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.
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.
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 immunity2,3. 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.
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
2. Tumor implantation
3. Vaccine preparation and injection of Flt3L-expressing B16-F10 (B16-Flt3L) cells
4. Intratumoral immune cell isolation
5. Flow cytometry analysis
NOTE: Cells collected from the leukocytes layer contain immune cells and tumor cells. Two independent staining panels are recommended.
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 cells9,10, 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.
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.
The authors have nothing to disclose.
We thank Dr. Stephen Schoenberger for providing B16-Flt3L cells and the staff of the LJI animal and flow cytometry facilities for excellent support.
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 |