High-content morphological profiling by Cell Painting in 3D spheroids.

Single-cell morphological profiling of 3D spheroids with Cell Painting reveals drug effects that are indistinguishable in 2D cultures, including divergence for 5-fluorouracil and olaparib.

• Why it matters: Understanding drug responses in physiologically relevant 3D models is crucial for better predicting clinical outcomes, but imaging challenges have limited their use. This work addresses the need for scalable, high-resolution methods to analyze 3D spheroids at the single-cell level.
• What they did: The authors developed a scalable workflow combining tissue clearing and a focusing routine to adapt Cell Painting for 3D spheroids, enabling single-cell imaging within intact spheroids on a standard confocal microscope.
• The result: Profiling 52 drugs demonstrated that single-cell fingerprints clustered by biological pathway and more robustly resolved drug effects than flattened spheroid views, with divergence observed for two drugs in 3D that was corroborated by additional assays.