Employing High-Resolution Microscopy to Identify Dormant Disseminated Tumor Cells in the Brain

Metastatic dormancy in the brain remains a major clinical problem and a biological challenge to study, as disseminated tumor cells (DTCs) can persist undetectable in a non-proliferative state for years before metastatic relapse. Here, we described an integrated methodological workflow to identify, isolate, and functionally characterize dormant brain metastatic clones taking advantage of high-resolution microscopy including light-sheet and intravital two-photon microscopy. Here, we present a method to generate dormant brain metastatic clones combined with high-resolution imaging for characterization of their dormant state. Briefly, tumor cells are delivered to the brain via intracarotid injection, and light-sheet microscopy is performed for whole-brain visualization of disseminated tumor cells. Individual clones are isolated and screened ex vivo by employing organotypic brain culture, where dormancy-associated phenotypes are assessed by multiphoton imaging. To validate their dormant behavior in vivo, clones exhibiting delayed outgrowth are then reintroduced via intracarotid injection, and brain tissue is analyzed ex vivo via multiphoton imaging. Finally, intracranial intravital imaging enables real-time visualization of dormant tumor cells and their interactions with the brain microenvironment. This pipeline provides a reproducible approach to isolate dormant clones and study brain metastatic dormancy, highlighting the versatility of multiphoton imaging for dynamic analysis of dormant DTCs in the brain.