A Novel Scale Biopsy Method to Collect Zebrafish Malignant and Non-Malignant Lymphocytes

Gilseung Park; Iman Owens; Jose-Juan Macias; Megan Malone-Perez; Clay A. Foster; J. Kimble Frazer

doi:10.3791/72153

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Method Article

A Novel Scale Biopsy Method to Collect Zebrafish Malignant and Non-Malignant Lymphocytes

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DOI:

10.3791/72153

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August 7th, 2026

Gilseung Park¹ , Iman Owens² , Jose-Juan Macias³ , Megan Malone-Perez² , Clay A. Foster² , J. Kimble Frazer¹^,²^,³

¹Department of Cell Biology, University of Oklahoma Health Sciences Center, ²Division of Pediatric Hematology-Oncology, Department of Pediatrics, Stephenson Cancer Center, ³Department of Microbiology & Immunology, University of Oklahoma Health Sciences Center

Summary

A novel scale biopsy method allows the collection of acute lymphoblastic leukemia cells from living zebrafish. Results show abundant lymphocytes in wild-type (WT) fish scales, and dramatically increased lymphocytes in scales from fish with ALL (acute lymphoblastic leukemia). This technique allows minimally invasive study of epidermal normal and malignant lymphocytes.

Abstract

Zebrafish (Danio rerio) are a powerful model for studying lymphopoiesis and lymphoid cancers, since zebrafish and humans share similar adaptive immune systems, including B and T cells. Several D. rerio T-cell acute lymphoblastic leukemia (T-ALL) models are described, including fish that express lymphoblast-specific human MYC (hMYC). We reported that rag2:hMYC fish also develop B-cell acute lymphoblastic leukemia (B-ALL), making them useful to study both ALL types. A limitation of zebrafish ALL models is that no non-lethal methods exist for serial sampling of ALL. To address this, we pioneered a novel ‘scale biopsy’ method to collect ALL cells from live zebrafish. However, knowledge about which lymphocytes reside in normal zebrafish scales is scarce. Thus, to define normal epidermal lymphocyte identities, we performed scale biopsies on lymphocyte-labeled transgenic lines lacking rag2:hMYC. We used conventional and confocal microscopy to analyze scales, revealing abundant epidermal lymphocytes. We also analyzed scales from fish with T- and B-ALL, which demonstrated markedly increased lymphocytes in confluent sheets of ALL cells in many scales. We next used fluorescence-activated cell sorting (FACS) to purify non-malignant and ALL lymphocytes from scales for ex vivo studies. Flow cytometric and expression analyses of ALL cells from scales or other tissues of the same fish showed that scale ALL cell gene expression resembled that of ALL cells elsewhere. We also quantified the number of normal T, B, T-ALL, and B-ALL cells that could be purified per scale. Overall, scale biopsy provides a non-lethal, minimally invasive technique to study zebrafish epidermal lymphocytes, enabling ex vivo studies, including analyses of malignant samples.

Introduction

Mammalian skin’s barrier function and roles in immunity are established¹^,²^,³. Mammalian lymphocytes contribute to cutaneous immunity, with cytotoxic T cells residing in the epidermis, and B, T, and natural killer (NK) cells present in the dermis¹^,³^,⁴^,⁵. Zebrafish have these lymphocytes, with surface immunoglobulins, T cell receptors, major histocompatibility complex (MHC) receptors, cytokines and their receptors, and other immune molecules, th....