Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Summary

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Abstract

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Introduction

The side population assay capitalizes on the enhanced ability of certain cells within a tissue to efflux the DNA binding dye Hoechst 33342 due to high levels of the ATP binding cassette (ABC) transporter proteins on the cell membrane. The cells that efflux the Hoechst 33342 dye can be identified using dual wavelength flow cytometric analysis after the dye is excited by a UV laser. This assay was first used to identify murine hematopoietic stem cells (HSCs)¹, but it has since been used to identify stem/progenitor cell populations in many tissues and cancers (reviewed in reference 2). However, not all populations of cells have a side population, and not all side populations are enriched for stem/progenitor cells.

The zebrafish is a powerful vertebrate genetic model system for studying human cancer^3,4, with a number of advantages over traditional murine models of cancer. Zebrafish embryos are externally fertilized and are optically clear, facilitating transgenesis and the in vivo observation of pathologic processes, including cancer initiation and progression. To date, the side population assay to detect potential stem or progenitor cells has only been applied to the kidney marrow in zebrafish to identify HSCs, and not to any zebrafish cancer models^5,6.

The zebrafish model of T-cell acute lymphoblastic leukemia (T-ALL) is morphologically and genetically similar to human T-ALL^7,8,11. T-ALL is an aggressive malignancy that, in humans, accounts for 10-15% of pediatric and 25% of adult ALL cases⁹. While the treatment of T-ALL has improved, relapse is still common and is associated with a poor prognosis. T-ALL tumors are heterogeneous and contain many different tumor cell subpopulations, including leukemia initiating cells (LICs). LICs are defined by their ability to regrow the entire tumor from a single cell, and the frequency of LICs within a tumor cell population can be calculated by transplanting varying cell doses into recipients via a limiting dilution transplantation assay (LDA). While LDA experiments have been performed in zebrafish to calculate the frequency of LICs^8,10,11, this determination is made in hindsight and does not allow for the prospective isolation of LICs. Therefore, a method to prospectively isolate a population enriched for cancer stem cell activity is lacking. Identifying and isolating side population cells from zebrafish T-ALLs is the first step towards addressing this deficiency.

The protocol presented here describes how to efficiently generate T-ALL tumors in zebrafish utilizing tol2-mediated transgenesis, harvest the T-ALL tumors cells, and stain them with Hoechst 33342 to identify the side population of cells. Future in vivo experiments with zebrafish T-ALL could include whether the side population cells are enriched for LICs or have other stem- or progenitor-like properties.

Protocol

All procedures with zebrafish have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago. The University of Chicago is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1. Generating and Isolating Fluorescently Labeled T-ALL Cells in Zebrafish

1. Microinjection into syngeneic zebrafish embryos
   1. Using commercially available kits, isolate circular rag2:c-myc and rag2:green fluorescent protein (rag2:GFP) plasmid DNA containing inverted terminal repeats recognized by tol2 transposase (see the Table of Materials for source of plasmid DNA)¹² and elute the DNA in sterile water.
      NOTE: GFP is used in this study, but any fluorescent protein will work.
   2. Prepare pCS2-transposase (tol2) RNA
      1. Isolate tol2 plasmid DNA using commercially available kits (see the Table of Materials for the source of plasmid DNA)¹² and elute the DNA in sterile water.
      2. Incubate 1 µg of tol2 DNA at 37 °C for 1 h with 5 units of NotI restriction enzyme in the appropriate buffer to linearize the plasmid. Heat-inactivate the NotI at 65 °C for 20 min.
      3. Transcribe the tol2 RNA with SP6 RNA polymerase from the linearized DNA using a commercially available RNA transcription kit¹³. Store the RNA in single-use aliquots at -80 °C.
   3. Prepare the injection mix on ice by adding 250 ng of rag2:c-myc plasmid DNA, 250 ng of rag2:GFP plasmid DNA, 150 ng of tol2 RNA, and 0.5 µL of phenol red. Adjust the volume to 10 µL with nuclease-free water.
   4. Inject ~1 nL of injection mix into the cell of a one-cell stage syngeneic zebrafish embryo using a microinjector and needle pulled from a glass capillary tube, following previously established methods^8,14.
      NOTE: Syngeneic zebrafish generated by parthenogenesis¹⁵ are used here to eliminate genetic heterogeneity and immune rejection after transplantation. However, wildtype zebrafish or any desired strain can also be used.
   5. Remove dead, underdeveloped, or malformed embryos at 24 h and 48 h post-injection¹⁶.
   6. Place the injected fish on the fish system at 6 days post-injection and raise them according to standard protocol⁶.
2. Screening injected zebrafish and monitoring tumor growth
   1. At 21 days post-injection, place a single injected larva in a droplet of embryo medium on a Petri dish and screen for GFP fluorescence using an epifluorescent microscope (excitation bandpass: 470/40, emission bandpass: 525/50)⁸.
      NOTE: Zebrafish larvae that have integrated both rag2:c-myc and rag2:GFP will have a green fluorescent thymus (positive fish) and may already exhibit the expansion of malignant green fluorescent cells beyond the thymus. All fish with a GFP+ thymus will go on to develop a GFP+ T-ALL. The larvae that do not demonstrate integration of the plasmids will not have any green fluorescent cells (negative fish).
   2. Separate the negative fish (based on a lack of fluorescence) into a different tank and monitor the fish again at 28 and 35 days post-injection. At 28 and 35 days post-injection, anesthetize the fish with 0.02% buffered tricaine (MS-222) in fish system water prior to screening.
   3. Place each zebrafish larva that is positive for a fluorescently labeled thymus or a fluorescently labeled tumor in an individual tank for monitoring tumor growth three times a week.
3. Collection of fluorescently labeled T-ALL cells from the primary tumor fish
   1. Prepare at least 20 mL of 10% heat-inactivated fetal bovine serum in 0.9x phosphate buffered saline (FBS/PBS).
   2. Add 1 mL of FBS/PBS to one vial of heparin sodium salt (300 units).
   3. Select fish bearing tumors in more than 50% of the body, or those fish having difficulty eating or swimming. Sacrifice the fish by overdosing them with tricaine or by ice water immersion. Confirm death by lack a of gill movement and heartbeat.
   4. Use a razor blade to remove the head of the fish.
   5. Wash the cavity of the fish with the heparin solution to remove excess red blood cells.
      1. Using a pipette, inject 100 µL of heparin solution (prepared in step 1.3.2) into the body cavity of the fish. Remove all liquid that spills out (it will contain blood cells) and pipette it into a 1.5 mL microcentrifuge tube.
      2. Use a fresh pipette tip to wash the body again. Perform at least three washes, or until the liquid that spills out appears free of blood.
      3. Discard all liquid from the washes in steps 1.3.5.1 and 1.3.5.2, as it will not be used in further analyses.
   6. Collect the leukemia cells.
      1. Inject 100 µL of FBS/PBS into the body cavity of the fish.
      2. Apply gentle pressure to the outside of the body of the fish with the pipette tip to squeeze/push out the tumor cells.
         NOTE: If this step is performed using a dissecting microscope, the tumor cells spilling out of the body cavity will be evident.
      3. Collect the liquid and cells from each wash into a 1.5 mL microcentrifuge tube.
      4. Repeat steps 1.3.6.1-1.3.6.3 until most of the tumor cells are collected (usually 5-10 times). Collect all tumor cells into the same 1.5-mL microcentrifuge tube. Keep the cells at room temperature.
   7. Mix the collected tumor cells from step 1.3.6 by gently pipetting them to dissociate the clumps of cells. Filter the cell suspension through a 40-µm mesh filter. Wash the filter 1-2 times with 100 µL of FBS/PBS. Keep the cells at room temperature.
   8. Mix 2 µL of cell suspension with 18 µL of trypan blue (0.4%, diluted 1:10 and filtered) in a new tube. Load 10 µL on a hemocytometer and count the number of live (non-blue) fluorescent cells to calculate the number of isolated tumor cells.
      NOTE: Non-blue GFP-negative cells are likely normal, non-malignant T-cells and should not be counted. At least 2 x 10⁶ cells are required to stain the cells with Hoechst 33342.

2. Staining the Fluorescently Labeled T-ALL Cells with Hoechst 33342 to Identify the Side Population

1. Dilute Hoechst 33342 1:10 with nuclease-free H₂O to make the 1 mg/mL working concentration. Store the dye at 4 °C and in the dark.
2. Prepare a 100-mM solution of verapamil by dissolving 49.1 mg in 1 mL of dimethyl sulfoxide (DMSO).
   NOTE: Verapamil is an inhibitor of ABC transporters and is an important control for side population experiments. Adding verapamil to samples will inhibit the ability of the ABC transporters to efflux the Hoechst 33342 dye. Therefore, a true side population will disappear when verapamil is added to the sample.
3. Set a water bath to 28 °C.
4. Prepare samples and controls in the dark by adding cells and solutions to fluorescence-activated cell sorter (FACS) tubes.
   1. Prepare the samples by adding 10⁶ cells, 15 µL of 1 mg/mL Hoechst 33342, and 2.5 µL of DMSO to a FACS tube. Adjust the volume to 1 mL with FBS/PBS.
   2. Prepare the controls by adding 10⁶ cells, 15 µL of 1 mg/mL Hoechst 33342, and 2.5 µL of 100 mM verapamil to a FACS tube. Adjust the volume to 1 mL with FBS/PBS.
      NOTE: A minimum of 10⁶ cells/sample is recommended, but staining more cells is preferred, especially if the side population cells will be sorted for further analysis. Do not exceed 10⁶ cells/mL, and maintain the Hoechst 33342 concentration at 15 µg/mL. If 10 x 10⁶ cells are stained, the total reaction volume is 10 mL.
5. Incubate in a 28 °C water bath in the dark for 120 min.
6. After the incubation, place the cells immediately on ice and keep them in the dark.
7. Pellet the cells at 4 °C for 6 min at 300 x g. Remove the supernatant. Wash with 1 mL of FBS/PBS.
8. Pellet the cells at 4 °C for 6 min at 300 x g. Remove the supernatant. Resuspend the cells in 1 mL of FBS/PBS.
9. Keep at 4 °C until cell sorting.
10. 15 min prior to cell sorting, add 2 µL of propidium iodide (PI) stock (1 mg/mL) to each 1 mL of FBS/PBS (final concentration: 2 µg/mL). Mix well.

3. Use FACS to Find the Side Population within the Fluorescently Labeled T-ALL Cell Population

1. Analyze the stained zebrafish T-ALL cell suspensions on a cell sorter equipped with a UV laser for Hoechst 33342 dye excitation and a 488-nm laser for PI excitation and for the detection of the GFP+ T-ALL cells.
2. Determine a gate for the live, GFP+ T-ALL cells.
   1. Use forward scatter (FSC) and side scatter (SSC) area to gate out debris.
      NOTE: FSC is used to distinguish cell size and SSC is used to distinguish the granularity of the cell.
   2. Use FSC and SSC width to discriminate doublets.
      NOTE: There are three components identified by the flow cytometer detectors: area, height, and width. Area is typically used to measure the fluorescence of the cell. Width measures the time of flight and is twice as wide if two cells are clumped together.
   3. For live tumor cells, draw a gate around the cells positive for GFP and negative for PI using the appropriate detectors (e.g., FITC for GFP and PerCP Cy5-5 for PI).
3. In the live cell gate, measure the Hoechst 33342 fluorescence through the Hoechst Blue and Hoechst Red bandpass filters (450/50 and 670/30, respectively).
   NOTE: The side population, if present, will be evident after this is set up. Importantly, the side population must disappear in the verapamil control in order for it to be considered a true side population.
4. If sorting the side population cells for further experiments, use the large nozzle tip (100 µm) and collect the cells into a FACS tube containing 3 mL of 100% FBS. Use a larger nozzle tip (100 µm) instead of the smaller tip (70 µm) to improve cell viability after sorting.

Results

To efficiently generate fluorescent myc-induced T-ALL tumors in zebrafish, circular DNA constructs flanked by tol2 transposase sites can be co-injected with tol2 RNA. Previous studies from the injection of linearized DNA into zebrafish embryos report a 5% transgenesis rate⁸. With the protocol adjusted to include tol2-mediated transgenesis, transgenesis rates ranging from 10-44% can be observed (Table 1).

Discussion

The side population assay is highly sensitive; therefore, small changes to the protocol can result in a difficulty in detecting side population cells. First, the temperature during the staining step is specific to each animal/cell system. For mammalian systems, the side population assay is typically performed at 37 °C². When the zebrafish T-ALL cells were incubated at 37 °C, many of the cells died, which made this incubation temperature unacceptable (data not shown). When Kobayashi and c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Syngeneic zebrafish (CG2)			See Mizgireuv et al., 2006
rag2:c-myc plasmid			Langenau Laboratory
rag2:GFP plasmid constructed in de Jong Lab			rag2 promoter from Langenau Laboratory
pCS2-transposase plasmid			Chien Laboratory
NotI -HF restriction enzyme	New England Bio-Labs Inc.	R3189S	500 units in 25 µL
mMessage Machine SP6 Transcription Kit	Ambion	Thermo Fisher Scientific - AM1340	25 reactions
QIAGEN Plasmid Midi Kit	Qiagen, Inc.	12643
SteREO Discovery V8 Microscope	Carl Zeiss Microscopy	495015-0008-000
Digital microinjector	Tritech Research	MINJ-D
Brass straight-arm needle holder	Tritech Research	MINJ-4
Magnetic base and stand	Tritech Research	MINJ-HBMB
Micromanipulator	Narishige International	MN153
Glass capillaries	Sutter Instrument Co.	B100-50-10	10 cm without filament
Flaming/Brown micropipette puller	Sutter Instrument Co.	P-97
Microloader pipette tips	Eppendorf North America Inc.	930001007
Phenol Red solution	Sigma Life Science	P0290	0.5% concentration 100 mL
Plastic Transfer pipettes	Fisherbrand	137119D	graduated 7.5 mL bulb
Tricaine methanesulfonate (MS-222)	Western Chemical, Inc.	Fisher Scientific - NC0342409	100 g
Fetal bovine serum (FBS)	HyClone Laboratories, Inc.	Fisher Scientific - SH3007103	500 mL
Phosphate-Buffered Saline (PBS)	Gibco by Life Technologies	1001-023	pH 7.4 (1x) - 500 mL
Heparin sodium salt	Sigma-Aldrich	2106	15 - 300 unit vials
40 µm mesh filter	Falcon Corning Brand	352340	Case of 50
Trypan blue	Sigma Life Science	T8154	20 mL - Solution (0.4%)
Hoechst 33342	Life Technologies	H3570	10 mL
Verapamil	Sigma Life Science	V4629	1 g
Dimethyl sulfoxide (DMSO)	Sigma Life Science	D8418	100 mL
Propidium Iodide	Life Technologies	P3566	10 mL
FACSAria Fusion cell sorter	BD Biosciences
BD FACSDiva 8.0.1	BD Biosciences
FlowJo v10.2	FlowJo, LLC