Bladder cancer remains one of the most prevalent causes of cancer-related mortality worldwide. Mumio, a naturally occurring substance with a long-standing history of use in traditional medicine, has demonstrated therapeutic potential, including anticancer properties. This study aimed to evaluate the effects of a pharmaceutical formulation of Mumio on the growth of normal (SV-HUC-1) and cancerous (T24, 5637) bladder cell lines in 3D culture.

Spheroids were generated using ultra-low attachment 96-well plates. Three concentrations of Mumio (LC₁₀, LC₅₀, LC₉₀), previously determined in 2D culture, were applied. Cells were incubated for 24, 48, and 72 h. Cell viability and caspase activity in 3D culture were assessed via luminescence-based assays. Hematoxylin–eosin (HE) and Ki-67 staining were performed to evaluate spheroid morphology and proliferation. Gene expression analysis was conducted to investigate molecular mechanisms.

Mumio exhibited dose-dependent cytotoxicity in 3D culture, with T24 cells showing the highest sensitivity compared to the control and the second bladder cancer cell line (5637) after all incubation times. Concentrations effective in 2D culture were less potent in 3D conditions. Proliferation rates decreased with increasing Mumio concentration and prolonged exposure. Molecular analysis revealed G1 phase cell cycle arrest and reduced DNA synthesis.

Mumio has potential in supportive bladder cancer treatment; however, its effectiveness, as observed in 2D culture, exhibited diminished efficacy when applied to 3D cultures. Further investigations are necessary, including the use of organoids or animal models, to confirm these properties.