Mechanical confinement plays an important role in regulating tumor growth and invasion; however, the quantitative, time-resolved, three-dimensional evaluation of confined tumor spheroids remains technically challenging. In this study, we developed a custom-built selective plane illumination microscopy (SPIM)-based monitoring platform for long-term volumetric imaging of tumor spheroids under mechanically confined conditions. This system integrates a culture housing unit and a transparent cuvette-based spheroid culture method optimized for SPIM observation. Colorectal adenocarcinoma-derived cell spheroids were embedded in agarose gels with defined concentrations to modulate the stiffness of the surrounding matrix. Bright-field imaging and viability analyses confirmed sustained spheroid growth without necrotic core formation over a 4-day culture period, demonstrating that the SPIM-based system maintained the physiological culture conditions. Three-dimensional imaging using SPIM enabled a quantitative evaluation of spheroid growth and anisotropic invasion. Volumetric expansion was observed under all confinement conditions. Notably, increasing the matrix stiffness enhanced both the volumetric growth rate and anisotropic invasion, indicating stiffness-dependent directional growth under mechanical confinement. The developed SPIM-based platform has the potential to serve as a practical tool for the time-resolved three-dimensional analysis of tumor spheroid growth and may provide a useful approach for investigating the mechanobiological regulation of tumor progression in confined microenvironments.
Oda et al. (Sat,) studied this question.