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

Summary

This protocol describes methods for generating compound transgenic zebrafish, imaging immune cells associated with tumors in larval and adult zebrafish, and quantifying immune cell frequency within the tumor microenvironment.

Abstract

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.

Introduction

The tumor microenvironment (TME) is an ecosystem composed of tumor, immune, fibroblast, and vascular cells¹. The TME plays an essential role in cancer progression, therapeutic response, and anti-tumor immunity. Within the TME, immune cells are key regulators of tumor fate, either participating in anti-tumor responses or inducing immunosuppression². Understanding the recruitment, distribution, and function of immune cells within the TME is critical for elucidating the mechanisms by which tumors evade immune surveillance. However, conventional approaches to studying the TME, such as endpoint histology, flow cytometry, and ....

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University Chobanian & Avedisian School of Medicine under protocol PROTO202100001. The overall workflow for generation of compound transgenic zebrafish, larval imaging, adult tissue preparation, and image analysis is summarized in Figure 1.

Results

The overall experimental workflow is shown in Figure 1. Adult Tg(cd4-1:mCherry) zebrafish were crossed with Tg(dβh:MYCN;dβh:EGFP) zebrafish to generate compound transgenic offspring in which CD4⁺ cells and dβh-expressing cells were differentially labeled with mCherry and EGFP fluorescence, respectively. Embryos were screened for the appropriate fluorescent markers and subsequently analyzed by either live confocal imaging during larval developmen.......

Discussion

The protocol described here utilizes zebrafish as an in vivo model to visualize and quantify immune cells within the TME. Several factors are critical for successful implementation of this approach. First, appropriate selection and validation of compound transgenic zebrafish lines are essential. Larvae should be screened for the expected fluorescent expression patterns and, if required, confirmed by genotyping. Immune-reporter and oncogene expression should be verified prior to experimentation to ensure accurate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 µL Gel Loading Tip	MilliporeSigma	1022-0600	Used for larval manipulation
Artificial Plants	The Hidden Reef	62874200000000000	Optional breeding enrichment
Bleach	Fisher Scientific	50371500	Used for embryo decontamination
Cell Counter Plugin	National Institutes of Health (NIH)	N/A	Fiji plugin used for cell quantification
Computer and Monitor	HP	2TB68A8#ABA	Used for image acquisition and analysis
Confocal Microscope	Zeiss	LSM 710	Used for larval and adult imaging
Cryomold, Plastic	Epredia	22-050-160	Used for OCT embedding
Cryostat	Thermo Fisher Scientific	95-664-0EC70	Used for cryosectioning
Dry Ice	Fisher Scientific	N/A	Used for sample transport
Fiji Software	National Institutes of Health (NIH)	Version 1.54p	Image analysis software
Fine-Mesh Net	Carolina Biological	651345	Used for embryo collection
Fish Incubator	VWR	35960-056	Maintained at 28.5°C
Forceps	Fisher Scientific	10-316B	Used for sample handling
Glass-Bottom Dish	IBIDI	81218-200	Used for imaging
Low-Melting Agarose	Prolab Supply	9012-36-6	Used for larval and adult mounting
Mating Tank (main tank, divider, grated section)	Carolina Biological	161937	Used for zebrafish breeding
Nutating Rocker	Midsci	R2D-30	Used for fixation during overnight incubation
OCT Compound	Tissue-Tek	4583	Embedding medium for cryosectioning
Paraformaldehyde (PFA)	Sigma Aldrich	158127-500G	Fixative
Petri Dish, Plastic	MilliporeSigma	BR452000	Used for embryo and larval handling
Phosphate-Buffered Saline (PBS)	Gibco	14080-055	Used for washing and fixation procedures
Plastic Transfer Pipette	Fisher Scientific	50-998-100	Used for embryo and larval transfer
Razor Blade	Fisher Scientific	12-640	Used during sample preparation
Conductivity Meter	YSI	EC300ACC-04	Used to verify rotifer water salinity
Rotifers	The Hidden Reef	116151603	Used for larval rearing
Sodium Thiosulfate	Thermo Fisher Scientific	AAA1762936	Used for bleach neutralization
Sucrose	Fisher Scientific	S25590A	Cryoprotection reagent
Tricaine Methanesulfonate	Western Chemical	NC0872873	Anesthetic; sold by Fisher Scientific
Tweezers	Fisher Scientific	12-000-122	Used for sample handling
Upright Fluorescence Microscope	Leica	M165	Used for fluorescence screening