Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model
April 13th, 2015
This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.
The overall goal of the following experiment is to perform a detailed analysis of the infiltrating leukocytes in a murine subcutaneous tumor model. This is accomplished by first inoculating murine B 16 F zero melanoma cells from female C 57 BL six mice to establish the growing tumor in vivo. Next B 16, tumor bearing mice are euthanized and the subcutaneous tumor is resected on block, including overlying and surrounding skin.
Then a single cell suspension of both tumor cells and tumor infiltrating leukocytes is isolated, which can then be analyzed by flow cytometry. Finally, the viable cell yield per volume is determined and the number of cells to be stained and analyzed by facts is calculated. Ultimately, flow cytometry is used to show the frequency and type of leukocyte subsets within the tumor microenvironment.
The implications of this technique extend towards the therapy or diagnosis of cancer because one can evaluate the effects of a therapy on the leukocytes within the tumor microenvironment. It is this balance of effector and suppressor cells that help determine whether or not the tumor grows or regresses To induce tumor growth. Subcutaneously inoculate murine B 16 F zero melanoma cells in a physiologic buffer such as phosphate buffered saline at or near the midline of the abdomen in female C 57 BL six mice according to the text protocol, seven to 14 days later, just prior to resecting tumors set up one 50 milliliter conical tube per tumor and place on ice.
Insert a 40 to 70 micron cell suspension filter into each tube. Prepare surgical forceps and find scissors for resecting and 70%ethanol to clean instruments between tumor samples after euthanizing a B 16 tumor bearing mouse, according to the text protocol, secure the mouse on a surgical stage, use 70%ethanol to spray the tumor area and wipe off the access to prevent spreading hair. Ensure topical ethanol is fully wiped and evaporated prior to resecting the tumor using forceps and scissors, resect the subcutaneous tumor on block, including overlying skin and approximately two to three millimeters of surrounding skin.
Weigh the resected tumors with and without the surrounding skin and place each section in a filter in the conical tube. To prepare a single cell tumor suspension, use forceps and scissors to mince the tumor and overlying and surrounding skin until all large sections of tissue are processed into one to two millimeter sections. Next, using a plunger from a disposable five or 10 milliliter syringe, mechanically disaggregate the minced tumor tissue against the secured filter.
Then with a pipette man and five to 10 milliliters of cold, medium, or buffer. Wash the processed tumor tissue. This will flush the single cells through the filter.
Repeat the disaggregation and washing steps several times, ensuring that the total number and volumes of washings are approximately the same For similarly sized tumors. After washing the tissue, verify that small fragments of skin and or other connective tissue are left behind in the filter to wash and pellet the cells add medium and centrifuge at 500 Gs and four degrees Celsius for five minutes. To carry out fax analysis, use ice cold fax buffer to re suspend the cells.
Then with trian blue and a hemo cytometer count, the viable cells determine the total number of viable cells per volume to be used to extrapolate absolute numbers of tumor infiltrating leukocytes or till in combination with facts. Data from the trian stained counting data calculate the volume of cells to be stained for facts. For example, if TIL typically constitute 5%of total tumor cells, factor this into the total number of tumor cells in suspension to be stained.
To stain the cells for live dead discrimination. Use a fixable or non fixable stain such as propidium iodide. Then use FBS in PBS with 1%rat serum and FC block to block the cells for 10 minutes before using a standard fact staining protocol to stain the cell suspension for specific leukocyte subsets.
After staining, use 4%paraform aldehyde to fix the samples and store at four degrees Celsius in the dark until time for collection and analysis. Use a flow cytometer to collect the samples with facts. Data analysis software begin by gating out dead cells, then with an antibody against a leukocyte specific marker such as anti CD 45, identify leukocytes within the live cells.
Next, use various gating schemes to identify specific subsets according to the text protocol. Analyze data and present in a number of ways to ensure consistency and identify patterns or trends as shown here. Forced overexpression of kein in murine B 16 tumors resected day 17 resulted in a significant increase in total CD 45 positive cells compared to controls in this figure using immunofluorescence on cryo sectioned B 16 tumors.
An increase in infiltrating CD 45 positive cells from tumors was confirmed. Further analysis of fax data shows a relative increase on average of NK cells, T cells, and conventional CD 11 C positive dendritic cells. In the tumors where kein was over expressed compared to the control tumors, there was also a relative decrease in the percentage of immune suppressing leukocytes, including myeloid derived suppressor cells and plasmacytoid dendritic cells.
Studies have shown that the ratio of leukocytes subsets within the tumor may be an important factor in terms of tumor growth and clinical outcomes, and in support of this, the number of CD three positive T cells and NK cells per tumor were increasingly represented in the kein expressing tumors compared to controls. This figure shows that in a direct comparison of effector and suppressor cell populations within the tumor microenvironment, significant increases in the ratios of effector to suppressor cell populations were evident within the kein expressing tumors. Compared to controls Follow this procedure.
Other methods like cell sorting or cell isolation can be performed in order to answer additional questions that address tumor infiltrating leukocyte functionality.
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Overview
This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice, and cells from the tumor microenvironment were analyzed by flow cytometry.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- Oncology
Background
- Understanding the tumor microenvironment is crucial for cancer research.
- Leukocyte subsets play significant roles in tumor progression and immune response.
- Flow cytometry allows for detailed analysis of cell populations.
- This study utilizes a murine model to investigate these dynamics.
Purpose of Study
- To analyze infiltrating leukocytes in a murine subcutaneous tumor model.
- To evaluate the frequency and type of leukocyte subsets within the tumor microenvironment.
- To establish a protocol for future studies on tumor immunology.
Methods Used
- Inoculation of murine B16 F0 melanoma cells into syngeneic mice.
- Euthanization of tumor-bearing mice and resection of tumors.
- Isolation of a single cell suspension from tumor and infiltrating leukocytes.
- Analysis of cell populations using flow cytometry.
Main Results
- Successful establishment of a murine tumor model for analysis.
- Detailed characterization of leukocyte subsets within the tumor microenvironment.
- Flow cytometry provided insights into immune cell dynamics.
- Viable cell yield was determined for accurate analysis.
Conclusions
- The protocol effectively evaluates leukocyte subsets in tumors.
- Findings contribute to understanding tumor-immune interactions.
- This method can be applied to other tumor models for broader insights.
Frequently Asked Questions
Executive Industry Relevance
Understanding the composition of tumor-infiltrating leukocyte subsets provides critical mechanistic insight into immune-mediated tumor control, enabling target validation and de-risking of immunomodulatory strategies. This method supports preclinical evaluation of effector-suppressor balance, informing go/no-go decisions in immuno-oncology pipeline advancement. By linking immune profiling to tumor growth outcomes, it enhances predictive confidence in translational biomarker identification and therapeutic mechanism validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying shifts in effector and suppressor leukocyte populations in response to immunomodulatory interventions.
- Operational Value: Provides a reproducible workflow for isolating and characterizing tumor-associated immune cells from subcutaneous tumor models.
- Predictive Value: Supports target confidence by correlating leukocyte subset ratios with tumor growth modulation, aiding in mechanistic de-risking of immuno-oncology candidates.
Screening & Assay Development
- Scientific Value: Generates standardized, quantitative flow cytometry readouts of leukocyte frequencies, enabling consistent comparison across experimental conditions.
- Operational Value: Establishes a scalable platform for immune profiling that can be integrated into screening cascades for immunomodulator lead identification.
- Assay Readiness: Produces single-cell suspensions suitable for multi-parameter staining and downstream functional validation of sorted subsets.
Translational & Preclinical Research
- Translational Continuity: Facilitates alignment with clinical biomarkers by enabling longitudinal tracking of immune infiltrates in syngeneic models relevant to human tumor immunology.
- Preclinical Validation: Supports risk-adjusted advancement decisions by demonstrating mechanistic effects on immune contexture, a known predictor of clinical response.
- Disease-Relevant System: Utilizes a well-established B16 melanoma model to study immune evasion and effector function, enhancing relevance to solid tumor immunotherapy development.
Pipeline & Workflow Integration
The method fits within the discovery-to-preclinical continuum, enabling immune profiling after in vivo compound or genetic modulation but before functional validation, supporting data-driven progression from target hit to lead candidate.
- Discovery Biology: Supports hypothesis testing by revealing changes in leukocyte composition following target perturbation, clarifying mechanisms of immune activation or suppression.
- Screening: Delivers reproducible, quantitative immune readouts that enable ranking of immunomodulators based on their ability to shift effector-suppressor balance.
- Analytics: Delivers multi-parametric flow cytometry data enabling statistical comparison of leukocyte subsets across treatment groups, supporting objective decision-making.
- Translational Research: Connects murine immune profiling to clinical relevance by modeling immune evasion and effector infiltration patterns observed in human tumors.
- Enterprise Reuse: Represents a modular, adaptable immune profiling workflow applicable across multiple tumor models and therapeutic modalities, maximizing resource efficiency.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing ambiguity in mechanistic interpretation of tumor growth effects through direct immune profiling.
- Operational Value: Enhances reproducibility and standardization of immune analysis across laboratories and studies through defined resection, dissociation, and staining protocols.
- Strategic Value: Improves capital efficiency by enabling early detection of immunomodulatory activity, reducing investment in candidates lacking mechanistic rationale.
- Portfolio Impact: Enables risk-based prioritization by identifying compounds that favorably alter tumor immune contexture, aligning with biomarkers of clinical response.
Implementation Considerations
- Requires expertise in murine tumor model handling, aseptic tissue resection, and flow cytometry sample preparation.
- Dependent on access to flow cytometers, cell dissociation tools, and validated leukocyte subset antibody panels.
- Necessitates standardization of tumor harvest timing and tissue processing to minimize variability in leukocyte yield and viability.
- Involves optimization considerations when adapting to different tumor types or model systems with varying stromal density or enzymatic sensitivity.
- Limited by the need for fresh tissue processing, which constrains batch size and requires coordinated scheduling of animal euthanasion and analysis.
