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: RPMI-1640 medium with L-Alanyl-Glutamine supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 &#956;g/mL).</li>
	<li>Prepare 50 mL of 0.1 M NaHCO3 in distilled water. Adjust the pH to 8.1, filter sterilize (0.22 &#956;m) and store at 4 &#176;C. 
	NOTE: For two 8-well extracellular flux analyzer plates, prepare 250 &#956;L of 0.1 M NaHCO3 pH 8.1.</li>
	<li>Prepare 1 mL of 2 M D-glucose in distilled water. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM Oligomycin A in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 250 &#956;L of 1 M 2-deoxy-D-glucose (2-DG) in Minimal DMEM (Dulbecco&#39;s Modified Eagle&#39;s medium). Warm to 37 &#176;C and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C). Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in DMSO. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM Rotenone in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mg/mL Antimycin A in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Just before the use, prepare 10 mL of Glycolysis stress test medium. Warm Minimal DMEM to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C).</li>
	<li>Prior to the use, prepare 10 mL of Cell Mito stress test medium with BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose, 1 mM HEPES (pH 7.4), 1 mM pyruvate and 0.1 % BSA (bovine serum albumin). Warm to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C).</li>
	<li>Prior to the use, prepare 10 mL of Cell Mito stress test medium without BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose,1 mM HEPES (pH 7.4) and 1 mM pyruvate. Warm Cell Mito stress test medium without BSA to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C). 
	NOTE: BSA is added to the Cell Mito stress test medium because the cells respond better to FCCP when the medium is supplemented with BSA (different conditions were tested). Cell Mito stress test medium without BSA is used for loading the ports as the manufacturer does not recommend using BSA in the medium.</li>
</ol>
2. Isolation of Mononuclear Cells from Bone Marrow
NOTE: Ideally, measurement of metabolic state of primary leukemia cells should start immediately after bone marrow collection and cell isolation. Nevertheless, relevant data could be also obtained from cells isolated after transportation from other hematology centers in the Czech Republic. Perform all sub-steps in a sterile tissue culture hood.
<ol>
	<li>Warm up PBS and the density gradient medium to room temperature.</li>
	<li>Dilute the bone marrow sample of leukemia patient with PBS, in a ratio of 1:1. Make sure that the sample contains at least 80% of leukemic blasts.</li>
	<li>Determine the percentage of blasts by flow cytometry using specific CD markers for immunophenotype characterization of cell types. After density gradient separation, detect nucleic acid positive events with a nuclear dye in order to establish the overall cell count.</li>
	<li>Then, determine leukemia cells using specific CD markers for each type of leukemia: B-ALL (CD19, CD45), T-ALL (CD3, CD4, CD8, CD5, CD7, CD99) and AML (CD45, CD33, and specific myeloid markers)24. Divide the number of leukemia cells positive for specific CD markers by all nuclear cells to determine the percentage of leukemia blast cells. 
	NOTE: The bone marrow has to be collected into the tubes with anticoagulants.</li>
	<li>Carefully layer 6 mL of diluted bone marrow sample over 6 mL of the density gradient medium in a 15 mL conical tube. Centrifuge at 400 x g for 35 min at 4 &#176;C in a swinging-bucket rotor without the brake. 
	NOTE: Larger volume of the sample could be divided into more aliquots or 50 mL conical tube can be used with the larger amount of density gradient medium.</li>
	<li>Using a Pasteur pipette, carefully transfer the interphase layer which consists of mononuclear cells (Figure 1) to a new 50 mL conical tube with 5 mL of PBS. Centrifuge at 400 &#215; g for 10 min at 4 &#176;C in a swinging-bucket rotor. 
	NOTE: Transfer all mononuclear cell layers into a single 50 mL conical tube with 5 mL of PBS.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 2 mL of sterile PBS. Count the cells using a hemocytometer. 
	NOTE: The number of leukemia cells in one mL of aspirated bone marrow differs significantly among patients25.</li>
</ol>
3. Overnight Cultivation of Mononuclear Cells
NOTE: Perform all sub-steps in a sterile tissue culture hood.
<ol>
	<li>Prepare two T75 flasks with 20 mL of RPMI. To each flask, add 30 x 106 isolated mononuclear cells. Incubate the cells with the flask standing up for 16&#8211;24 h at 5% CO2 and 37 &#176;C.</li>
</ol>
4. Preparation of Cell Adhesive-coated Plates
<ol>
	<li>Coat two 8-well extracellular flux analyzer plates. 
	NOTE: Perform the coating in a sterile tissue culture hood.</li>
	<li>Add 2.2 &#956;L of cell adhesive(density: 2.54 mg/ml) to 250 &#956;L of 0.1 M NaHCO3, pH 8.1, and pipette immediately 12.5 &#956;L of the solution into each well. 
	NOTE: Cell adhesive stock solutions can differ in their density, adjust the volume added to NaHCO3 accordingly.</li>
	<li>Let the plates sit in the hood for about 20 min, then aspirate the cell adhesive and wash each well twice using 200 &#956;L of sterile water. Let it sit in the hood with the lid open until wells are dry.</li>
	<li>Use the plates right away or save up to 1 week at 4 &#176;C with the rim wrapped in paraffin film to avoid condensation. Ensure that the plates are warmed up to room temperature (for about 20 min) in the hood before seeding the cells.</li>
</ol>
5. Hydration of Sensor Cartridge
NOTE: Hydrate two 8-well extracellular flux analyzer cartridges.
<ol>
	<li>Separate the utility plate and the sensor cartridge. Place the sensor cartridge upside down on the lab bench.</li>
	<li>Fill each well of the utility plate with 200 &#956;L calibrant. Fill each moat around the outside of the wells with 400 &#956;L of calibrant.</li>
	<li>Return the sensor cartridge to the utility plate that now contains the calibrant.</li>
	<li>Place the cartridge assembly in a humidified, non-CO2, 37 &#176;C incubator overnight.</li>
	<li>Turn on the extracellular flux analyzer and let it warm to 37 &#176;C overnight.</li>
</ol>
6. Seeding Cells in Cell Adhesive-coated Plates
NOTE: For Glycolysis stress test, use Glycolysis stress test medium. For Cell Mito stress test, use Cell Mito stress test medium with BSA.
<ol>
	<li>Seed cells for the Glycolysis stress test and the Cell Mito stress test in separate plates. For each test, use cells from one flask with overnight culture.</li>
	<li>Centrifuge cells at 200 x g for 5 min at room temperature. Resuspend cells in 1 mL of the appropriate medium and count them.</li>
	<li>Add 4 x 106 of live cells to the final volume of 400 &#956;L (use appropriate medium).</li>
	<li>Plate 50 &#956;L of the cell suspension into wells B-G. Ensure 500,000 cells are seeded in one well. 
	NOTE: It is crucial to seed exactly 500,000 cells per well as no other normalization is performed. That way, the results from different patients can be compared. The optimal number of replicates is six, as is described here. Using less replicates is not recommended since primary cells could sometimes behave erroneously.</li>
	<li>Add 180 &#956;L of the appropriate medium into wells A and H (these wells will serve as a background correction).</li>
	<li>Centrifuge the plate at 400 x g for 5 min at room temperature with brake set to 1.</li>
	<li>Add 130 &#956;L of appropriate medium to wells B-G in two 8-well extracellular flux analyzer plates slowly and carefully. Visually confirm that the cells are stably adhered to the bottom of the wells by viewing under the microscope.</li>
	<li>Place the plate into a humidified, non-CO2, 37 &#176;C incubator for 30 min.</li>
</ol>
7. Loading the Sensor Cartridge
<ol>
	<li>For the Glycolysis stress test, prepare 250 &#956;L each of 100 mM glucose, 20 &#956;M Oligomycin A and 1 M 2-DG, all in Glycolysis stress test medium.</li>
	<li>For the Cell Mito stress test, prepare 250 &#956;L each of 20 &#956;M Oligomycin A, 15 &#956;M FCCP, 30 &#956;M FCCP and a mixture of 10 &#956;M Rotenone and 10 &#956;g/ml Antimycin A, all in Cell Mito stress test medium without BSA. 
	NOTE: Injecting two concentrations of FCCP in one assay is recommended since there is not enough patients&#39; material for the FCCP titration. Nevertheless, the concentrations should be determined by the researcher.</li>
	<li>Load the compounds into the appropriate injector ports of the cartridge as follows (Table 1):</li>
</ol>
8. Setting Up the Program
<ol>
	<li>For the Glycolysis stress test, set up the program as described in Table 2.</li>
	<li>For the Cell Mito stress test, set up the program as described in Table 3.</li>
	<li>Start the program. Replace the calibrant plate with the assay plate (when prompted).</li>
</ol>
9. Evaluation and Interpretation of the Results
<ol>
	<li>In the Glycolysis stress test results, subtract the lowest ECAR value after 2-DG injection from all other ECAR values. 
	NOTE: This lowest value represents the non-glycolytic acidification. Usually, the lowest value is from the 4th measurement.</li>
	<li>Calculate Basal acidification, Glycolysis, Maximal glycolysis and Glycolytic reserve parameters from glycolytic function (Figure 2A). After subtracting the lowest ECAR value, calculate Basal acidification as a mean of ECAR from the first three measurement points (omit the first ECAR value if it significantly differs from the other two), calculate Glycolysis as a mean of ECAR from three measurement points after glucose injection and calculate Maximal glycolysis as a mean of ECAR from three measurement points after Oligomycin A injection. Calculate Glycolytic reserve as Maximal glycolysis minus Glycolysis. 
	NOTE: Alternatively, for Maximal glycolysis calculation, use the highest ECAR value from the three points measured.</li>
	<li>In the Cell Mito stress test results, subtract the lowest OCR value after Rotenone/Antimycin A injection from all other OCR values.</li>
	<li>Calculate Basal respiration, ATP production, Maximal respiration and Spare capacity parameters from mitochondrial function (Figure 2B). After subtracting the lowest OCR value, calculate Basal respiration as a mean of OCR from the first three measurement points. 
	NOTE: Maximal respiration is the highest OCR value after FCCP injection.</li>
	<li>For ATP production calculation, subtract the mean of the three OCR measurement points after Oligomycin A injection from Basal respiration. Calculate Spare capacity as the Maximal respiration minus the Basal respiration. 
	NOTE: Always calculate the Maximal respiration from the highest OCR value, regardless of the FCCP concentration used.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

10.2K Views.  Charles University, Prague. 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.

Video: Assessment of the Metabolic Profile of Primary Leukemia Cells

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

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 ...