Comprehensive Approach to Heterogenous Tumor Cell Dynamics Analysis for Intravital Microscopy

Intravital microscopy (IVM) enables observation of single-cell behaviors in live animal models, allowing detailed analysis of cancer cell dynamics within native 3D environments. However, comprehensively analyzing the extensive and complex data generated by IVM requires robust tools that exceed conventional approaches. This chapter presents a protocol for profiling heterogeneous cancer cell behavior within IVM movies, facilitating the study of cellular movement patterns and their associations with microenvironmental cues. The protocol allows researchers to systematically quantify single-cell (morpho)dynamics, providing unbiased analysis of cellular heterogeneity without requiring advanced computational expertise. The framework is adaptable to various tumor and non-tumor models, supporting insights into processes such as invasion, migration, and spatial relationships with distinct cell types, offering an accessible approach to studying heterogeneous cell behavior within its microenvironment.