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.

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			<td height="20" style="height:21px;width:363px;">Pancoll</td>
			<td style="width:189px;">PAN-Biotech</td>
			<td style="width:128px;">P04-60500</td>
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			<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>
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			<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>
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			<td height="20" style="height:21px;">AutoMACS Running Buffer &#8211; MACS Separation Buffer</td>
			<td>MACS Buffer Miltenybiotec</td>
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			<td>B36289</td>
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			<td height="20" style="height:21px;">Anti-CD90-APC (clone 5E10)</td>
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			<td>A07770</td>
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			<td height="20" style="height:21px;">Anti-CD34-AA700 (clone 581, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92417</td>
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			<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>
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			<td height="20" style="height:21px;">Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92396</td>
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			<td height="20" style="height:21px;">Anti-CD45-KO (clone J.33, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
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			<td>IM1451</td>
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			<td height="20" style="height:21px;">Anti-CD20-PE</td>
			<td>Beckman Coulter</td>
			<td>A07747</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
			<td></td>
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			<td height="20" style="height:21px;">Flow-Set</td>
			<td>Beckman Coulter</td>
			<td>A63492</td>
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			<td height="20" style="height:21px;">Flow-Check</td>
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			<td>A63493</td>
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			<td height="20" style="height:21px;">Navios</td>
			<td>Beckman Coulter</td>
			<td>DS-14644A</td>
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			<td height="20" style="height:21px;">LSR Fortessa X20</td>
			<td>BD Biosciences</td>
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			<td>BD Biosciences</td>
			<td>655051</td>
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			<td height="20" style="height:21px;">FacsFlow&#160;</td>
			<td>BD Biosciences</td>
			<td>336911</td>
			<td></td>
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			<td height="20" style="height:21px;">Anti-hCD45-FITC</td>
			<td>Biolegend</td>
			<td>BLE304006</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Anti-mCD45-APC</td>
			<td>Biolegend</td>
			<td>BLE103112</td>
			<td></td>
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		<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>
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		<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>
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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 ...

The overall goal of this procedure is to estimate leukemia stem cells in acute myeloid leukemia and in patient-derived-xenografts by flow cytometry at diagnosis and at treatment follow up. This method can answer key questions in the hematology/oncology field, such as the clinical relevance of the quantitative assessment of leukemia stem cells by flow cytometry in acute myeloid leukemia. The main advantage of this technique is that it is applicable to most hematology/oncology laboratories with commonly used antibodies.This procedure will be demonstrated by Thomas, a post-doc, Fanny, a grad student, and Adeline, an engineer from my laboratory. To begin, collect two milliliters of bone marrow from de novo AML in tubes containing 1.8 milligrams per milliliter of the anti-coagulant EDTA. Isolate mononuclear cells from red blood cells and granulocytes by first diluting bone marrow in three volumes of PBS.Then, carefully overlay one volume of ficol with one volume of diluted bone marrow. Spin the density gradient at 300g for 30 minutes without a break. Then, transfer the white buffy coat layer of mononucleated cells into a new sterile tube.To remove the supernatants contaminating serum components, add 10 milliliters of PBS and centrifuge the cells at 300g for five minutes before repeating the wash. Lyse the red blood cells by adding two milliliters of lysis buffer to the cell pellet and gently vortex. Then, incubate the cells at room temperature for five minutes.Centrifuge the cells at 300g for five minutes and remove the supernatant. Then, use PBS to wash the cells. After isolating blast cells from bone marrow according to the text protocol, under a laminar flow hood, insert an unconditioned NSG mouse into a restraining device and inject five times 10 to the sixth AML blast cells into the tail vein.Return the mouse to an individual ventilated cage under conventional and specific pathogen-free conditions. Every two weeks, use a submandibular collection method with a hematocrit capillary to draw 60 microliters of blood. Transfer the capillary into a five milliliter tube then with a sterile gauze pad, apply gentle pressure to stop the bleeding.Add one milliliter of lysis buffer to the tube and gently vortex. Then, incubate the cells at room temperature for five minutes. Centrifuge the cells at 300g for five minutes.Then, after removing the supernatant, add 100 microliters of PBS with 10%BSA, three microliters of anti-human CD45 FITC, incubate for 30 minutes. Then, wash the cell pellet two times in PBS, two milliliters, with 10%BSA and centrifuge at 300g for five minutes. Every two weeks, use flow cytometry to analyze the engraftment by chimerism analysis of human versus murine CD45 expression.When the blast count is greater than 70%or severe clinical signs of sickness are observed, sacrifice the animal and collect mononuclear blast cells from a crushed spleen and from bone marrow by using PBS to flush tibias and femurs as previously described. To carry out panel staining, combine conjugated antibodies with human primary or patient-derived-xenograft, or PDX samples, and vortex the tubes. Then, incubate the cells in the dark at room temperature for 15 to 30 minutes.Remove the unbound antibodies by using two milliliters of PBS to wash the cells. Re-suspend the cells in 450 microliters of PBS, then proceed to flow cytometry analysis and MRD monitoring. To accurately and reproducibly assess membrane of cytoplasmic markers by flow cytometry, verify optical alignment.Proper tuning of the 3D system and optical sensitivity and stomatization using stomazine calibration beats every day. Use normal bone marrow to set the gates for CD38. A minimum of 500, 000 events are required and it is verified that each flow run is regular and homogenous.In addition, doublets, as well as dead cells and cell debris are eliminated. Immature hematopoietic cells are defined by being CD45 intermediate, SSC low. And the CD38 positive control or hematogones, are CD19/CD38 double positive.Then P8 or CD38 positive, P7 or CD38 intermediate, and P6 or CD38 negative are defined. The P6 cells can be further subdivided into HSC, MPP, and LSC. Since it is a normal bone marrow it is LSC negative.Using multiparametric flow cytometry, it was found that the CD34 positive, CD38 negative blast fraction was higher in PDX compared to the diagnostic patient sample. Interestingly, a higher percentage of putative LSC was found in the PDX compared to the primary patient sample, suggesting a possible enrichment of malignant progenitor cells in the hematologic tissues in this mouse model. Upon comparison of the blast progenitor populations in two different patient samples, the CD34 positive, CD38 negative fraction increased from 0.06%at diagnosis to 3.33%at the first treatment follow-up in the first patient.In contrast, the CD34 positive, CD38 negative fraction decreased in the second patient from 7.8%at diagnosis to 2.27%at the first treatment follow-up. Accordingly, the putative LSC fraction increased in the first patient from 0%at diagnosis to 0.65%and decreased in the second patient from 6.57%at diagnosis to 1.44%at treatment follow-up. Minimal residual disease or MRD monitoring, using as molecular markers the NPM1 mutation in patient one and the CBF-beta MYH11 gene fusion for patient two showed that molecular MRD decreased less in the first patient compared to the second patient for the first treatment follow-up point.Once mastered, this technique can be done in two hours if it is performed properly. While you're attempting this procedure, it is important to verify flow cytometric settings and CD38 gauging. Following this procedure, additional methods can be performed, like flow cytometric cell sorting in order to answer additional questions like functional leukemia stem cell assess.After watching this video you should have a good understanding of how to detect and quantify leukemia stem cells in patient samples in patient-derived-xenografts. Don't forget that working with patient samples can be hazardous and precautions such as GOP guidelines and institutional safety procedures should always be taken while you are performing this procedure.

Research

JoVE Journal

Cancer Research

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

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

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

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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 ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...