Imaging and Quantification of Immune Cells in the Tumor Microenvironment Using a Zebrafish Model of Neuroblastoma

The recruitment of immune cells to the tumor microenvironment (TME) is an important component of immune regulation and tumor progression. Studying the spatial and temporal relationships between immune and tumor cells in vivo can provide insight into interactions within the TME. However, visualizing and quantifying these interactions in intact organisms remains challenging using conventional imaging and histological approaches. Zebrafish provide a valuable model for cancer research owing to their genetic tractability, rapid development, and optical transparency during early developmental stages, enabling visualization of fluorescently labeled cells in vivo. Here, we present a protocol to generate, identify, image, and quantify immune and tumor cells that are differentially labeled with fluorescent markers in a zebrafish model of neuroblastoma. The protocol includes breeding and screening of compound transgenic zebrafish, live imaging of larvae, preparation and imaging of adult tumor-bearing fish, and quantification of immune cells surrounding and within tumor regions. Using transgenic reporter lines, CD4⁺ immune cells and MYCN-driven neuroblastoma cells can be visualized and analyzed at different developmental stages. The workflow enables assessment of immune cell localization relative to tumors in both larval and adult fish and can be used to compare immune cell abundance across disease stages. This protocol provides a reproducible approach for imaging and quantifying immune cells within the TME in vivo using zebrafish and may facilitate studies of immune-tumor interactions in other cancer models.