A workflow for quantifying cell quiescence in 3D spheroids.

Quiescence is a reversible, tightly regulated state in which cells exit the cell cycle while retaining the potential to re-enter proliferation upon an appropriate stimulus. In solid tumors, quiescent cancer cells are thought to contribute to therapy resistance and disease relapse, especially in poorly vascularized regions. Detecting these dormant cells could be key to predicting therapeutic outcomes and personalizing treatment. To assess the metabolic state of individual cells, we developed a fluorescence-based method using acridine orange (AO) staining, paired with algorithm-driven quantification. By measuring AO-RNA and AO-DNA signals, we distinguished metabolic differences in 3D non-small cell lung cancer spheroids. The RNA/DNA staining ratio correlated with transcriptional activity, validating this approach as a reliable readout of cell state. This method provides a powerful tool to uncover hidden pockets of drug-resistant, quiescent cancer cells in 3D culture.