Wang, Z.; Tian, L.; Li, B.

Understanding early tumor formation offers critical insights for cancer prevention at the earliest stages. However, most in vivo studies focus on later stages, providing limited information on initial tumorigenesis. Hypoxia, a common physiological condition, is a primary microenvironmental factor in cancer. While molecular studies indicate significant yet sometimes conflicting roles of hypoxia in cancer progression, experimental data on hypoxia\'s impact on tumor formation from a single cell, especially regarding cellular dynamics, remain scarce. To address this gap, we developed an integrated platform for live-cell imaging under hypoxic conditions, enabling, for the first time, observation of 3D tumor spheroid development from a single cell. Our results show that tumorigenesis is markedly accelerated under hypoxia. Beyond linear growth, we demonstrate that directional movement enhances fusion events, leading to larger spheroids that, in turn, stimulate further proliferation - a positive feedback loop between spheroid dynamics and growth. Actin polymerization and cell-cell adhesion are optimized to promote spheroid fusion. Additionally, hypoxia induces cells with outreaching F-actin co-localized with smaller nuclei to bridge gaps between spheroids, facilitating fusion, while the appearance of tiny nuclei suggests genetic instability. These findings provide direct insights into hypoxia\'s role in early tumorigenesis, highlighting the potential of modulating dynamic, mechanical, and physiological factors for cancer prevention.