Comparative Evaluation of Volume Estimation Methods and Growth of Osteosarcoma Tumor Spheroids

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults, characterized by marked biological heterogeneity and aggressive behavior. The early development of metastases and high mortality rates highlight the need for physiologically relevant models that accurately recapitulate complexity and tumor growth dynamics. Tumor spheroids represent a valuable in vitro model of the early avascular phase of solid tumors, enabling the study of diffusion-limited proliferation, hypoxia, and growth deceleration. Accurate spheroid volume estimation is essential for mathematical modeling of growth kinetics. This study evaluated whether a simple standard axial volume estimation method (SAVE) provides volume estimates comparable to those obtained using the Z-stack-based volume estimation method (ZSVE). Spheroids were generated from a patient-derived, stem cell-enriched population of chondroblastic OS cells and monitored longitudinally to establish growth curves. Representative spheroids were analyzed using both methods while accounting for technical variability in image acquisition and analysis. Spheroids exhibited a characteristic sigmoidal growth pattern, with progressive growth deceleration as size increased. The two methods showed comparable volume estimates, indicating that SAVE provides a robust and reliable approach for longitudinal spheroid volume quantification and mathematical modeling of clonal osteosarcoma spheroid growth.