Visualizing extracellular vesicles in cancer: from biogenesis to theranostic applications.

Within the complex landscape of the tumor microenvironment(TME), extracellular vesicles(EVs) function as sophisticated nanoscale signaling hubs that govern the intercellular crosstalk driving metastatic progression and immune modulation. Despite their burgeoning potential as next-generation biomarkers and biocompatible nanocarriers, the clinical translation of EVs is currently impeded by an incomplete elucidation of their complex spatiotemporal kinetics in vivo. This review provides a critical synthesis of the EV pipeline, from microenvironment-modulated biogenesis to advanced theranostic applications. We begin by analyzing how specific microenvironmental cues, such as hypoxia, acidosis, and radiation, modulate the cargo and surface markers of EVs to reveal new opportunities for bioengineering. Subsequently, we critically evaluate current isolation strategies and, more importantly, label-based and label-free imaging modalities, comparing their resolution and sensitivity for tracking EV biodistribution in real-time. Finally, we discuss the integration of these imaging technologies with therapeutic strategies, highlighting the transition of EVs from biological entities to engineered nanomedicines for liquid biopsy and targeted delivery. By identifying current technical bottlenecks in quantification and off-target labeling, we propose an integrated roadmap to accelerate the clinical translation of EV-based cancer nanotheranostics.