Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Summary

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Abstract

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

Introduction

Acute myeloid leukemia (AML) is the most common cause of leukemia-associated mortality. Initially, AML is a clonal disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of blast cells, and reduced production of functional hematopoietic elements. Recently, clonal heterogeneity of the blast cell population has been established essentially by using next generation sequencing (NGS) strategies and showing the existence of intra-clonal evolution of tumor cells¹.

Heterogeneity extends to the presence of leukemic stem cells (LSC). It is hypothesized that this dynamic cell compartment evolves to overcome various selection pressures imposed upon during leukemia progression and treatment. Therefore LSC are supposed to be resistant to current chemotherapeutic regimens and mediate disease relapse, with profound clinical implications². Various markers have been described to characterize LSC like CD123³, CLL-1⁴, CD97⁵ or TIM-3⁶. The CD34+CD38- compartment of blast cells is considered to be enriched for LSC⁷ but also includes normal HSC. CD90 and CD45RA expression applied to the CD34+CD38- (P6) compartment (Figure 1) permits to segregate several stages of normal and malignant hematopoietic precursors⁸. Specifically, normal CD34+CD38-CD90+CD45RA- HSCs, multipotent progenitors (MPP) like CD34+CD38-CD90dimCD45RA- cells and CD34+CD38-CD90-CD45RA+ lymphoid-primed multipotent progenitor (LMPP) cells can be determined in the majority of AML cases^8,9. The mean fluorescence intensity (MFI) of CD38 is particularly important to be considered within the CD34+ cell compartment. Because CD38 intensity defines three new cell compartments thereof: CD38 negative (P6), CD38 dim (P7) and CD38 bright (P8) (Figure 1). The MFI of CD38 may be determined using either hematogones and/or plasma cells as an internal positive control as these cells strongly express CD38.

The immunodeficient NOD/SCID/IL2Rγc^null (NSG) mouse model is widely used for engraftment of normal and malignant human hematopoietic cells^10,11. Serial xenotransplantation assays are used to experimentally validate LSC or HSC function. These studies have been extensively analyzed by large scale sequencing approaches. Nevertheless, less is known about the LSC and HSC immunophenotype of AML blast population engrafted into NSG mice compared to its primary AML (Figure 2).

The numbers of LSC are inherently low thus, identification and quantification of LSC are challenging and the method described here may be used as a flow cytometric assay at diagnosis and at clinical follow up to evaluate chemotherapy response (as residual LSC may cause AML relapse). The presence of LSC may indicate positive minimal residual disease (MRD). To date,  MRD monitoring in AML mostly rely on molecular methods (i.e., RT-PCR, NGS)^10,12. However, in this protocol we detect simultaneous protein expression of several HSC/LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry^2,8,9. This combination of antibodies may be applied in any standard flow cytometry laboratory and this flow MRD assay is particularly interesting when MRD by real-time polymerase chain reaction (RT-PCR) is not possible, i.e., if  leukemia specific molecular markers are not detectable in the diagnostic AML sample. Furthermore, this protocol, is complementary to the more sensitive molecular MRD techniques, as it aims to detect and quantify the abnormal hematopoietic progenitor cells which represents a functional cell characteristic (Figure 3).

We show how to quantify three hematopoietic progenitor populations and the putative LSC compartment with different degree of maturation by flow cytometry with antibodies available in the majority of clinical laboratories. Furthermore, we confirmed the presence of these cell compartments in corresponding patient-derived-xenografts (PDX) and at treatment follow up.

Protocol

We follow European standards for the use of animals for scientific purposes. This study was approved by the local Ethics committee (#3097-2015120414583482v3).

1. Sample preparation

1. Collect bone marrow (2 mL) from de novo AML in tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA) at a concentration of 1.8 mg per mL of bone marrow.
2. Perform Ficoll separation, a density gradient separation to isolate mononuclear cells from red cells and granulocytes, as followed. Dilute bone marrow in 3 volumes of phosphate buffer saline (PBS: NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10mM, KH₂PO₄ 1.8 mM). Carefully overlay 1 volume of Ficoll with 1 volume of diluted bone marrow. Spin 30 minutes at 300 x g without brake. Transfer the white buffy coat layer of mononucleated cells into a new sterile tube.
3. Wash cells twice in 10 mL of PBS) and centrifuge at 300 x g for 5 min, in order to remove contaminating serum components.

2. Lysis of red blood cells (RBC)

1. Add 2 mL of lysis buffer (ammonium chloride 0.8%) to the cell pellet, vortex gently and incubate at room temperature for 5 min. Centrifuge at 300 x g for 5 min.
   Note: if necessary, repeat this step.
2. Wash cells in PBS as previously described (1.3.).

3. Patient derived xenografts.

1. Obtain blast cells from bone marrow after Ficoll separation and after lymphocytes depletion using immunomagnetic negative selection (CD3 for T-, and CD20 for B-lymphocytes). Follow the manufacturer's instructions.
2. Inject 5 x 10⁶ AML blast cells into the tail vein of unconditioned NSG mice. Use a restraining device to facilitate tail vein injection. Keep mice under conventional conditions and especially under specific-pathogen-free conditions at all times, by using individual ventilated cages and by manipulation under laminar flow hood. Every two weeks, take 60 µL of blood by the submandibular collection method using a hematocrit capillary. Place the capillary into a 5 mL round bottom tube. Stop the bleed by applying gentle pressure using a small sterile gauze pad. Expulse blood with a clean syringe. 
3. Add 1ml of lysis buffer and incubate for 5min. Then centrifuge at 300 x g for 5min. Wash with PBS and centrifugre at 300 x g for 5 minutes. Remove supernatant, the add 100 µL PBS with 10% bovine serum albumin (BSA) stain with 3 µL anti-human CD45-FITC and 1 µL anti-murine CD45-APC and incubate at 4°C for 30 minutes.
4. Wash cell pellet two times in PBS (2 mL) with 10% BSA and centrifuge at 300 x g for 5 min.
5. Aliquot up to 1 x 10⁶ cells per 100 µL of PBS into flow cytometry tubes.
6. Survey engraftment every two weeks by chimerism analysis of CD45 expression (human versus murine) using flow cytometry (Figure 2A and B).
7. At sacrifice (peripheral blast count greater than 70% or severe clinical signs of sickness), collect mononuclear blast cells from crushed spleens and from bone marrow by flushing tibias and femurs with PBS as previously described¹³. Euthanize mice by cervical dislocation following international guidelines.   
8. If cell pellet contains RBC, perform lysis of red blood cells with lysis buffer (Step 2.1).
9. Wash cells pellet two times in PBS (2 mL) with 10% BSA and centrifuge at 300 x g for 5 minutes.
10. Collect cells and aliquot up to 1 x 10⁶ cells per 100 µL of PBS into flow cytometry tubes.

4. Staining

1. Add conjugated antibodies (see Step 4.2) and vortex. Incubate cells for 15 minutes in the dark at room temperature.
2. Use the following panel for human primary or PDX samples consisting of 10 µL of anti-CD36-FITC, 5 µL of anti-CD19-ECD, 5 µL of anti-CD33-PC5.5, 5 µL of anti-CD90-APC, 5 µL of anti-CD34-AA700, 5 µL of anti-CD45RA-APC-H7, 5 µL of anti-CD38-Pacific Blue , 5 µL of anti-CD123-PC7 and 2.5 µL of anti-CD45-KO.
3. Remove unbound antibodies by washing the cells in 2 mL of PBS by centrifugation at 300 x g for 5 min.
4. Re-suspend cells in 450 µL of PBS for final flow cytometric analysis.

5. Gating strategy for flow cytometry analysis

1. Perform data acquisition (at least 500,000 cells) on a flow cytometer equipped with red, blue and violet lasers. Verify cytometer settings every day for 1) optical alignment, 2) fluidic system, 3) optical sensitivity, and 4) standardization using fluorospheres
2. Follow the sequential gating strategy to define CD38 expression (Figure 1).
   1. Use cells from normal bone marrow samples to define hematogones or plasma cells which will serve as positive control  for CD38 expression.
      Note: this gate is especially important to assess as subsequent putative LSC populations depend on its definition.
   2. Define physiologic precursors cells (normal blast cells) by CD45dim/SSC (side scatter) low population criteria (Figure 1A). Within this blast population, define hematogones, which display a CD38++CD19+ phenotype (Figure 1B) and finally, use CD36 and CD33 expression to define myeloblasts, monoblasts, and erythroblasts (Figure 1C). Determine CD34 expression on hematogones as shown in (Figure 1E). Assess several subpopulations of precursors within the blast cells defined as P6-P10 (Figure 1D). Separate the CD34 compartment of blast cells into P6, P7, P8 progenitor subpopulations based on preset CD38 gates. Ensure that the P8 compartment contains the blasts that have the same CD38 intensity as the hematogones, that P6 includes cells which are CD38-based on the CD38 fluorescence minus one (FMO) control and that P7 cells are in between the two extremes with regard to CD38 expression (Figure 1D).
3. From the CD34+38- (P6) gated cells, separate HSC from putative LSC using CD90 and CD45RA expression (Figure 1F).
   Note: the HSC phenotype is CD34+CD38-CD90+CD45RA- and the multipotent progenitors (MPP) are defined as CD34+CD38-CD90-CD45RA-. The putative LSC phenotype is CD34+CD38-CD90dimCD45RA+ and the more immature downstream population are the lymphoid-primed progenitors (LMPP) defined as CD34+CD38-CD90-CD45RA+.

6. MRD monitoring by RT-PCR

1. Monitor MRD levels by RT-PCR as described previously¹⁴.

Results

Here we present a method to determine progenitor populations in normal and malignant human bone marrow samples. We collected bone marrow and spleen from a successful PDX and performed multiparametric flow cytometry as described above. We compared several subpopulations of blast cells between the diagnostic patient sample and the corresponding PDX and particularly, the CD34+CD38- progenitor compartment, notably enriched in putative LSC. We have found that the CD34+CD38- blast fraction was higher in PDX compared to the dia...

Discussion

An accurate and reproducible assessment of membrane or cytoplasmic markers by flow cytometry requires that instrument settings are verified every day for optical alignment, proper functioning of the fluidic system,  optical sensitivity, and standardization using calibration beads.

Detection of blast cell populations in AML using CD90 and CD45RA by multi-parametric flow cytometry analysis is a relatively simple and reliable method. A critical step in this analysis is an accurate assessment...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pancoll	PAN-Biotech	P04-60500
RBC Lysis Buffer (10x)	Ozyme	BLE420301	RBC Lysis solution A : 8.29 g of NH4Cl + 0.037 g of Na2EDTA in 100 mL of sterile H2O and RBC Lysis solution B: 1 g of KHCO3 in 100 mL of sterile H2O
NOD/SCID/IL2Rγcnull  (NSG)  mice	JACKSON LABORATORY	Stock No: 005557
PBS	Gibco by life technologies	14190-144
AutoMACS Running Buffer – MACS Separation Buffer	MACS Buffer Miltenybiotec	130-091-221
Anti-CD36-FITC (clone FA6-152, Iotest)	Beckman Coulter	PN IM0766U
Anti-CD33-PC5.5 (clone D3HL60.251, Iotest)	Beckman Coulter	B36289
Anti-CD90-APC (clone 5E10)	Biolegend	328114
Anti-CD19-ECD (clone J3-119, Iotest)	Beckman Coulter	A07770
Anti-CD34-AA700 (clone 581, Iotest)	Beckman Coulter	B92417
Anti-CD45RA-APC-H7 (clone HI100)	Beckton Dickinson	560674
Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)	Beckman Coulter	B92396
Anti-CD45-KO (clone J.33, Iotest)	Beckman Coulter	B36294
Anti-CD3-PE	Beckman Coulter	IM1451
Anti-CD20-PE	Beckman Coulter	A07747
Anti-CD123-PC7	Beckman Coulter	B13647
Flow-Set	Beckman Coulter	A63492
Flow-Check	Beckman Coulter	A63493
Navios	Beckman Coulter	DS-14644A
LSR Fortessa X20	BD Biosciences
CST BD	BD Biosciences	655051
FacsFlow	BD Biosciences	336911
Anti-hCD45-FITC	Biolegend	BLE304006
Anti-mCD45-APC	Biolegend	BLE103112
EasySep human PE selection kit	Stem Cell Technologies	18000
EasySep  magnet	Stem Cell Technologies	18551