Name: assessment of the metabolic profile of primary leukemia cells
Uploaded: 2018-11-21T13:00:00
Description: Watch this Scientific Journal Video about Assessment of the Metabolic Profile of Primary Leukemia Cells at JoVE.com

We would like to thank the Czech Pediatric Hematology Centers. This work was supported by the Grant of Ministry of Health (NV15-28848A), by Ministry of Health of Czech Republic, University Hospital Motol, Prague, Czech Republic 00064203 and by Ministry of Education, Youth and Sports NPU I nr.LO1604.

All samples were obtained with the informed consent of the children&#39;s parents or guardians and approval of Ethical committee of Charles University in Prague, Czech Republic, the study no. NV15-28848A.
1. Preparation of Reagents
<ol>
	<li>Prepare 500 mL of PBS by dissolving 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, in ddH2O. Adjust the pH to 7.4 with HCl. Sterilize by autoclaving.</li>
	<li>Prepare 100 mL of RPMI medium...

<ol>
	<li>DeBerardinis, R. J., Lum, J. J., Hatzivassiliou, G., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biology+of+Cancer:+Metabolic+Reprogramming+Fuels+Cell+Growth+and+Proliferation.">The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation.</a> Cell Metabolism. 7, 11-20 (2008).</li><li>Ward, P. S., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Reprogramming:+A+Cancer+Hallmark+Even+Warburg+Did+Not+Anticipate.">Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate.</a> Cancer Cell. 21, 297-308 (2012).</li><li>Pavlova, N. N., Thompson, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+InsP6-kinases+as+InsP6-dephosphorylating+enzymes+provides+a+new+mechanism+of+cytosolic+InsP6+degradation+driven+by+the+cellular+ATP/ADP+ratio.">Discovery of InsP6-kinases as InsP6-dephosphorylating enzymes provides a new mechanism of cytosolic InsP6 degradation driven by the cellular ATP/ADP ratio.</a> The Biochemical Journal. 462, 173-184 (2014).</li><li>Morciano, G., 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=Use+of+luciferase+probes+to+measure+ATP+in+living+cells+and+animals.">Use of luciferase probes to measure ATP in living cells and animals.</a> Nature Protocols. 12, 1542-1562 (2017).</li><li>Chau, M. D. L., Gao, J., Yang, Q., Wu, Z., Gromada, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+21+regulates+energy+metabolism+by+activating+the+AMPK-SIRT1-PGC-1alpha+pathway.">Fibroblast growth factor 21 regulates energy metabolism by activating the AMPK-SIRT1-PGC-1alpha pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. , 12553-12558 (2010).</li><li>Lee, K. -. H., 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=Targeting+energy+metabolic+and+oncogenic+signaling+pathways+in+triple-negative+breast+cancer+by+a+novel+adenosine+monophosphate-activated+protein+kinase+(AMPK)+activator.">Targeting energy metabolic and oncogenic signaling pathways in triple-negative breast cancer by a novel adenosine monophosphate-activated protein kinase (AMPK) activator.</a> The Journal of Biological Chemistry. 286, 39247-39258 (2011).</li><li>Godlewski, 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=MicroRNA-451+Regulates+LKB1/AMPK+Signaling+and+Allows+Adaptation+to+Metabolic+Stress+in+Glioma+Cells.">MicroRNA-451 Regulates LKB1/AMPK Signaling and Allows Adaptation to Metabolic Stress in Glioma Cells.</a> Molecular Cell. 37, 620-632 (2010).</li><li>Zhang, 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=Measuring+energy+metabolism+in+cultured+cells,+including+human+pluripotent+stem+cells+and+differentiated+cells.">Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells.</a> Nature Protocols. 7, 1068-1085 (2012).</li><li>Kaplon, 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=A+key+role+for+mitochondrial+gatekeeper+pyruvate+dehydrogenase+in+oncogene-induced+senescence.">A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence.</a> Nature. 498, 109-112 (2013).</li><li>Pardee, T. S., 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+phase+I+study+of+the+first-in-class+antimitochondrial+metabolism+agent,+CPI-613,+in+patients+with+advanced+hematologic+malignancies.">A phase I study of the first-in-class antimitochondrial metabolism agent, CPI-613, in patients with advanced hematologic malignancies.</a> Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 20, 5255-5264 (2014).</li><li>Stein, 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=A+defined+metabolic+state+in+pre+B+cells+governs+B-cell+development+and+is+counterbalanced+by+Swiprosin-2/EFhd1.">A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.</a> Cell Death and Differentiation. 24, 1239-1252 (2017).</li><li>Hru&#353;&#225;k, O., Porwit-MacDonald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+expression+patterns+reflecting+genotype+of+acute+leukemias.">Antigen expression patterns reflecting genotype of acute leukemias.</a> Leukemia. 16, 1233-1258 (2002).</li><li>Amin, H. 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The above-described protocol allows for the measurement of the metabolic activity assessed by OCR and ECAR values in primary leukemic blasts derived from patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). The advantage of measurement using an extracellular flux analyzer is that it enables the detection of metabolic profile in the real time in the live cells. Essentially, every step in the provided protocol could be adjusted depending on the cell type one plans to study. Here, we will discus...

The metabolic profile is one of the main characteristics of cells and altered bioenergetics are now considered one of the hallmarks of cancer1,2,3. Moreover, changes in the metabolic setup could be used in the treatment of cancer by targeting signal transduction pathways or enzymatic machinery of cancer cells4,5,6. Knowing the metabolic predisposition of cancer cells is thus an advantage and can help improve the current therapy.
There are a plenty of already established methods which can assess the metabolic activity of cells in culture. Regarding glycolysis, glucose uptake can be measured by the radioactive labeling, using 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) or extracellular lactate levels measured enzymatically7,8. Fatty acid oxidation rate is another metabolic parameter measured by isotopically labeled palmitate9,10. Oxygen consumption rate is a method widely-used for determining mitochondrial activity in cells11,12, together with the mitochondrial membrane potential evaluation13,14, ATP/ADP (adenosine 5&#8242;-triphosphate/Adenosine 5&#8242;-diphosphate) ratio measurement15 or total intracellular ATP measurement16. Signaling pathways known to regulate metabolic processes could be determined by protein quantifications and can improve the understanding of metabolic measurements17,18,19.
However, all these methods measure only one or, in the best scenario, a few metabolic parameters in one sample simultaneously. Importantly, simultaneous measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) can be achieved by the extracellular flux analysis by, for example, Seahorse XFp Analyzer. OCR is an indicator of mitochondrial respiration and ECAR is mainly the result of glycolysis (we cannot ignore CO2 production possibly elevating ECAR of cells with high oxidative phosphorylation activity)20. So far, various cell types have been studied using these analyzers21,22,23.
Here we describe the protocol for the extracellular flux analysis of primary blasts (leukemia cells derived from the immature hematopoietic stage) from leukemia patients. To the best of our knowledge, a specific protocol for primary blasts is not available yet.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biosera</td>
			<td>FB-1001/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco, ThermoFisher Scientific</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+) Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>Sigma-Aldrich</td>
			<td>75351-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Deoxy-D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D8375-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2920-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin A from Streptomyces sp.</td>
			<td>Sigma-Aldrich</td>
			<td>A8674-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF Base Medium, 100 mL</td>
			<td>Agilent Technologies</td>
			<td>103193-100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution, 200 mM</td>
			<td>Sigma-Aldrich</td>
			<td>G7513-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution, 1 M, pH 7.0-7.6</td>
			<td>Sigma-Aldrich</td>
			<td>H0887-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>Sigma-Aldrich</td>
			<td>GE17-1440-02</td>
			<td>Density gradient medium</td>
		</tr>
		<tr>
			<td>Seahorse XFp FluxPak</td>
			<td>Agilent Technologies</td>
			<td>103022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell-Tak Cell and Tissue Adhesive</td>
			<td>ThermoFisher Scientific</td>
			<td>CB40240</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse Analyzer XFp</td>
			<td>Agilent Technologies</td>
			<td>S7802A</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFp Cell Culture Miniplate</td>
			<td>Agilent Technologies</td>
			<td>103025-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Figure 3 shows the curves after Glycolysis stress test and Cell Mito stress test measurements of leukemic blasts from the BCP-ALL (B-cell precursor acute lymphoblastic leukemia) and AML (acute myeloid leukemia) patients. The calculation of metabolic parameters from these measurements is also indicated. 500,000 cells per well were seeded and all measurements were done in hexaplicates.
In the Glycolys...

Watch this Scientific Journal Video about Assessment of the Metabolic Profile of Primary Leukemia Cells at JoVE.com

Assessment of the Metabolic Profile of Primary Leukemia Cells

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

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

This method can help answer key questions regarding metabolic pathway setup in leukemia cells. The main advantage of this technique is that it allows measurement of the metabolism of primary leukemia cells in real time in live cells. Begin by diluting a bone marrow sample obtained from a leukemia patient in PBS at a one to one ratio.Carefully, layer six milliliters of the diluted bone marrow sample over six milliliters of freshly prepared density gradient medium in a 15 milliliter conical tube and separate the cells by density gradient centrifugation. Use a Pasteur pipet to carefully transfer the interface layer of mononuclear cells to a new 50 milliliter conical tube containing five milliliters of PBS for a centrifugation wash. Then, resuspend the mononuclear cell pellet in two milliliters of sterile PBS for counting.Dilute the cells to a three times 10 to the seven cells per milliliter concentration. And add one milliliter of cells to each of two T75 flasks containing 20 milliliters of RPMI medium for a 16 to 24 hour incubation at 37 degrees Celsius and 5%CO2. Meanwhile to prepare the plates for extracellular flux analysis, add 12.5 microliters of cell adhesive solution into each well of two, eight well extracellular flux analyzer plates.After 20 minutes, aspirate the cell adhesive and wash each well two times with 200 microliters of sterile water per wash. After the second wash, leave the plates in the hood until the wells are dry. And place the sensor cartridge upside down on the lab bench.Separate the utility plate and the sensor cartridge of the flux analyzer. And fill each well of the utility plate with 200 microliters of calibrant and each moat around the outside of the wells with 400 microliters of calibrant. Return the sensor cartridge to the utility plate that now contains the calibrant and place the cartridge assembly in a humidified 37 degrees Celsius incubator without CO2 overnight.Then turn on the extracellular flux analyzer and let it warm to 37 degrees Celsius overnight. The next morning, transfer the cells from the flask to 50 milliliter conical tubes for centrifugation. And resuspend the pellets in one milliliter of the appropriate experimental medium for counting.Resuspend the cells at four times 10 to the six cells in 400 microliters of experimental medium concentration. And add 50 microliters of cells into wells B through G of the flux analyzer plate. And 180 microliters of the experimental medium into wells A and H as the background control wells.After centrifugation slowly and carefully add 130 microliters of the experimental medium to wells B through G.And visually confirm stable adherence of the cells to the bottom of each well under the microscope. Then return the flux analysis plate to the humidified 37 degrees Celsius incubator without CO2 for 30 minutes. 20 minutes before the end of the incubation, load the compounds into the appropriate injector ports of the cartridge according to the experimental protocol as indicated in the table.Then set up the appropriate extracellular flux analysis program. Start the program and replace the calibrant plate with the assay plate when prompted. In a glycolysis stress test, only basal medium is used so that the cells are deprived of nutrients.The first parameter obtained is the basal acidification which should reflect the amount of glucose stored in cells. After the first injection, the extracellular acidification rate is increased as the cells utilize the glucose and can ferment the glucose to lactate. The oligomycin A in the second injection inhibits ATP synthase and thus directs the cells to produce ATP mainly by a glycolysis causing further elevation of the extracellular acidification rate.While the injection of 2-Deoxy-D-glucose completely inhibits glycolysis and the extracellular acidification rate drops. In the cell mito stress test, a medium supplemented with glutamine and glucose is used so that the cells are not deprived of all nutrients. The basal respiration parameter reflects their basal metabolic state.After the first injection with oligomycin A, the cells inhibit mitochondrial respiration and switch to glycolysis which is represented as a decrease in the oxygen consumption rate. The second and third injections of FCCP however, uncouple the ATP production from the respiration so that the cells now consume oxygen at a maximal rate. And the oxygen consumption rate rises to its highest value.The last injection of the rotenone and antimycin A mixture completely inhibits the mitochondrial respiration decreasing the oxygen consumption rate to almost zero. While attempting this procedure, it's important to assess the percentage of blasts in the sample to ensure that the metabolic parameters of the leukemiablasts only are measured. When implementing this method, a careful optimization has to be applied to cultivation conditions and data normalization.

assessment-of-the-metabolic-profile-of-primary-leukemia-cells

Research

JoVE Journal

Cancer Research

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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 deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

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

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.

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 ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

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 healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

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 cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

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

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 (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

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 (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection 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 ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...