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Oct 2020
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Multi-color Flow Cytometry for Comprehensive Analysis of the Tumor Immune Infiltrate in a Murine Model of Breast Cancer
多色流式细胞术综合分析乳腺癌小鼠模型中肿瘤免疫渗透   

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Abstract

Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, γδT cells, and regulatory T cells.

Keywords: Flow cytometry (流式细胞术), Immune cell subsets (免疫细胞亚群), Breast cancer (乳腺癌), Tumor microenvironment (肿瘤微环境), Mouse model (小鼠模型), Immunophenotyping (免疫表型), Antibodies (抗体)

Background

Tumor-infiltrating immune cells comprise a major part of the tumor microenvironment and play a crucial role in controlling cancer, with both anti- and pro-tumorigenic effects (Dunn et al., 2002; Grivennikov et al., 2010). Inflammation and infiltration of innate immune cells, including macrophages and neutrophils, are necessary to fight infections but, in the case of cancer, often promote the progression of the disease. Cytotoxic T cells and natural killer (NK) cells can destroy tumors, but cancer cells have developed several mechanisms to evade immune destruction (Dunn et al., 2002; DeNardo et al., 2010). For instance, cancer cells can secrete cytokines that directly inhibit cytotoxic CD8+ T cells and recruit regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (Beatty and Gladney, 2015).


Tumoral immune cell infiltration can be prognostic in some breast cancer subtypes (DeNardo et al., 2011). For instance, high lymphocyte infiltration is associated with increased survival in breast cancer patients and a favorable prognosis (Ménard et al., 1997). In addition, clinical studies have shown that the accumulation of tumor-associated macrophages (TAMs) is strongly correlated with poor prognosis in breast cancer (Noy and Pollard, 2014).


Over the past two decades, advances in multi-color flow cytometry have allowed researchers to gain insights into the role of immune cells within the tumor microenvironment. Flow cytometry is widely used to characterize and quantify different cell types in heterogeneous cell populations, such as tumors, by detecting cell surface and intracellular molecules (Perfetto et al., 2004).


Here, to investigate immune cell infiltration during tumor development, we utilized the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) model of luminal B breast cancer, where the polyoma virus middle T antigen is expressed under the direction of the mouse mammary tumor virus promoter (Lin et al., 2003; Fein et al., 2020). This protocol was optimized for use with mouse mammary tumors, where immune cells may represent 20-40% of total cells. This protocol has two major steps: first, we prepare a single-cell suspension of the mouse mammary tumors, and second, we use multi-color flow cytometry to identify different immune cell subsets. We used this protocol to identify and characterize the following tumor-infiltrating immune cell populations: TAMs, conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, NK cells, CD4+ T cells, CD8+ T cells, γδT cells, and Tregs. Other immune cell types can be studied (e.g., B cells), depending on the aim of the experiment. This protocol can also be used with other mouse models of breast cancer (i.e., orthotopic transplantation models based on cell lines, such as the 4T1) and can be easily adapted to other murine cancer models.


Materials and Reagents

  1. Multi-channel pipette 200 μl + 200 μl tips

  2. Multi-dispenser pipette 1,000 μl + 1,000 μl tips

  3. 60 mm × 15 mm Petri dish (Sigma, catalog number: P5481)

  4. Cell strainers 70 μm or 100 μm nylon (Falcon, catalog number: 352350 or 352360)

  5. Falcon conical tubes (15 ml, 50 ml) (Falcon, catalog numbers: 352196 and 352070)

  6. 1 ml sterile syringes (Fisher Scientific, catalog number: 15889142)

  7. 5 ml, 10 ml, and 25 ml pipettes (VWR, catalog numbers: 612-3702, 612-3700, and 612-3698)

  8. Eppendorf tubes 1.5 ml

  9. Scalpels

  10. Parafilm

  11. Fluorescence-activated cell sorting (FACS) tubes with cell strainer (Corning, catalog number: 352235)

  12. FACS tubes polystyrene 5 ml round bottom 12 × 75 mm (Corning, catalog number: 352052)

  13. UltraComp eBeads compensation beads (eBiosciences, catalog number: 01-2222-42), store at 4 °C

  14. 96-wells V-/conical-bottom plates (Sarstedt, catalog number: 82.1583.001)

    Note: Alternatively, we have used round-bottom plates with similar results.

  15. 1× Dulbecco’s Phosphate-Buffered Saline (PBS) (sterile, without Ca++ and Mg++) (Gibco, catalog number: 14190144), store at room temperature

  16. Hank’s balanced salt solution (HBSS) (Gibco, catalog number: 14170112), store at 4 °C

  17. 0.02% Sodium Azide (Sigma, catalog number: S2002), store at 4 °C

  18. Bovine serum albumin (BSA) lyophilized powder, suitable for cell culture (Sigma, catalog number: A9418), store at 4 °C

  19. RPMI 1640 (Gibco, catalog number: 21875034), store at 4 °C

  20. Red Blood Cell Lysing (RBCL) buffer Hybri-Max (Sigma, catalog number: R7757), store at room temperature

  21. Collagenase IV (Sigma, catalog number: C5138), store at -20 °C

  22. DNase I recombinant, RNase-free, 10 U/μl (Roche, catalog number: 4716728001), store at -20 °C

  23. Trypan blue solution (Gibco, catalog number: 15250061), store at room temperature

  24. Purified anti-mouse CD16/CD32 (Fc block) (Biolegend, catalog number: 101302), store at 4 °C

  25. Zombie Red Fixable Viability Kit (Biolegend, catalog number: 423109), store at 4 °C

  26. True-Nuclear Transcription Factor Buffer Set (Biolegend, catalog number: 424401), store at -20 °C

  27. Dissociation buffer (see Recipes)

  28. DPBS (or HBSS) with 0.5% BSA (see Recipes)

  29. 10% Sodium azide stock solution (see Recipes)

  30. Ice-cold FACS buffer (see Recipes)

  31. FACS antibodies (see Table 1)


    Table 1. Flow antibodies information

    Antibody Fluorophore Host Clone IgG subtype Catalog no. Producer
    CD45 eFluor450 Rat 30-F11 IgG2b, kappa 48-0451-82 eBiosciences
    MHC II APC-eFluor780 Rat M5/114.15.2 IgG2b, kappa 47-5321-82 eBiosciences
    CD103 PE Armenian Hamster 2E7 IgG 121405 Biolegend
    CD3 FITC Rat 17A2 IgG2b, kappa 100203 Biolegend
    CD4 PerCP/Cy5.5 Rat RM4-4 IgG2b, kappa 116011 Biolegend
    CD11b FITC Rat M1/70 IgG2b, kappa 101205 Biolegend
    CD11c APC Armenian Hamster N418 IgG 117309 Biolegend
    F4/80 PerCP/Cy5.5 Rat BM8 IgG2a, kappa 123127 Biolegend
    CD8 AF700 Rat 53-6.7 IgG2a, kappa 100729 Biolegend
    NKp46/CD335 PE Rat 29A1.4 IgG2a, kappa 137603 Biolegend
    γδTCR APC Armenian Hamster GL3 IgG 118115 Biolegend
    FOXP3 PE Rat NRRF-30 IgG2a, kappa 14-4771-80 eBiosciences

Equipment

  1. Pipettor (e.g., Pipet-Aid)

  2. Counting chamber slides or a hemacytometer

  3. Dissection tools (Fine Science Tools)

  4. Caliper (to measure tumor size) (Fine Science Tools, catalog number: 30087-00)

  5. BD LSRII flow cytometer (BD, Franklin Lakes, NJ)

    Four-laser setup: Violet (403 nm), Blue (488 nm), Yellow/Green (561 nm), and Red (640 nm). This protocol is written for analysis on a BD LSRII flow cytometer, but it can be easily adapted for use with any 4-laser cytometer. The availability of the lasers and the configuration of the mirrors in the user’s cytometer will determine which fluorochromes can be used.

  6. Shaker incubator at 37 °C

  7. Table-top centrifuge (with plates adaptor) at room temperature and 4 °C

  8. Automated cell counter (Invitrogen) or a light microscope

  9. Bench-top vortex with a 96-well plate adaptor (optional)

Software

  1. FlowJo (BD, version 10, https://www.flowjo.com/)

  2. GraphPad Prism (GraphPad, version 8, https://www.graphpad.com/)

Procedure



Figure 1. Schematic of the main steps of the protocol


Before starting:

Prepare the Dissociation buffer (see Recipes and Notes below). Use 10 ml per tumor (for diameters of 0.6-1.2 cm), filter it, and warm it at 37 °C (e.g., in a water bath). Turn on the shaker incubator at 37 °C. The dissociation buffer should be prepared fresh before each experiment.

Animal euthanasia must be performed according to the instructions of the local Institutional Animal Care and Use Committee (IACUC).


  1. Preparation of a single cell suspension from mammary tumors (Figure 1)

    1. Measure the tumor with a caliper and take note of the tumor size.

    2. Surgically remove the tumor from the mouse and place it on a sterile 60 mm × 15 mm Petri dish containing 3 ml of ice-cold RPMI or ice-cold DPBS/0.5% BSA. Take care to avoid taking the lymph node embedded within the mammary gland/tumor tissue.

    3. Mince the tumor into 1-2 mm3 pieces using two scalpels (5-10 min, depending on the tumor size) and pour the minced tumor in the buffer into a 15 ml conical tube.

    4. Add 10 ml of pre-warmed Dissociation buffer (Recipe 1).

    5. Incubate for 30 min at 37 °C in a shaker incubator. Tighten the 15 ml conical tube cover and wrap it with parafilm. Disrupt tissue every 10 min by vigorously pipetting up and down with a 5 ml pipette.

      Note: The sample should be cloudy but not stringy. If it is stringy at this step, the DNase I concentration in the dissociation buffer can be increased by adding more DNase I solution.

    6. Pipette cell suspension up and down for 2 min using a 5 ml pipette; it is very important to resuspend the cells completely as red blood cells tend to attach to myeloid cells.

    7. Centrifuge at 300 × g (1,200 rpm) for 10 min at room temperature.

      Note: Do not centrifuge at speeds higher than 300 × g if working with lymphocytes as they are sensitive to high g-forces.

    8. Aspirate the supernatant and gently resuspend the pellet in 10 ml of DPBS (or HBSS) supplemented with 0.5% BSA.

    9. Place a 100 μm cell strainer directly on top of a fresh 50 ml conical tube. Filter the resuspended cell suspension through the cell strainer. Lift the cell strainer from the tube to allow the content to go through. If necessary, rinse the cell strainer once with 2-3 ml of DPBS (or HBSS) supplemented with 0.5% BSA.

    10. Centrifuge at 300 × g (1,200 rpm) for 10 min at room temperature. Aspirate and discard the supernatant.

    11. [Skip Steps A10-A14 if tumor cell suspension is not bloody]. Resuspend the pellet in 2 ml of Red Blood Cell Lysing (RBCL) buffer. Gently mix for 1 min at room temperature.

      Note: Do not place cells on ice during this incubation step.

    12. Add 20 ml of DPBS (or HBSS) supplemented with 0.5% BSA.

    13. Centrifuge at 300 × g (1,200 rpm) for 7 min at room temperature. Discard the supernatant.

      Note: If red blood cell lysis is incomplete, which will be evident macroscopically as a red-colored cell pellet, repeat Steps A10-A12.

    14. Resuspend the pellet in 20 ml of DPBS (or HBSS) supplemented with 0.5% BSA.

    15. Centrifuge at 300 × g (1,200 rpm) for 7 min at room temperature. Discard the supernatant.

      Note: This washing step is to ensure that no RBCL buffer remains in the sample.

    16. Resuspend the pellet in 1 ml of DPBS (or HBSS) supplemented with 0.5% BSA.

    17. Place a 70 μm cell strainer directly on top of a fresh 50 ml conical tube. Filter the resuspended cell suspension through the cell strainer.

      Note: The cells tend to clump; therefore, ensure you resuspend the cells carefully and filter them before counting.

    18. Count the cells to calculate the concentration of live cells using Trypan blue. Keep the cells on ice or at 4 °C.


  2. Stain cell populations for FACS analysis (Figure 1)

    Note: Use ice-cold reagents/solutions and perform all steps at 4 °C (on ice); low temperatures prevent the modulation and internalization of surface antigens, which can reduce fluorescence intensity.

    1. Plate cells in a 96-well V-/conical-bottom plate. Each well should contain approximately 1 × 106 cells. Be sure to include wells for single-color controls for compensation (see Figure 2 for example).

    2. Centrifuge the plate of cells at 300 × g (1,200 rpm) for 4 min at 4 °C.

    3. Discard the supernatant by flicking the plate.

      Note: Ensure there are cells collected at the bottom of each inoculated well as a visible pellet.

    4. Resuspend the cells in 50 µl of ice-cold FACS Buffer with Fc Block CD16/CD32 (1:50 dilution for a concentration of 0.5 µg per well).

      Note: This blocking step is important to prevent non-specific binding and background fluorescence.

    5. Incubate on ice for 10 min.



      Figure 2. Ninety-six-well plate layout. Example of a suggested layout for Mix 1, including unstained, single stain, and FMO controls. For single-color controls, compensation beads (e.g., UltraComp eBeads) can be used. Unstained and FMO controls must be done with the sample of interest to account for auto-fluorescence. Single-color controls must be included for each antibody/fluorophore used.


Cell-Surface Staining:

  1. While cells are incubating in Fc block, prepare the antibody solutions (see Table 1) with the FACS buffer. Suggested antibody combinations and dilutions (based on a total volume of 100 µl/well, 50 µl of Fc block + 50 µl of antibody mix) are shown in Table 2. Prepare 50 µl antibody mixtures per sample.


    Table 2. Suggestion of three different antibody panels

    Note: Antibodies are added as 50 µl solutions to final total volumes of 100 µl. Therefore, the final dilution represents half of the dilutions prepared. * Intracellular antibody


    From this step on, keep the plate in the dark and on ice.


  2. Add 50 µl of antibody solution (Table 2) to the appropriate wells (it is unnecessary to wash the FC block off the cells; the total volume of the well will be 100 µl, with final dilution factors indicated in Table 2) and mix by pipetting up and down. Incubate the cells in the dark and on ice for 30 min.

  3. For flow cytometer compensation, prepare a sample of unstained cells (control well) and samples with cells (or beads) stained with each antibody-fluorophore combination used in the experiment. In control wells that do not receive antibody, it is critical to add the 50 µl of FACS buffer to avoid letting the cells dry out. Compensation beads can also be used for single-cell staining. We suggest the use of UltraComp eBeads (follow the suggested protocol from the manufacturer).

  4. Wash the cells by adding 150 µl of ice-cold FACS buffer to each well.

  5. Centrifuge the plate with cells at 300 × g (1,200 rpm) for 4 min at 4 °C.

  6. Discard the supernatant by flicking the plate. Wash 2× with 200 µl of ice-cold FACS buffer.


Intracellular (nuclear) staining:

(If no intracellular staining is to be performed, go directly to Step B23)

To stain for FOXP3, an intracellular antigen, we used the True-NuclearTM Transcription Factor Staining Buffer from Biolegend, following the exact instructions provided by the manufacturer. Other kits might be used instead of the protocol is adapted for staining of intracellular cytokines (e.g., IL-6).


  1. After the last wash, discard the supernatant and gently vortex the samples (or pipette up and down) to dissociate the cell pellet.

  2. Prepare fresh True-Nuclear Fix working solution by diluting the 4× Fix Concentrate (1 part) with the Fix Diluent (3 parts)

  3. Add 200 µl of the True-Nuclear 1× Fix working solution to each well. Gently pipette up and down to ensure cells are fully resuspended. Incubate at room temperature in the dark for 45-60 min.

    Note: A longer fixation period can help reduce high background.

  4. Centrifuge the plate at 300 × g (1,200 rpm) for 4 min at room temperature. Discard the supernatant.

  5. Prepare a 1× working solution of the Perm Buffer by diluting the 10× Perm Buffer with distilled water.

  6. Add 200 µl of the True-Nuclear 1× working solution Perm Buffer to each well.

  7. Centrifuge the plate at 300 × g (1,200 rpm) for 4 min at room temperature. Discard the supernatant.

  8. Repeat Steps B14-B15 for two additional times, for a total of three washes using the True-Nuclear 1× Perm Buffer.

  9. Add the appropriate amount of FOXP3 antibody diluted in True-Nuclear 1× Perm Buffer to each well and incubate in the dark at room temperature for at least 30 min.

  10. Add 200 µl of the True-Nuclear 1× Perm Buffer to each well. Repeat Steps B15-B16.

  11. Resuspend the cells in 150 µl of ice-cold FACS Buffer.

  12. Transfer the cells to labeled 12 × 75 mm polystyrene test tubes and add 350 µl of FACS Buffer (so that the final volume is 500 µl).

  13. Acquire the samples on a flow cytometer as fast as possible (keep the cells in the dark and on ice or at 4 °C). We typically acquire 500,000-750,000 events or cells per sample.

  14. Analyze data using software such as FlowJo or FACSDiva. Immune cell populations are identified based on the expression of cell surface molecules (see Table 3); an illustrative gating scheme is shown in Figure 3.


    Table 3. Definition of immune cell populations based on the expression of cell surface markers

    Immune cell Population Cell Surface Markers
    Total leukocytes CD45+

    Conventional Dendritic Cells
    (myeloid lineage)

    CD45+ CD11b+ MHCII+ CD11c+
    CD103+ Dendritic cells CD45+ CD11b- MHCII+ CD11c+ CD103+
    Tumor-associated macrophages CD45+ CD11b+ MHCII+ F4/80+
    Inflammatory Monocytes CD45+ CD11b+ MHCII- F4/80- Ly6Chigh

    Myeloid-derived suppressor cells
    (monocytic)

    CD45+ CD11b+ MHCII- Ly6Chigh Ly6G-

    Neutrophils/Myeloid-derived Suppressor cells
    (granulocytic)

    CD45+ CD11b+ MHCII- Ly6Clow Ly6G+
    T lymphocytes CD45+ CD3+
    CD8+ T cells CD45+ CD3+ CD8+
    CD4+ T cells CD45+ CD3+ CD4+
    FOXP3+ T regulatory cells CD45+ CD3+ CD4+ FOXP3+
    NK cells CD45+ CD3- CD335/NKp46+
    NK T cells CD45+ CD3+ CD335/NKp46+
    Gamma-delta T cells CD45+ gdTCR+



    Figure 3. Illustrative gating strategy for immune cells. First, cells from tumors are plotted on a Forward Scatter (FSC) versus Side Scatter (SSC) plot to discriminate intact cells from debris. Then, single cells are selected by displaying a plot of FSC area (FSC-A) versus FSC height (FSC-H). Live/dead cells are discriminated by selecting the Zombie Red low (live cells) or high (dead cells) population. Live single cells are then evaluated for the expression of selected cell surface markers (Table 3). Epithelial-derived cancer cells can be further discriminated from CD45+ cells by expression of EpCAM. The plots in this figure are representative of data presented in Fein et al. (2020) and adapted with permission.

Data analysis

Raw data output files of a flow cytometry experiment are generated as .fcs. These can be easily opened in the FlowJo software (https://www.flowjo.com/).

The gating strategy used is depicted in Figure 3 (adapted from Fein et al., 2020).

Statistical analyses can be performed using GraphPad Prism Version 8 software (https://www.graphpad.com/).

Figures 1 and 2 were created with BioRender.com.

Notes

  1. Proper compensation using single-color controls is necessary to account for bleed-through fluorescence being measured in a detection channel other than the primary channel. Compensation parameters can be automatically calculated “live” after signal detection by the cytometer or after the data have been collected (“offline”). In this protocol, we performed “live” compensation using the FACSDiva software.

  2. We strongly recommend the use of fluorescent-minus-one (FMO) controls. This is a sample that has been stained with all the reagents except one; the analysis of these samples allows for the precise definition of cells that have fluorescence above background levels. For example, in the myeloid panel 1 (Table 2), the FMO controls would be 1) all antibodies minus CD45, 2) all antibodies minus Ly6G, 3) all antibodies minus CD11b, and so on (see Figure 2).

  3. Isotype controls should also be used when first using a new antibody. Isotype controls are primary antibodies that lack specificity to the target but match the class and type of the primary antibody. They are used as negative controls to help differentiate non-specific background signals from specific antibody signals. However, isotype controls do not replace FMO controls (Maecker and Trotter, 2006; Cossarizza et al., 2017).

  4. For inexperienced users, we highly recommend discussing with the institution’s flow cytometry core manager before planning the experiment.

Recipes

  1. Dissociation buffer

    Collagenase IV (final concentration 2 mg/ml)

    DNase I 10U/ml (final concentration 4 U/ml) in RPMI

    For 10 ml of RPMI, use 20 mg of collagenase IV + 4 μl of 10 U/μl DNase I

  2. DPBS (or HBSS) with 0.5% BSA

    1. Dissolve 2.5 g of BSA in 500 ml of DPBS

    2. Filter the solution and keep the buffer at 4 °C

  3. 10% Sodium azide stock solution

    1. Dissolve 10 g of sodium azide in 100 ml of distilled H2O

    2. Prepare 1 ml aliquots and store at -20 °C

  4. Ice-cold FACS buffer (filtered)

    DPBS/0.5%BSA/Sodium Azide 0.02% w/v

    Keep at 4 °C for up to 2 months.

    Notes:

    1. Use Ca/Mg2+ free PBS. The absence of these ions reduces cation-dependent cell-to-cell adhesion and prevents clumping.

    2. Use 0.1 to 1% BSA. Serum proteins protect cells from apoptosis, prevent non-specific staining and prevent cells from sticking.

    3. EDTA prevents cation-based cell-to-cell adhesion and should be included in the buffer if dealing with sticky and adherent cells, like macrophages, and if these cells are to be sorted for functional cell culture assays. In that case, we recommend including 0.5-5 mM EDTA (the optimum concentration should be determined in pilot experiments to avoid cell toxicity). For characterization of immune cell infiltration into tumors using this protocol, the use of EDTA is optional.

    4. Sodium azide (0.01-1%) at low concentrations reduces bacterial contamination, prevents photobleaching, and blocks antibody shedding. The optimum concentration should be determined to avoid cell toxicity. If cells are to be collected for functional assays, do not use sodium azide because it inhibits metabolic activity.

Acknowledgments

The authors would like to acknowledge support from the Cold Spring Harbor Cancer Center Support Grant (CCSG, P30-CA045508) shared resources, the Animal Facility, and P. Moody in the Flow Cytometry Facility. This work was supported by funds from the Simons Foundation to CSHL. This protocol was adapted from previous work (Fein et al., 2020).

Competing interests

M. Egeblad holds stocks in Agios Pharmaceutical and has received personal fees from MPM, CytomX, and Insmed for consulting services, outside the submitted work.

Ethics

All animal procedures and studies were approved by the Institutional Animal Care and Use Committee at CSHL and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (ID 18-15-12-09-6, valid from July 2018 to July 2021). This protocol was modified from Fein et al. (2020).

References

  1. Beatty, G. L. and Gladney, W. L. (2015). Immune escape mechanisms as a guide for cancer immunotherapy. Clin Cancer Res 21(4): 687-692.
  2. DeNardo, D. G., Andreu, P. and Coussens, L. M. (2010). Interactions between lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity.Cancer Metastasis Rev 29(2): 309-316.
  3. DeNardo, D. G., Brennan, D. J., Rexhepaj, E., Ruffell, B., Shiao, S. L., Madden, S. F., Gallagher, W. M., Wadhwani, N., Keil, S. D., Junaid, S. A., Rugo, H. S., Hwang, E. S., Jirstrom, K., West, B. L. and Coussens, L. M. (2011). Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov 1(1): 54-67.
  4. Dunn, G. P., Bruce, A. T., Ikeda, H., Old, L. J. and Schreiber, R. D. (2002). Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3(11): 991-998.
  5. Fein, M. R., He, X. Y., Almeida, A. S., Bružas, E., Pommier, A., Yan, R., Eberhardt, A., Fearon, D. T., Van Aelst, L., Wilkinson, J. E., Dos Santos, C. O. and Egeblad, M. (2020). Cancer cell CCR2 orchestrates suppression of the adaptive immune response. J Exp Med 217(10).
  6. Grivennikov, S. I., Greten, F. R. and Karin, M. (2010). Immunity, inflammation, and cancer. Cell 140(6): 883-899.
  7. Lin, E. Y., Jones, J. G., Li, P., Zhu, L., Whitney, K. D., Muller, W. J. and Pollard, J. W. (2003). Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. Am J Pathol 163(5): 2113-2126.
  8. Ménard, S., Tomasic, G., Casalini, P., Balsari, A., Pilotti, S., Cascinelli, N., Salvadori, B., Colnaghi, M. I. and Rilke, F. (1997). Lymphoid infiltration as a prognostic variable for early-onset breast carcinomas. Clin Cancer Res 3(5): 817-819.
  9. Noy, R. and Pollard, J. W. (2014). Tumor-associated macrophages: from mechanisms to therapy. Immunity 41(1): 49-61.
  10. Perfetto, S. P., Chattopadhyay, P. K. and Roederer, M. (2004). Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4(8): 648-655.
  11. Cossarizza, A., Chang, H., Radbruch, A., Akdis, M., Andrä, I., Annunziato, F., Bacher, P., Barnaba, V., Battistini, L., Bauer, W.M., et al. (2017). Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 47: 1584-1797.
  12. Maecker, H.T., and Trotter, J. (2006). Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69(9): 1037-1042.

简介

[摘要]流式细胞术是一种流行的基于激光的技术,它允许在高表型和各个单元的功能表征-通量方式。在这里,我们描述了制备从乳腺肿瘤的单细胞悬浮液中的详细过程的的小鼠乳腺肿瘤病毒-多瘤中间T(MMTV- PyMT )和由多色流式细胞术分析这些细胞。该协议可用于研究以下由细胞表面分子表达定义的浸润肿瘤的免疫细胞群:总白细胞,肿瘤相关巨噬细胞(噬细胞),常规树突状细胞(DC),CD103表达的DC,肿瘤相关的嗜中性粒细胞,炎性单核细胞,自然杀伤(NK)细胞,CD4 + T细胞,CD8 + T细胞,γδ Ť细胞,和调节性T细胞。

[背景]肿瘤浸润的免疫CE LLS包括肿瘤微环境的重要组成部分,在控制癌症的重要作用,既防和亲致瘤性的影响(邓恩等人,2002; Grivennikov等,2010)。先天免疫细胞(包括巨噬细胞和嗜中性粒细胞)的炎症和浸润是抵抗感染所必需的,但在癌症的情况下,通常会促进疾病的发展。细胞毒性T细胞和自然杀伤(NK)细胞可以破坏肿瘤,但癌细胞已发展出多种逃避免疫破坏的机制(Dunn等,2002;DeNardo等,2010)。例如,癌细胞可以分泌直接抑制细胞毒性CD8 + T细胞并募集调节性T细胞(Tregs)和髓样来源的抑制细胞(MDSCs)的细胞因子(Beatty和Gladney,2015年)。

肿瘤免疫细胞浸润可以预后某些乳腺癌亚型(DeNardo等,2011)。例如,高淋巴细胞浸润与乳腺癌患者的存活率增加和预后良好有关(Ménard等,1997)。此外,临床研究表明,肿瘤相关巨噬细胞(TAM)的积累与乳腺癌的不良预后密切相关(Noy and Pollard,2014)。

在过去的二十年中,多色流式细胞术的进步使研究人员能够洞悉免疫细胞在肿瘤微环境中的作用。流式细胞术被广泛用于表征和量化异质细胞群不同的细胞类型小号,例如肿瘤,通过detecti纳克细胞表面和细胞内分子(普菲等人,2004)。

在这里,为了研究肿瘤发展过程中的免疫细胞浸润,我们利用了管腔B型乳腺癌的小鼠乳腺肿瘤病毒-多瘤中期T(MMTV- PyMT )模型,其中多瘤病毒中T抗原在小鼠乳腺的方向上表达肿瘤病毒启动子(Lin等,2003; Fein等,2020)。该协议经过优化,可用于小鼠乳腺肿瘤,其中免疫细胞可能占总细胞的20-40%。该协议有两个主要步骤:首先,我们准备了小鼠乳腺肿瘤的单细胞悬液,其次,我们使用了多色流式细胞仪来识别不同的免疫细胞亚群。我们使用此协议来识别和表征以下肿瘤浸润免疫细胞群:TAM,常规树突状细胞(DC),表达CD103的DC,肿瘤相关嗜中性粒细胞,炎性单核细胞,NK细胞,CD4 + T细胞,CD8 + T细胞,γδ Ť细胞和调节性T细胞。根据实验目的,可以研究其他免疫细胞类型(例如B细胞)。该协议还可以与其他乳腺癌小鼠模型(即基于细胞系的原位移植模型,例如4T1)一起使用,并且可以轻松地适应其他鼠类癌症模型。

关键字:流式细胞术, 免疫细胞亚群, 乳腺癌, 肿瘤微环境, 小鼠模型, 免疫表型, 抗体



材料和试剂


多通道移液器200μl + 200μl吸头
多分配器移液管1 ,000微升+ 1 ,000微升的提示
60 mm × 15 mm培养皿(Sigma,目录号:P5481)
细胞过滤网70μm或100μm尼龙(Falcon,目录号:352350或352360)
猎鹰锥形管(15 m l ,50 m l )(猎鹰,目录号s :352196和352070)
1米升无菌注射器(Fisher Scientific公司,目录号:15889142)
5米升,10米升,25米升移液管(VWR,目录号小号:612-3702,612-3700,和612-3698)
Eppendorf管1.5 m l
手术刀
封口胶卷
带细胞过滤器的荧光激活细胞分选(FACS)管(Corning,目录号:352235)
FACS管聚苯乙烯5米升圆底12 × 75毫米(康宁,目录号:352052)
UltraComp eBeads补偿珠(eBiosciences ,目录号:01-2222-42) ,小号撕4 ℃下
96孔V型/锥形底部平板(Sarstedt ,目录号:82.1583.001)
注意:或者,我们使用了圆底板,结果相似。


1 × Dulbecco氏磷酸盐缓冲盐水(PBS)(无菌的,不含Ca ++和Mg ++ )(GIBCO,目录号:14190144),小号撕在室温下
Hank氏平衡盐溶液(HBSS)(Gibco公司,目录号:14170112),小号撕4 摄氏
0.02%钠叠氮化物(Sigma,目录号:S2002),小号撕4 ℃下
牛血清白蛋白(BSA)的冻干粉末,适合于细胞培养物(Sigma,目录号:A9418),小号撕4 ℃下
RPMI 1640(GIBCO,目录号:21875034),小号撕4 ℃下
红细胞溶解(RBCL)缓冲液Hybri -Max(Sigma,目录号:R7757),小号撕在室温下
胶原酶IV(Sigma公司,目录号:C5138) ,小号撕在-20 ℃下
DNA酶我重组的,无RNase,10U /微升(罗氏,目录号:4716728001) ,小号撕在-20 ℃下
台盼蓝溶液(Gibco,目录号:15250061),小号撕在室温下
纯化的抗小鼠CD16 / CD32(Fc封闭)(Biolegend公司,目录号:101302) ,小号撕4 ℃下
僵尸红可定影活力试剂盒(Biolegend公司,目录号:423109),小号撕4 ℃下
真核转录因子缓冲器组(Biolegend公司,目录号:424401),小号撕在-20 ℃下
解离缓冲区(请参阅食谱)
含0.5%BSA的DPBS(或HBSS)(请参阅食谱)
10%叠氮化钠储备溶液(请参阅食谱)
冰冷的FACS缓冲液(请参阅食谱)
FACS抗体(请参阅表1)


表1.流抗体信息


设备


移液器(例如,移液器)
计数室载玻片或血细胞计数器
解剖工具(精细科学工具)
卡尺(用于测量肿瘤大小)(Fine Science Tools,目录号:30087-00)
BD LSRII流式细胞仪(BD,新泽西州富兰克林湖)
四激光设置:紫色(403 nm),蓝色(488 nm),黄色/绿色(561 nm)和红色(640 nm)。该协议是为在BD LSRII流式细胞仪上进行分析而编写的,但可以很容易地适用于任何4激光细胞仪。激光器的可用性以及用户细胞仪中反射镜的配置将决定可以使用哪些荧光染料。


摇床培养箱在37 °C
室温和4 °C的台式离心机(带平板适配器)
自动细胞计数仪(Invitrogen)或光学显微镜
带有96孔板适配器的台式涡流(可选)


软件


FlowJo (BD,版本10,https: //www.flowjo.com/ )
GraphPad Prism(GraphPad,版本8,https://www.graphpad.com/)


程序




图1.协议主要步骤示意图


在开始之前:


准备解离缓冲液(见配方小号和注意事项如下图)。使用10米升每肿瘤(直径为0.6-1.2厘米),过滤它,并在37暖℃下(例如,在水浴)。打开37 °C的振荡器培养箱。解离缓冲液应在每次实验前新鲜制备。


动物安乐死必须按照当地动物保护和使用机构委员会(IACUC)的指示进行。


从乳腺肿瘤制备单细胞悬液(图1)
用卡尺测量肿瘤并记下肿瘤大小。
通过外科手术从小鼠身上取出肿瘤,并将其放置在无菌的60 mm × 15 mm培养皿中,该培养皿中装有3 ml冰冷的RPMI或冰冷的DPBS / 0.5%BSA。注意避免淋巴结被嵌入乳腺/肿瘤组织内。
使用两个手术刀将肿瘤切成1-2 mm 3的碎片(5-10分钟,取决于肿瘤的大小),然后将切碎的肿瘤倒入缓冲液中,放入15 ml锥形管中。
添加10米升预热解离缓冲液(配方1)的。
在摇床培养箱中于37 °C孵育30分钟。拧紧15 ml锥形管盖,并用封口膜包裹。每5分钟用5 ml移液管上下吸打,以破坏组织。
注意:样品应该是混浊的,但不能是丝状的。如果它是在该步骤拉丝,所述在解离缓冲液的DNase I浓度可以通过添加增加更多的DNase I溶液。


用5 ml移液管上下移液2分钟; 完全重悬细胞非常重要,因为红细胞倾向于附着在髓样细胞上。
离心300 ×克(1 ,200rpm)下10分钟,在室温下。
注意:不要离心速度高于300 ×如果有淋巴细胞工作摹因为他们是高G力敏感的小号。


吸出上清液并轻轻悬浮在10米粒料升的补充有0.5%BSA的DPBS(或HBSS) 。
将100μm细胞过滤器直接放在新鲜的50 ml锥形管上。通过细胞过滤器过滤重悬的细胞悬液。从管中提起细胞过滤器,以使内容物通过。如果必要的话,R樱雪细胞滤网一旦与2 - 3米升的补充有0.5%BSA的DPBS(或HBSS) 。
在室温下以300 × g (1200 rpm)离心10分钟。吸并丢弃的上清液。
[如果肿瘤细胞悬液不带血,则跳过步骤A 10- A 14]。将沉淀物重悬于2 ml的红细胞溶解(RBCL)缓冲液中。在室温下轻轻混合1分钟。
注意:待办事项不置于冰上细胞在此孵育步骤。


加入补充有0.5%BSA的20 ml DPBS(或HBSS)。
在室温下以300 × g (1200 rpm)离心7分钟。丢弃上清液。
注意:如果红细胞裂解不完全,从宏观上看是红色细胞沉淀,则重复步骤A10-A12。


将沉淀重悬于补充有0.5%BSA的20 ml DPBS(或HBSS)中。
在室温下以300 × g (1200 rpm)离心7分钟。丢弃上清液。
注意:该洗涤步骤是Ë nsure没有RBCL缓冲器保持小号于样品中。


将沉淀重悬于补充有0.5%BSA的1 ml DPBS(或HBSS)中。
将70μm细胞过滤器直接放在新鲜的50 ml锥形管上。通过细胞过滤器过滤重悬的细胞悬液。
注意:细胞趋于结块;因此,ê nsure你仔细悬浮细胞,并将其筛选计数之前。


计数细胞以使用锥虫蓝计算活细胞浓度。将细胞放在冰上或在4 °C下。


用于FACS分析的染色细胞群体(图1)
注意:使用冰冷的试剂/溶液并在4 °C (在冰上)上执行所有步骤;低温会阻止表面抗原的调节和内在化,从而降低荧光强度。


1.将细胞铺在96孔V型/锥形底平板中。每个孔应包含大约1 × 10 6个细胞。是一定要包括用于补偿单色彩控制(见图井URE例如2)。     

2.在4 °C下以300 × g (1,200 rpm)离心细胞板4分钟。     

3.轻弹培养板,弃去上清液。     

注意:È nsure有在每个孔接种作为可见光粒料底部收集细胞。


4.将细胞重悬于含有Fc Block CD16 / CD32的50 µl冰冷FACS缓冲液中(1:50稀释,每孔浓度为0.5 µg)。     

注意:此阻断步骤对于防止非特异性结合和背景荧光很重要。


5.在冰上孵育10分钟。     





图2.九十- 6 -嗯板布局。Mix 1建议布局的示例,包括未染色,单一污渍和FMO控件。对于单色彩控制,补偿珠(例如,UltraComp eBeads )都可以使用。必须对感兴趣的样品进行未染色和FMO对照,以说明自发荧光。单-颜色控制必须包含对于每种抗体荧光团使用/。


细胞表面染色:


6.当细胞在Fc区域中孵育时,用FACS缓冲液制备抗体溶液(请参见表1)。小号uggested antibod ÿ组合小号和稀释液(基于微升/孔的100总体积,50μl的Fc封闭+50μl的抗体混合物的)示于表2中所示,准备50μl的抗体混合物小号每个样品。     

版权所有©20 21作者;保留所有权利。独家被许可人生物协议有限责任公司。1个                                                                                                                           



说明:logonew                                                             



表2.三种不同抗体组的建议


注意:抗体以50 µl溶液s的形式添加至最终总体积s为100 µl。因此,最终稀释度是所制备稀释度的一半。*细胞内抗体








           



版权所有©20 21作者;保留所有权利。独家被许可人生物协议有限责任公司。1个                                                                                                                           



说明:logonew               

从这一步开始,将板放在黑暗和冰上。


7.加入50μl抗体溶液(表2)至所述适当的孔中(不需要洗FC挡掉所述细胞;井的总体积为100微升,以最终稀释因子小号表2所示)并用上下移液混合。在黑暗中和冰上孵育细胞30分钟。     

8.对于流式细胞仪补偿,请准备未染色细胞的样品(对照孔)和细胞(或珠子)的样品,并用实验中使用的每种抗体-荧光团组合染色。在对照孔中那些不接受抗体,它是临界添加50微升FACS缓冲液,以避免让所述细胞干。补偿珠也可用于单细胞染色。我们建议使用UltraComp eBeads (遵循制造商的建议规程)。     

9.洗涤细胞通过加入150微升的冰冷FACS缓冲液到每个孔中。     

10.离心板与在300个细胞×克在4(1200 rpm)离心4分钟℃。 

11.轻弹培养板,弃去上清液。洗涤2 ×用200μl的冰冷的FACS缓冲液。 



细胞内(核)染色:


(如果不进行细胞内染色,则直接转到步骤B 23)


染色的FOXP3,细胞内抗原,我们使用了道道通核TM转录因子从染色缓冲Biolegend的,遵循荷兰国际集团由制造商提供的确切说明。如果该规程适用于细胞内细胞因子(例如IL-6)的染色,则可以使用其他试剂盒代替。


12.最后一次洗涤后,弃去上清液并轻轻涡旋样品(或上下吸液管)以解离细胞沉淀。 

13.通过用固定液稀释剂(3份)稀释4倍固定液(1份),准备新鲜的True-Nuclear Fix工作溶液。 

14.在每个孔中加入200 µl True-Nuclear 1 × Fix工作溶液。轻轻地上下移液以确保细胞完全重悬。在黑暗中于室温下孵育45-60分钟。 

注意:较长的注视时间可以帮助减少高背景。


15.在室温下以300 × g (1200 rpm)离心平板4分钟。丢弃上清液。 

16.用蒸馏水稀释10 × Perm Buffer,制备1 × Perm Buffer的工作溶液。 

17.在每个孔中加入200 µl True-Nuclear 1 ×工作溶液的Perm Buffer。 

18.在室温下以300 × g (1,200 rpm)离心平板4分钟。丢弃上清液。 

19.使用True-Nuclear 1 × Perm Buffer重复步骤B14-B15两次,共进行3次洗涤。 

20.向每个孔中加入在True-Nuclear 1 ×彼尔姆缓冲液中稀释的适量FOXP3抗体,并在室温下于黑暗中孵育至少30分钟。 

21.向每个孔中添加200 µl真核1 ×彼尔姆缓冲液。重复St eps B15- B16。 

22.将细胞重悬于150 µl冰冷的FACS缓冲液中。 

23.将细胞转移到标记的12 × 75 mm聚苯乙烯试管中,并加入350 µl FACS缓冲液(以使最终体积为500 µl)。 

24.获取关于流式细胞仪尽可能快地将样品(保持在细胞中的黑暗和在冰上或在4 ℃下)。我们通常会获得50万-每个样品750000的事件或细胞。 

25.使用FlowJo或FACSDiva等软件分析数据。根据细胞表面分子的表达鉴定免疫细胞群(见表3);图3显示了一个示例性的门控方案。 



表3 。基于细胞表面标志物表达的免疫细胞群定义


图3.免疫细胞的示例性门控策略。首先,将肿瘤细胞绘制在前向散射(FSC)与侧面散射(SSC)图上,以区分完整细胞与碎片。然后,通过显示FSC面积(FSC-A)对FSC高度(FSC-H)的图来选择单个单元格。活/死细胞通过选择僵尸红低(活细胞)或高(死细胞)种群来区分。然后评估活的单个细胞中所选细胞表面标志物的表达(表3)。通过表达EpCAM,可以进一步将上皮来源的癌细胞与CD45 +细胞区分开。 该图中的p个批次代表了Fein等人提供的数据。(2020 )并经许可改编。


数据分析


流式细胞术实验的原始数据输出文件生成为.fcs。这些可以很容易地打开了的FlowJo软件(https://www.flowjo.com/)。


使用的门控策略如图3所示(改编自Fein等人,2020年)。


可以使用GraphPad Prism Version 8软件(https://www.graphpad.com/)进行统计分析。


图1和2是使用BioRender.com创建的。


笔记


使用单适当的补偿-色彩控制是必要的帐户渗出通过荧光比主信道之外的信道检测被测量为。补偿参数可以在通过流式细胞仪检测信号后或收集数据后自动“实时”计算(“离线”)。在此协议中,我们使用FACSDiva软件执行了“实时”补偿。
我们强烈建议使用荧光减一(FMO)控件。这是一种样品,除一种试剂外,所有试剂均已染色。通过对这些样品的分析,可以对荧光高于背景水平的细胞进行精确定义。例如,在骨髓面板1(表2)时,FMO对照将是1)的所有抗体减去CD45 ,2)所有抗体减去Ly6G,3)所有抗体减去CD11b的,依此类推(见图URE 2)。
首次使用新抗体时,也应使用同种型对照。同型对照是对靶标缺乏特异性但与一抗的种类和类型相匹配的一抗。它们被用来作为阴性对照,以帮助区分非特异性背景信号小号的特异性抗体信号小号。但是,同型对照不能替代FMO对照(Maecker和Trotter,2006; Cossarizza等人,2017)。
对于没有经验的用户,我们强烈建议您在计划实验之前与该机构的流式细胞仪核心经理进行讨论。


菜谱


解离缓冲液
胶原酶IV(终浓度2 mg / ml )


RPMI中的DNase I 10U / ml (终浓度4 U / ml )


对于10米升RPMI的,使用20mg的胶原酶IV + 4微升10单位的/ μl的DNA酶I


具有0.5%BSA的DPBS(或HBSS)
溶解2.5克BSA在500米升DPBS的
过滤溶液并将缓冲液保持在4 °C
10%叠氮化钠原液
将10克叠氮化钠溶于100毫升l蒸馏过的H 2 O中
制备1米升等分试样并储存在-20 ℃下
冰冷的FACS缓冲区(已过滤)
DPBS / 0.5%BSA /叠氮化钠0.02%w / v


保持在4 °C最多2个月。


注意小号:


使用不含Ca / Mg 2+的PBS。在一个这些离子的bsence减少阳离子依赖性细胞-至-细胞粘附和防止结块。
使用0.1%到1%的BSA。血清蛋白可保护细胞免于凋亡,防止非特异性染色并防止细胞粘连。
EDTA防止基于阳离子的细胞-细胞粘附,并且如果处理粘和贴壁细胞,如巨噬细胞应被包括在缓冲器中,并且如果这些细胞被以被分类为功能细胞培养物测定。在那种情况下,我们建议加入0.5-5 mM EDTA(最佳浓度应在实验中确定,以避免细胞毒性)。为了使用该方案表征免疫细胞浸润到肿瘤中,可以选择使用EDTA。
小号憎恨叠氮化物(0.01〜1%)在低浓度下减少了细菌污染,防止光致漂白,并阻止抗体脱落。应该确定最佳浓度以避免细胞毒性。如果要收集细胞进行功能测定,请勿使用叠氮化钠,因为它会抑制代谢活性。


致谢


作者要感谢冷泉港癌症中心支持补助金(CCSG,P30-CA045508 )的共享资源,动物设施以及流式细胞仪的穆迪(P. Moody)。这项工作得到了西蒙斯基金会(Simons Foundation)向CSHL的资助。该协议改编自以前的工作(Fein等人,2020年)。


利益争夺


M. Egeblad持有Agios Pharmaceutical的股票,并已从MPM,CytomX和Insmed处获得个人费用,用于在提交的工作之外提供咨询服务。


伦理


所有动物程序和研究均由CSHL的机构动物护理和使用委员会批准,并根据《 NIH实验室动物的护理和使用指南》(ID 18-15-12 -09-6,于2018年7月生效)进行到2021年7月)。该协议是从Fein等人修改而来的。(2020)。


参考


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引用:Almeida, A. S., Fein, M. R. and Egeblad, M. (2021). Multi-color Flow Cytometry for Comprehensive Analysis of the Tumor Immune Infiltrate in a Murine Model of Breast Cancer. Bio-protocol 11(11): e4012. DOI: 10.21769/BioProtoc.4012.
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