Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

Summary

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

Abstract

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

Introduction

The tumor microenvironment (TME) is comprised of rapidly proliferating neoplastic cells and a surrounding heterogeneous stromal cell compartment. As growing tumors are often poorly vascularized, the TME is a peripheral site uniquely characterized by hypoxia, nutrient deprivation, and acidosis¹. To survive in this landscape, tumor stress responses and metabolic reprogramming result in the secretion of soluble factors that promote tissue remodeling and angiogenesis as well as the selective recruitment of immune cells². As myeloid cells are one of the most abundant type of hematopoietic cells in the TME, there is increasing interest in examining the role of tumor-infiltrating myeloid cells in the TME.

Myeloid cells are a heterogenous and plastic group of innate immune cells including monocytes, macrophages, dendritic cells, and granulocytes. Although they have critical roles in tissue homeostasis and adaptive immune response regulation, their function can be polarizing depending on the composition of activation signals within the local microenvironment³. Tumors take advantage of myeloid cell characteristics through the secretion of soluble factors within the TME. These alternative signals can divert myelopoiesis towards immature differentiation and skew the function of existing tumor-infiltrating myeloid cells³. Indeed, myeloid cells within the TME often promote cancer progression and can suppress antitumor immune responses, leading to adverse effects on cancer therapy.

Although therapeutic strategies promoting the depletion of immunosuppressive myeloid cells have been shown to delay tumor growth⁴, the lack of target specificity risks the removal of immunostimulatory myeloid cells, which by contrast, aid in the resolution of cancer. These inflammatory myeloid cells can exert profound antitumor effects including direct tumor cell killing and activation of cytotoxic CD8⁺ T cells⁵. Alternatively, strategies normalizing the composition and function of myeloid cells in the TME have shown therapeutic success⁶; however, the biological mechanisms underlying their re-education towards an antitumor phenotype have still not been fully understood. Ultimately, a comprehensive characterization of tumor myeloid cells is necessary for further improvement of cancer therapy.

Unfortunately, reproducible disaggregation of tumors for myeloid cell isolation is challenging. Tumor-derived myeloid cells are sensitive to ex vivo manipulation compared to other leukocyte subsets, and the aggressiveness of tumor processing can lead to enzymatic epitope cleavage and reduced viability of recovered cells⁷. The purpose of this method is to provide a reliable means of tumor dissociation to preserve surface marker integrity for analysis and cellular vitality for functional study. In comparison to tumor-infiltrating leukocyte (TIL) isolation protocols that favor harsher enzymatic mixes to enhance the reproducible release of various cellular subsets, this method favors more conservative enzymatic digestion to maximize myeloid cell recovery. High-level multi-color flow gating strategies are also provided to identify murine tumor myeloid cell subsets for further characterization and/or sorting.

Protocol

NOTE: All animal studies complied with the Canadian Council on Animal Care guidelines and were approved by McMaster University's Animal Research Ethics Board.

1. Tumor harvest and dissociation

1. Inoculate 6-8-week-old, female, C57BL/6 mice intradermally/subcutaneously with 2 × 10⁵ B16 melanoma cells as described by Nguyen et al.⁸ Allow tumors to grow for 7 days before harvesting.
2. Euthanize the mouse by cervical dislocation while making sure to not disrupt the tumor when doing so. Spray the mouse down with 70% ethanol before harvesting.
3. Using a scalpel and scissors, surgically remove the intradermal/subcutaneous tumor from surrounding tissue (including attached tumor-draining lymph nodes), and place the tumors into a preweighed microfuge tube. Keep on ice.
   NOTE: Conduct tumor harvest in an animal-use biosafety cabinet. Use a 15 mL conical tube for larger tumors.
4. Weigh the tumors, and add 500 µL of RPMI-1640 medium with 10% fetal bovine serum (FBS) to each tube, using scissors to cut the tumors into small pieces within the tube or in a 6-well plate.
   NOTE: The tumor pieces should be small enough to be mixed by an electric pipettor once the digestion medium has been added.
5. Prepare the dissociation mix by dissolving collagenase type IV at 0.5 mg/mL and DNase at 0.2 mg/mL in RPMI-1640 medium with 10% FBS and 5 mM calcium chloride.
   NOTE: Dissociation mix must be prepared fresh to maximize collagenase activity.
6. Transfer the minced tumor suspension to a 15 mL conical tube, and add 10 mL of dissociation mix per 0.25 mg of tumor. Place the tube in a temperature-controlled orbital shaker for 30 min at 37 °C with 200 rpm agitation. Neutralize the collagenase activity by adding two volumes of cold RPMI-1640 medium with 10% FBS and 2 mM ethylenediamine tetraacetic acid (EDTA), and refrigerate for 10 min at 4 °C.
7. Briefly vortex and pipette the suspension into a 40 µm strainer on a 50 mL conical tube. Use a syringe plunger and neutralizing media to disaggregate the residual tumor tissue, and wash it through the strainer. Centrifuge the suspension for 5 min (500 × g, 4 °C), discard the supernatant, and resuspend the pellet in phosphate-buffered saline (PBS) with 2% FBS and 1 mM EDTA.

2. TIL enrichment and flow cytometric staining (FACS)

1. To enrich TILs for myeloid cell characterization, use a magnetic cell separation kit designed for biotin-positive selection with biotinylated CD45.2 antibodies according to the manufacturer's instructions (see the Table of Materials).
2. Resuspend the cells in 200 µL of FACS buffer (PBS with 0.5% w/v bovine serum albumin (BSA)), and transfer them to a 96-well U-bottom plate.
   NOTE: Do not exceed a staining concentration of 1 × 10⁸ cells/mL. Adjust the volume, and split samples into multiple wells to compensate for high cell numbers.
3. Centrifuge the plate for 5 min (500 × g, 4 °C), and discard the supernatant. Add 50 µL of Fc block solution (1:200 dilution of purified rat anti-mouse CD16/CD32 [see the Table of Materials) in FACS buffer, final concentration of 2.5 µg/mL), and resuspend the cells by pipetting. Incubate for 10 min at 4 °C.
4. Add 50 µL of FACS buffer containing 2x concentration of surface-staining antibody (1:50 dilution of CD45.2, NK1.1, CD11c, F4/80, CD8a, Ly6C, CD11b, CD4, Ly6G) and fixable viability stain (FVS, 1:500 dilution), and mix the cells by pipetting. Incubate for 20 min at 4 °C.
   NOTE: Cover the plate with aluminum foil to minimize light exposure. Antibodies should be titrated prior to the experiment to empirically determine the optimal dilution.
5. Wash the cells twice by adding 200 µL of FACS buffer to each well, centrifuging the suspension (5 min, 500 × g, 4 °C), and discarding the supernatant.
6. Add 100 µL of fixation/permeabilization solution (see the Table of Materials) to each well, mix the cells by pipetting, and incubate for 20 min at 4 °C.
7. Add 100 µL of 1x permeabilization buffer (see the Table of Materials) to each well, centrifuge the plate for 5 min (500 × g, 4 °C), and discard the supernatant.
8. Wash the cells by adding 200 µL of 1x permeabilization buffer to each well, centrifuging the suspension (5 min, 500 × g, 4 °C), and discarding the supernatant.
   NOTE: The experiment can be paused overnight after resuspending the cells in permeabilization buffer. Store the sample at 4 °C and protected from light. Resume after briefly mixing the cells before centrifuging.
9. Add 100 µL of permeabilization buffer containing 1x concentration of intracellular staining antibody (1:100 dilution of nitric oxide synthase 2 (NOS2), arginase 1 (Arg1)), and mix the cells by pipetting. Incubate for 20 min at 4 °C.
   NOTE: Cover the plate with aluminum foil to minimize light exposure. Antibodies should be titrated prior to the experiment to empirically determine the optimal dilution.
10. Add 100 µL of 1x permeabilization buffer to each well, centrifuge the plate for 5 min (500 × g, 4 °C), and discard the supernatant.
11. Wash the cells by adding 200 µL of 1x permeabilization buffer to each well, centrifuging the suspension (5 min, 500 × g, 4 °C), and discarding the supernatant.
12. Resuspend the cells in 300 µL of FACS buffer. Filter the sample through a 5 mL round-bottom polystyrene tube with 40 µm strainer cap before performing flow cytometry analysis.

3. Tumor myeloid cell sorting for functional studies

1. After identifying the desired myeloid cell populations by flow cytometry analysis, pre-enrich bulk myeloid cells for sorting with a magnetic cell separation kit designed for CD11b- or CD11c-positive selection according to the manufacturer's instructions (see the Table of Materials).
2. Using surface-staining antibodies specific for the desired myeloid cell subsets (1:100 dilution of CD11b, Ly6C, Ly6G), stain the pre-enriched cells as described in steps 2.2-2.5.
   NOTE: Include fixable viability stain to ensure the sorting of live myeloid cells. Do not exceed a staining concentration of 1 × 10⁸ cells/mL. Adjust the volume, and split the samples into multiple wells to compensate for high cell numbers.
3. Resuspend the cells in cold sorting buffer (PBS with 1% w/v BSA, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1 mM EDTA). Filter the sample through a 5 mL round-bottom polypropylene tube with a 40 µm strainer cap. Keep the cells on ice, and cover the tube with aluminum foil.
   NOTE: Adjust the concentration of cells/volume to the desired instrument specification for sorting.
4. Prepare a sample collection tube with capture medium (5 mL round-bottom polypropylene tube containing PBS with 50% FBS).
   NOTE: Coat tubes with 5 mL capture medium overnight prior to sorting. Discard all but 1-2 mL of the capture medium on the next day before running the sample.
5. Modify the sorter instrument settings to decrease sample pressure and prevent perturbations in droplet formation. Equip the 130 µm nozzle tip, and utilize the 10 psi setting. Run the sample at a low flow rate with periodic sample agitation at 100 rpm, ensuring that the deposition of droplets is in the center of the tube. After the sort, keep the sample on ice.
6. Incubate the sample for 10 min at 4 °C. Centrifuge the tube for 5 min (500 × g, 4 °C), discard the supernatant, and resuspend the pellet in the desired medium for functional assays or adoptive transfer.

4. Adoptive transfer of purified tumor myeloid cells

1. Inoculate 6-8-week-old, female, C57BL/6 mice intradermally with 1 × 10⁶ B16 melanoma tumor cells resuspended in 30 µL of PBS.
   NOTE: Inoculate mice in advance such that tumor growth does not exceed 100 mm³ by the time of adoptive transfer.
2. Resuspend the sorted tumor myeloid cells in PBS with 25 mM HEPES and 1 mM EDTA at a concentration of 2 × 10⁶ cells/mL. Filter the sample through a 5 mL round-bottom polystyrene tube with a 40 µm strainer cap. Keep on ice.
3. Induce and maintain mice under anesthesia with 3% isoflurane. Apply ophthalmic ointment to prevent ocular dryness/injury.
4. Load a 31 G syringe with 50 µL of cell suspension. Dislodge air bubbles by gently flicking the syringe. Clean the injection site using an alcohol swab.
5. Using sterile forceps, lift the skin at the base of the tumor. Insert the needle into the subcutaneous space at a slight upward angle to enter the tumor from below the skin. Use the forceps to pinch the skin surrounding the needle, and slowly dispense the syringe volume. Continue to pinch the skin with the forceps while removing the needle slowly, and use a cotton swab to clean potential leakage.
   NOTE: Proceed with any additional therapeutic treatments if desired.
6. Allow the mice to recover from anesthesia.

Results

The results demonstrate that this method produces a high yield of myeloid cells from solid murine tumors. The preservation of receptor integrity and cellular viability facilitates reliable functional analysis of the desired myeloid subsets. These improvements to myeloid cell isolation allowed the discernment of the changing function of intratumoral myeloid cells upon normalization of the TME with the class I histone deacetylase inhibitor (HDACi), MS-275, during adoptive T cell therapy. TIL isolation protocols typically d...

Discussion

Although tumor-infiltrating myeloid cells exist in varying activation and differentiation states within the tumor, several subsets have been identified including tumor-associated DCs (TADCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs)¹². Unfortunately, the overlapping expression of cell-surface markers used to identify these myeloid cell subsets makes it currently challenging to phenotypically differentiate tumor myeloid c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 700 Mouse Anti-Mouse CD45.2	BD Biosciences	560693	1:100
APC-Cy7 Mouse Anti-Mouse NK-1.1	BD Biosciences	560618	1:100
Biotin Mouse Anti-Mouse CD45.2	BD Biosciences	553771
BV421 Hamster Anti-Mouse CD11c	BD Biosciences	562782	1:100
BV650 Rat Anti-Mouse F4/80	BD Biosciences	743282	1:100
BV711 Rat Anti-Mouse CD8a	BD Biosciences	563046	1:100
Collagenase, Type IV, powder	Gibco	17104019
DNase I	Roche	10104159001
EasySep Mouse CD11b Positive Selection Kit II	Stemcell technologies	18970
EasySep Mouse CD11c Positive Selection Kit II	Stemcell technologies	18780
EasySep Release Mouse Biotin Positive Selection Kit	Stemcell technologies	17655
FITC Rat Anti-Mouse Ly-6C	BD Biosciences	553104	1:100
Fixable Viability Stain 510	BD Biosciences	564406	1:1000
Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm)	BD Biosciences	554714
PE Rat Anti-CD11b	BD Biosciences	557397	1:100
PE-Cy7 Rat Anti-Mouse CD4	BD Biosciences	552775	1:100
PerCP-Cy5.5 Rat Anti-Mouse Ly-6G	BD Biosciences	560602	1:100
Perm/Wash (BD Perm/Wash)	BD Biosciences	554723
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Biosciences	553141
iNOS Monoclonal Antibody (CXNFT), APC	Thermo Fisher	17-5920-82	1:100
Human/Mouse Arginase 1/ARG1 Fluorescein-conjugated Antibody	R&D Systems	IC5868F	1:100