Name: flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up
Uploaded: 2018-03-26T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

This work was in part supported by the French Association Laurette Fugain, LigueContre le Cancer (North Center), Fondation de France (Leukemia Committee), SIRIC Oncolille, and French National Cancer Institute.

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
<ol>
	<li>Collect bone marrow (2 mL) from de novo AML in tubes containing the anticoagulant ethylenediaminetetraacetic acid&#160;(EDTA) at a concentration of 1.8 mg per mL of bone marrow.</li>
	<li>Perform Ficoll separation, a density gradient separation to isolate mononuclear cells from red cells and gra...

<ol>
	<li>Shlush, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+origins+of+relapse+in+acute+myeloid+leukaemia+to+stem+cells.">Tracing the origins of relapse in acute myeloid leukaemia to stem cells.</a> Nature. , (2017).</li><li>Terwijn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemic+stem+cell+frequency:+a+strong+biomarker+for+clinical+outcome+in+acute+myeloid+leukemia.">Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.</a> PLoS One. 9 (9), e107587 (2014).</li><li>Jordan, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interleukin-3+receptor+alpha+chain+is+a+unique+marker+for+human+acute+myelogenous+leukemia+stem+cells.">The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.</a> Leukemia. 14 (10), 1777-1784 (2000).</li><li>van Rhenen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+AML+stem+cell+associated+antigen+CLL-1+aids+in+discrimination+between+normal+and+leukemic+stem+cells.">The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.</a> Blood. 110 (7), 2659-2666 (2007).</li><li>Bonardi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteomics+and+transcriptomics+approach+to+identify+leukemic+stem+cell+(LSC)+markers.">A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.</a> Mol Cell Proteomics. 12 (3), 626-637 (2013).</li><li>Kikushige, Y., Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+TIM-3+as+a+Leukemic+Stem+Cell+Surface+Molecule+in+Primary+Acute+Myeloid+Leukemia.">Identification of TIM-3 as a Leukemic Stem Cell Surface Molecule in Primary Acute Myeloid Leukemia.</a> Oncology. 89, 28-32 (2015).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat Med. 3 (7), 730-737 (1997).</li><li>Kersten, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD45RA,+a+specific+marker+for+leukaemia+stem+cell+sub-populations+in+acute+myeloid+leukaemia.">CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.</a> Brit J Haematol. 173 (2), 219-235 (2016).</li><li>Goardon, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+LMPP-like+and+GMP-like+leukemia+stem+cells+in+acute+myeloid+leukemia.">Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia.</a> Cancer Cell. 19 (1), 138-152 (2011).</li><li>Ng, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+17-gene+stemness+score+for+rapid+determination+of+risk+in+acute+leukaemia.">A 17-gene stemness score for rapid determination of risk in acute leukaemia.</a> Nature. 540 (7633), 433-437 (2016).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174 (10), 6477-6489 (2005).</li><li>Roloff, G. W., Lai, C., Hourigan, C. S., Dillon, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Advances+in+the+Measurement+of+Residual+Disease+in+Acute+Myeloid+Leukemia.">Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia.</a> J Clin Med. 6 (9), (2017).</li><li>Swamydas, S., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification+and+Labeling+of+Mouse+Bone+Marrow+Neutrophils+for+Functional+Studies+and+Adoptive+Transfer+Experiments.">Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments.</a> J Vis Exp. (77), e50586 (2013).</li><li>Gabert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+and+quality+control+studies+of+'real-time'+quantitative+reverse+transcriptase+polymerase+chain+reaction+of+fusion+gene+transcripts+for+residual+disease+detection+in+leukemia+-+a+Europe+Against+Cancer+program.">Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.</a> Leukemia. 17 (12), 2318-2357 (2003).</li></ol>

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,&#160; 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...

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 cases8,9. 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/IL2R&#947;cnull (NSG) mouse model is widely used for engraftment of normal and malignant human hematopoietic cells10,11. 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,&#160; 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 cytometry2,8,9. 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&#160; 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.

<table border="0" cellpadding="0" cellspacing="0" width="1146">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:363px;">Pancoll</td>
			<td style="width:189px;">PAN-Biotech</td>
			<td style="width:128px;">P04-60500</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="75">
			<td height="75" style="height:75px;">RBC Lysis Buffer (10x)</td>
			<td>Ozyme</td>
			<td>BLE420301</td>
			<td style="width:467px;">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</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">NOD/SCID/IL2R&#947;cnull&#160; (NSG)&#160; mice</td>
			<td>JACKSON LABORATORY</td>
			<td>Stock No: 005557</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">PBS</td>
			<td>Gibco by life technologies</td>
			<td>14190-144</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">AutoMACS Running Buffer &#8211; MACS Separation Buffer</td>
			<td>MACS Buffer Miltenybiotec</td>
			<td>130-091-221</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD36-FITC (clone FA6-152, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>PN IM0766U</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD33-PC5.5 (clone D3HL60.251, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36289</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD90-APC (clone 5E10)</td>
			<td>Biolegend</td>
			<td>328114</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD19-ECD (clone J3-119, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>A07770</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD34-AA700 (clone 581, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92417</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD45RA-APC-H7 (clone HI100)&#160;</td>
			<td>Beckton Dickinson</td>
			<td>560674</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92396</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD45-KO (clone J.33, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD3-PE</td>
			<td>Beckman Coulter</td>
			<td>IM1451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD20-PE</td>
			<td>Beckman Coulter</td>
			<td>A07747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flow-Set</td>
			<td>Beckman Coulter</td>
			<td>A63492</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flow-Check</td>
			<td>Beckman Coulter</td>
			<td>A63493</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Navios</td>
			<td>Beckman Coulter</td>
			<td>DS-14644A</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">LSR Fortessa X20</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">CST BD</td>
			<td>BD Biosciences</td>
			<td>655051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">FacsFlow&#160;</td>
			<td>BD Biosciences</td>
			<td>336911</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-hCD45-FITC</td>
			<td>Biolegend</td>
			<td>BLE304006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-mCD45-APC</td>
			<td>Biolegend</td>
			<td>BLE103112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">EasySep human PE selection kit</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">EasySep&#160; magnet&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>18551</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up

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.

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...

Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

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

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.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...