Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Summary

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Abstract

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Introduction

Cancer immunotherapy has been recognized as a promising therapeutic strategy based on its less toxic side effects and more durable responses. Several types of immunotherapies have been developed, including oncolytic virus therapies, cancer vaccines, cytokine therapies, monoclonal antibodies, adoptive cell transfer (CAR-T cells or CAR-NK), and immune checkpoint blockade¹.

For cancer vaccines, there are different forms of therapeutic vaccines, such as whole cell-based vaccines, protein or peptide vaccines, and RNA or DNA vaccines. Vaccination relies on the ability of antigen-presenting cells (APCs) to process tumor antigens, including tumor-specific antigens, and present them in an immunogenic form to T cells. Dendritic cells (DCs) have been known to be the most potent APCs and are believed to play an important role in antitumor immunity^2,3. These cells take up and process tumor antigens, and then migrate to the draining lymph nodes (dLN) to prime and activate tumor-specific T effector (Teff) cells through engagement of the T-cell receptor (TCR) and costimulatory molecules. This results in differentiation and expansion of tumor-specific cytotoxic T cells (CTL), which infiltrate the tumor and kill tumor cells⁴. Consequently, activation and maturation of DCs represent attractive strategies to stimulate immunity against tumor antigens.

Flt3L is known to promote the maturation and expansion of functionally mature DCs that express MHC class II, CD11c, DEC205, and CD86 proteins⁵. Intratumoral, but not intravenous, administration of an adenovirus vector incorporating the Flt3L gene (Adv-Flt3L) has been shown to promote immune therapeutic activity against orthrotopic tumors⁶. Flt3L has also been used in tumor cell-based vaccines consisting of irradiated B16-F10 cells stably expressing retrovirally transduced Flt3L as a way of enhancing the cross-presentation of tumor antigens by DCs and, thus, increasing antitumor responses. The protocol of B16-Flt3L tumor vaccination described here is based on a study published by Dr. James Allison's group⁷. In this paper, they reported that a B16-Flt3L vaccine combined with CTLA-4 blockade synergistically induced the rejection of established melanoma, resulting in increased survival.

The goal of this protocol is to provide a preclinical immunotherapy model for melanoma. Here, detailed procedures of how to prepare and implant tumor vaccines, and how to analyze the composition and function of intratumoral immune cells from solid tumor are described.

Protocol

All mice used in the study were maintained and housed in the vivarium of the La Jolla Institute for Immunology (LJI) under specific pathogen-free conditions with controlled temperature and humidity. Animal experiments were performed with 8-14 weeks old female C57BL/6 mice according to guidelines and protocols approved by the LJI Animal Care Committee.

1. Preparation of cultured tumor cells for implantation

1. Culture B16-F10 melanoma cells in Iscove's Modified Dulbecco's medium (IMDM) containing 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino acids, and 100 U/mL each of penicillin and streptomycin. Maintain the cell line at 37 °C under 5% CO₂.
2. Seed 1.5-2 x 10⁶ B16-F10 cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent.
3. Remove the culture medium and wash the flask once with PBS. Aspirate PBS and add 5 mL of 0.25% trypsin-EDTA followed by harshly tapping the rim of the culture flask.
4. Add 15 mL of culture medium to neutralize the trypsin-EDTA and pour the contents of the flask into a 50 mL centrifuge tube. Wash the dish surface with 10 mL of PBS and pour into the same 50 mL tube.
5. Centrifuge the cells for 5 min at 200 x g. Discard the supernatant and break the cell pellet by finger tapping the bottom of the tube.
6. Add cold 10 mL of PBS and gently pipet the cell suspension; then, manually count cells using hemocytometer. Keep the cells on ice before injection.

2. Tumor implantation

1. Gas anesthetize mice with 5% isoflurane at gas flow rate of 1.0 L per min in the fume hood. Change the flow rate to the maintenance dose of 2% isoflurane once the mice are fully anesthetized. For this protocol, anesthetization was performed by the veterinarian following the institutional animal care and use guidelines.
2. Shave the hair on the left flank of the mice and sterilize the injection site using alcohol wipes. Intradermally (i.d.) implant B16-F10 tumor cells at 5 x 10⁵ cells in 50 µL of cold PBS in the left flank using a 30 G needle.
   NOTE: The dose of implanted B16-F10 tumor cells may need to be adjusted in the range of 0.5-5 x 10⁵ cells for successful tumor development.
3. After implantation, measure the tumor length and width three times a week using an electronic digital caliper. Calculate the tumor volume (mm³) using the formula:
   Tumor volume (mm³) = width² × length × 0.5
   Treat mice with tumor vaccine when tumors have reached a size of ≥2 mm.
   ​NOTE: Tumors usually can be measured on day 3 after implantation of 5 x 10⁵ tumor cells. Faster B16-F10 tumor growth rate was observed in male C57BL/6, Rag1^-/-, or Rag2-/-γc-/-mice. A similar observation was described in other studies⁸. Keeping the gender of the mice consistent is recommended. However, note that the NIH places emphasis on sex as an important biological variable in biomedical research.

3. Vaccine preparation and injection of Flt3L-expressing B16-F10 (B16-Flt3L) cells

1. Maintain the B16-Flt3L cells in DMEM containing 8% heat-inactivated FBS, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin at 37 °C under 5% CO₂.
2. Seed 1 x 10⁶ B16-Flt3L cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent as described in steps 1.3 to 1.6 and suspend in 1 mL of cold PBS.
3. Irradiate cells at 150 Gy dose of gamma rays using X-ray Irradiator with 160 kV and 25 mA parameter setting. Count and check the cell viability by trypan blue staining before injection.
4. Gas anesthetize mice as described earlier and sterilize the injection site using alcohol wipes. Intradermally inject the mice with 1 x 10⁶ irradiated B16-Flt3L cells in 50 µL of cold PBS on the same flank as the original tumor implantation, ~1 cm away from the site of the primary tumor on days 3, 6, and 9 after the initial cell implantation.
5. Mark the vaccine injection sites with a colored pen to distinguish it from the primary tumor.
   ​NOTE: If 0.5 x 10⁵ B16-F10 cells are initially implanted, it is recommended to perform vaccine treatment on days 8, 11, and 14.

4. Intratumoral immune cell isolation

1. Sacrifice mice using CO₂ and cervical dislocation in the fume hood at the end of the experiment (on day 15 after tumor implantation; Figure 1).
2. Surgically remove the tumor with the skin from each mouse and put it into 24-well plate with 1 mL 10% FBS/RPMI-1640 medium. Dry the tumors using a paper towel before weighing it.
3. Cut the tumors into small pieces. Add 2 mL of digestion buffer (100 µg/mL TL Liberase and 200 µg/mL DNase I in RPMI-1640 medium) and incubate for 25 min at 37 °C.
4. Add 10 mL of 10% FBS/RPMI-1640 medium to stop the digestion. Transfer the cells using a 25 mL serological pipette and use the plunger of a 1 mL syringe to grind tissue on a 40 µm cell strainer.
5. Centrifuge the cells at 500 x g for 5 min at 4 °C. Resuspend the pellet in 5 mL of 40% density gradient specific medium in PBS, diluted to 1x concentration).
6. Add the cell suspension slowly on top of 5 mL of 80% density-gradient-specific medium containing PBS. Centrifuge the cells at 325 x g with low brake setting for 23 min at room temperature (RT).
7. After centrifugation, carefully collect the leukocytes layer at the interface between 40% and 80% density-gradient-specific medium and pass it through a 40 µm cell strainer. Centrifuge the cells at 500 x g for 5 min at 4 °C.
8. Incubate the pellet in 2 mL of red blood cell (RBC) lysis buffer for 5 min at RT. After incubation, add 10 mL of 10% FBS/RPMI-1640 medium to quench the RBC lysis buffer.
9. Centrifuge the cells at 500 x g for 5 min at 4 °C. Resuspend the cells in 0.5 mL of 10% FBS/RPMI-1640 medium and count the total number of cells before use for further analysis.
   ​NOTE: Collect the spleen or dLN as controls for gating strategy of immune cell subsets by flow cytometry analysis. Follow the cell isolation method as described above with the following changes: Use the plunger of a 1 mL syringe to grind tissue on a 70 µm mesh filter. Wash the tissue with 10% FBS/RPMI-1640 medium to get single-cell suspensions. Lyse the RBC as described.

5. Flow cytometry analysis

NOTE: Cells collected from the leukocytes layer contain immune cells and tumor cells. Two independent staining panels are recommended.

1. Surface staining
   1. Transfer the cells into a 96-well V-shape-bottom plate. Wash the cells with PBS and stain them with cell viability dye (50-100 µL/well) for 15 min at RT.
   2. Centrifuge the cells at 500 x g for 5 min at 4 °C. Stain the surface markers with mixed antibodies (detailed dilution is provided in Table of Materials) in FACS buffer (1% FBS and 0.05% NaN₃ in PBS) for 30 min on ice (50-100 µL/ well).
   3. Centrifuge the cells at 500 x g for 5 min and wash them with FACS buffer twice.
   4. Fix the cells with cell fixation buffer (50-100 µL/ well) for 35 min on ice. Centrifuge the cells at 500 x g for 5 min and wash them twice with FACS buffer.
   5. Store the samples in FACS buffer (150-200 µL/tube) at 4 °C and protect from light. Acquire the samples on a flow cytometer. For DCs and T cells population, follow the gating strategies provided in Figure 2B and Figure 3A.
      NOTE: For the staining of myeloid DCs, incubating FC-blocker (Rat anti-mouse CD16/CD32) for 15 min on ice before surface antibodies incubation (step 5.1.2) is recommended.
2. Cytokine analysis upon ex vivo re-stimulation
   1. Plate the cells in complete medium (RPMI-1640 supplemented with 10% FBS, 10 mM HEPES, pH 7.2-7.6, 0.1 mM non-essential amino acid, 1 mM sodium pyruvate, 100 U/mL each penicillin and streptomycin, 50 μM 2-mercaptoethanol, and 2 mM L-glutamine) and stimulate with 50 ng/mL of PMA plus 1 µM ionomycin in the presence of protein transport inhibitor for 4 h at 37 °C under 5% CO₂.
   2. Perform surface staining for surface markers as described above in step 5.1.
   3. Add the permeabilization solution (50-100 µL/well) and incubate for 5 min at RT for intracellular staining. Incubate the cells with antibodies specific for cytokines or nuclear proteins in permeabilization solution (50-100 µL/well) for 60 min on ice or overnight at 4 °C.
   4. Centrifuge the cells at 500 x g for 5 min and wash them with FACS buffer twice. Store the samples at 4 °C and protect from light. Acquire the samples on a flow cytometer.
      NOTE: Antibody dilution may have to be adjusted as necessary to accomplish optimal staining.

Representative Results

A visible black dot of the implanted B16-F10 cells is usually observed on the skin surface ~3 days after tumor implantation. Mice are treated with the tumor vaccine 3, 6, and 9 days after the tumor nodule has reached a size of ≥2 mm. We observed a significant reduction in tumor growth in vaccinated mice group ~2 weeks after tumor implantation (Figure 1). At the end of the experiment, we isolated the intratumoral immune cells and analyzed their number and cell surface marker expression, as well as cytokine production after a short in vitro stimulation as described above. Cells collected from the leukocytes layer still contain many tumor cells, making it somewhat difficult to readily define the lymphocyte population. Therefore, using in parallel splenocytes is recommended for proper gating of intratumoral immune cell subsets in flow cytometry analysis (Figure 2A). Here, gating strategies of CD103⁺CD11c⁺DC, CD8⁺, CD4⁺, and Treg are shown (Figure 2B and Figure 3A) along with the compensation matrix (Table 1 and Table 2). Representative data of acquired counts and frequency of each population are also provided in Figure 2C and Figure 3B.

Intratumoral CD103⁺CD11c⁺DCs, which represent the most potent tumor antigen-processing and presenting cells^9,10, from vaccinated mice displayed a significantly elevated expression of the costimulatory ligand CD86 (Figure 4). Vaccinated mice also displayed an increase in tumor-infiltrating CD8⁺ and CD4⁺Foxp3⁻ T cells (Figure 5A), as well as in CD8⁺GzmB⁺ and IFN-γ⁺ CTLs (Figure 5B). These results suggest that this tumor vaccination promotes DC maturation and induces stronger antitumor immunity.

Figure 1: Analysis of tumor growth in a therapeutic model of cell-based tumor vaccine using Flt3L-expressing B16-F10 melanoma cells. Female C57BL/6 mice were implanted i.d. with B16-F10 cells (5 x 10⁵) and injected with irradiated (150 Gy) B16-Flt3L cells (1 x 10⁶) in an adjacent site on the same flank. The arrowheads indicate the time points of vaccination. Cumulative data of four experiments are shown (Control, n = 12; B16-Flt3L, n = 10). Data are presented as the mean ± SEM. Statistical analysis by two-way repeated measures ANOVA test with Bonferroni post-test. *P < 0.05; ***P < 0.001. This figure has been modified from¹¹. Please click here to view a larger version of this figure.

Figure 2: Gating strategies of lymphocytes and CD103⁺CD11c⁺DCs. (A) Gating strategies of lymphocytes in spleen and tumor samples. (B) Gating strategies of intratumoral CD103⁺CD11c⁺DCs in tumor sample. (C) Acquired counts and frequency of each population from a representative unvaccinated control mouse are shown. Please click here to view a larger version of this figure.

Figure 3: Gating strategies of CD8⁺, CD4⁺, and Treg. (A) Gating strategies of T cells in tumor sample. (B) Acquired counts and frequency of each population from a representative unvaccinated control mouse are shown. Please click here to view a larger version of this figure.

Figure 4: CD86 surface expression on CD103⁺ intratumoral DCs. Expression is reported as median fluorescence intensity (MFI) normalized to average MFI in the control group (= 1). Control, n = 8; B16-Flt3L, n = 10. Data are presented as the mean ± SEM. Statistical analysis by unpaired Student's t-test. **P < 0.01. This figure has been modified from¹¹. Please click here to view a larger version of this figure.

Figure 5: Analysis of intratumoral T cells in control and vaccinated mice. (A) Enumeration of tumor infiltrating CD8⁺ (left) and non-Treg CD4⁺ (right) per gram of tumor tissue. (B) Enumeration of intratumoral GzmB⁺ (left) and IFNγ⁺ (right) CD8⁺ T cells. Control, n = 11; B16-Flt3L, n = 10. Data are presented as the mean ± SEM. Statistical analysis by unpaired Student's t-test. *P < 0.05; **P < 0.01. This figure has been modified from¹¹. Please click here to view a larger version of this figure.

Table 1: Compensation matrix of Figure 2. Please click here to download this Table.

Table 2: Compensation matrix of Figure 3. Please click here to download this Table.

Discussion

The protocol described here is based on the study by Allison's group. They demonstrated that combination of B16-Flt3L vaccine with CTLA-4 blockade showed a synergistic effect on survival rate and tumor growth, whereas no reduction of tumor growth was seen in mice receiving the B16-Flt3L vaccine or anti-CTLA-4 antibody treatment alone⁷. Recent studies have revealed a novel Treg-intrinsic CTLA4-PKCη signaling pathway that plays an important obligatory role in regulating the contact-dependent suppressive activity of Treg¹¹. Both B16-Flt3L vaccine treatment alone or vaccine combination with Treg-specific PKCη deletion significantly reduced the growth of tumor when higher number (0.5-5 x 10⁵) of B16-F10 melanoma cells than in the Allison's study (1 x 10⁴) cells were implanted. Subtle differences in the source of melanoma cells or mice may account for this difference. Therefore, titrating the number of implanted tumor cells is recommended. Increased infiltration of CD8⁺ T cells in the primary tumor was similarly observed in the previous study⁷. In addition, we observed increased CD86 expression on intratumoral CD103⁺CD11c⁺DCs, which likely accounts for a stronger activation and expansion of tumor infiltrating CD8⁺ CTLs.

This protocol, using a cell-based tumor vaccine expressing Flt3L, is convenient and straightforward to use, and serves as a reliable model to study intratumoral immune cell infiltrates, including DCs. For example, PKCη signaling is required for the contact-dependent suppressive activity of Treg. Thus, Prkch^−/− Treg displayed higher conjugation efficiency with APCs in a Treg -DC in vitro coculture system, indicating a defect in the ability of Prkch^−/− Treg to break contact and disengage from attached DCs¹². B16-Flt3L vaccination likely recruits more mature DCs that present tumor-specific antigens more efficiently and, thus, it is likely to facilitate observation of the interaction between tumor-infiltrating Treg and DC by live cell imaging.

Keeping a sufficient distance between the primary tumor and the tumor vaccine is an important feature of this protocol. This physical separation is critical in order to allow room for growth of the primary tumor and avoid potential fusion between the two tumor implants. Alternatively, the tumor vaccine can be implanted in the opposite flank relative to the primary tumor, as this was also reported to inhibit tumor growth⁷. One limitation of the study is the lack of a commercial source of B16-Flt3L cell line, but the same concept can be applied to other tumor types, for example, TRAMP-C2 prostate adenocarcinomas⁷.

Although the protocol described here uses B16-F10 melanoma cells as a tumor model, the underlying principles can be adapted and modified as necessary to establish immunotherapeutic models for other solid tumors. Furthermore, the vaccination protocol we used can be readily combined with other therapeutic modalities, e.g., CTLA-4- or PD-1- based checkpoint blockade, in order to achieve additive or synergistic effects that can potentially result in more potent antitumor immunity and increased survival.

Materials

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco	25200-056
10% heat-inactivated FBS	Omega Scientific	FB-02	Lot# 209018
30G needle	BD Biosciences	305106
96 well V-shape-bottom plate	SARSTEDT	83.3926.500
B16 cell line expressing Fms-like tyrosine kinase 3 ligand (B16-Flt3L)	Gift of Dr. Stephen Schoenberger, LJI		Flt3L cDNAs were cloned into the pMG-Lyt2 retroviral vector, as in refernce 5, Supplemental Figure 1
B16-F10 cell lines	ATCC	CRL-6475
Centrifuge 5810R	Eppendorf
Cytofix fixation buffer	BD Biosciences	BDB554655	Cell fixation buffer (4.2% PFA)
Cytofix/Cytoperm kit	BD Biosciences	554714	Fixation/Permeabilization Solution Kit
DNase I	Sigma	11284932001
Dulbecco's Modified Eagle Medium  (DMEM)	Corning	10013CV
Electronic digital caliper	Fisherbrand	14-648-17
FlowJo software	Tree Star		Flow cytometer data analysis
GolgiStop (protein transport inhibitor)	BD Biosciences	554724	1:1500 dilution
HEPES (1M)	Gibco	15630-080
Ionomycin	Sigma	I0634
Iscove’s modified Dulbecco’s medium (IMDM)	Thermo Fisher	12440053
LSR-II cytometers	BD Biosciences		Flow cytometer
MEM nonessential amino acids	Gibco	11140-050
penicillin and streptomycin	Gibco	15140-122
Percoll	GE Healthcare Life Sciences	GE17-0891-02	density gradient specific medium
PMA	Sigma	P1585
Red Blood Cell Lysing Buffer Hybri-Max liquid	Sigma	R7757-100ML
RPMI 1640 medium	Corning	10-040-CV
RS2000 X-ray Irradiator	Rad Source Technologies
sodium pyruvate	Gibco	11360-070
Sterile cell strainer 40 μm	Fisherbrand	22-363-547
Sterile cell strainer 70 μm	Fisherbrand	22-363-548
TL Liberase	Roche	477530
Zombie Aqua fixable viability kit	BioLegend	423101
Antibodies
Anti-mCD45	BioLegend	103135	Clone: 30-F11 Fluorophore: BV570 Dilution: 1:200
Anti-mCD3ε	BioLegend	100327	Clone: 145-2C11 Fluorophore: PerCP-Cy5.5 Dilution: 1:200
Anti-mCD8	BioLegend	100730 100724	Clone: 53-6.7 Fluorophore: Alexa Fluor 700, Alexa Fluor 647 Dilution: 1:200
Anti-mCD4	BioLegend	100414	Clone: GK1.5 Fluorophore: APC-Cy7 Dilution: 1:200
Anti-mFoxp3	Thermo Fisher Scientific	11577382	Clone: FJK-16s Fluorophore: FITC Dilution: 1:100
Anti-m/hGzmB	BioLegend	372208	Clone: QA16A02 Fluorophore: PE Dilution: 1:100
Anti-mIFNg	BioLegend	505826	Clone: XMG1.2 Fluorophore: PE-Cy7 Dilution: 1:100
Anti-mCD19	BioLegend	115543	Clone: 6D5 Fluorophore: BV785 Dilution: 1:100
Anti-mGr1	BioLegend	108423	Clone: RB6-8C5 Fluorophore: APC/Cy7 Dilution: 1:200
Anti-mCD11b	BioLegend	101223	Clone: M1/70 Fluorophore: Pacific blue Dilution: 1:100
Anti-mF4/80	BioLegend	123114	Clone: BM8 Fluorophore: PECy7 Dilution: 1:100
Anti-mCD11c	BioLegend	117328	Clone: N418 Fluorophore: PerCP Cy5.5 Dilution: 1:100
Anti-mMHCII	BioLegend	107622	Clone: M5/114.15.2 Fluorophore: AF700 Dilution: 1:400
Anti-mCD103	BioLegend	121410	Clone: 2E7 Fluorophore: Alexa Fluor 647 Dilution: 1:200
Anti-mCD86	BioLegend	105007	Clone: GL-1 Fluorophore: PE Dilution: 1:200
FC-blocker (Rat anti-mouse CD16/CD32)	BD Biosciences	553141	Clone: 2.4G2 Dilution: 1:200