Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Summary

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Abstract

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils ≥ 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Introduction

Innate immune cells constitute an essential proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer¹. Recently, it has become more widely appreciated that chronic immune responses play critical roles in promoting tumor progression, metastasis and resistance to chemotherapies². Macrophages are important innate immune cells that have been shown to directly regulate tumor cell response to chemotherapy ^3,4. However, the role of neutrophils, key players in the innate immune system, in regulating tumor response to anti-cancer treatment is not known. The aims of these protocols are to use a fast and credible method to separate neutrophils from CLL patient's blood samples and to differentiate HL60 cells along the granulocytic pathway in order to study their role in regulating the sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant cellular component of the innate immune system in blood ⁵ and act as a first line of defense against invading microorganisms ⁶. Neutrophils have an essential role in rising effective innate immune responses in addition to variable effector functions in several pathological conditions ⁷. Therefore, a fast and credible method to isolate neutrophils from other blood cells, such as density gradient separation method, is required for in vitro studies. Using this method for neutrophil isolation will facilitate further research on neutrophil-mediated immunological functions in vivo and ex vivo.

The ability to obtain pure populations of neutrophils is an important first step for the investigation of patients with immunological diseases ⁸. Density gradient separation method is an ideal technique in which a high yield of cells is obtained. The method involves the addition of density gradient solution in the bottom of a tube containing diluted human blood followed by centrifugation at 300 g for 35 min without break. The ring of the mononuclear cells appears at the interface and the neutrophils reside below the former. This method have significant advantages with respect to other available methods such as neutrophil isolation kits which are much more expensive⁹. In addition, isolating neutrophils from human blood by commercial kits using antibodies directed to a surface marker specific for human neutrophils, increase the risk of cell activation or differentiation. Density gradient separation method allows the isolation of neutrophils within a short period of time. Within the same step, mononuclear cells are also separated and recovered. It's a fundamental technique in which a high yield of pure cells is obtained in order to achieve functional integrity.

In order to mimic the tumor microenvironment, 3D experiments were performed. Given the short half-life of neutrophils in vitro, the 3D experiments with fresh human neutrophils are not conclusive. For this reason, promyelocytic (HL60) cells are induced to differentiate into neutrophil-like cells using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). Using differentiated HL60 cells (HL60diff) will prevent having different responses of neutrophils due to isolation from different donors.

In vitro 3D culture models represent an intermediate stage between in vitro 2D models and in vivo models. In 2D culture, the cells spread on plastic surface forming unnatural cell attachments to deposited proteins that are denatured on this synthetic surface. Conversely, the cells in 3D culture form natural cell-cell attachments since the cells and the extracellular matrix they synthesize are the natural material to which they are attached. For this reason, 3D co-culture models, especially between cancer cells and other cell types, have been very useful for indicating their contribution to tumor growth, angiogenesis, and metastasis. As a result, 3D cultures make the cell culture mimic the physiological conditions that exist in vivo ¹⁰.

Protocol

1. Neutrophil Isolation and Co-culture with Primary Leukemic Cells

NOTE: Procedures were conducted under the approval of Lyon Hospital Ethics committee with all patients signing informed consent.

1. Isolation of Primary Leukemic Cells and Neutrophils
   1. Collect tubes of peripheral blood on EDTA (1.8 mg EDTA per milliliter of blood) from patients diagnosed with chronic lymphocytic leukemia (CLL).
   2. Add each 15 ml of blood to a sterile 50 ml tube and dilute with 15 ml RPMI (dilution 1:1), then carefully and slowly add 15 ml of density gradient solution to the bottom of the tube without mixing the phases. Ensure that the density gradient solution is at RT for the preparation.
   3. Centrifuge at 300 x g for 35 min at RT and without brake. The blood should separate into four distinct phases as shown in Figure 1A, top to bottom: platelet and plasma, mononuclear cells (white ring), density gradient solution, granulocytes and erythrocytes.
   4. Collect the white ring which represents the primary leukemic cells with a plastic Pasteur pipette and transfer to a new 50 ml tube.
      1. Fill the tube with PBS (contains calcium and magnesium) up to 50 ml in total and centrifuge at 300 g for 10 min at RT.
      2. Resuspend the pellet with 5 ml PBS then add PBS up to 50 ml in total and centrifuge at 300 g for 10 min at RT.
      3. Resuspend the pellet with complete RPMI medium (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin) for counting using cell viability counter.
   5. Aspirate the upper phases leaving the granulocytes and erythrocytes phase and add PBS up to 25 ml then add 3% dextran in 0.9% NaClup to 50 ml in total. Mix the tubes 10 times and keep them for 30 min at RT without mixing.
   6. Collect the upper RBC-poor neutrophil layer and place them into a clean sterile 50 ml tube and centrifuge the tubes at 300 g for 10 min at RT. Resuspend each pellet with 5 ml PBS then add red cell lysis buffer up to 50 ml in total.
   7. Keep the tubes in dark for 15 min at RT then centrifuge at 500 g for 10 min at RT. Wash the pellets with PBS (add PBS up to 50 ml in total) then centrifuge at 500 g for 10 min at RT.
   8. Resuspend the pellets with complete RPMI medium for counting using cell viability counter. Keep 3 x 10⁵ of each cell type in 50 µl PBS-FBS (4%) in 5 ml plastic tubes to determine the purity of their isolation using flow cytometry.
2. Analyze the Morphological Appearances of the Isolated Cell Populations
   1. To do so, resuspend each 3 x 10⁴ neutrophils and 3 x 10⁴ mononuclear cells in 150 µl complete RPMI medium. Assemble the glass slides, filter cards, and sample chambers in the cytospin centrifuge, ensuring that the centrifuge is well balanced.
   2. Place each cell type into sample chamber and cyto-centrifuge at 750 x g for 10 min at RT Remove the slides, filter cards, and sample chambers from the centrifuge. Disassemble carefully, so as not to damage the cells on the slide. Discard filter cards and sample chambers.
   3. Stain the cells using Giemsa staining kit and keep the slides for 1 hr to dry. Examine the cells by microscope with 100X magnification.
3. Test the Purity of Isolated Cells by FACS
   1. Label the isolated primary leukemic cells with anti-human CD19 antibody conjugated to APC (5 µl/10⁶ cells). Label the purified neutrophils with mixture of anti-human antibodies listed in Table 1 (5 µl/10⁶ cells). Incubate the cells in dark for 30 min at 4 ^oC.
   2. Wash the cells with 500 µl PBS-FBS (4%) the centrifuge the tubes at 300 g for 5 min at RT.
   3. Resuspend each pellet in 200 µl PBS-FBS (4%).
   4. Analyze on a flow cytometer using the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PerCP-Cy5.5 (695/40 BP), PE (575/26 BP); 633 nm laser: APC (660/20 BP), APC-Cy7 (780/60 BP). Ensure the color compensation.
4. Coculture of Primary Leukemic Cells with Autologous Neutrophils
   1. Seed 2 x 10⁵ cells/ml of primary leukemic cells alone or with autologous neutrophils at ratio 1:10 in complete RPMI medium. To do so, adjust cell numbers by diluting cell suspensions and add 2 x 10⁵ cells/ml of primary leukemic cells to 2 x 10⁶ cells/ml neutrophils. Mix carefully.
   2. Add 25 µM of Bruton's tyrosine kinase (Btk) inhibitor (ibrutinib) to the cells. Incubate for 24 hr at 37 °C with 5% CO₂.
5. Cell Harvest and FACS Analysis
   1. Collect the cells in 5 ml plastic tubes for FACS analysis and centrifuge the tubes at 300 g for 5 min at RT. Wash the pellets with 1 ml PBS-FBS (4%) then centrifuge at 300 g for 5 min at RT.
   2. Resuspend pellets in Annexin V and PI using commercial kit and incubate the cells in dark for 10 min at RT. Analyze on a flow cytometer using the gating strategy of Figure 2A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PI (610/20 BP). Ensure the color compensation.

2. Differentiation of HL60 Cells along the Granulocytic Pathway and Their Coculture with RL Lymphoma B Cells in 3D Model

1. Differentiation of Human Promyelocytic (HL60) Cells into Neutrophil-like Cells
   1. Adjust HL60 cell number to 3 x 10⁵cells/ml then add 1 µM retinoic acid (RA) and 1.25% DMSO to induce their differentiation. Seed each 3 x 10⁵ HL60 cells per well in 48-well plate then incubate for 48 hr at 37 °C with 5% CO₂.
   2. Later, collect the cells and centrifuge at 300 g for 5 min at RT. Resuspend the cells with complete RPMI medium for counting using cell viability counter.
   3. Then dilute cell suspension in complete RPMI medium to adjust HL60 cell number to 3 x 10⁵ cells/ml and induce again their differentiation by adding 1 µM retinoic acid (RA) and 1.25 % DMSO. Seed each 3 x 10⁵ HL60 cells per well in 48-well plate and incubate for another 48 hr at 37 °C with 5% CO₂.
2. Analysis of HL60 differentiation (HL60diff)
   1. Collect the cells and centrifuge the tubes at 300 g for 5 min at RT. Resuspend the pellet with complete RPMI medium for counting using cell viability counter.
   2. To analyze the changes in cell-surface markers expression by flow cytometry. Place each 3 x 10⁵ undifferentiated HL60 cells (from cell culture) and 3 x 10⁵ HL60diff cells in 5 ml plastic tubes for FACS analysis and centrifuge the tubes at 300 g for 5 min at RT.
   3. Resuspend the pellets in 50 µl PBS-FBS (4%). Label the cells with anti-human CD11b conjugated to AF700 or anti-human CD38 conjugated to APC (5 µl/10⁶ cells). Incubate the cells in dark for 30 min at 4 ^oC.
   4. Wash with 500 µl PBS-FBS (4%) then centrifuge at 300 g for 5 min at RT. Resuspend the pellets in 200 µl PBS-FBS (4%).
   5. Analyze on flow cytometer using the gating strategy of Figure 3A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP); 633 nm laser: APC (660/20 BP), Alexa Fluor 700 (730/45 BP).
   6. To test the morphological changes in HL60diff, immobilize the cells onto glass slides for microscopic examination.
      1. To do so, resuspend each 3 x 10⁴ undifferentiated HL60 cells (from cell culture) and 3 x 10⁴ HL60diff in 150 µl complete RPMI medium. Assemble the glass slides, filter cards, and sample chambers in the cytospin centrifuge, ensuring that the centrifuge is balanced.
      2. Place each cell type into sample chamber and cyto-centrifuge at 1,500 rpm for 10 min at RT. Remove the slides, filter cards, and sample chambers from the centrifuge. Disassemble carefully, so as not to damage the cells on the slide. Discard filter cards and sample chambers.
      3. Stain the cells using Giemsa staining kit and keep the slides for 1 hr to dry. Examine the cells by microscope with 100X magnification.
3. 3-dimensional (3D) Culture
   NOTE: Ensure that the materials used in this experiment are cold and the experiment is taking place on ice.
   1. Resuspend each 5 x 10⁴ RL cells, either alone or mixed with HL60diff at ratio RL:HL60diff 1:10, with 300 µl basement membrane matrix using 1 ml pipette tip cut with a sterile scissor in order to widen the opening to about 2 to 3 mm. Avoid bubbles during this step.
   2. Seed each 300 µl of cell suspension/well in 24-well plate. Avoid bubbles during this step. Incubate the plate for 30 min at 37 °C with 5% CO₂then add 1 ml complete RPMI medium for each well and incubate for 7 days at 37 °C with 5% CO₂. Change the medium every two days. Add 10 nM vincristine at day 5.
4. FACS Analysis
   1. After 7 days of culture, aspirate the medium and wash each well with 1 ml ice-cold PBS twice. Add 3 ml/well of ice-cold PBS-EDTA (5 mM). Detach the gel from the bottom of the well by scraping using the bottom of 200 µl pipette tip. Shake the plate gently on ice for 30 min.
   2. Transfer cell suspension into sterile 15 ml tube and shake the tubes gently on ice for another 30 min. Check for the appearance of homogeneous cell suspension. (If it's not the case, then shake the cells for longer time or add more PBS-EDTA).
   3. Centrifuge the tubes at 300 x g for 10 min at RT. Wash the pellets with PBS then centrifuge at 300 x g for 10 min at RT.
   4. Resuspend with PBS-FBS (4%) and label with anti-human CD19 antibody conjugated to PE-Cy7 and anti-human CD38 antibody conjugated to APC (5 µl/10⁶ cells). Incubate in dark for 30 min at 4 ^oC.
   5. Wash with 500 µl PBS-FBS (4%) then centrifuge tubes at 300 g for 5 min at RT. Resuspend the cells with Annexin V and PI using commercial kit.
   6. Analyze on flow cytometer using the gating strategy of Figure 4A and the following optical configuration: 488 nm laser: FSC-A (488 nM), SSC-A (488/10BP), FITC (530/30 BP), PI (610/20 BP), PE-Cy7 (780/60 BP); 633 nm laser: APC (660/20 BP). Ensure the color compensation.

Results

Density gradient separation method described here provides primary leukemic cells and unstimulated neutrophils isolated from the blood of CLL patients. Figure 1A represents the different blood layers obtained after density gradient centrifugation (from top to bottom: platelets and plasma, white ring represents the mononuclear cells, density gradient solution, granulocytes and erythrocytes). Figure 1B and 1C show the differences in the morphological appear...

Discussion

Described here, an effective, simple, fast and inexpensive protocol for the isolation of neutrophils from human blood with high purity using density gradient centrifugation approach and within the same step mononuclear cells are also separated and recovered. The isolated cell populations are ≥90% pure.

Several methods are available for neutrophil isolation from human blood. These include similar methods using discontinuous gradients^11,12, or using commercial kits for neutrophi...

Materials

Name	Company	Catalog Number	Comments
RPMI 1640	Gibco Invitrogen	21875-034
Fetal bovine serum (FBS)	Gibco Invitrogen	10270-106
Phosphate-buffered saline (PBS contains calcium and magnesium)	Gibco Invitrogen	14040-091
N-acetyl-L-alanyl-L-glutamine (L-Glutamine)	Life technologies	25030-024
Penicillin streptomycin (Pen Strep)	Life technologies	15140-122
Bruton's tyrosine kinase (Btk) inhibitor (Ibrutinib)	CliniSciences	A3001
Vincristine	EG labo
BD Matrigel basement membrane matrix	BD Biosciences	354234	Put at 4 oC overnight (O/N) before the day of the experiment
Red cell lysis buffer	BD Biosciences	555899
Ficoll (Pancoll)	PAN Biotech	P04-60500
Dextran	Sigma-Aldrich	D8906
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Retinoic acid	Sigma-Aldrich	R2625
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E5134
Sodium chloride (NaCl)	Euromedex	S3014
Annexing V-FLOUS staining kit	Roche	11 988 549 001
Kit RAL 555 Modified Giemsa staining kit	Cosmos Biomedical	CB361550-0000
LSRII flow cytometry	BD Biosciences
Cytocentrifuge	Thermo Scientific
Leica DMR-XA microscope	Leica Microsystems
Cellometer Auto T4 Cell Viability Counter	Nexcelom  Bioscience