Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Summary

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Abstract

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Introduction

The suppressive TIME has recently been recognized as a hallmark of cancer, and is known to play a significant role in the development, progression, and protection of solid tumor cancers as well as mitigate their susceptibility to immunotherapy¹. The TIME is composed of numerous cellular subsets and phenotypes, all of which provide critical insight into the immunologic state of the tumor. These immunologic subsets can be further stratified into cells of lymphocyte or myeloid origin, which together constitute the majority of the innate and adaptive immune responses^2,3. Recent advances in the field of cancer immunotherapy have demonstrated that immunotherapeutic strategies (i.e., immune checkpoint inhibitors, chimeric antigen receptor T-cells, etc.) have the potential to induce durable cancer regressions; however, they remain relatively ineffective in solid tumor cancers^4,5. Numerous groups have shown the critical hurdle that the TIME can play on treatment success^6,7, and thus, there remains a need to accurately evaluate new immunotherapies in the pre-clinical setting specifically focusing on their ability to modulate or overcome the TIME⁸.

Current efforts to characterize the TIME typically utilize either microscopy or flow cytometry along with antibody labeling strategies to identify immune cellular subsets and their features^9,10. These two strategies provide uniquely different information, as microscopy allows spatial appreciation of cellular subsets and flow cytometry provides high throughput and broader quantification of cellular changes. Despite the recent improvements in fluorescent multiplex immunohistochemistry optimizing spectral imaging systems, which now can support up to 7 parameters, limited panel size makes broad level immune profiling difficult and thus this technique is often reserved for more focused analyses. As a result, flow cytometry remains one of the most widely used immune profiling techniques. Despite its widespread use in TIME characterization, the methods used for processing and staining are quite variable. Most often protocols utilize a tumor dissociating enzyme (i.e., collagenases, DNase, etc.) and manual dissociation methods to achieve single-cell suspensions, followed by antibody staining and analysis⁷. Despite the benefits of each method, numerous groups have shown the extensive variability that can be induced through these techniques¹¹. This makes cross-study comparisons of tumor microenvironment profiling extremely difficult, even when assessing the same murine tumor model. Furthermore, these methods provide limited potential to assess tumor cellular and tumor immune infiltrate components independently, since both components are interspersed after digestion. Sample quantity then limits multi-panel staining and analysis, which becomes a major issue when attempting to characterize rare immunologic subsets (i.e., tumor-specific T-cells)¹². More recent techniques such as mass cytometry, or cytometry by time of flight (CyTOF), allow high-dimensional phenotypic analysis of cellular subsets with some systems supporting panel designs of greater than 42 independent parameters¹³. Despite the tremendous power of CyTOF technology in immune profiling, it remains limited because of the expense, analysis expertise, and the access to the equipment. In addition, many CyTOF protocols recommend purification of immune subsets to improve signal-to-noise ratios¹⁴, and thus we suggest that our enrichment method could be used upstream of CyTOF analysis to improve data quality.

Herein we describe a tumor microenvironment digestion and analysis method that incorporates tumor immune infiltrate separation. The purpose of this method is to allow independent high-throughput profiling of the tumor immune infiltrate and tumor cellular fractions for broader characterization of the tumor microenvironment. Using this method, we further demonstrate the importance of performing whole tumor analysis, as a subcutaneous solid tumor model was found to have significant spatial immunologic heterogeneity. Overall, this method can more accurately and consistently compare between samples because it enriches for immune cellular subsets within the tumor and allows for independent profiling of tumor cell and immune fractions of a tumor.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine.

1. Tumor Harvest and Digestion

NOTE: The time required for harvest is ~3 - 5 min/tumor, and the time required for processing is ~1 h + 2 - 3 min/tumor.

1. Euthanize mice by carbon dioxide inhalation and spray each mouse down with 70% ethanol before harvesting to prevent hair contamination. Surgically remove subcutaneous tumors from the mice and ensure that the tumors are free of any contaminating tissue (i.e., hair, skin, peritoneum, etc.). Place each tumor in 1.8 mL of base RPMI-1640 media without fetal bovine serum (FBS) in a 24-well plate. For larger harvests, keep plates cold on ice to preserve cellular viability.
   NOTE: If sterility is required, all tumor dissections will need to be performed in a biosafety cabinet with sterile surgical tools (i.e., scissors, forceps, etc.), as well as any further steps. Tumors with the longest diameter between 0.5 - 1 cm are optimal, and larger or smaller tumors will require scaling of reagents.
2. Use scissors to cut the tumors into less than 1 mm³ pieces within each well.
3. Prepare a 10x digestion cocktail by dissolving collagenase I at 10 mg/mL, collagenase IV at 2,500 U/mL, and DNase I at 200 U/mL in base RPMI-1640 media (without FBS).
   NOTE: The digestion cocktail can be prepared a day in advance and stored at -20 °C; however, prolonged storage is not recommended as enzymes will lose activity over time once dissolved.
4. Add 200 µL of 10x dissociation cocktail containing collagenase I, collagenase IV, and DNase I to each well with the 1 mm³ pieces.
5. Allow the samples to digest for 1 h at 37 °C while lightly shaking at 60 - 100 rpm using an orbital plate shaker.
   NOTE: The elevation of temperature to 37 °C during the enzymatic digestion may cause immune cell activation.
6. After 1 h, neutralize the reaction by adding 1 mL of RPMI-1640 media supplemented with 5% FBS and 2 mM EDTA to each well.
7. Pipette the tumor digestion and the remaining tumor pieces into a 40 µm cell strainer seated on top of a 50 mL centrifuge tube.
   NOTE: Cut the tip off of a 1 mL pipette tip for easier transfer of the tumor digestion to the strainer.
8. Use a 10 mL syringe plunger to mechanically disaggregate the tumor pieces through the strainer. Periodically rinse the strainer with 2 mL of supplemented RPMI-1640 media (containing 5% FBS) and repeat until the cell strainer is clear of tumor.
   NOTE: Approximately 4 - 10 mL of total media should be sufficient to adequately rinse the strainer of tumor.
   NOTE: Place samples on ice until this step is completed for all samples to preserve cellular viability.
9. Adjust each 50 mL centrifuge tube to the same volume using the supplemented RPMI-1640 media.
   NOTE: The adjustment of volume is critical for centrifuge balancing. Skipping this step could result in centrifuge damage or personal injury.
10. Centrifuge the cell suspensions (5 min, 805 x g, 4 °C), discard the supernatants, and re-suspend in 2 mL of supplemented RPMI-1640 media.
    NOTE: A cell aggregate may form following the centrifugation that is difficult to resuspend. Use a 5 mL serological pipette and mix rapidly to break it up before proceeding.

2. Separation of Immune and Tumor Cellular Fractions

NOTE: The time required for this step is approximately 1 h.

1. Add 3 mL of density gradient medium to the bottom of a 15 mL centrifuge tube. Prepare and label enough tubes for each tumor.
2. Layer the 2 mL of tumor cell suspension on top of the density gradient medium by pipetting slowly down the side of the tube to prevent mixing of the two layers.
3. Carefully place the layered tubes in the centrifuge and spin for 20 min at 805 x g, 20 °C, with no brake.
   NOTE: It is extremely important to verify proper balancing and ensure that no brake is used during centrifugation. Any disruption of this process will result in layer mixing and the full sample recovery will be difficult.
4. Carefully transfer the tumor infiltrating leukocyte (TIL) layer and the top media layer to a new 15 mL tube using a transfer pipette. Discard all remaining density gradient medium and resuspend the tumor pellet at the bottom of the tube in 2 mL of supplemented RPMI-1640.
   NOTE: After centrifugation there will be four visible layers from the top to bottom which respectively correspond to: media, TILs, density gradient medium, and tumor cell pellet. See Figure 2A for an example of the various layers.
5. Centrifuge both the TIL and tumor samples in separate tubes (5 min, 805 x g, 4 °C) and discard the supernatant.
6. Resuspend the TIL sample in 200 µL and the tumor pellet in 2 mL of supplemented RPMI-1640 media. Keep on ice to preserve viability.

3. Plating and Staining

Note: The time required for plating is 30 s/tumor; the time required for surface staining is 1 h; the time required for intracellular staining is 40 min; the time required for flow cytometry analysis is 1 - 4 min/tumor.

1. Prepare and label a 96-well U-bottom plate for each immune staining panel and an additional plate for cell counts.
   NOTE: Typically, three plates are prepared in total: a lymphocyte-focused panel plate, a myeloid-focused panel plate, and a cell count plate.
2. Aliquot the single-cell suspensions into each of the staining panel plates and a set volume into the count plate.
   NOTE: The count plate is used to calculate the total number of cells added to each staining panel, thereby allowing accurate cell population counts. A flow cytometer with 96-well plate sampling and volumetric analysis capabilities can provide the number of cells per µL in each sample, and therefore, calculate the number of cells added to each staining panel. Count plates can be acquired the following day after fixation overnight at 4 °C, rinsing with FACs buffer (phosphate buffered saline (PBS) with 2% FBS), and resuspending in a set volume of FACs buffer.
3. Wash the cells twice with 200 µL of Dulbecco's phosphate buffered saline (DPBS) per sample by centrifuging (5 min, 805 x g, 4 °C) and discarding the supernatant between each rinse to remove any free FBS.
4. Add 50 µL of Fc block, mix the samples using a multi-channel pipette, and incubate for 20 min at 4 °C.
5. Add 50 µL of 2x concentrated extracellular targeting antibody and fixable viability stain mixture, mix using a multi-channel pipette, and incubate for 30 min in the dark at RT or 4 °C.
   NOTE: The exact staining conditions for the antibody depend on the manufacturer's instructions. All staining dilutions must be in DPBS, with no added FBS to prevent saturation of the fixable viability dye. Please see the Table of Materials/Equipment for the optimal dilutions of the staining panel used in this manuscript. Antibody and fixable viability staining solution is prepared at a 2x concentration to provide a final 1x staining concentration in 100 µL of total staining volume when added to the Fc block.
6. Wash the samples twice with FACs buffer by centrifuging (5 min, 805 x g, 4 °C) and discarding the supernatants between each rinse.
7. Resuspend in 200 µL of 1x fixation/permeabilization solution and incubate overnight at 4 °C protected from light.
   NOTE: The experiment can be paused overnight at this point: keep samples at 4 °C protected from light.
8. The following day, centrifuge the plates (5 min, 805 x g, 4 °C) and discard the supernatant.
9. Rinse once with 200 µL of 1x permeabilization buffer, centrifuge (5 min, 805 x g, 4 °C), and discard the supernatant.
10. Add 100 µL of 1x concentrated intracellular antibody staining panel prepared in 1x permeabilization buffer and incubate for 30 min in the dark at RT or 4 °C (this depends on staining recommendations from the manufacturer).
11. Rinse once with 1x permeabilization buffer (5 min, 805 x g, 4 °C), discard the supernatant, and rinse a second time with FACs buffer (PBS with 2% FBS).
12. Resuspend the samples in 300 µL of FACs buffer (PBS with 2% FBS), transfer the samples to flow cytometry tubes, and perform flow cytometry analysis.

Results

Our results demonstrate the significant benefit of TIL separation from non-immune tumor components, as explained in the protocol. Additionally, using the described method we demonstrate the significant immunologic heterogeneity of established solid tumors.

A significant problem with many tumor dissociation techniques is the loss of sample viability, most often as a result of harsh digestion conditions (i.e., elevated te...

Discussion

The TIME is composed of diverse and complex cellular components and molecules. Recent evidence suggests that accurate characterization of this environment can provide a better understanding of treatment success or failure, and can even help identify mechanisms of therapeutic resistance. For example, increasing intratumoral levels of various immunosuppressive cells (i.e., MDSCs, T regulatory cells, etc.) can mitigate effector immune responses and abrogate immunotherapeutic effects. On the other hand, the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 to 12 week old male C57BL/6 mice	Jackson Laboratory	N/A	Mice used in our tumor studies
MEER Murine Syngeneic Cancer Cell line	Acquired from collaborator	N/A	E6/E7 HPV antigen expressing murine tonsillar epithelial cancer cell line
70% isopropyl alcohol	EMD Milipore	PX1840
Murine surgical tool kit	World Precision Instruments	MOUSEKIT	Primarily only need scissors, tweezers, and a scalpel.
24-well plate	Denville Scientific Inc.	T1024	Or any 24-well flat bottom plate.
RPMI-1640 media	Sigma-Aldrich	R0883
Collagenase I	EMD Milipore	234153
Collagenase IV	Worthington Biochemical Corporation	LS004189
DNase I	Sigma-Aldrich	11284932001
Low Speed Orbital Shaker	BioExpress (supplier: Genemate)	S-3200-LS	Or any orbital shaker.
Thermoregulating incubator	Fisher Scientific	13-255-27	Or any other thermo-regulated incubator
FBS	Sigma-Aldrich	F8192
EDTA	AMRESCO	E177
1 mL Pipette and tips	Eppendorf	13-690-032	Or any other pipette and tips
40 um Cell Strainers	Fisher Scientific	22363547
50 mL Centrifuge Tubes	Denville Scientific Inc.	C1062-P
15 mL Conical Tubes	Denville Scientific Inc.	C1012
10 mL Luer-Lok Syringe without needles	BD	309604
Lymphoprep Density Gradient Medium	STEMCELL Technologies	7811
Transfer Pipettes	Denville Scientific Inc.	P7212
96-well U-bottom plates	Denville Scientific Inc.
DPBS	Sigma Aldrich	D8573
FoxP3 Transcription Factor Staining Buffer Set	ThermoFisher Scientific	00-5523-00	Fix/Permeabilization Buffer and Permeabilization Buffer
Thermoregulated Centrifuge	Eppendorf	5810 R
Attune NxT Flow Cytometer	ThermoFisher Scientific	N/A	For 96-well cell volumetric counting
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) Clone 2.4G2	ThermoFisher Scientific	553141	Fc Block
LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation	ThermoFisher Scientific	L34962	Fixable viability stain
CD45 Monoclonal Antibody (30-F11), APC-eFluor 780	ThermoFisher Scientific	47-0451-80
TCR beta Monoclonal Antibody (H57-597), APC	ThermoFisher Scientific	17-5961-82
CD8-α Antibody (KT15), PE	Santa Cruz Biotechnology	sc-53473
CD4 Monoclonal Antibody (GK1.5), PE-Cyanine5	ThermoFisher Scientific	15-0041-81
MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), Alexa Fluor 700	ThermoFisher Scientific	56-5321-82
CD11c Monoclonal Antibody (N418), PE-Cyanine5	ThermoFisher Scientific	15-0114-82
F4/80 Monoclonal Antibody (BM8), eFluor 450	ThermoFisher Scientific	48-4801-80
CD11b Monoclonal Antibody (M1/70), APC	ThermoFisher Scientific	17-0112-81
Ly-6G (Gr-1) Monoclonal Antibody (RB6-8C5), PE	ThermoFisher Scientific	12-5931-82
CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7	ThermoFisher Scientific	25-5982-80
CD273 (B7-DC) Monoclonal Antibody (122), PerCP-eFluor 710	ThermoFisher Scientific	46-9972-82
Ki-67 Monoclonal Antibody (B56), BV711	BD Biosciences	563755