Protocol to study the immune profile of syngeneic mouse tumor models

Summary Flow cytometry, single-cell RNA sequencing, and other analyses enable us to capture immune profiles of the tumor microenvironment. Here, we present a protocol to characterize the immune profile of tumor-bearing mice. We describe steps for establishing mouse models and preparing single-cell suspensions from tumor tissue and other immune-related organs, which can be further analyzed by flow cytometry and other omics assays. We then detail procedures for staining, flow cytometry analysis, and phenotyping of the immune cell populations. For complete details on the use and execution of this protocol, please refer to Miyauchi et al.1

detectors.Staining panels need to be designed based on the configuration of your flow cytometer and your proteins/markers of interest.
Note: Examples of staining panels for myeloid cells and lymphoid cells are shown in Table 1 and Table 2, respectively.
4. Know the configuration of your flow cytometer: number of lasers, number of detectors, and range of wavelengths.5. Design your staining panel.
a. Use brighter fluorochromes for rare/lowly expressed proteins and use dimmer fluorochromes for highly expressed proteins.b.If possible, use different lasers for markers of the same cell population.For example, Treg markers CD25 and Foxp3 are best detected using fluorochromes excited by different lasers.
In the example lymphoid panel (Table 2), we used PE (excited by blue laser) for CD25 and APC (excited by red laser) for Foxp3.c. Test and optimize your staining panels.Note: Sodium azide helps to prevent shedding and internalization of cell surface antigens, but it also inhibits metabolic activities.Therefore, do not add sodium azide if cells are to be collected for functional assays (i.e. if sorted live cells will be used for downstream analysis).This section details how to inject tumor cells into syngeneic mice subcutaneously at the flank.

KEY RESOURCES
Note: Cells should be cultured in different sizes of dishes or flasks depending on the desired cell number for your experiment.Solution volume should be scaled up or down accordingly.
Here, the protocol describes the procedure for a 150 mm dish.
1. Culture cancer cell line in appropriate culture medium.(See before you begin, cell culture).
2. Trypsinize cells.a. Remove culture medium, wash cells with sterile PBS, and then remove the PBS.b.Add 2-4 mL of trypsin-EDTA for a 150 mm dish and incubate the dishes at 37 C for 3-5 min or until all the cells are detached from the culture dish.Optimal trypsin incubation times will vary on a per-cell line basis.
Note: Add 1-2 mL of trypsin-EDTA for a 100 mm dish.
Note: Dishes may be gently tapped to promote cell detachment during trypsin incubation.
Cell detachment can be confirmed by visual inspection or with a microscope if not distinguishable by eye.3][4][5] In this protocol, 1 3 10 5 cells of B16F10 or 5 3 10 5 cells of LLC or MC38 in 100 mL were subcutaneously injected into the flank.
Note: Adjust the volume of cell suspension per mouse according to your tumor model.For example, 25-30 mL would be appropriate for the tongue orthotopic tumor model.
Note: Subcutaneous tumor models are easier for tumor cell implantation and tumor monitoring compared to orthotopic tumor models.However, ideally, the cell lines should be injected into the organ of origin to mimic the native tumor microenvironment.For example, E0771 and 4T1 cells are better to be injected into the mammary fat pad.

Protocol
Optional: If the injected cell lines show unstable growth, the addition of Matrigel at 50% of the final volume would help with in vivo growth.
5. Inject cells into mice.a.Put a mouse into an isoflurane chamber to anesthetize it.b.Shave the fur at the injection site.c.Load cell suspension into a 1 mL syringe with a 25G needle.d.Sanitize injection site by spraying with 70% ethanol or an equivalent sanitizing agent that is approved in your institution's animal protocol.
CRITICAL: Ensure that the mouse is under sedation during the injection by using a nose cone to avoid it waking up in the middle of the injection.
Note: An ophthalmic ointment can be used to protect their eyes during sedation although it is not necessary to use it for a brief sedation.
e. Insert the needle subcutaneously and slowly inject 100 mL of cell suspension.
Part 2: Establishment of tumor-bearing mouse models

Timing: 3-4 weeks
This section describes the process of tumor monitoring and measurement.
6. Start measuring the tumors when they become palpable.
Note: Tumors may take 5-8 days to become palpable depending on the cell line and the number of cells injected.
7. Measure tumor size every 2-3 days with a caliper until sacrifice.The longest diameter is ''Length'' and the transverse diameter is ''Width''.8. Calculate tumor volume using this formula: Tumor volume (mm 3 ) = (Length 3 Width 2 )/2.Note: Measure tumors as frequently as required for their size as specified in your institution's policy.
Note: Sacrifice timing depends on the tumor model (growth speed).Depending on the scientific question you would like to address, it may be preferable to have multiple time points to capture certain anti-tumor immune responses and tumor microenvironments.This section explains how to obtain different tissues including blood, tumor, tumor-draining lymph nodes, and spleen from a mouse.9. Euthanize mice by CO 2 inhalation followed by cervical dislocation in accordance with IACUC policy.(Please follow your institution's IACUC guidelines on animal euthanasia.).10.Place the mouse on its back and spray it with 70% ethanol.11.Open the thorax and expose the heart.Collect blood via cardiac punctures with a 25G needle syringe.
Note: This step needs to be done quickly as the blood coagulates rapidly.
Note: It is recommended to collect 50-100 mL of blood for flow cytometric analysis.The required volume varies depending on the downstream assay.
12. Place the blood in EDTA-or heparin-coated tubes.
Note: Detach the needle from the syringe before expelling blood into EDTA/heparin tubes to avoid hemolysis.
Note: Ensure that the volume of blood does not exceed the maximum, manufacturer-recommended volume of the collection tube.Excess volume causes blood clotting.Split into multiple tubes as necessary.Invert tubes to fully mix blood with EDTA or heparin.
13. Carefully dissect the mouse and harvest organs of interest.

Note:
The anatomical location of the tumor-draining lymph node varies depending on your tumor model.Inguinal lymph nodes are the tumor-draining lymph nodes for flank tumor models.
14. Keep the harvested organs in flow cytometry buffer on ice while preparing to proceed to the next step.
Note: Alternatively, other buffers (PBS, HBSS, culture medium, etc.) may also be used to store the harvested tissue.
Note: If the harvested tissue will be processed with collagenase at Step 15, buffer without EDTA should be used because EDTA inhibits the enzymatic activity of collagenase.

Part 4: Preparation of single-cell suspension
Timing: 0.5-3 h Single-cell suspensions are required for flow cytometry analysis and other omics assays.This section explains how to process different organs to obtain single-cell suspensions.(Figure 1).

Timing: 2-3 h
Note: Enzymatic tissue dissociation is required only for firm tumors (e.g.LLC lung carcinoma, MC38 colon carcinoma, 4T1 mammary cancer, etc.).Soft tumors (e.g.B16F10 melanoma) can skip this step and start from the manual tissue dissociation (Step 16).
Note: Density-gradient centrifugation (Step 18) can be skipped when tumors do not contain much necrotic tissue.

Note:
In this protocol, we used collagenase D for enzymatic tissue dissociation followed by manual dissociation.Alternative options are available including gentleMACS Dissociator with Tumor dissociation kit (Miltenyi Biotec).However, some enzymes in the kit reagents alter the cell surface expression of certain proteins.Refer to the epitope preservation list before use.See also troubleshooting 1 and 2.

Note:
The cell strainers can be also placed over 50 mL centrifuge tubes instead of 6-well plates.
Note: See also troubleshooting 1 and 2. Note: If you still see a red pellet at this step, it is acceptable to repeat Steps 28b and 28c.However, please keep in mind that over-incubation in ACK buffer can kill some cells.Even if a small amount of RBCs remains after a total of two rounds of Steps 28b and 28c, proceed to the next step to limit the loss of other cells.This section details how to stain cells with fluorescent-conjugated antibodies.Stained cells can be analyzed by flow cytometry.This protocol can also be used for cell sorting followed by cell population-specific single-cell RNA sequencing or other assays.
Note: This protocol describes how to stain the cells in 96-well plate format.You can also stain cells in 5 mL flow cytometry tubes or 1.5 mL microcentrifuge tubes.When cells are stained in the tubes, a one-time wash with a larger volume of buffer should be enough to wash out reagents or excess antibodies.The wash steps throughout Part 5 can be modified as follows; add 1 mL of appropriate buffer (please follow the instructions at each step) to the tube, centrifuge, aspirate the supernatant, and move onward to the next step.56.Prepare compensation beads (e.g., UltraComp eBeads Plus Compensation Beads) for fluorescent-conjugated antibodies according to the manufacturer's protocol.57.Prepare amine-reactive compensation beads (e.g., ArC Amine Reactive Compensation Bead Kit) for amine-reactive viability dyes according to the manufacturer's protocol.
Note: Cells may also be used for single-color-stained samples, but the use of compensation beads is preferred and recommended to obtain optimal compensation.
Pause point: The fixed samples can be stored at 4 C.It is recommended to acquire samples within 3 days of staining and fixation.
Note: Some fluorochromes, especially tandem dyes, might degrade faster in storage.Signal stability needs to be tested before assuming that all signals are stable for 3 days.

Timing: 1-4 h depending on the number of samples
This section explains how to acquire stained samples and controls on a flow cytometer.
Optional: Tumor samples can be passed through a 25-30 mm cell strainer immediately before sample acquisition.This will help to clean up the tumor samples and prevent clogging on the flow cytometer.(See also troubleshooting 3).

Adjust the voltage on each detector as needed.
Note: This step is required for flow cytometers from BD or other companies.It is not usually required for a NovoCyte flow cytometer.

Perform automatic compensation.
a. Acquire single-color stained compensation beads or samples for each fluorochrome.
Note: For compensation beads, it is recommended to record a total of 10,000 events (5,000 events for both positive and negative populations).For cells, the number of events should be higher; at least 50,000 cells.
b. Calculate a compensation matrix by using an automatic compensation function in the software.60.Acquire FMO samples and a full-color-stained sample, then draw plots to identify different immune populations.
Note: Use FMOs to set proper gating.
61.Once the flow cytometer has been properly configured, acquire all samples (wells).
Note: Recording 100,000 events of the CD45 + population is recommended, however, this should be adjusted depending on your populations of interest.It is recommended to record at least 1,000 events of the population of interest.If the population is rare, you need to record 1 million or even more events of the CD45 + population accordingly.

Protocol EXPECTED OUTCOMES
The expected results of this protocol are shown in Figure 3.
Immune cell profiles are different depending on the organ or tissue (Figure 3A).Interestingly, different tumor types (cell lines) have distinct immune cell profiles.For example, LLC and MC38 tumors have myeloid cell-enriched profiles whereas B16F10 tumors have lymphoid cell-enriched profiles, indicating the uniqueness of the tumor microenvironment.
The development of memory T cell subsets in lymph nodes is a crucial step for the induction of antigen-specific anti-tumor immune response.The subsets of memory CD8 + T cells in tumor-draining lymph nodes and tumors are shown in Figure 3B.As expected, the majority of the CD8 + T cells in the tumor-draining lymph nodes are naı ¨ve and central memory subsets whereas most of the CD8 + T cells in the tumors are the effector memory subset, indicating the migration of the memory T cells developed in the tumor-draining lymph nodes into the tumor site.
Effects of treatments or your genes/proteins of interest on the tumor microenvironment can be investigated by analyzing the alterations in the immune cell profile induced by drug administration or gene knockout.

QUANTIFICATION AND STATISTICAL ANALYSIS
The acquired events are first gated on FSC-A vs. SSC-A to remove debris and cell aggregations, then single cells are gated on FSC-A vs. FSC-H to remove doublets.Doublet discrimination can also be achieved by gating on FSC-H vs. FSC-W and then SSC-H vs. SSC-W.After excluding dead cells by gating viability dye-negative live cells, gate immune cells by CD45 positivity.The following strategies are used to identify different immune cell populations.

LIMITATIONS
The staining panels in this study were designed to provide an immune profile overview, and the panels cover a majority of the main immune populations.Therefore, the information on their subpopulations is missing.For example, subsets of B cells (including memory B cells, regulatory B cells, and plasma cells) and subsets of dendritic cells (including plasmacytoid DCs and conventional DC1) cannot be distinguished by staining panel.Also, functional markers need to be added to analyze the activation/exhaustion status or phenotype of some populations of immune cells including macrophages (anti-tumor or pro-tumor) and myeloid-derived suppressor cells (MDSCs) (monocytic-MDSC or polymorphonuclear-MDSC).The information can be obtained by adding/replacing markers specific to the cell types and cell states.
The configuration of the flow cytometer affects the results because each flow cytometer has a different spillover matrix.The designed staining panels need to be carefully tested.

TROUBLESHOOTING Problem 1
After the enzymatic dissociation step, the tumor tissue does not pass through the cell strainer.

Potential solution
Some tumor types require a longer incubation time for enzymatic reaction.Cutting tumors into smaller pieces to increase the surface area exposed to the enzyme solution could improve dissociation efficiency.Make sure to add enough enzymes.Adding more enzymes could help with efficient dissociation.Also, using additional enzymes (e.g., hyaluronidase) could help the dissociation.However, some enzymes are known to affect the cell surface expression of proteins.Make sure the enzyme does not affect your proteins of interest.
If the cell suspension becomes viscous due to DNA release from digested cells, adding DNase I can alleviate this problem.Also, make sure the dissociation enzyme solution does not contain EDTA as it is a collagenase inhibitor.Using a bigger size of cell strainer (100 mm) before passing through smaller ones (40 or 70 mm) could also resolve the problem (Step 15).Problem 2 Some of the surface markers cannot be detected or lower frequencies than expected.

Potential solution
Make sure to use appropriate dissociation enzymes.It has been reported that some dissociation enzymes decrease cell surface protein expression.For example, dispase decreases cell surface expression of CD8, which leads to a misreading of the immune profile 20 (Step 15).

Problem 3
The cells in single-cell suspension aggregate during the staining steps.

Potential solution
Adding EDTA to buffers helps to prevent cell aggregation mediated by DNA released from dead cells.Passing suspension through a smaller size (25-30 mm) of cell strainer immediately before running samples on the flow cytometer could help remove cell aggregation and prevent clogging the lines in the flow cytometer (Step 58).

Problem 4
The signal intensity of some markers is low.

Potential solution
Make sure to add a sufficient concentration of antibodies.Adding more antibodies will help to get higher signals.It is recommended to titrate antibodies to find an optimal concentration.It can be different depending on the organs of interest (Steps 38 and 49).

Problem 5
Infiltration of immune cells in the tumor is low and the frequency of immune cells in the single-cell suspension is too low to process for single-cell RNA sequence analysis or other assays.

Potential solution
The frequency of immune cell infiltration depends on the immunogenicity of the tumor models.If the tumor type of interest is low immunogenicity and shows low immune cell infiltration, it might be better to sort CD45 + hematopoietic cell populations from tumor tissue.This will allow more sufficient numbers of immune cells to be analyzed and help you characterize the tumor microenvironment (Step 41).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dong-Er Zhang (d7zhang@health.ucsd.edu).

Technical contact
Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Sayuri Miyauchi (smiyauchi@health.ucsd.edu).

Materials availability
This study did not generate new unique regents.

c. Add 5 4 .
mL of fetal bovine serum (FBS)-containing medium to stop the trypsin reaction, and collect cell suspension in a sterile centrifuge tube.d.Centrifuge the cells for 5 min at 400-600 3 g at 20 C-25 C. 3. Wash cells with sterile PBS.a. Remove the supernatant and resuspend the cells in PBS.Add PBS up to the maximum volume of the centrifuge tube.b.Centrifuge the cells for 5 min at 400-600 3 g at 20 C-25 C. c. Remove the supernatant and resuspend the cells in PBS.Recommended cell concentration is 1 3 10 5 -1 3 10 7 cells/mL for accurate cell counting.Note: If the cell suspension contains clumps, filtering the cell suspension is recommended to ensure a single-cell suspension.Adjust the concentration of the cell suspension.a. Determine cell number by using an automated cell counter or hematocytometer.b.Adjust cell suspension to the desired concentration based on the cell counts.c.Keep the cell suspension on ice until use.

Part 3 :
Dissection of mice and harvesting tissueTiming: 15 min per mouse

Protocola.
In a petri dish, cut tumor tissue into small pieces (1-3 mm) with a scalpel or razor blade.Note: You may process half of the tumor for single-cell suspension and fix or snap-freeze the other half for histological analysis or any other desired assays.b.Transfer cut tissues into a 12-well plate.Add 2-3 mL of 1x collagenase D solution (1 mg/mL).Note: If tumor mass exceeds 500 mg, split tissue into 2 wells or use a 6-well plate.c.Incubate the tissue for 1 h at 37 C with gentle shaking.CRITICAL: You may need longer incubation times depending on the tumor type.However, over-incubation can damage and kill the cells.d.Transfer the digested tissue and solution to a 70 mm cell strainer placed in a new 6-well plate by using tip-cut transfer pipettes, serological pipettes, or wide-bore micropipette tips.

Figure 1 .
Figure 1.Workflow for preparation of single-cell suspensions Preparation of single-cell suspension for (A) tumor, (B) tumor-draining lymph node, (C) spleen, and (D) peripheral blood.Created with BioRender.com.

16 .Figure 2 .
Manual tissue dissociation.a. Smash the tumor tissue against the 70 mm cell strainer using a syringe plunger to pass it through.Most of the tissue should go through the cell strainer.b.Transfer dissociated cell suspension into 15 mL centrifuge tubes.c.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard the supernatant by aspiration or flicking of the tube.17.Red blood cell (RBC) lysis.Note: The density-gradient centrifugation (Step 18) will also isolate RBCs.However, this step is recommended to clean up the single-cell suspension if the tumor tissues contain a lot of RBCs.Skip this step if the tumor tissues do not contain many RBCs.a. Resuspend cells in 3-5 mL of 1x ACK buffer.b.Incubate for 3-5 min at 20 C-25 C. Note: Incubation temperature can be either 20 C-25 C or on ice.Please follow the instructions from the manufacturer of the ACK buffer.c.Add PBS up to 15 mL to wash cells at 20 C-25 C. d.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard the supernatant by aspiration or flicking of the tube.18. Density-gradient centrifugation for debris removal.Note: See also troubleshooting 4. a. Resuspend cells in 8 mL of 40% Percoll solution.b.Insert a glass Pasteur pipet to the bottom of the 15 mL centrifuge tube containing the 40% Percoll cell suspension.Slowly layer 5 mL of 80% Percoll solution under 40% Percoll by slow addition through the glass Pasteur pipet.(Figure 2A).c.Centrifuge at 600 3 g for 20 min at 20 C-25 C with slow acceleration and no brake.d.Aspirate the layer of debris and 40% Percoll solution down to 1-2 cm above the interface.Note: The separation (interface) is visible after centrifugation as shown in Figure 2B.e. Transfer the immune cell-containing interface into a new 15 mL centrifuge tube with a transfer pipet.Percoll density-gradient centrifugation (A) Layering 80% Percoll solution under 40% Percoll solution using Pasteur pipet.(B) Density-gradient separation after centrifugation.f.Add PBS up to 15 mL at 20 C-25 C. g.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard the supernatant by aspiration or flicking of the tube.19.Resuspend cells in flow cytometry buffer and keep cells on ice.20.Determine cell number.Tumor-draining lymph node Timing: 30 min 21.Manual tissue dissociation (Figure 1B).a. Smash the lymph nodes against the 70 mm cell strainer using a syringe plunger to pass it through into a 6-well plate containing 5-6 mL of flow cytometry buffer per well.b.Transfer cell suspension into 15 mL centrifuge tubes.c.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard supernatant.22. Resuspend cells in flow cytometry buffer and keep cells on ice.23.Determine cell number.Spleen Timing: 30 min 24.Manual tissue dissociation (Figure 1C).a. Smash the spleen against the 70 mm cell strainer using a syringe plunger to pass it through into a 6-well plate containing 5-6 mL of flow cytometry buffer per well.b.Transfer cell suspension into 15 mL centrifuge tubes.c.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard supernatant.25.RBC lysis.a. Resuspend cells in 3-5 mL of 1x ACK buffer.b.Incubate for 3-5 min at 20 C-25 C. Note: Incubation temperature can be either 20 C-25 C or on ice.Please follow the instructions from the manufacturer of the ACK buffer.c.Add PBS up to 15 mL to wash cells at 20 C-25 C. d.Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard supernatant.26.Resuspend cells in flow cytometry buffer and keep cells on ice.27.Determine cell number.Blood Timing: 30 min 28.RBC lysis (Figure 1D).a. Add 30-50 mL of whole blood in 1.5 mL of 1x ACK buffer to a 1.5 mL microcentrifuge tube.Note: The volume of whole blood and 1x ACK buffer can be scaled up depending on the desired cell number.b.Incubate for 3-5 min at 20 C-25 C. Note: Incubation temperature can be either 20 C-25 C or on ice.Please follow the instructions from the manufacturer of the ACK buffer.STAR Protocols 5, 103139, September 20, 2024 Protocol c. Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard supernatant.
d. Resuspend cells in 1 mL of PBS for washing at 20 C-25 C. e. Centrifuge cells at 400-600 3 g for 5 min at 20 C-25 C and discard supernatant.29.Resuspend cells in flow cytometry buffer and keep cells on ice.30.Determine cell number.Part 5: Staining of cells Timing: 3-4 h

Fixable live/dead staining 31 .
Plate 1 3 10 6 cells per well in a v-bottom 96-well plate.32.Wash cells with PBS.Note: Residual FBS or BSA will increase background signals.a. Add PBS up to 200 mL per well.Note: This protocol uses 96-well plates that can hold up to 350 mL/well.If 96-well plates with a lower maximum volume are being used, reduce the volume of PBS or other buffers used down to 2/3 of the maximum volume per well throughout the protocol.b.Centrifuge the plate at 400-600 3 g for 3-5 min at 20 C-25 C. c. Discard the supernatant by flipping the plate.33.Resuspend cells in 100 mL/well of Live/Dead staining solution.(See materials and equipment).34.Incubate for 15 min at 20 C-25 C or 30 min on ice in the dark.35.Wash the cells twice with PBS.a. Add 100 mL of PBS per well.(Total volume will be 100 mL per well).b.Centrifuge the plate at 400-600 3 g for 3-5 min at 20 C-25 C. c. Discard the supernatant by flipping the plate.d.Resuspend the cells in 200 mL of PBS.e. Centrifuge the plate at 400-600 3 g for 3-5 min at 20 C-25 C. f.Discard the supernatant by flipping the plate.
62. Export your data in FCS file format.63.Analyze the samples with FlowJo or a similar software.The gating strategies are shown in the Quantification and statistical analysis section.64.Perform statistical analyses with GraphPad Prism software or similar.STAR Protocols 5, 103139, September 20, 2024

Figure 3 .
Figure 3.Immune profiling of different tissues (A) Immune profile of different tissues from tumor-bearing mice on Day 12. (B) Memory subsets of CD8 + T cells in tumor-draining lymph nodes and tumors.

Figure 4 .
Figure 4. Gating strategy for myeloid cell populations Samples collected from the spleen are shown as an example.

Figure 5 .
Figure 5. Gating strategy for lymphoid cell populations Samples collected from the spleen are shown as an example.
If there are low intracellular marker signals, increasing the incubation time for permeabilization and intracellular staining steps could enhance the signals (Step 51).If there are low tumor tissue signals, removing debris/necrotic tissue by Percoll gradient will help clean up your samples and improve the signal-to-noise ratio (Step 18).