The 4T1 murine mammary carcinoma cell line harboring the luciferase construct was provided by the Pathology Department of the College of Basic Medical Sciences, Jilin University, China. 4T1 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA) and incubated at 37°C in an atmosphere containing 5% CO₂. In the logarithmic growth phase, 4T1 cells were collected and diluted to a concentration of 5×10⁶ cells/ml with phosphate-buffered saline (PBS). Next, 0.2 ml of the cell suspension was injected subcutaneously into the right flanks of 6–8-week-old female BALB/c mice, provided by the Laboratory Animal Center of the College of Basic Medical Sciences, Jilin University. All procedures followed the animal care regulations of the University Health Network and were approved by the Research Ethic Board of Jilin University.

Tumor nodules reached 50 mm³ on day 3. Mice were randomly divided into four groups as follows: vehicle, anti-CTLA-4 antibody alone (a-CTLA-4); MMPI alone, and combination therapy (a-CTLA-4 plus MMPI). Mice were treated with 100 µg anti-CTLA-4 antibody (clone, 9H10; BioXCell, West Lebanon, NH, USA) by intraperitoneal (i.p.) injection once every two day and/or with 0.1 MMPI (1 mg/kg) by subcutaneous injection once every two days. The MMPI potassium ferricyanide {K₃[Fe(CN)₆ (10,11), was kindly provided by Professor Xuexun Fang (Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, China). Mice in the vehicle group were treated with an equal volume of normal saline and PBS. The dosages used in the combined treatment group were equal to those used in the monotherapy groups. The longest (a) and shortest (b) diameters of the tumor were measured with calipers every 3 days, and tumor volume was estimated by the formula V = ab²/2. On days 7, 21 and 35, tumors were measured using an Ultrasound Biomicroscopy InviVue instrument (PanoView β1500; Taiwan).

Mice were anesthetized by i.p. injection of 4% chloral hydrate and then subjected to i.p. injection with 200 µl D-luciferin (15 mg/ml; GoldBio, St. Louis, MO, USA). After 10 min, the mice were imaged using a small animal in vivo optical imaging system (IVIS Spectrum; Caliper Life Sciences, USA).

Mice were sacrificed. The lungs and livers were immediately placed in Bouin's fixation and 10% formaldehyde. After 48 h, metastatic lesions on the surface of the lungs (faint yellow in color) were counted without the use of a microscope. The lungs and livers of each mouse were then embedded in paraffin, sectioned, stained with H&E staining, and examined histologically for evidence of metastatic lesions within the lung and liver tissue. For each lung and liver, three consecutive sections, separated by 200 µm, were collected. The sections was randomized and coded, and the total number of metastatic foci was counted.

Peripheral blood was obtained from each mouse and centrifuged at 500 × g for 20 min after standing at room temperature for 20 min. The supernatant was collected as serum. Total protein, serum albumin, serum globulin, albumin/globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALT/AST, alkaline phosphatase, urea, creatinine, and uric acid in serum were measured using an automatic biochemical analyzer (Beckman Coulter, USA).

The spleens of mice were placed in cold RPMI-1640 medium immediately and then cut into small pieces with eye scissors. Single-cell suspensions of spleen tissues were acquired after filtration with 300- and 100-mesh filters. Single-cell suspensions of bone marrow tissues were obtained from the thigh bones of mice after being washed with cold PBS. The red blood cells were then disrupted within the cell suspension. For treatment, 2×10⁶ cells were stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA; 50 ng/ml) plus 2 µg/ml ionomycin (Sigma) and monensin (Sigma; 5 µg/ml) for 5 h. The cells were then collected for Th17 cytokine staining. Subsequently, they were fixed and/or permeabilized using fixation/permeabilization kits (BD, USA) according to the manufacturer's instructions, and incubated with anti-interleukin (IL)-17A-Alexa Fluor (BD), anti-CD4-PE (BD), anti-CD4-APC (BD), anti-CD25-PE-CyTM7 (BD), anti-FoxP3-PE (eBioscience, San Diego, CA, USA), anti-CD3e-FITC (BD), anti-CD4-PE-CyTM⁷ (BD), anti-CD8-PE (BD), anti-CD11b-APC (BD), and anti-Gr-1-PE (BD) antibodies for 40 min at 4°C. The cells were incubated with rat anti-mouse CD16/CD32 (BD) for 30 min before CD11b and Gr-1 staining at 4°C. Flow cytometry (BD) was performed, and data were analyzed using FlowJo software.

For immunohistochemical analysis of microvessel density (MVD), 2-µm-thick sections of formalin-fixed, paraffin-embedded mammary tumor tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. For antigen retrieval, slides were immersed and boiled for 5 min in citrate buffer (pH 6.0). Slides were incubated with a peroxidase inhibitor and horse serum blocking solution for 30 min and then subjected to overnight staining with rat anti-mouse CD34 primary monoclonal antibodies (1:100 dilution; ab81289; Abcam, USA) at 4°C. Slides were then washed three times with PBS-T, incubated with horse anti-rat secondary antibodies for 30 min at room temperature, washed again with PBS-T three times, and incubated in DAB peroxide substrate solution. Counterstaining was performed with Harris hematoxylin counterstain. MVD was determined as the mean number of microvessels under five microscopic fields (200×).

Immunofluorescence staining of slides was performed as described for immunohistochemistry until antigen retrieval. Then, slides were treated with or without 1% Triton X-100 for 20 min, followed by blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, slides were incubated with anti-CD3e-FITC (BD), anti-CD8a-PE (BD), anti-CD4-PE (BD), anti-CD11b-FITC (BD), anti-Gr-1-PE (BD), anti-Foxp3-PE (BD), and anti-IL-17A-Alexa Fluor 647 (BD) for 30 min at room temperature. Slides were then washed three times with PBS-T, incubated with Hoechst-33342 for 5 min at room temperature, washed again with PBS-T three times, and sealed with glycerin.

All values were expressed as means ± standard deviations (SDs). All statistical analyses were performed using Student's t-tests, non-parametric tests, or Spearman correlation analyses. Differences or correlations with P-values of ≤0.05 were considered significant.

First, we established a breast cancer model by subcutaneous inoculation of 4T1 cells in order to easily monitor tumor growth using ultrasound and calipers. By day 3, tumor nodules reached ~50 mm³; therefore, treatments, including anti-CTLA-4 antibody and MMPI, were applied beginning on day 3. Our results demonstrated that treatment with the MMPI or MMPI plus anti-CTLA-4 antibody inhibited the growth of transplanted tumors compared with vehicle or anti-CTLA-4 antibody alone to varying degrees. Indeed, according to analysis of the total days, combined treatment caused a significant reduction in the growth of breast tumors compared with that in vehicle-treated mice (P<0.05; Fig. 1A). Additionally, ultrasonography on days 7, 21 and 35 revealed that tumors in mice treated with anti-CTLA-4 antibody and MMPI were significantly smaller than those in mice treated with vehicle, MMPI alone, or anti-CTLA-4 antibody alone (Fig. 1B). However, the combination treatment had no effect on the survival of tumor-bearing mice (P>0.05; Fig. 1C).

In order to visualize tumor metastasis in live mice, we constructed a mouse model of breast cancer using 4T1 cells stably expressing luciferase. D-luciferin was applied on day 42 for detection of tumor metastasis using a small animal in vivo optical imaging system. The results showed that the mice in the vehicle group had metastatic lesions, whereas the mice in the other groups had no metastatic lesions (Fig. 2A).

Observation of fixed lungs showed that the number of tumor metastases on the lung surface was significantly reduced after combined treatment (P<0.05; Fig. 2B). However, there was no significant reduction after treatment with anti-CTLA-4 antibody alone or MMPI alone (P>0.05; Fig. 2B). Additionally, analysis of paraffin-embedded lung and liver sections stained with H&E revealed that both tissues had reduced numbers of metastases after the combined treatment (P<0.001; Fig. 2C). However, there was no significant difference in the number of metastases between the anti-CTLA-4 antibody group and vehicle group (Fig. 2C).

MMPIs have been reported to have toxic effects. Therefore, in this study, we examined changes in liver and kidney function in mice after treatment with the MMPI. The results showed that the liver and kidney functions of tumor-bearing mice treated with the MMPI did not differ from those of control mice (P>0.05; Fig. 3), indicating that the MMPI used in this study did not damage the liver or kidneys of mice.

Our data showed that MMPI could enhance the therapeutic effects of anti-CTLA 4 antibodies in a model of breast cancer in mice. The mechanism through which the anti-CTLA-4 antibody improves the antitumor immune response involves prompt T-cell activation and proliferation. Therefore, in order to determine whether the MMPI affected the immune microenvironment of tumor-bearing mice to enhance the therapeutic effects of the anti-CTLA-4 antibody, we used flow cytometry to measure changes in the percentages of CD4⁺ T cells, CD8⁺ T cells, Tregs, Th17 cells, and MDSCs in the spleens of mice and to evaluate changes of MDSCs in the bone marrow. The results showed that there were no significant differences in the number of CD4⁺ and CD8⁺ T cells between the groups (Fig. 4). However, the ratio of CD8⁺ T cells to CD4⁺ T cells was significantly increased after MMPI treatment (P<0.05; Fig. 4). Moreover, combined treatment caused significant decreases in Tregs and the Treg/Th17 ratio in mouse spleens (P<0.01; Fig. 4), and the percentage of MDSCs in spleens and the bone marrow was significantly reduced after combination treatment (P<0.01; Fig. 4).

Thus, taken together, these data suggested that the anticancer mechanism of the MMPI may be related to the increased proportion of CD8⁺ T cells, relieving immune suppression and enhancing the antitumor function of the immune system.

The primary target of MMPIs is the TME, which is generally enriched in immunosuppressive cells. Thus, we examined the infiltration of immune cells into tumor tissues using immunofluorescence. The results showed that while there were no obvious increases in the percentage of CD4⁺ and CD8⁺ T cells in the TME, the ratio of CD8⁺ T cells to CD4⁺ T cells was increased after MMPI treatment and after combined treatment as compared with that in vehicle-treated mice (P<0.05; Fig. 5). Moreover, the percentages of Tregs, Th17 cells, and MDSCs in the tumors were significantly decreased after combined treatment (P<0.05 for Tregs and Th17 and P<0.01 for MDSCs; Fig. 5). However, the Treg/Th17 ratio did not differ significantly compared with that in vehicle-treated mice. Therefore, these data showed that immunosuppression of the TME was improved to a certain extent after combination treatment with the MMPI and anti-CTLA-4 antibody.

Tumor vessels are important elements of the TME, and MVD within the tumor is often associated with tumor progression. We selected paraffin-embedded sections of tumors, stained them with anti-CD34 antibodies to mark tumor stromal vascular endothelial cells, and determined the MVD. Immunohistochemical staining showed that the MVD was higher in tumors from vehicle-treated mice than in tumors from mice treated with the MMPI and anti-CTLA-4 antibody (P<0.01; Fig. 6). Thus, the MMPI may reduce immunosuppression and inhibit neovascularization in the TME.

Immune cells are associated with angiogenesis in the tumor stroma (12). Therefore, we next analyzed the relationship between infiltration of immune cells and MVD within the tumor tissue. Spearman correlation analyses showed that CD8⁺ T cells, CD4⁺ T cells, and the CD8⁺/CD4⁺ T cell ratio were negatively correlated with MVD in tumor tissues (correlation coefficients: −0.800, −0.800, and −0.100, respectively; P>0.05), whereas Tregs, the Treg/Th17 ratio, Th17 cells, and MDSCs were positively correlated with MVD (correlation coefficients: 0.400, 0.400, 0.800 and 0.800); the correlation between MDSCs and MVD was statistically significant (P<0.05; Fig. 7).