BEHAV3D Tumor Profiler to map heterogeneous cancer cell behavior in the tumor microenvironment

Intravital microscopy (IVM) enables live imaging of animals at single-cell level, offering essential insights into cancer progression. This technique allows for the observation of single-cell behaviors within their natural 3D tissue environments, shedding light on how genetic and microenvironmental changes influence the complex dynamics of tumors. The complexity of data generated by IVM often surpasses the capabilities of conventional analyses accessible to biomedical scientists, thereby neglecting single-cell heterogeneity and limiting the exploration of microenvironmental influences on cellular behavior without bias. To address this challenge, here we introduce BEHAV3D Tumor Profiler (BEHAV3D-TP), a user-friendly computational framework designed for the comprehensive analysis of single tumor cell behaviors and their interactions with the tumor microenvironment (TME). BEHAV3D-TP facilitates unbiased profiling of cancer cell dynamics without requiring advanced computational expertise. Here, we apply BEHAV3D-TP to study diffuse midline glioma (DMG), a highly aggressive pediatric brain tumor characterized by invasive growth. Our analysis reveals that distinct migratory behaviors of DMG cells correlate with specific TME components such as tumor-associated macrophages and vasculature. This approach, initially aimed at uncovering tumor invasive patterns and their interactions with the TME, holds promise for understanding additional cancer cell behaviors like intravasation and metastasis. BEHAV3D-TP represents a significant advancement in democratizing the analysis of heterogeneous cancer cell behaviors and their TME interactions, providing accessible computational insights into tumor dynamics.