Background
Microwave ablation (MWA) is a relatively new minimally invasive therapy that generates electromagnetic heating and causes focal hyperthermic injury to destroy tumor tissues [
1]. MWA shows several advantages over radiofrequency ablation (RFA), the most commonly used thermal ablation technique [
2‐
5], and has proven to be an effective and safe treatment option for liver, lung, and kidney tumors [
6]. Our group has previously reported that MWA is feasible for small breast cancer [
7].
Tumor debris produced by thermal ablation provide pro-inflammatory signals and serve as tumor antigens to induce adaptive antitumor immunity [
1,
8]. Recent study has reported that MWA of osteosarcoma can elicit tumor-specific T-cell immune response in a rat model [
9]. T-cell immune responses induced by MWA have also been observed in hepatoma patients [
10]. However, the tumor recurrence rate after MWA is similar to that after curative surgical resection [
11,
12]. These results indicate that the antitumor immunity induced by MWA is not strong enough to prevent the recurrence of cancers. Thus, additional immunomodulatory strategies are needed to enhance the antitumor immunity.
OK-432, a penicillin-inactivated and lyophilized preparation of a low-virulence strain Su of Streptococcuspyogenes (group A), was approved by Japan administration in 1975 as an immunotherapeutic agent in cancers [
13]. Previous studies have reported OK-432 induce pro-inflammatory cytokines to activate T-cell-mediated immunity [
14,
15]. It has been reported that OK-432 augment antitumor T-cell response in both animal models and patients [
16‐
19].
Despite growing attention, the immune responses induced by MWA in breast cancer are still unclear. The purpose of this study was to investigate the antitumor immunity against breast cancer induced by MWA and whether these immune responses could be promoted by administration of OK-432.
Methods
Cell line
The murine breast cancer cell line 4T1 was obtained from Chinese Academy of Sciences (Shanghai, China). The cells were grown (37 °C incubator with 5% CO2) in RPMI 1640 supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 unit/mL penicillin. All cells culture reagents were purchased from Invitrogen (Shanghai, China).
Animal models
5 × 104 4T1 cells in 100 μL of PBS were injected subcutaneously into the right inguinal mammary fat pads of 6- to 8-week-old female Balb/c mice (Vital River Laboratories, Beijing, China). The mice were euthanized when the tumor diameter exceeded 20 mm in diameter or when they became moribund during the observation period, and the time of euthanization was recorded as the time of mortality. All protocols and studies involving animals were approved by Nanjing Medical University Institutional Animal Care and Use Committee.
Study design
Animals with established tumors were randomized to four groups. Mice in MWA group were treated with microwave ablation alone. About 30 min after MWA, 1 Klinishe Einheit (KE; i.e., 0.1 mg) OK-432 (Lukang Pharmaceutical, Shandong, China) in 100 μL PBS were injected peritumorally in the combination treatment group, whereas mice treated with MWA alone were injected with 100 μL of PBS. The injection was repeated once 3 days later. Mice in OK-432 group received OK-432 only. No-treatment group served as control.
For rechallenge test, mice that survived for 25 days after MWA and age-matched healthy mice were injected subcutaneously with 2 × 104 4T1 cells in 100 μL of PBS into the left inguinal mammary fat pads. Subsequently, the sizes of second tumors were measured with calipers every 5 days, and tumor volumes were calculated using the formula 1/2 (Length × Width2).
Microwave ablation
On day 35 after tumor implantation, when tumor size reached 8–10 mm in diameter, tumor-bearing mice were treated with MWA using a microwave generator (ECO-100E, Yigao Microwave Electric Institute, Nanjing, China). Mice were anesthetized with isoflurane (RWD Life Science, Shenzhen, China) inhalation. After disinfected the tumor area with alcohol, a 17-gauge MWA antenna (Yigao Microwave Electric Institute, Nanjing, China) was inserted into the center of the tumor. According to the results of the pre-test, MWA was performed at a power output of 5 W for 3 min to achieve complete ablation of primary tumors. The microwave irradiation frequency is 2450 MHz.
Immunohistochemical analysis
The removed tumor tissues were fixed in 4% formalin solution and paraffin embedded. Paraffin sections were stained with rat anti-mouse CD4 (4SM95, eBioscience, San Diego, USA) and rat anti-mouse CD8 (4SM15, eBioscience) followed by horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz, USA) and diaminobenzidine visualization (DAB kit, Beyotime, Nanjing, China). Nuclei were counterstained with hematoxylin. The numbers of positive cells were counted in five randomly selected fields at 400-fold magnification. Results from the five areas were averaged and used in the statistical analysis.
Flow cytometric analysis
The harvested spleen was minced gently with the plunger of a 10 mL syringe and passed through a 70-μm nylon mesh cell strainer (Falcon, New Jersey, USA) to achieve a single cell suspension. Red blood cells were removed by cell lysis buffer (BD Biosciences, San Jose, USA). For intracellular cytokine staining, harvested cells were stimulated with phorbol myristate acetate (PMA), ionomycin, and brefeldin A (Cell Activation Cocktail, Biolegend, San Diego, USA) for 5 h. FITC-anti-CD4 (RM4-5), APC/Cy7-anti-CD8a (53-6.7), and matched isotype control antibodies were purchased from BD Biosciences. PerCP/Cy5.5-anti-CD4 (RM4-5), FITC-anti-IFN-γ (XMG1.2), APC-anti- interleukin (IL)-4 (11B11), and matched isotype control antibodies were purchased from Biolegend. Flow cytometric analysis was performed using a FACS flow cytometer (Beckman Coulter, Miami, USA), and analyzed by Kaluza software (version: 1.1; Beckman Coulter).
ELISPOT assay
The enzyme-linked immunospot (ELISPOT) assays were performed with Mouse Interferon-γ (IFN-γ) ELISPOT Set (BD Biosciences) according to the manufacturer’s manual. Splenocytes (5 × 105 cells/well) were added to plates precoated with anti-mouse IFN-γ antibody together with Mitomycin C treated 4T1 or CT26 cells (2.5 × 105 cells/well). After cultured at 37 °C with 5% CO2 for 48 h, the plates were washed, further incubated with biotinylated detection antibody. Finally, the IFN-γ producing cells were visualized using AEC substrate set (BD Biosciences). The numbers of spots were counted automatically by BioSys BioReader 4000 PRO (Biosys, Karben, Germany).
ELISA assay
Blood was collected via cardiac puncture into tubes containing EDTA. Plasma was obtained by centrifugation at 1000g for 10 min and stored at −70 °C until analysis. Concentrations of IL-2, IL-4, IL-10 and IFN-γ in plasma were measured by enzyme-linked immunosorbent assay (ELISA) using High Sensitivity ELISA Kit (eBiosciences). IL-12p70 and IL-18 were quantified using mouse IL-12p70 Quantikine Reagent Kit (R & D Systems, Minneapolis, USA) and IL-18 Platinum ELISA Kit (eBiosciences) respectively.
Statistical analysis
The significance of differences between four experimental groups was assessed by one-way ANOVA followed by Student’s t-test with Bonferroni’s correction or Dunnett’s test for multiple comparisons. Student’s t-test was used for rechallenge test. Survival was analysed with the Kaplan–Meyer method comparing survival curves by log-rank test. Analyses were done with SPSS software (version: 22; SPSS, Chicago, USA). A P value of less than 0.05 was considered statistically significant.
Discussion
As a minimally invasive technique, MWA of breast cancer may achieve complete tumor ablation with less surgical complications, better cosmetic outcomes, shorter recovery times, and reduced health-care costs [
1]. Our prior study has shown that MWA was a feasible approach for small breast cancer [
7]. In addition to hyperthermic injury, the immunological effect induced by thermal ablation may contribute another mechanism of tumor cell death and destruction [
1].
During tumor destruction caused by thermal ablation, intracellular tumor-specific antigens are released and captured by antigen-presenting cells in lymphatic tissue [
8,
21]. These antigens are processed and presented to T cells to activate antitumor immunity [
22,
23]. Recent research has shown that both local and systemic T-cell responses were elicited by MWA alone in hepatoma and osteosarcoma [
9,
10]. Although MWA induced T-cell responses in breast cancer were firstly reported by us, the median survival of MWA treated mice was only 5 days longer than that of untreated mice. Previous study has reported that T-cell response induced by MWA is not enough to protect patients from relapse, while immunocyte infiltration extent in ablated tissue is inversely correlated with overall survival and local recurrence rate [
10]. Thus, combining MWA with immune adjuvants to enhance antitumor immune response may achieve additional clinical benefit.
Several studies have explored the combination strategies to augment antitumor immunity by administration of immunostimulant, such as IL-2, IFN-β, and staphylococcal enterotoxin C (SEC) [
24‐
26]. Nevertheless, these adjuvants are still under preclinical development. In contrast, OK-432 has been used as immuno-oncology agents in clinical practice for over 40 years [
13]. Previous studies have reported the ability of OK-432 to improve the survival of patients after resection of lung or gastric cancers [
27,
28]. Our data showed that local injection of OK-432 after MWA markedly prolonged the survival of mice and protected most surviving mice from tumor rechallenge, suggesting that this combination therapy could provide augmented clinical benefit and may prevent tumor recurrence. Similar to the results of Behm et al and Iida et al who used RFA in animal models [
20,
29], we observed increased number of T cells infiltrated into ablated tissues. Moreover, we showed that OK-432 could further enhance CTL rather than Th cell infiltration that induced by MWA. In addition, our findings showed that OK-432 plus MWA not only promote peripheral T-cell responses, but also synergistically elicit systemic tumor-specific immunity. This augmented antitumor immunity might result in the tumor rejection in rechallenge test.
Next, we explored the role of CD4
+ Th cell upon this synergistic effect on immune system. Th cell can be classified generally into two predominant subtypes as Th1 and Th2 cell, which can cross-inhibit each other [
30]. Although Th2 cell has been reported to promote the recruitment of eosinophils and macrophages into the tumor microenvironment [
31], it appears to contribute to tumor progression in another research [
32]. On the other hand, Th1 cell promotes durable tumor-specific CTL responses and induces strong immunological memory against tumor rechallenge [
31,
33]. In this study, we found that Th1 to Th2 ratio in the combination treatment group was significantly higher than that of other groups. These data may suggest that skewing of Th1/Th2 balance toward Th1-dominated immunity is responsible for the augmented T-cell responses that observed in the combination treatment group. Additional, plasma levels of Th1-type cytokines, including IL-12, IL-18, IL-2, and IFN-γ, were increased in mice treated with MWA plus OK-432, whereas Th2-type cytokines were not affected. As previously mentioned, OK-432 could augment the production of IL-12 and IL-18 [
14,
15]. These two cytokines were shown to synergistically induce T cells to differentiate into Th1 cells [
34,
35], and stimulate IFN-γ production from Th1 cells [
36]. Th1 cell secreting IL-2 is crucial to the expansion of CD8
+ T cells and is particularly important for the functional maturation of activated T cells [
37,
38]. These results may indicate that combining OK-432 with MWA could produce various Th1-type cytokines to activate Th1-type response, and this process might be potentially an important mechanism underlying the synergistic effect of this combination therapy.
Several limitations in our study should be noted. First, the optimal dose or schedule of OK-432 has yet to be determined. Based on the data from previous animal experiments [
39], mice in our study received two doses of OK-432 (1 KE per mouse) given 3 days apart. However, the synergistic effect on Th cell was not observed 14 days after MWA. Thus, a longer duration of OK-432 treatment might provide greater therapeutic benefits. Second, the progression and immune responses in metastatic lesions were not evaluated when primary tumors were ablated. Moreover, the antitumor effects of Th cell and CTL could be further demonstrated through additional experiments, such as CD4
+ or CD8
+ T cell depletion. Also, the changes in regulatory T cell and myeloid-derived suppressor cell were not examined to determine whether MWA affects the tumor immunosuppressive environments. Finally, antitumor immunity may vary widely with different tumor host systems. Thus, the immunological effect of MWA and OK-432 should be validated in other animal models and humans.
In conclusion, the present study showed that MWA in breast cancer could elicit T-cell infiltration and systemic T-cell responses. Subsequent administration of OK-432 induced multiple Th1-type cytokines and polarized T-cell responses to Th1 dominance. Furthermore, the combination treatment augmented both local and systemic T-cell responses, and synergistically elicited strong tumor-specific immune responses. Lastly, MWA plus OK-432 prolonged the survival of 4T1 breast cancer bearing mice and resulted in complete rejection of tumor rechallenge in most surviving animals. These data together indicate that the combination of OK-432 and MWA is a potent activator of antitumor immunity. Considering the feasibility and efficiency of either MWA or OK-432 in the treatment of tumors [
6,
7,
28], this combination offers a novel treatment option for breast cancer.
Authors’ contributions
LL, WW, HP, WZ and SW conceptualized and designed the research. LL, WW, HP, GM, XS, HX, XL and QD performed the experiments. LL, WW and HP analysed the data. LL, WW and HP interpreted results of the experiments. LL, WW, HP, WZ and SW edited and revised the manuscript. All authors read and approved the final manuscript.