Isolation of Myeloid-derived Suppressor Cells from Mouse Tumor and Determining Their Migration Potential In Vitro

Summary

This article outlines the procedures to isolate myeloid-derived suppressor cells from mouse solid tumors and perform an in vitro assay with the cells to determine their response migration potential to certain soluble factors like cytokines and chemokines.

Abstract

The importance of the immune response in cancer and other diseases (like diabetes mellitus, alzheimers, cystic fibrosis) is now known, and the manipulation of the immune system as a therapy to treat cancer is gaining attention. The immune system regulates tumorigenesis both negatively and positively. The myeloid-derived suppressor cells (MDSCs) are a population of immune cells that are increased during cancer, inflammation, and infection. These cells influence the immune response and effectively suppresses the anti-tumor T cell response. They serve as potential targets for therapeutic intervention to effectively use the immune system to inhibit tumorigenesis. To better understand how such intervention can be applied it is important to study these cell types. Using mouse ovarian tumors, we describe the isolation of MDSCs from solid tissue using gentle dissociation techniques. We further describe how MDSCs are isolated from such dissociated tissue based on the expression of cell surface markers with the help of flow cytometry. Additionally, we describe the procedure to perform an in vitro MDSC migration assay to determine the migration potential of these cells in response to soluble factors like cytokines and chemokines.

Introduction

In recent years a number of studies have focused on understanding the role of immune cells in cancer development and progression. One way by which tumor cells evade the immune system is through the expression of immunosuppressive factors that activate, upregulate, and attract immune suppressive cells like MDSCs in the tumor microenvironment¹.

MDSCs are a population of immature myeloid cells that are generated in the bone marrow. Under normal conditions, these immature cells differentiate into mature myeloid cells like macrophages, monocytes, or dendritic cells². Under pathologic condition, these cells fail to differentiate completely into mature myeloid cells. Instead, they are expanded and activated by factors secreted by activated T cells, tumor cells, and stromal cells. Secretory proteins like stem-cell factor³, IL-6, granulocyte/macrophage CSF (G-CSF;M-CSF)⁴, vascular endothelial growth factor⁵,IFNγ⁶, ligands for Toll-like receptors (TLRs)⁷, IL-4⁸, IL-13⁹, and transforming growth factor β (TGFβ)¹⁰ promote MDSC proliferation and activation. Upon activation, these cells upregulate expression of factors like arginase 1¹¹, inducible nitric oxide synthase¹², and reactive oxygen species^{13,14,15,16,17}, resulting in their remarkable potential to suppress the T-cell response. In addition to inhibiting the adaptive immune response, MDSCs have been reported to negatively influence the innate immune response through macrophages¹⁸ and NK cells¹⁹.

These cells lack markers of mature myeloid cells. In mice, MDSCs are characterized by the expression of the cell surface markers GR1 and CD11b. Based on morphology, they are further characterized as granulocytic (CD11b⁺LY6G⁺LY6C^low) ormonocytic (CD11b⁺LY6G⁻LY6C^hi) MDSCs. Both subtypes have immunosuppressive properties, but in mouse tumors, the granulocytic MDSCs are the major population¹⁷. In humans, MDSC markers are not well characterized. Often, the monocytic MDSCs are identified as CD11b⁺ CD14⁺ CD33^+high HLA-DR^neg/Low and CD66b^neg, while granulocytic MDSCs are identified as CD11b⁺ CD14^neg CD33^+low HLA-DR^neg CD66b.

A number of functional studies in mouse tumor models have established the importance of MDSCs in tumor development and T cell suppression. Hence, they are potential targets for therapeutic intervention and it is critical to study this subset of immune cells to understand immune suppression in cancer. We need to identify new factors that may promote or inhibit MDSC expansion and activation, which can be utilized for therapy. To perform such tests, we need to isolate MDSCs in a simple and timely manner to obtain live, non-contaminated cells. Here, we outline the methods to isolate a viable MDSC population from mouse tumor. This method can be used to isolate MDSC from any other tissue following the same method. Cell types other than MDSCs can also be isolated by this method using the appropriate cell-specific markers. We also outline methods to determine MDSC migration potential towards a cytokine gradient. Here we use tumor necrosis factor α (TNFα) as an example. Other cytokines can also be tested. Instead of conditioned media from cell lines, regular media supplemented with specific cytokines in varying concentrations can be used to test the role of those cytokines in MDSC migration from a particular tumor type.

Protocol

All procedures were performed under the guidance of University of Texas at MD Anderson IACUC review board.

1. Reagent Preparation

1. Preparation of medium for migration assay
   1. Prepare 500 mL of complete RPMI media for the migration assay: RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% pen-strep (penicillin 500 U/mL and streptomycin 500 µg/mL).
2. Preparation of buffers for staining MDSCs
   1. Prepare the tumor dissociation buffer with 100 U/mL Collagenase type IV and 100 µg/mL DNase in RPMI with 10% FBS. Store the buffer on ice.
   2. Prepare the 1X RBC lysis buffer by diluting the 10X stock 1:10 in sterile water.
   3. Prepare the FACS buffer by adding 1% heat-inactivated FBS to 1X PBS.
   4. Add 1 µL of amine reactive viability dye (binds and fluoresces non-viable or necrotic cells more strongly than live cells) to 1 mL of FACS buffer to stain for dead cells. Store the solution in the dark on ice or at 4 °C.
   5. Prepare the staining buffer by adding 20 µL each of the following antibodies (α-FITC-GR1, α-BV510-CD11b, α-APC-Cy7-CD45, α-PE-Cy7-Ly6C, and α-APC-Ly6G) in 940 mL of FACS buffer with cell viability dye. Keep the antibody cocktail covered on ice or 4 °C.
3. Generation of murine tumors
   NOTE: When syngeneic mice are injected with a murine ovarian cancer cell line 333 derived from TPP²⁰ mice, they will spontaneously form tumors 8-12 weeks post injection. These cells can be modified to stably express PRKCI by lentiviral infection²¹.
   1. Grow 333 cells in DMEM with glutamine supplementation on 15 cm tissue culture plates in a tissue culture incubator at 37 °C and 5% CO₂.
   2. To passage the cells: aspirate the media, add 5 mL of 2.5% trypsin with EDTA, and incubate for 1-2 min at 37 °C and 5% CO₂. When the cells detach from the tissue culture plate, neutralize trypsin with 5 mL of DMEM and collect the cells in a 15 mL tube. Centrifuge the cells at 300 x g for 5 min at room temperature.
   3. Discard the supernatant and resuspend the cell pellet in 10 mL of HBSS media. Count the cells using trypan blue (4%, 1:1 cell suspension to dye ratio) and a cell counter.
   4. Aliquot the cell suspension according to cells required and concentration of cells. Inject intraperitoneally, 10 million cells per mice into 4-6 week old syngeneic BL/6 mice. Let the tumors grow for 8-12 weeks.

2. Isolation of Single Cells from Tumor Tissue and Staining Cells for FACS

1. When the mice develop tumors 8-12 weeks post injection (determined by bloated abdomen cavity), euthanize the mice by CO₂ asphyxiation.
2. Pin down all four limbs of a mouse on a polystyrene foam board with the abdominal cavity facing up. Cut open the abdominal cavity using sharp scissors and expose the tumor tissue. If the tissue is isolated from a large tumor, avoid using the center mass of the tumor if it appears necrotic.
3. Aseptically collect the freshly dissected murine intraperitoneal tumor from the mice in a 50 mL tube with 2.5 mL/g of tumor dissociation buffer to tissue.
   NOTE: This protocol can be used for subcutaneous xenograft tumors or for the isolation of single cells from most mouse tissue. The percentage of MDSCs in tumors varies and needs to be determined for the specific tumor type. To collect enough MDSCs from the isolation, multiple tumor samples from the same experimental background can be combined.
4. In a tissue culture hood, transfer the tissue to a culture dish with a small amount of tumor and dissociation buffer and chop the tumor with a razor blade into smaller pieces, about 2 mm in size.
5. Transfer the chopped tissue back into the tube and incubate the partially dissociated tissue at 37 °C with continuous rotation on a rotator at approximately 12 rpm for 45 min.
6. After incubation, centrifuge the dissociated tissue in the tube at 300 x g for 5 min at room temperature. Discard the supernatant.
7. Resuspend the dissociated tissue in 25 mL of RPMI and filter through a 70-µm cell strainer placed on a 50 mL tube to collect single cells from the dissociated tissue.
8. Centrifuge the single cell suspension at 300 x g for 5 min at room temperature and discard the supernatant.
9. Add 25 mL of 1X RBC lysis buffer to the tube and incubate on ice for 5 min to lyse RBCs in the cell suspension.
10. Centrifuge the cells at 300 x g for 5 min at room temperature and discard the red supernatant. If the cell pellet still appears red or dark brown, repeat the RBC lysis step (step 2.9).
11. Add 25 mL of PBS to the cell pellet and centrifuge at 300 x g for 5 min at room temperature. Discard the supernatant.
12. Resuspend the isolated single cells in 1 mL of FACS buffer and proceed to stain the immune cells.

3. Staining and Isolation of MDSCs from Single Cells Isolated from Mouse Tumor

1. Aspirate two 50 µL aliquots of the single cell suspension into centrifuge tubes for the controls. To one tube, add 100 µL of FACS buffer. To the other tube, add 100 µL FACS buffer containing cell viability dye (see step 1.2.4). Keep the controls on ice until use.
2. Centrifuge the remaining cell suspension at 300 x g for 5 min and then incubate the cell pellet with FACS staining buffer (see step 1.2.5) containing anti-CD45 (4 µg/mL), anti-Gr1 (10 µg/mL), anti-Cd11b (4 µg/mL), anti-Ly6C (4 µg/mL), and anti-Ly6G (4 µg/mL) antibodies.
   1. Incubate the cell suspension with the antibody solution on ice in the dark for 30 min.
   2. Centrifuge the antibody conjugated cells at 300 x g for 5 min at 4 °C and discard the supernatant. Keep the tubes covered with aluminum foil to avoid light exposure.
   3. Resuspend the cells in FACS buffer, count them using a cell counter, centrifuge the cells at 300 x g for 5 min at 4 °C, and discard the supernatant. Repeat this step once more and discard the supernatant.
   4. Resuspend the stained cells in 1 mL of FACS buffer and keep on ice covered with aluminum foil before FACS.
3. For the color compensation controls for flow cytometry, take 100 µL of compensation beads in a centrifuge tube and add 1 mL of FACS buffer. Centrifuge the beads at 300 x g for 5 min and discard the supernatant.
   1. Resuspend the beads in 150 µL of FACS buffer and divide them equally into three parts (50 µL/tube). To each part, add anti-FITC-GR1 (1.6 µg/mL), anti-BV510-CD11b (0.6 µg/mL), and anti-APC-Cy7-CD45 (0.6 µg/mL) for the staining. Incubate for 15 min on ice in the dark.
   2. Centrifuge the beads at 300 x g for 5 min and discard the supernatant.
   3. Add 1 mL of FACS buffer and centrifuge the beads at 300 x g for 5 min. Discard the supernatant.
   4. Resuspend the beads in 500 µL of FACS buffer and store on ice before flow cytometry and cell sorting.

4. Flow cytometry and Cell Sorting of the MDSCs from Mouse Tumor

NOTE: See Figure 1. Here, we performed flow cytometry and cell sorting in a core facility.

1. Sort the stained MDSCs using fluorescence-activated cell sorting (FACS). For color compensation, use the compensation beads stained with individual antibodies (step 3.3). Use the cells stained with only the viability dye for the compensation of dead and live cells (step 3.1).
2. Use the unstained cells (step 3.1) as a negative control before running the stained samples.
3. For sorting the samples, gate on the total events based on forward and side scatter of the density plots to remove all doublets and cellular debris.
4. Exclude all dead cells based on the positive viability dye staining.
5. Gate on live single cells positive for CD45 staining (total leukocytes), followed by cells double positive for CD11b⁺ and Gr1⁺ to collect MDSCs. CD11b⁺/Gr1⁺ MDSCs can be further gated for monocytic and granulocytic markers with Ly6C and Ly6G as shown in Figure 1. For the purpose of the MDSC migration assay, CD11b⁺/Gr1⁺ MDSCs are sufficient.
6. Sort the cells into a collection tube with complete RPMI media.

5. MDSC Migration Assay

1. Centrifuge the sorted MDSCs at 300 x g for 5 min at room temperature. Discard the supernatant.
2. Resuspend the cell pellet with 10 mL of RPMI media with pen-strep and without FBS. Centrifuge at 300 x g for 5 min at room temperature and discard the supernatant. Repeat this step once.
3. Prepare a cell suspension of the sorted cells at 0.3-0.5 x 10⁶ cells per 0.5 mL of RPMI media with pen-strep and without FBS.
4. Count the cells using trypan blue (4%, 1:1 cell suspension to dye ratio) and a cell counter.
5. Add 500 mL/well of complete RPMI media (with pen-strep and FBS; see step 1.1) of a 24-well plate. Add desired concentrations of the cytokine to be tested. As a control, add neutralizing antibody corresponding to the cytokine tested as one set of conditions. Replicate each condition in at least 3 wells.
6. Gently place a membrane-insert into each well to avoid the formation of any bubbles under the membrane.
7. Add 500 mL of cell suspension containing 0.3-0.5 x10⁶ sorted MDSCs into each membrane insert and incubate the plate for 6 h at 37 °C, 5% CO₂ in a tissue culture incubator.
8. Count the migrated MDSCs in the bottom chamber of the transwell system by counting the absolute number of FITC positive cells using flow cytometry.

Results

Here, we present results obtained from the isolation of MDSCs from mouse ovarian tumors²⁰. Following the procedure described above, we isolated single cells and stained them for MDSCs. MDSCs in the tumors were labeled with APC-Cy7-CD45, FITC-GR1, PE-CD11b. To elucidate the MDSC population, these cells can be further stained with Ly6C and Ly6G, as shown in the gating strategy in Figure 1. Labeled cells were sorted by flow cytometry. Lab...

Discussion

We have described the methodology to isolate MDSCs from mouse ovarian tumor. The same method can be utilized for isolating MDSCs or other immune cells from any solid normal tissue or solid tumor using cell-specific markers. Additionally, depending on the nature of the tissue the incubation time with the dissociation buffer will need to be optimized.

The isolation of viable immune cells from tumor tissue depends on performing the different isolation steps from dissecting tumors to obtain the so...

Materials

Name	Company	Catalog Number	Comments
Collagenase IV	Thermo Fisher	17104019	Tumor dissociation
Cell strainer	Falcon	352350	Cell strain
RBC lysis buffer	Biolegend	420301	Cell culture medium
DMEM+Glutamax	Gibco	10569010	Cell culture medium
RPMI	Gibco	11875093	Cell culture medium
FBS	Gibco	10082147	Serum for Cell culture
Phosphate Buffered Saline (PBS)	Sigma	D8537-500ML	PBS
Trypsin	Invitrogen	25200-056	Cell dissociation
Penstrep	HyClone	SV30010	Antibiotic
Sterile water	Invitrogen	10977-015	For dilution of buffers
BV605-CD11b	Biolegend	101237	Antibody for MDSC labeling
FITC-GR1	Biolegend	108405	Antibody for MDSC labeling
APC-Cy7-CD45	Biolegend	103115	Antibody for lymphocyte labeling
PE-Cy7-Ly6C	Biolegend	128017	Antibody for MDSC labeling
APC-Ly6G	Biolegend	127613	Antibody for MDSC labeling
TNFα	Sigma	T7539	Cytokine
TNFα neutralizing antibody	Biolegend	506309	neutralizing antibody
Ghost Dye Violet 450	Tonbo Biosciences	13-0863	Cell viability dye
UltraComp Beads	Invitrogen	01-2222-42	Compensation beads
Cell culture inserts	Corning	353097	Migration chamber
LSR Fortessa X-20	BD Biosciences	LSR Fortessa X-20	Fluorescent Cell analyzer
BD FACSAria Fusion	BD Biosciences	BD FACSAria Fusion	Fluorescent Cell sorter
Cell countess	Cell countess	Cell countess	Cell countess
Flow Jo			Software for Analysis of flow data
Prism			Plotting of graph and statistical analysis
C57BL/6 mice	Taconic	B6-F	mice for tumor generation