Abstract
The advent of volume electron microscopy (vEM) has provided unprecedented insights into cellular and subcellular organization, revolutionizing our understanding of cancer biology. This study presents a previously unexplored comparative analysis of the ultrastructural disparities between cancer cells cultured as monolayers and tumorspheres. By integrating a robust workflow that incorporates high-pressure freezing followed by freeze substitution (HPF/FS), serial block face scanning electron microscopy (SBF-SEM), manual and deep learning-based segmentation, and statistical analysis, we have successfully generated three-dimensional (3D) reconstructions of monolayer and tumorsphere cells, including their subcellular organelles. Our findings reveal a significant degree of variation in cellular morphology in tumorspheres. We observed the increased prevalence of nuclear envelope invaginations in tumorsphere cells compared to monolayers. Furthermore, we detected a diverse range of mitochondrial morphologies exclusively in tumorsphere cells, as well as intricate cellular interconnectivity within the tumorsphere architecture. These remarkable ultrastructural differences emphasize the use of tumorspheres as a superior model for cancer research due to their relevance to in vivo conditions. Our results strongly advocate for the utilization of tumorsphere cells in cancer research studies, enhancing the precision and relevance of experimental outcomes, and ultimately accelerating therapeutic advancements.