Biochemical state in tissue can be detected through ultrasound signal

Three-dimensional (3D) cell cultures, such as spheroids, are indispensable models for investigating cellular behaviors and responses under conditions that closely resemble in vivo environments. Conventional imaging techniques, including optical microscopy, are often limited by penetration depth, complicating the analysis of structural and biochemical changes within dense 3D systems. This study demonstrates the application of ultrasound imaging for the non-invasive evaluation of internal dynamics in cancer spheroids. Scattering-based acoustic parameters revealed spatial variations in amplitude and brightness density, correlating with cellular proliferation, apoptosis, and necrosis. Amplitude in central regions progressively decreased after Day 3, approaching near-zero by Day 15, reflecting necrotic core formation. Artificial inhibition of myosin contractility significantly influenced these patterns, providing insights into biomechanical contributions to spheroid organization. The findings establish ultrasound imaging as a label-free, high-penetration technique capable of addressing critical challenges in 3D culture analysis, offering new opportunities for studying cellular dynamics in spheroids and organoid models.