Introduction

Breast cancer is one of the main deadly cancers in women. After lung cancer, breast cancer is the second most frequent and widespread cancer type (Veisy et al. 2015). According to the World Health Organization (WHO) data, the number of diagnosed breast cancers in 2020 was 2.3 million, and it is estimated to reach 3.2 million by 2050. Breast cancer also makes up almost 29.8% of all types of cancer in women in 2020, and 685,000 women died of breast cancer worldwide.

Most traditional cancer treatments, including surgery, chemotherapy, and radiotherapy, regularly fail to achieve a complete remission of cancer. Furthermore, significant side effects of chemotherapy or radiotherapy are well-known. So, cancer treatment needs to be developed into innovative and more effective methods (Lopes et al. 2019).

Immunotherapy has received increasing attention as a strategy for cancer treatment, and many different approaches are being developed to improve the clinical outcome in cancer patients (Veisy et al. 2015; Hung et al. 2001). Recently, genetic vaccines composed of DNA or RNA with some advantages serve as novel strategy for vaccination (Behzadi et al. 2016). DNA vaccine has emerged as an attractive approach for vaccine development against cancer and some infectious diseases (Jahangiri et al. 2011, 2018; Khalili et al. 2015; Sefidi-Heris et al. 2020; Perabo et al. 2005; Sundstedt et al. 2009; Yousefi et al. 2015; Sawant et al. 2020; Choi et al. 2017). DNA vaccine is the third-generation vaccine and is a modern tool composed of DNA that encodes the desired protein or specific antigens of a pathogen. This vaccine stimulates cellular and humoral responses against different diseases like cancer, autoimmune, and infectious diseases (Choi et al. 2017). DNA vaccine has been shown to generate long-term cell-mediated immunity (Halabian et al. 2009). The potency of presented several antigen genes in the same construct is one of advantages of DNA vaccines to combat different types of cancer. A polyepitope DNA vaccine could be present of immunodominant and promote the CTL response (Lopes et al. 2019).

Employing bacteria or their toxins for the control and treatment of malignant tumors appears promising. Research records also show the ability of Staphylococcus aureus enterotoxins to induce apoptosis in different cancer cell lines (Zhang et al. 2017). Moreover, staphylococcal enterotoxins (SEs) are bacterial superantigens (SAgs) which could stimulate T cells (Sedighian et al. 2018a; Nodoushan et al. 2022). SEs of the enterotoxin gene cluster (egcSEs) activate peripheral blood mononuclear cells (PBMCs) that kill a wide range of cancer cells, depending on time and concentration (Choi et al. 2017; Terman et al. 2013; Sedighian et al. 2018b). Staphylococcal enterotoxin B (SEB) which establishes interaction between Fas and its receptor could stimulate apoptosis in the RT112 and RT4 cancer cell lines (Perabo et al. 2005; Hedayati et al. 2016). SEB strongly induces cytotoxic T cell activity and cytokine production in vivo. Less than 0.1 pg/ml of superantigen is sufficient to stimulate T cells. The SEB forms a complex with MHCII molecule on the antigen-presenting cells outside the peptide-binding groove and then binds to the TCR via the β-chain variable region. Following this binding, SEB activates all T cells that express a specific set of Vβ-TCR regardless of actual antigen specificity (clonality), resulting in activation of more than 25% of peripheral T cells. Both TCD8+ and TCD4+ cells proliferate as a result of the exposure to this superantigen. Increased T cell activity is associated with increased production of Th1 cell cytokines such as IFN-γ, IL-2, and TNF-α. The increased T cell activity usually occurs within 24 h after superantigen exposure (Imani Fooladi et al. 2014). It had been shown that SEB-activated peripheral blood mononuclear cells extensively secrete the cytokines IL-2, IFN-γ, and TNF-α and induce apoptosis in bladder cancer cells (Xu et al. 2011). SEB has been reported to be effective against fibrosarcoma. Tumor cell death is attributed to increased cytotoxic T cell activity and increased cytokine levels due to intravenous injection of SEB (Imani Fooladi et al. 2014).

Although antitumor activity of SEB protein has been established in previous studies, this toxin causes systemic toxicity; so, applying this toxin in high doses and in one step can be dangerous. When using DNA vaccine, the protein production must be performed in a long run and during a slower process. Moreover, the longer half life of the drug can improve the cancer-fighting process. Hence, this method could induce an appropriate response (Sundstedt et al. 2009). Our in silico study suggested that SEB as a DNA vaccine can be effective against cancer (Jahangiri et al. 2018). However, experimental investigation must confirm the in silico results. The present study is conducted to evaluate SEB as a DNA vaccine for the treatment of breast cancer in the murine model.

Materials and methods

Restriction endonuclease enzymes and silica-based DNA gel extraction kits were purchased from Fermentas (Thermo Fisher Scientific, Inc., Waltham, MA). PCR purification kit and DNA extraction kit were purchased from Bioneer (Bioneer, Korea). Deoxynucleoside triphosphate was purchased from Fermentas (Fermentas, Germany). The 10 × thermophilic buffer, pfu DNA polymerase, and MgSO4 got from Promega (Promega, Germany). Diaminobenzidine and nitrocellulose membrane were purchased from Sigma (Sigma, Germany) and Schleicher (USA). Oligonucleotides were used as primers for PCRs synthesis by Bioneer and ShineGene Molecular Biotech, Inc. (China).

In vitro experiments

SEB gene construct preparation

The SEB optimized gene was synthesized and cloned into the pGH vector by Nano Zist Aria. The cleavage site for XhoI and HindIII enzymes was embedded at upstream of the gene, and BamHI, EcoRI, and SalI enzyme sites were embedded at downstream of the gene. The final length of the gene was 831 bp. The recombinant vector was transformed into the competent Escherichia coli Top10F' host by the CaCl2 method to obtain high copy number of the construct. Since the vector contained an Amp resistance gene, recombinant bacteria were then cultured on LB agar Amp + plate.

The transformants were cultured in LB broth containing Amp (100 µg/ml) overnight at 37 °C. Enzymatic digestion reaction was performed by EcoRI and HindIII to retrieve and confirm the SEB optimized gene.

The pVAX plasmid (Invitrogen, Germany) was selected as the DNA vaccine vector. This vector contains a kanamycin resistance gene. Therefore, the E. coli Top10F' host containing the vector was cultured in LB broth Kan+ medium to amplify desired vector (Agheli et al. 2016). Plasmid purification was performed to extract the vector. For sub-cloning, the pGH recombinant vector containing the SEB gene and pVAX vector was digested by BamHI and HindIII restriction enzymes.

Then, the ligation was done in final volume of 20 μl as follows: 1 μl T4 DNA ligase (~ 100 U/μl), 2 μl buffer (10 ×), 5 μl vector (~ 100 ng), 7 μl target fraction (~ 300 ng), and 5 μl distilled water. The reaction was carried out overnight (16 h) at 10 °C. Then, the ligation product was transformed using a heat shock method to the competent cells. The cells were grown on a LB agar containing kanamycin (70 µg/ml) for 16 h. The SEB gene presence/absence within the pVAX plasmid was evaluated by digestion with BamHI and HindIII restriction enzymes.

Re-cloning of SEB gene construct with histidine-tagged peptide in the pVAX vector

To detect the expressed recombinant SEB in the eukaryotic cell lines, a 6 × His (His-tag) was added to the SEB gene. In this regard, new primers containing 6 × His (His-tag) codons (Table 1) were designed to amplify the SEB gene. The amplicon was digested and ligated to as described above pVAX expression vector. The re-cloned construct was harboring a SEB gene with His tag. For re-cloning, the amplicon (SEB + His tag) and pVAX vector were digested by BamHI and HindIII restriction enzymes.

Table 1 Forward and reverse primers for SEB gene cloning
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The re-cloned construct was confirmed by enzymatic cleavage as previously described. Finally, the construct was sequenced to be additionally confirmed.

Transfection of HEK293T cell line with pVAX-SEB cassette

HEK293T cell line that purchased from the Pasteur Institute of Iran Cell Bank cultured in RPMI (Gibco,Germany) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Germany) at 37 °C and 5% CO2. After cells reached around 70% confluent, transfection was performed using FuGENE 6 kit (Roche, Germany) according to manufacturer instruction, with different amounts of FuGENE 6 and a vector containing gene (pVAX-SEB), in ratios of 3:2, 3:1, and 6:1. The same steps were performed for the pEGFP vector as a positive transfection control.

Evaluation of SEB gene expression in HEK293T cell line with RT-PCR and Western Blot

Total RNA (500 ng) of HEK293T cells transfected with blank vector (control), and HEK293T cells transfected with pVAX-SEB (expressed SEB gene) used to construct cDNA by RT-PCR (SuperScript Double-Stranded cDNA Synthesis Kit, invitrogen, Germany). Then, PCR was done with specific primers and β-actin primers. The PCR product was electrophoresed on a 2% agarose gel.

To confirm the expression of SEB in pVAX-SEB-transfected cells, SDS-PAGE was performed and then blotting was performed. For this purpose, the cells transfected with the pVAX-SEB construct were cultured in 6-well plates and compared to the control. Three days later, the cell culture medium and the cell lysate were collected and the samples were electrophoresed on the SDS-PAGE gel. Then, the gel transferred onto the PVDF membrane with a blotting action. The membrane was affected by the specific antibody of histidine and the staining process.

Purification of the pVAX-SEB cassette with LPS-free plasmid extraction kit

To amplify the prepared cassette and prepare it for injection into mice, E. coli Top10 was transformed with pVAX-SEB cassettes, and then, the bacteria containing these cassettes were cultured in 1500 ml LB broth medium. Next, the plasmid extraction was accomplished using LPS-free kit GigaEndo (Qiagen, Germany).

In vivo experiments

Therapeutic setting in mice

Five to 6-week-old female BALB/c mice were provided by the Center of Medical Experimental Animals of Baqiyatallah Medical Science University (Tehran, Iran). Animals were treated according to the Animal Care and Use Committee of the university. The animals were kept in the standard condition, in plastic cages, on a 12:12-h light and dark cycle, with free access to food and water, and at 25 °C and 50% humidity. The mice were divided into groups based on the type of injected antigen, and the injection route determined according to Table 2. The sample size in each group was 10 BALB/c female mice of 5–6 weeks.

Table 2 Mice groups, type of antigen, and the route of injection
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Tumor induction in mice

The 4T1 cancerous cell lines were purchased from the Pasteur Institute (Tehran, Iran.) and cultured in RPMI1640 with 10% FBS and 1% antibiotics (all purchased from Gibco, Darmstadt, Germany) in T75 culture flasks. Mouse breast cancer 4T1 is a transplantable tumor cell line (Sztalmachova et al. 2015). Before injection, the 4T1 cells were washed twice, counted, and resuspended in PBS at 2 × 105/100 µl. Cells were injected to subcutaneous in right flank to induce a tumor.

Measurement of tumor size during the growth process

For analyzing the tumor growth process, tumor size (width and length) was measured every 24 h with a digital caliper. Then, the tumor volume was calculated consistent with the formula: Volume (mm3) = (Length (mm) × Width (mm)2)/2 (Euhus et al. 1986; Tomayko and Reynolds 1989; Faustino-Rocha et al. 2013).

Mice immunization

Mice with 4T1 tumor cells were immunized one or three times at 7-day intervals, first in the in plantar and later in lower-left flank with pVAX-1-SEB, pVAX-1, or PBS by subcutaneous injection on days 0, 7, and 14. Generally, the first injection transfers more gene cassette to the host cells. However, the subsequent subcutaneous injections stimulate the immune system (Imani Fooladi et al. 2014).

After preparing the DNA vaccine by a large-scale Endofree kit (Qiagen, Germany), its concentration was measured by a NanoDrop spectrophotometer. The concentration used in gene vaccine based on similar researches is around 100 ng/µl. Reagan-Shaw et al. and the Web Site (https://dtp.cancer.gov) have revealed a renowned mode to change the dose of medication used from one species to another (Reagan-Shaw et al. 2008; https://dtp.cancer.gov/organization/tpb/drug_development.htm2015). The maximum safe starting dose and alteration between animals and humans are predictable according to the following formula: human equivalent dose (mg/kg) = animal dose (mg/kg) × animal Km/human Km.

Specific Km values of species are coefficient of body surface area (for a 60-kg adult human, Km = 37, for a 20-g mouse, Km = 3) (Rattan et al. 2011; Saadh et al. 2020).

Evaluation of lymphocyte proliferative responses

Two weeks after the last injection of synthesized immunogens, two mice from each group were sacrificed. Their spleen was removed from the body, washed, gently grounded with a syringe and suspended in PBS. The suspension was washed twice in cold PBS by centrifugation at 300 g at 4 °C for 10 min.

Lymphocyte proliferative responses by cell counting

After the spleen was isolated and cutted, the suspension was washed twice in cold PBS; 5 ml of RBC lysis buffer (0.1 M ammonium chloride) was added to the cell mass and kept at room temperature (RT) for 5 min to lyse the erythrocytes. After washing the cells, the suspension was resuspended in a complete RPMI-1640 medium. Immediately, 10 μl of suspension was stained with 10 μl of trypan blue dye and counted using a Neobar slide (Li et al. 2008). Then, 2 × 105 cells were added to each well of two 96-well culture plates for performing MTT assay and ELISA. One microgram of antigen was added to each well, negative control consisted of cells only, and positive control included PHA at a concentration of 5 μg/ml.

Evaluation of cytokine response pattern

Evaluation of cytokine response patterns was performed by ELISA assay in the supernatants of the 4T1 cell culture (in vitro) and the cultured spleen cells (in vivo). A kit of ELISA Antibodies (R&D System) was used to determine the levels of IL-4 and IFN-γ, keeping with the manufacturer’s protocol with some modifications (Imani Fooladi et al. 2014). Briefly, a 96-well microtiter ELISA plate (MaxiSorb, Nunc, Denmark) was coated with a capture antibody of the relative cytokine and incubated overnight at 4 °C. Following washing three times with PBST wells were blocked ultimately with 200 µl of 2% BSA for 2 h at 37 °C. After the incubation period, wells were washed three times and incubated with the supernatants, harvested from the 4T1 cell culture, for 2 h at RT. The wells were washed five times and incubated with the antimouse IgG-horseradish peroxidase for 1 h at RT. Then, wells were washed seven times and incubated with 100 µl of substrate solution for 30 min in the dark at RT. The reaction was inhibited by adding 50 µl of stop solution (DMSO) to each well. The absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad Laboratories) 30 min after completing the reaction. The concentrations of cytokines in the culture supernatants were calculated using a linear regression equation following the standards given by kit manufacturer (Imani Fooladi et al. 2014). Levels of IL-4 and IFN-γ in the supernatant of cultured spleen cells were assessed by the mouse ELISA Kit (R&D System), according to the manufacturer’s protocol for ELISA assay of the 4T1 cells (Imani Fooladi et al. 2014).

Histopathology

Tumor isolation for pathological diagnostic tests

After isolation of the tumor, the central part of the tumor may develop spontaneous necrosis due to lack of blood supply, stiff mass, and the tumor’s peripheral regions used for the study. Isolated tumor fragments of mice were fixed with 10% formalin and placed in a processing machine for 12 h. Then, they embedded paraffin in special molds and with a microtome, sections of 4 μm thick prepared on glass slides impregnated with poly-lysine. Then, the prepared smears were deparaffined and fixed by the standard method. Hematoxylin and eosin (H&E) staining was used to study histopathology and the extent of necrosis in tumor tissues (Elmore et al. 2016). Also, the quantity of metastasis was measured in the bone marrow, lung, and lymph nodes.

Survival time test in tumor mice

Assessment of survival time was recruited to determine the effect of recombinant SEB construct on the survival time of tumor mice. For this purpose, five groups of 5 mice were examined daily after vaccination and tumor formation and the death of each mouse was recorded separately. Finally, the survival rates were compared within groups (Saeedi et al. 2019).

Statistical data analysis

The specific interleukin levels were statistically analyzed with SPSS 20.0.0 software (SPSS, Inc., IBM Co, USA). One-way ANOVA test determined the differences between control and experimental groups. Dunn’s test was carried out following Kruskal–Wallis’s test for multiple pairwise comparison between the groups. The p < 0.05 of all results was considered significant.

Results

In vitro experiments

SEB gene construct preparation

SEB optimized gene approved for synthesis. The final length of the gene was 831 bp after adding the sequence of cleavage sites which was cloned into the pGH vector (Figure S1). The recombinant vector pGH-Jah containing the SEB synthetic gene construct was transformed into the cloning host E. coli Top10. After confirmation, the plasmid containing the fragment was stored, as well as the bacteria containing the plasmid.

Subcloning the SEB gene into the pVAX eukaryotic expression vector

The pVAX plasmid containing the kanamycin resistance gene was selected as the vector of the DNA vaccine. The pVAX DNA plasmid and the vector containing SEB gene were cleaved by HindIII and BamHl restriction enzymes. The DNA fragment ligated into linear plasmid DNA. The enzymatic digestion reaction was performed using two BamHI and HindIII enzymes for 4 out of 5 cases. The presence of the fragment was verified in each of the 4 vectors (Figure S2). Once verified, hosts containing these 4 vectors were stored.

Transfection of HEK293T cell line with pVAX-SEB cassette

SEB gene expression in HEK293T cell line

After optimizing the transfection and transfection conditions of the cells with a 3:1 ratio of vector to insert, the results showed that the vector transfected correctly into the HEK293T cell line. RNA extraction from transfected cells was performed using a TriPure™ isolation reagent. The quality of the extracted RNA of transfected cells was evaluated and run on a 2% agarose gel. Three bands related to 28S rRNA, 18S rRNA, and 5.8S rRNA were observed, indicating the absence of nonspecific bands and DNA interference in the extracted RNA (Figure S3a). The concentration of extracted RNAs was evaluated using a NanoDrop™ spectrophotometer that showed to be 1126.1 ng/µl (Figure S3b). The total RNA extracted from HEK293T transfected cells and an empty vector (control) and HEK293T transgenic cells transfected with pVAX-SEB were utilized for cDNA synthesis. The PCR product samples were electrophoresed on 1% agarose gel, and the results are shown in Figure S4. The results indicated an 845-bp band in the pVAX-SEB transfected specimen that proved the expression of the SEB gene in transfected cells. The absence of the band in control samples confirmed the test. There was also a band that corresponds to the 115-bp of the β-actin.

Also, the cell culture medium and the cell lysate were collected and the samples were electrophoresed on the SDS-PAGE gel. Then, the gel was transferred onto the PVDF membrane with a blotting action. The membrane was affected by the specific antibody of histidine and after the staining process; the results are displayed in Fig. 1. There are very sharp blots of 28 kD on PVDF. The absence of the SEB-specific band in the control sample and its presence in SEB-transfected cells represent the expression of SEB protein in recombinant vector-transfected cells.

Fig. 1
figure 1

Western blotting of SEB protein expression. Expression of SEB protein in cells containing pVAX-SEB was demonstrated in comparison with control using Western blotting. Lanes 1 and 2 are related to the expression of SEB protein obtained from cell lysis, and lanes 3 and 4 are related to the SEB expression in cell culture medium of SEB transfected cells. Lanes 5 and 6 show the high product of cells transfected with empty vector (control). The expression of the SEB protein was observed in cells containing SEB gene

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The effect of SEB on the proliferation of the isolated lymphocytes from the immunized mice,in vitro

Following immunization of mice with prepared vaccines, immune responses were assessed. The mice groups were as follows: PBS group, negative control group that received only PBS; pVAX group, the recipient group (vaccinated) with blank vector; SEB.St.Vac group, the group that received standard SEB; SEB.Vac (1) group, the group that received (vaccinated) one dosage of vector containing SEB gene construct; SEB.Vac (2) group, the group that received (vaccinated) two dosages of vector containing SEB gene construct; and SEB.Boost.Vac group, the group received (vaccinated) vector containing SEB gene construct, and then, two doses of SEB protein was injected as a booster.

The MTT assay was performed for investigating the proliferating ability of the spleen lymphocytes, reoccurring with the tumor cells. The results showed that after 72 h exposure of the separated cells of the spleen to tumor cells, lymphocyte proliferation significantly increased in the SEB-immune mice group SEB.Vac (2) compared to the four control groups PBS, pVax control, SEB.Boost.Vac, and SEB.St.Vac group (p < 0.001). The proliferation rate in SEB Vac group received DNA vaccine increased to 98.9%. The group received recombinant SEB showed the lowest proliferation rate (Fig. 2).

Fig. 2
figure 2

The effect of SEB vaccination on the proliferation of lymphocytes isolated from vaccinated mice. After exposure of splenic lymphocytes to tumor cells, a significant increase in the proliferation of lymphocytic cells was observed in the group vaccinated with the recombinant vector containing the SEB gene construct compared to PBS control (p < 0.001). PBS group, negative control group that received only PBS; pVAX group, the recipient group (vaccinated) with blank vector; SEB.St.Vac group, the group that received standard SEB; SEB.Vac (1) group, the group that received (vaccinated) one dosage of vector containing SEB gene construct; SEB.Vac (2) group, the group that received (vaccinated) two dosages of vector containing SEB gene construct; SEB.Boost.Vac group, the group received (vaccinated) vector containing SEB gene construct, and then, two doses of SEB protein were injected as a booster

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The DNA vaccine increased the release of IL-4 and IFN-γ cytokines fromthe isolated in vitro immunized mice lymphocytes

The isolated lymphocytes from the spleen of the mice were exposed to the specific antigen (purified SEB), after the level of IL-4 and IFN-γ were measured by the ELISA method. The IL-4 production in the group that received single dose of the DNA vaccine did not change significantly in comparison with the control group (viz. pVAX) (Fig. 3a). However, the level of IL-4 increased in the group that received two doses of DNA vaccine.

Fig. 3
figure 3

a Graph of IL-4 level measured in cultured lymphocytes isolated from spleen of vaccinated mice. The concentration of IFN-γ in SEB-Vac group showed a significant increase compared to the PBS and pVAX groups (p < 0.001). b Measured level of IFN-γ in cultured splenic lymphocytes of mice. The production of IL-4 in the group received single dose of the DNA vaccine did not change significantly compared to the control group (viz. pVAX)

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The concentration of IFN-γ cytokine in all groups showed a significant increase compared to the control groups viz. PBS and pVAX (p < 0.001) (Fig. 3b). The production of IFN-γ in the spleen lymphocytes was higher in the group that was stimulated with specific antigens in DNA vaccine in comparison with two other groups that received booster and SEB.St (p < 0.01); the higher level of IFN-γ indicates that more Th1 cells are stimulated, and cellular immune responses are involved in this process.

The DNA vaccine harboring SEB gene retarded the death of tumor mice

Vaccine immunization with the SEB-containing gene construct significantly increased the survival time of tumor mice (54 days) (p < 0.001) (Fig. 4). Survival time in SEB.St.Vac, SEB.Boost.Vac, pVAX, and PBS groups was 46, 41, 38, and 37 days, respectively.

Fig. 4
figure 4

The effect of recombinant SEB gene construct on survival time in vaccinated mice

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The group received the pVAX-SEB gene construct reduced the tumor size

The tumor size in the groups was monitored for 30 days. A significant reduction in tumor size was observed in the mice twice immunized with the construct containing SEB gene (SEB.Vac (2) and SEB.Boost.Vac) compared to the PBS group (p < 0.001) (Fig. 5), while tumor volume in the SEB.Vac (1) group was significantly increased.

Fig. 5
figure 5

The effect of recombinant SEB gene construct on tumor size in vaccinated mice

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Immunization with pVAX-SEB and its effect on tumor necrosis

The results of the pathologic test of tumor tissues obtained from the mice showed that in the SEB-VAC group, the incidence of necrosis significantly increased compared to the pVAX group (p < 0.01) (Fig. 6). The percentages of necrosis in the SEB.St.Vac and SEB.Boost.Vac groups are 48 and 12/55%, respectively. The PBS and pVAX control levels were 28.65% and 34%, respectively.

Fig. 6
figure 6

The effect of recombinant SEB gene construct on tumor tissue necrosis in vaccine-treated mice and control groups

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Pathological observations

Pathological manifestation showed metastases to the lung were higher in SEB.Vac (1), pVAX, PBS, SEB.stVac, SEB.Vac (2), and SEB.Booster and were 81.5, 69.1, 52.3, 48.2, 41.2, and 34.3 respectively. In SEB.Vac (1) and pVAX groups, the metastasis level was significantly higher than the other groups. Furthermore, metastasis was detected in the lymph node and bone marrow of two mice in SEB.Vac (1) group (Figs. 7 and 8).

Fig. 7
figure 7

Bright-field microscopy images of metastatic tissues. a–b, c–d, e–f Metastases to the bone marrow, lung tissue, and lymph node, respectively. Arrows show the tumor cells. All the images were taken at magnifications × 40 and × 100

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Fig. 8
figure 8

Bright-field microscopy images of tumor tissues. a Tumor tissue (× 40). b, c Angiogenesis in tumor tissue at different magnifications × 100 and × 400, respectively. Arrows show the angiogenesis

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Discussion

Superantigens are bacterial or viral proteins that activate numerous T cells regardless of their antigenic specificity (Imani Fooladi et al. 2014). SAs could release large volumes of cytokines from T cells and monocytes and are able to enhance the antitumor activity of the immune system (Imani Fooladi et al. 2014). The cellular receptors for superantigens are MHCII and TCR molecules. Superantigens can bind to the β subunit of the TCR molecule and, when delivered by MHCII molecules, activate T cells independent of their CD4+ or CD8+ phenotype (Xu et al. 2010). All staphylococcal superantigens are strong T cell mitogens. They bind differently to the MHCII molecules by specifically recognizing TCR Vβ (Xu et al. 2010). Activated cells produce a variety of cytokines, such as TNF-α, IFN-γ, IL-1, IL-2, IL-6, IL-8, and IL-12 (Xu et al. 2010).

Activation of peripheral blood mononuclear cells (PBMCs) occurs after the exposure to staphylococcal enterotoxins of the gene cluster (egcSEs) enabling these cells to kill a wide range of human tumor cells, depending on time and concentration (Terman et al. 2013). SEB induces cytotoxic T cell activity and cytokine production in vivo. Less than 0.1 pg/ml of superantigen is sufficient to stimulate T cells. The SEB forms a complex outside the peptide-binding groove with the MHCII molecule on the antigen-presenting cells and then binds to the TCR via the β-chain variable region. Following this binding, SEB activates all T cells that express a specific set of Vβ-TCR regardless of actual antigen specificity (clonality), resulting in the activation of more than 25% of peripheral T cells. Both TCD8+ and TCD4+ cells proliferate due to the presence of the superantigen. Increased T cell activity is associated with increased production of Th1 cell cytokines such as IFN-γ, IL-2, and TNF-α. The increased T cell activity usually occurs within 24 h after superantigen exposure (Imani Fooladi et al. 2014).

Antitumor activity of SEB protein has been established in previous studies. Our in silico study suggested that SEB used in the DNA vaccine could be effective against cancer (Jahangiri et al. 2018). However, experimental validation is mandatory to confirm the performed in silico study.

In this study, pVAX plasmid vector was used to express SEB in eukaryotic cells. The pVAX plasmid can stimulate humoral and cellular immunity and has been approved by the FDA for the clinical trials. Various methods have been used to improve the efficacy of DNA in vaccines such as immunization with primary boosters, designing multivalent vaccines, using genetic adjuvants, and codon optimization. In addition, SEB gene construct has been used as a promising vaccine for breast cancer treatment. The effect of primary booster for the treatment of breast cancer was evaluated compared to the gene construct alone. Findings showed that using the booster did not improve the gene activity. The gene construct alone had a better effect on the immunization of mouse models than the booster injection along with gene vaccine. Nucleotide vaccines have been reported to be used as immunogens and can increase the serum cytokines including IL-12, TNF-α, and IFN-γ (Higgs et al. 2006). The ratio of IFN-γ and IL-4 in the immunogen-receiving groups was significantly different. The DNA vaccinator had higher IFN-γ levels; therefore, the immune response in this group could stimulate the cellular/immune arm. As shown in the results section, there is a significant difference (p < 0.001) between the immunogen receiving group and the control. Also, the observations made in the microscopic experiment with hematoxylin–eosin staining on tumor tissue showed well that the rate of necrosis was higher in the vaccinated DNA groups than in the control and PBS groups.

The results indicate that the SEB gene construct could induce the process of apoptosis. It may produce a cytostatic compound that suppresses the immune response (Bernardes et al. 2013). As explained, the unique point of the SEB gene construct in immunotherapy is related to its immune system stimulation potential. Following vaccination, the number of cytokines in the mice cancer group receiving the recombinant vector increased.

SEB can induce apoptosis in the RT112 and RT4 cancer cell lines by establishing interaction between Fas and its receptor (Perabo et al. 2005).

Perabo et al. showed that SEB-activated peripheral blood mononuclear cells extensively secrete the cytokines IL-2, IFN-γ, and TNF-α and could induce apoptosis of bladder cancer cells. It is because cytotoxicity induced by superantigens is mediated primarily by apoptosis (Xu et al. 2011).

In 2011, Xu et al. created a mutant SEC2 molecule called SAM-3 to reduce toxicity and increase the superantigenicity of SEC2 as an antitumor agent by creating a point mutation in the Thr20 and Gly22 positions, introduced as a strong candidate in the treatment of cancer (Xu et al. 2011; Issa et al. 2013). In 2008, Imani Fooladi et al. reported that SEB is affective for the treatment of fibrosarcoma. Their results suggested that tumor cell death is caused by increased cytotoxic T cell activity and increased cytokine levels due to intravenous injection of SEB, and it is a proper candidate for the treatment of fibrosarcoma (Imani Fooladi et al. 2014).

The results of RT-PCR and Western blotting indicated that the protein was expressed after 3 to 5 days in HEK293T cell line. The 4T1 cell line selected for the in vivo experiments is a model of basal-like breast cancer with the ability to metastasize. This cell line creates a cancer model equivalent to grade IV human breast cancer in terms of growth and metastasis, which can metastasize spontaneously to other areas such as the lungs, liver, brain, and bones (Miller et al. 1983; Newell et al. 1991). On the other hand, these cells are more resistant to the immune system than other cancer cells and can be an appropriate model for examining the effect of SEB on breast cancer.

The results showed that all test groups received two doses of SEB.Vac (SEB.Vac (2) and SEB.Boost.Vac) significantly reduced tumor size. Interestingly, those groups received pVAX or single dose of SEB.Vac showed significantly increased tumor size. These results indicate at least two doses of the DNA vaccine are needed to be effective. It seems that this tumor volume could be attributed to the DNA vector (pVAX) effects which is superior to the SEB dose expressed in single-dose (SEB.Vac (1)).

However, survival of the group receiving the SEB gene construct was more prolonged than the group receiving SEB-booster and the other groups. This issue could indicate that the DNA vaccine alone has a better effect in inhibiting cancer cells. Pathological results also confirm the crucial role of SEB gene construct in the destruction of tumor tissue. The rate of necrosis caused by the injection of SEB gene construct was higher than other groups, which could be due to robust activation of cytotoxic T cells.

Vaccination with the SEB gene construct before forming a tumor in mice can produce memory lymphocytes, so that the re-exposure of splenic lymphocytes with tumor cells significantly decreased the proliferation of these cells in the vaccinated group with the SEB DNA vaccine compared to the control group. In addition, the cytokine levels after re-exposure of splenic lymphocytes to nonactive tumor cells demonstrated a significant increase in the production of IL-4 and IFN-γ in the group treated with SEB gene construct compared to the negative control group. These results suggest that increased T cell proliferation and activation may stimulate specific antitumor immunity. The results of the pathological study on the tumor tissue of vaccinated mice showed a significant increase in the group receiving SEB DNA construct compared to the negative control group. In addition, infiltration of lymphocyte cells into tumor tissue has been also reported. The infiltration of T cells into tumor tissue and the production of cytotoxic cytokines may cause lysis of tumor cells and their necrosis.

Conclusion

To the best of our knowledge, the present study represents the first report of the cytostatic property of the SEB gene construct on breast cancer cell lines. Overall, the SEB gene construct designed in the present study is a new model for immuno-apoptotic therapeutic purposes. The SEB DNA vaccine could enhance lymphocyte proliferation, in vitro secretion of IFN-γ from the lymphocytes of immunized mice, and survival time of the mice. Moreover, it could reduce the size of tumors. Hence, it could be considered as an effective therapeutic agent against breast cancer. Further comprehensive studies are needed to qualify this agent for future clinical trials.