Contributed equally
Adjuvant immunotherapy has recently emerged as a potential treatment strategy for breast cancer. The tumor-associated protein mucin 1 (MUC1) has received increasing attention due to its high expression in numerous types of common tumors, in which MUC1 acts as a cancer antigen. However, the simple mixed composition of an adjuvant and a peptide is not a sufficient rationale for a MUC1 peptide-based vaccine. The present study developed a novel Toll-like receptor 7 (TLR7) agonist-conjugated MUC1 peptide vaccine (T7-MUC1), which elicited an effective immune response and a robust antitumor effect in a mouse breast cancer model.
Mucin 1 (MUC1) is a membrane-associated glycoprotein involved in the protection of mucous membranes and modulation of immune system (
Toll-like receptors (TLRs) are a family of integral membrane proteins that are primarily localized on immune cells, such as dendritic cells (DCs) and macrophages (
The present study conjugated a novel T7 and MUC1 peptide together (T7-MUC1) for use as a vaccine and examined its immune responses and anti-tumor effects. It was hypothesized that systemic administration of T7-MUC1 may induce antitumor immune responses and elicit an antitumor effect in a mouse breast cancer model by enhancing CTL activity and antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, it was speculated that the therapeutic effect of T7-MUC1 may occur due to non-specific anti-tumor responses elicited by the adjuvant T7, and specific cellular and humoral immune responses elicited by the MUC1 peptide.
4T1 mouse breast cancer cells, MCF-7 human breast cancer cells, MB231 human breast cancer cells and K562 human leukemia cells (American Type Culture Collection) were cultured in RPMI-1640 medium (K562 cells) or DMEM (4T1, MCF-7 and MB231 cells) (both HyClone; Cytiva), supplemented with 10% FBS (HyClone; Cytiva), 100 µg/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO2. All experiments were performed with mycoplasma-free cells.
Female 4-week-old BALB/c mice (n=150; weight, 15–20 g) were purchased from the Medical Laboratory Animal Centre of Guangdong Province. All mice were housed in constant specific pathogen-free laboratory conditions at 18–22°C and 50–60% humidity with a 12 h light/dark cycle and
The MUC1 peptide used in the present study is a well-documented murine MUC1 epitope (
Bone marrow dendritic cells (BMDCs) were generated from the femurs and tibiae of one BALB/c mouse as previously described (
All experiments were routinely performed in groups of eight mice. On days 0, 14 and 28, the mice were intraperitoneally (i.p.) administered PBS, 20 µg MUC1 peptide, 8.5 µg T7 or 28 µg T7-MUC1. A total of 0.2 ml blood was collected from the tail of the mice 7 days after the last vaccination, and serum was harvested from the blood for serologic assays. Anti-MUC1 IgG, IgG1, IgG2a and IgM antibody titers were determined by ELISA as previously described (
Whole cell protein was extracted from K562, MCF-7 and MB231 cells using RIPA lysis buffer with 1 mM PMSF (Beyotime Institute of Biotechnology), and protein concentrations were detected using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were first blocked in 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 1 h, and then incubated with rabbit anti-human MUC1 (cat. no. 14161) or β-actin (cat. no. 4970) monoclonal antibodies (1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight, and finally with goat anti-rabbit peroxidase conjugated-secondary antibodies (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 1 h. Antibodies bound to the blots were detected using the Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.).
Ethics approval was obtained for the use of human tissues in the present study by the Medical Ethics Committee of the Third Affiliated Hospital of Shenzhen University (approval no. 2019-SZLH-LW-009), and informed consent was provided by the healthy donors. PBMCs (peripheral blood mononuclear cells) were isolated from human blood samples of 3 male healthy donors (age range, 25–35 years; mean age, 30 years), and separated by density gradient centrifugation at 400 × g for 30 min, according to the manufacturer's instructions (Tianjin Haoyang Biological Co., Ltd.). DCs and CIKs were cultured from PBMCs as previously described (
On day 21, mice were subcutaneously injected in the mid-back region with 100 µl 4T1 cell suspension (1×106 cells/ml in PBS). The tumor dimensions were measured twice a week, and tumor volume was calculated according to the equation Volume = LxW2/2, where L is length (mm) of the long axis of the tumor. On day 42, the mice were sacrificed by CO2 inhalation to minimize animal suffering, and the tumors were surgically dissected, weighed and measured. Additionally, another 30 mice were used for the evaluation of the long-term survival until the mice died naturally or the tumor diameter reached 15 mm.
The tumor tissues were cut into thin slices and stained with a Hematoxylin and Eosin Staining kit, according to the manufacturer's protocol (Beyotime Institute of Biotechnology) and observed under a light microscope at ×200 magnification.
At the time of sacrifice, the spleens were removed and lymphocytes were obtained by filtering the organs through a sterile nylon mesh (70 µm). Then, 4T1 tumor cells used as target cells and lymphocytes used as effect cells were seeded in 96-well culture plates (Corning, Inc.) at an effector/target cell ratio of 50:1 at 37°C for 4 h. The levels of LDH released by the target cells in the supernatants were determined as aforementioned.
4T1 tumor cells (5,000 cells/well) were incubated with serum (1:20 dilution) obtained from the vaccinated mice for 30 min at 37°C. NK cells separated from the spleen were used as effectors and were seeded with the antibody-labeled tumor cells in 96-well culture plates (Corning, Inc.) at an effector/target cell ratio of 50:1 at 37°C for 4 h. The levels of LDH in the supernatants were determined as aforementioned.
At the time of sacrifice, the mouse spleen was collected, and splenocytes were prepared by removing the red blood cells (RBCs) with RBC lysis buffer (BioLegend, Inc.) after separating the cells by a 70-µm cell strainer. Subsequently, ~1×106 cells were stained with the corresponding florescence antibodies and analyzed using a FACScalibur flow cytometer (BD Biosciences) and the FlowJo v10 software (BD Biosciences). The rat anti-mouse CD4-FITC (cat. no. 100406), CD8-PE (cat. no. 100708) and CD3-APC (cat. no. 100236) monoclonal antibodies (1:100 dilution) for flow cytometry were purchased from BioLegend, Inc.
Data are presented as the mean ± SEM from the indicated number of independently performed experiments. Two-way ANOVA with Bonferroni post hoc test was used to compare the tumor volumes in different groups collected over all time points. One-way ANOVA with Bonferroni post hoc test was used for the determination of statistical significance for all other experiments. P<0.05 was considered to indicate a statistically significant difference.
Structures of all of the vaccines used in this study are presented in
In order to evaluate the immunological activity of T7-MUC1, BMDCs and spleen-derived lymphocytes were incubated with different concentrations of T7, MUC1 and T7-MUC1, and ELISA was used to determine the release of cytokines. The results indicated that the levels of IL-12 and TNF-α remained unchanged when BMDCs were incubated with MUC1 alone (
The anti-MUC1 IgG, IgG1, IgG2a and IgM antibody titers were determined by ELISA. The results demonstrated that T7 and MUC1 alone had no effect on IgG and IgM antibody responses, but the T7-MUC1 conjugate significantly elicited the IgG total, IgG1, IgG2a and IgM antibody responses (
To observe the antigen effect of the T7-MUC1 conjugate, the present study developed a DC-CIK co-culture procedure using human samples. DCs and CIKs were cultured separately, and the T7-MUC1 conjugate was added as an antigen on day 3 and 5 to the DC culture. Then, DCs and CIKs were co-cultured on day 7, and the CTL effect was measured on day 14. Western blotting results for the three types of human tumor cells, MCF-7, MB231 and K562, demonstrated high protein expression levels of MUC1 in MCF-7 cells, whereas K562 and MB231 cells exhibited relatively low MUC1 expression; MB231 cells expressed undetectable levels of MUC1 protein (
To assess the antitumor effect of the T7-MUC1 conjugate, BALB/c mice were injected subcutaneously with 4T1 mouse breast cancer cells after two immunizations. Then, 21 days after the 4T1 cell injection, the mice were sacrificed, and the tumors were dissected, weighed and measured. The results demonstrated a significant growth inhibition in the T7, MUC1 and conjugate-treated groups compared with that in the control group (P<0.05;
To determine the capacity of the T7-MUC1 vaccine to elicit a tumor-specific immune response, lymphocytes were isolated from the spleen in the different groups, and the percentages of CD3+/CD4+ and CD3+/CD8+ T cells were measured by flow cytometry. No changes in the percentages of CD3+/CD4+ T cells were observed among the groups (
Breast cancer is a major cause of cancer mortality among women, especially in the least developed countries, and the risk factors include reproductive and endocrine dysfunction, obesity and physical inactivity (
In recent years, multimodal cancer vaccines have received increased attention due to their ability to simultaneously stimulate different aspects of the immune system (
TLR ligands are widely used as adjuvants in the design of vaccines to boost the immunogenicity of antigens, producing strong and long-lasting immunity. For example, TLR2 peptide-overexpressing
The results of the present study suggested that the T7-MUC1 conjugate caused a rapid induction of inflammatory mediators TNF-α, IFN-γ and IL-12 in mouse BMDCs and spleen lymphocytes
Following mouse immunization with the vaccines, the present study generated a tumor challenge model in BALB/c mice via subcutaneous implantation of mouse 4T1 breast cancer cells in order to investigate the
DC-CIK adoptive cellular immunotherapy is significant due to its strong antitumor activity against a broad spectrum of solid tumor types, including breast cancer, as DCs are stimulators of tumor-specific T cell responses, and CIKs are
In conclusion, the novel T7 was conjugated with the unglycosylated MUC1 peptide to develop a safe and effective immunotherapeutic vaccine against breast cancer. The results of the present study demonstrated that T7 elicited non-specific immune responses and strengthened the specific humoral and cellular immune responses to the MUC1 antigen. In addition, the vaccine induced CTLs and ADCC-mediating antibodies recognizing MUC1, and overall exhibited a potential to inhibit tumor growth. Therefore, this vaccine candidate may have beneficial effects for the prevention of tumor recurrence in patients with breast cancer.
Not applicable.
The present study was supported by the Guangdong Science and Technology Department (grant nos. 2017A030310400 and 2018A0303130225) and the Shenzhen Science and Technology Innovation Commission (grant nos. JCYJ20180305163318492 and JSGG20160331161046511).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
XW, GJ, YL and LT designed the study. YL, LT, NG, YWD and JZ performed the experiments. YL, LT, NG, YQD and ZW analyzed the data. XW, GJ, YL and LT wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval was obtained for the use of human tissues in the present study by the Medical Ethics Committee of the Third Affiliated Hospital of Shenzhen University (approval no. 2019-SZLH-LW-009), and informed consent was provided by the healthy donors. The protocols of animal experiments were approved by the Laboratory Animal Ethics Committee of Shenzhen University (approval no. AEWC-201712025).
Not applicable.
The authors declare that they have no competing interests.
Chemical structures of the synthetic vaccines.
T7-MUC1 conjugate induces a cytolytic response in cells expressing high levels of MUC1 protein. (A) MUC1 expression in MCF-7, MB231 and K562 cells. (B-D) Induction of the CTL effect on (B) MCF-7, (C) K562 and (D) MB231 cells. The groups are as follows: A, T7-MUC1 conjugate added on day 3; B, MUC1 added on day 3; C, T7-MUC1-conjugate added on day 5; D, MUC1 added on day 5; and E, control with no added antigen. Data are presented as the mean ± SD; n=5. *P<0.05 vs. E. MUC1, mucin 1; T7, Toll-like receptor 7 agonist; PBMCs, peripheral blood mononuclear cells; CTL, cytotoxic T lymphocytes.
T7-MUC1-conjugate inhibits 4T1 mouse breast tumor growth after two immunizations. (A) Tumor growth curves of tumor volumes measured twice a week until day 21 after tumor implantation. (B) Tumor weights were determined at the time of sacrifice; the mean tumor weight in the PBS control group was 0.75±0.10 g. (C) Survival curves of the tumor-bearing mice. (D) Representative H&E staining results of tumor tissues for the PBS control and T7-MUC1 groups. (E) Representative images of the excised tumors from all groups. Data are presented the mean ± SD; n≥5. *P<0.05, ***P<0.001 vs. PBS. H&E, hematoxylin and eosin; MUC1, mucin 1; T7, Toll-like receptor 7 agonist.
T7-MUC1 conjugate induces tumor-specific immune responses. (A and B) Percentages of (A) CD4+/CD3+ T cells and (B) CD8+/CD3+ T cells in the total splenocytes were measured using flow cytometry. (C) Induction of cytolytic T cells in the tumor model mice was measured using a CTL assay. (D) Induction of ADCC in the tumor model mice was measured using an ADCC assay. (E and F) Representative flow cytometry results of CD8+/CD3+ T cells in splenocytes in the (E) PBS and (F) T7-MUC1 groups. Data are presented as the mean ± SD; n=8. *P<0.05 vs. PBS. MUC1, mucin 1; T7, Toll-like receptor 7 agonist; NK, natural killer; ADCC, antibody-dependent cell-mediated cytotoxicity; CTL, cytotoxic T lymphocytes.
ELISA anti-MUC1 antibody titers after three immunizations with T7-MUC1.
Treatment | IgG total | IgM | IgG1 | IgG2a |
---|---|---|---|---|
PBS | 0 | 0 | 0 | 0 |
T7 | 55 | 13 | 8 | 15 |
MUC1 | 1,719 | 303 | 920 | 688 |
T7-MUC1 | 11,147 | 9,552 | 12,890 | 13,257 |
Titers were defined as the highest dilution yielding an optical density of ≥0.1 relative to normal control mouse sera. MUC1, mucin 1; T7, Toll-like receptor 7 agonist; Ig, immunoglobulin.