiologically relevant models - especially for studying drug perturbations - they remain challenging to image because light scatters before it reaches the cultures' interior. Here, we introduce a scalable method to adapt Cell Painting for 3D spheroids. We show that tissue-clearing, a technique that makes spheroids transparent, combined with a focusing routine that locates spheroids on the fly, enables Cell Painting at single-cell level inside intact spheroids. By profiling 52 clinically relevant drugs, we show that single-cell fingerprints clustered by biological pathway and resolved drug effects more robustly than those of flattened spheroid-wide views. Notably, the effect of two drugs, 5-fluorouracil and olaparib, nearly indistinguishable in 2D monolayers, diverged in spheroids. This finding was then corroborated by DNA-damage, cell-cycle, and gene-expression assays. The workflow runs on a spinning-disk confocal microscope in a standard 384-well plate format, making single-cell 3D profiling accessible to laboratories already performing Cell Painting.
Chemical Biology Consortium Sweden [Technology development]
PubMed 42907469
DOI 10.1038/s41467-026-78596-5
Crossref 10.1038/s41467-026-78596-5
pii: 10.1038/s41467-026-78596-5