Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Summary

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

Abstract

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.

Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.

Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Introduction

Adult stem cells are responsible for the regeneration of differentiated cell types in a given organ and are predominantly present in a dormant, non-dividing state^1,2. For example, hematopoietic stem cells (HSCs), which maintain the blood, largely remain quiescent, and only a small fraction enters the cell cycle to self-renew or differentiate to generate mature blood components³. Similarly, in cancers, a rare subpopulation of cells called cancer stem cells (CSCs) possess the self-renewal ability and are responsible for the long-term maintenance of the malignancy⁴. Cancer stem cells exist in vivo in a state of quiescence or slow growth, which may enable them to escape the anti-proliferative cancer treatments⁵, evade clearance by the immune system⁶, reduce oxidative stress, and enhance their DNA repair pathways⁷. Even a low number of CSCs left behind after treatment can potentially repopulate the tumor, resulting in a patient's relapse⁸. Accordingly, understanding cellular quiescence holds great promise for the identification of potential vulnerabilities of CSCs and the development of new ways to target them.

Cell proliferation dyes, such as the carboxyfluorescein succinimidyl ester (CFSE) stain and its derivatives, are commonly used to track the frequency of cell divisions⁹. The dye permeates across the cell membrane and, once inside the cell, undergoes activation by intracellular esterases into a fluorescent product. The resultant fluorescent compound is retained inside the cell through the covalent amide bonds formed between the succinimidyl moiety and the amine functional groups of intracellular proteins¹⁰. With each cellular division, the fluorescent compound is divided equally between the two resulting cells, causing a two-fold signal dilution. This dye enables the detection of up to 10 cell divisions through flow cytometry analysis¹¹.

This approach has been previously utilized to enrich CSC populations in vitro by identifying slow-cycling populations of cells with high retention of the dye^11,12. In T-ALL, CFSE has been used to track tumor growth in vivo in patient-derived xenografts in mice. Following cell labeling and three weeks of transplant, flow cytometry analysis showed a rare population of cells that still retained CFSE fluorescence. This population was associated with stemness, treatment resistance, and high similarity to relapse-causing cells in patients¹³. Accordingly, this dye provides a useful tool for the study of leukemia stem cell (LSC) phenotypes in T-ALL.

The aim of the work is to extend the application of the cell proliferation dye to study quiescence in vivo using a zebrafish T-ALL model. In particular, the rag2:Myc-driven zebrafish T-ALL model¹⁴ provides an excellent venue for the study of self-renewal due to the high frequency of LSCs compared to mouse models and human disease¹⁵. In addition, the use of zebrafish allows for large-scale transplantation studies, which can be done at a much lower cost of care and maintenance compared to their mouse counterparts¹⁶. Zebrafish are also excellent for live imaging applications, as fluorescently labeled tumor cells can be readily viewed using a simple fluorescence microscope to estimate the rate of tumor development¹⁶.

In this protocol, we describe the workflow for staining zebrafish T-ALL cells with cell proliferation dye followed by in vivo propagation of stained cells in syngeneic CG1 zebrafish. Upon leukemia development, we describe the sorting of cells that retained the dye and their use for a subsequent limiting dilution transplantation experiment to quantify rates of LSC self-renewal. This protocol can be extended for additional applications, including in vivo drug screening of potential compounds for the targeting of quiescent LSCs. In addition, collected cells can be used for different downstream analyses, such as transcriptomic profiling, proteomics, and metabolomics, offering unique insights into the behavior of quiescent LSCs in T-ALL.

Protocol

In this protocol, we are using GFP-labeled zebrafish T-ALL cells that were generated previously in the CG1 strain and thus can be directly injected into recipient syngeneic CG1 zebrafish15. Briefly, leukemia was generated by DNA microinjection of rag2:Myc and rag2:GFP into single-cell CG1 zebrafish embryos. Animals were monitored for leukemia development starting at 3 weeks post injection, using fluorescence microscopy. GFP-positive leukemia cells were FACS isolated and serially …

Representative Results

We followed the above-described protocol to sort cells that have retained the cell proliferation dye, CT-FR, and used them for a Limiting dilution assay (LDA) to estimate LSC frequency in the CT-FR High and CT-FR Low populations. To set the gating for the flow cytometry experiment, we used a no fluorophore (no color) control in addition to single-color controls (Figure 2A). Those quadrants were used to identify FITC- and PE-positive and -negative populations for the set…

Discussion

LSCs are known to be resistant to conventional, anti-proliferative chemotherapy treatments, and finding targeted therapies against these cells holds great promise in reducing the occurrence of relapse and improving patient prognosis²⁰. Previous research described the use of fluorescent cell proliferation stains to identify a small population of quiescent cells associated with drug resistance and stemness in T-ALL PDX models¹³. In this work, we describe the use of a similar …

Materials

26 G/2” micro-syringe	Hamilton	87930	NA
35 µm filter cap FACS tubes	Falcon	352235	NA
40 µm cell strainer	CELLTREAT	229482	NA
96-well skirted PCR plate	Thermo Fisher Scientific	AB0800	NA
Cell sorter	Sony Biotechnology	SY3200	NA
CellTrace Far Red	Thermo Fisher Scientific	C34564	NA
Conical tubes	VWR	10026-078	NA
DAPI	Thermo Fisher Scientific	62248	NA
DMSO	Sigma-Aldrich	D4818	NA
Dulbecco'sPhosphate-buffered saline (PBS)	Caisson Labs	22110001	NA
Epifluorescence stereo microscope	Nikon	SMZ25	NA
Fetal Bovine Serum (FBS)	Sigma-Aldrich	12306C	NA
Fish system water	N/A	N/A	0.03-0.05% salinity, pH 6.5-8, buffered with sodium bicarbonate
Microcentrifuge tubes	Thermo Fisher Scientific	C2171	NA
MS-222	Pentaire	TRS-1	tricaine mesylate, an anesthetic
Petri dishes	Corning	07-202-011	NA
Razor blades	American Line	66-0089	NA
Trypan Blue	Thermo Fisher Scientific	T10282	NA