STAT3-blocked whole-cell hepatoma vaccine induces cellular and humoral immune response against HCC

Male BALB/c, C57BL/6 mice, and T cell-deficient nude (BALB/cA-nu) mice were obtained from Beijing HFK Bioscience (Beijing, China). All procedures were performed in accordance with the Institutional Animal Care and Use Committee Protocols of Shandong University. H22 cells (BALB/c-derived hepatoma, obtained from the Shandong Academy of Medical Sciences) were cultured in Roswell Park Memorial institute (RPMI) 1640 medium (Life Technologies BRL, Gaithersburg, MD, USA), and Hepa1–6 cells (C57BL/6-derived hepatoma, purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies BRL, Gaithersburg, MD), all media were supplemented with 10% fetal bovine serum (FBS; Sijiqing, Hangzhou, China).

The STAT3 decoy oligonucleotide (ODN) sequences were 5′-CATTTCCCGTAAATC-3′, and 5′-GATTTACGGGAAATG-3′, whereas the scrambled sequences were 5′-CATCTTGCCAATATC-3′ and 5′-GATATTGGCAAGATG-3′ [16]. These ODNs were modified with phosphorothioate, and the sense and antisense strands were annealed and purified using high performance liquid chromatography (Takara, Dalian, China). All ODNs were prepared at a concentration of 100 mM.

H22 cells or Hepa1–6 cells were transfected with STAT3 decoy or scrambled ODN using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), as previously described [15, 16], and then cultured in complete medium containing 10% FBS. Twenty-four hours later, the cells were inactivated through repeated cycles of liquid nitrogen freeze-thawing. After centrifugation at 1200 rpm, the cell lysates were used as HCC vaccines.

Male BALB/c mice were immunized with 100 μL of lysate from 2 × 10⁶ H22 cells treated with Lipofectamine 2000, scrambled-ODN or decoy-ODN, via subcutaneous (s.c.) injection in the right flank once a week for 3 weeks. Mice injected (s.c.) with an equal volume of phosphate-buffered saline (PBS) in their right flank were defined as the control (Ctrl). One week after the last immunization, all mice were injected (s.c.) with 2 × 10⁶ H22 cells in their left flank. C57BL/6 mice were immunized as the same manner, and challenged with Hepa1–6 cells.

Splenocytes and liver mononuclear cells were isolated as described previously [16]. Flow cytometric analysis was performed using BD FACSCalibur and FACSAria III instruments. Antibodies used in this study included fluorescein isothiocyanate (FITC)-labeled anti-mouse CD49b (DX5), anti-NK1.1, anti-CD4, and anti-CD11c; Phycoerythrin (PE)-labeled anti-mouse-CD69, anti-CD107a, anti-CD44, anti-CD86, and anti-IFN-γ; PE-cyanine 5.5-labeled anti-mouse CD3e and anti-CD8; and allophycocyanin (APC)-labeled anti-mouse CD314 (NKG2D), anti-CD69, anti-CD25, anti-CD80, anti-CD62L, and anti-TNF-α, these antibodies were obtained from eBioscience (San Diego, CA, USA). FITC-B540-labeled anti-CD3, APC-cy7-labeled anti-NK1.1, PE-YG582-labeled anti-CTLA4, PE-cy7-YG780-labeled anti-TIGIT, APC-R660-labeled anti-PD-1, V450-labeled anti-LAG-3, Percpcy5.5-B695-labeled anti-Tim-3, YG780-labeled anti-Granzyme B, and B540-labeled anti-perforin were obtained from Biolegend (California,USA) and BD (New York, USA). For the analysis of intracellular molecules, cells stained with anti-CD4 and anti-CD8 antibodies were fixed using Fix/Perm Buffer (eBioscience, San Diego, CA, USA) for 30 min, incubated with anti-mouse-IFN-γ and -TNF-α for 30 min at 4 °C, and then analyzed using flow cytometry.

After being treated with STAT3 decoy ODN or scrambled ODN for 12 h, cells were harvested, washed with PBS at 4 °C, and resuspended in 100 μL binding buffer (1 × 10⁶ cells/mL) containing 5 μL of Annexin V-FITC and 10 μL of PI (propidium iodide) using an Annexin V–FITC kit (BestBio, Shanghai, China). After incubation for 10–15 min in the dark at room temperature, these cells were analyzed by flow cytometry.

One week after the last immunization, splenocytes were isolated from BALB/c mice and plated into 96-well plates. The splenocytes were then co-cultured with inactivated H22 cells at a ratio of 50:1. After 5 days, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to test the proliferation of the splenocytes.

To detect tumor-reactive serum IgG, H22 cells were cultured with serum harvested from BALB/c mice immunized with HCC-vaccines for 4 h, washed thoroughly, and stained with an FITC-labeled anti-mouse IgG antibody, and analyzed by flow cytometry using a FACSCalibur flow cytometer.

H22 cells were cultured with serum harvested from HCC-vaccine immunized mice for 4 h, stained with carboxyfluorescein succinimidyl ester, washed thoroughly, and plated into 12-well plates. The splenocytes harvested from untreated mice were added at effector/target cell (E:T) ratio of 50:1. Twenty-four hours later, these cells were harvested and stained with 7-Aminoactinomycin D (7-AAD) for 15 min, and then analyzed by flow cytometry using a FACSCalibur flow cytometer.

For the transfer experiment, 100 μL of serum harvested from HCC-vaccine immunized mice was injected (i.v.) into the healthy mice. The recipient mice were then challenged with 2 × 10⁶ H22 cells (s.c.) in their right flank at day 2. The tumor volume was measured at day 20.

The levels of IL-10 and TGF-β in the serum of mice were assayed by enzyme-linked immunosorbent assay (ELISA) (ExCell Bio, Shanghai, China).

Statistical analysis was performed using a paired Student’s t test. Statistical significance was determined as ***p < 0.001, **p < 0.01 and *p < 0.05 compared with the control.

To determine whether blocking STAT3 could increase the immunogenicity of HCC and apply a tumor vaccine against HCC, we prepared the tumor vaccine using two murine hepatoma cell lines H22 and Hepa1–6. First, HCC cells were efficiently transfected with the STAT3 decoy-ODN (Additional file 1: Fig. S1A), which competitively blocked STAT3 with high specificity and promoted HCC apoptosis (Additional file 1: Fig. S1B). HCC cells treated with the scrambled-ODN or Lipofectamine were used as controls. These hepatoma cells were inactivated by repeated liquid nitrogen freeze-thawing, and the cell lysates were acquired and used as HCC vaccines; PBS was used as a blank control.

To investigate the protective effect of the vaccines against HCC, BALB/c mice and C57BL/6 mice were immunized with the H22- or Hepa1–6-based whole cell lysate vaccine once a week for 3 weeks. These mice were then challenged with untreated-HCC cells 1 week after the last immunization, and the tumor volumes of mice were monitored ever 2–3 days. The results showed the rate of formation rate of H22 tumors in BALB/c mice immunized with STAT3-blocked HCC vaccine was 50%, which was significantly lower than that of the other groups (p < 0.001), while in C57BL/6 mice the tumor formation rate was not affected by immunization with Hepa1–6-based tumor vaccines (Additional file 1: Table S1). Furthermore, as shown in Fig. 1a, immunization with STAT3-blocked whole-cell hepatoma vaccine resulted in significant antitumor activity in BALB/c mice challenged with H22 tumor cells, showing significantly lower tumor growth rates compared with the other groups (Fig. 1b ). Meanwhile, mice in PBS group all died within 4 weeks. Similar results were observed in C57BL/6 mice challenged with Hepa1–6 cells (Fig. 1c), and the survival of mice immunized with STAT3-blocked HCC vaccine was prolonged markedly (Fig. 1d). These findings indicated that blocking STAT3 in HCC cells increased the immunogenicity of HCC.

To explore the potential mechanisms by which STAT3-blocked HCC vaccine displayed its anti-tumor activity, BALB/c mice were immunized with the HCC vaccine three times, and then the major anti-tumor effector cells of these mice were analyzed 1 week after the last immunization. First, we observed that the CD3⁺DX5⁻T cell population increased while the CD3⁻DX5⁺NK cell population decreased significantly in peripheral blood mononuclear cells (PBMCs) from mice immunized with STAT3-blocked HCC vaccine compared with the other groups (Fig. 2a). In contrast, in the C57BL/6 mice model, immunization with STAT3-blocked HCC vaccine did not affect the CD3⁺DX5⁻T cell population, while the CD3⁻DX5⁺NK cell population increased significantly compared with the control mice (Additional file 1: Fig. S2A). CD69 molecule, a sensitive indication of lymphocyte activation, was elevated in T cells and NK cells of PBMCs from both mouse models immunized with STAT3-blocked HCC vaccine compared with the control mice (Fig. 2c, Additional file 1: Fig. S2B). In addition, as shown in Fig. 2d , the proportion of CD11c⁺ Dendritic Cells (DCs) increased in mice immunized with STAT3-blocked HCC vaccine, accompanied by the upregulation of costimulatory molecules CD80 and CD86 compared with mice immunized with scrambled-ODN-treated HCC vaccine or mice injected with PBS only (Fig. 2e). These results indicated that immunization with STAT3-blocked HCC vaccine could activate the immune system.

Immune memory plays a critical role in the anti-tumor effect of a vaccine. Memory lymphocytes, including memory T cells and memory B cells, are the executors of immune memory [17, 18]. In the present study, we observed that significantly more CD3⁺CD44^highCD62L⁺ memory T cells were induced in PBMCs from mice immunized with STAT3-blocked HCC vaccine compared with those from the other groups (Fig. 3a & b ). Similar results were observed in C57BL/6 mice immunized with HCC vaccines (Fig. 3c). Furthermore, to characterize the properties of these cells, the proliferative capacity of splenocytes from immunized mice in response to H22 cells was assessed. The splenocytes from BALB/c mice immunized with STAT3-blocked (Decoy) or Scrambled ODN-treated HCC vaccine were cultured in the presence or absence of mitomycin C-treated H22 cells, respectively, for 5d in vitro, after which the proliferation of the splenocytes was detected. The results showed that the proliferative ability of splenocytes from mice immunized with STAT3-blocked HCC vaccine was significantly higher than that of the control mice (Fig. 3d). These results suggested that immunization with STAT3-blocked HCC vaccine could induce the generation of immune memory against HCC.

BALB/c mice immunized three times with HCC vaccines as above were inoculated subcutaneously with 2 × 10⁶ H22 cells. One week later, as shown in Fig. 4a, the population of CD3⁺DX5⁻T cells from BALB/c mice immunized with STAT3-blocked HCC vaccine had increased, while there was no difference in the population of CD3⁻DX5⁺ NK cells among the different groups. Meanwhile, the expression of CD69 on both T cells and NK cells in PBMCs from mice immunized with STAT3-blocked HCC vaccine were significantly higher than those from the control mice (Fig. 4b). In addition, production of degranulation molecule CD107a was induced in T cells by the STAT3-blocked HCC vaccine (Fig. 4c). In addition, the number of CD3⁺CD4⁺CD25⁺Tregs in PBMCs from the tumor-bearing BALB/c mice immunized with STAT3-blocked HCC vaccine decreased (Fig. 4d). Similar results were shown in the C57BL/6 mice (Additional file 1: Fig. S3). One month after tumor challenge, only one mouse in the PBS group survived, whereas only two mice immunized with STAT3-blocked HCC vaccine established HCC tissue (Fig. 4e, f). Moreover, the proportion of tumor-infiltrated T cells and NK cells in mice immunized with the STAT3-blocked HCC vaccine was significantly increased compared with those in the other groups (Fig. 4e), while CD69 expression on tumor-infiltrated T cells and NK cells was not affected by immunization with the STAT3-blocked HCC vaccine (Fig. 4f). These results proved that the secondary immune response was induced in the immunized mice inoculated with the homologous tumor cells.

Previously, we confirmed that STAT3-blocked HCC could augment NK cell cytotoxicity against HCC and increase the expression of molecules associated with NK cell activation and cytotoxicity [16]. Therefore, to determine whether the STAT3-blocked HCC vaccine-induced anti-tumor immune response was mediated by NK cells, we performed an immunization strategy in nude mice that lack mature T cells. As shown in Fig. 5a-b , the STAT3-blocked HCC vaccine failed to delay tumor progression in the nude mice, and there was no significant difference in the tumor size and weight among the different groups. The population of NK cells, and the levels of CD69 and NKG2D on the NK cells were not changed significantly by immunization with the STAT3-blocked HCC vaccine (Fig. 5c-e). These results indicated that the STAT3-blocked HCC vaccine could not activate NK cells under deficiency of T cells. Subsequently, we explored whether humoral immunity was involved in the anti-HCC effect elicited by the STAT3-blocked HCC vaccine. We found that the levels of tumor-specific IgG antibodies from mice immunized with the STAT3-blocked HCC vaccine were significantly increased (Fig. 5f-g). Furthermore, the cytotoxicity of spleen lymphocytes against H22 cells was stronger in the presence of serum from mice immunized with the STAT3-blocked HCC vaccine, suggesting that tumor-specific IgG was essential for inhibiting tumor growth (Fig. 5h). Finally, the serum from mice immunized with the HCC vaccine was transferred to naive BALB/c mice, and these recipient mice were inoculated subcutaneously with H22 cells. The results showed that serum from mice immunized with the STAT3-blocked HCC vaccine significantly inhibited tumor growth compared with serum from the controls (Fig. 5i). These results indicated that the anti-tumor effect induced by the STAT3-blocked HCC vaccine was dependent on both cellular and humoral immunity.

Previous studies demonstrated that T cell dysfunction or exhaustion in tumor-bearing mice would hinder the production of anti-tumor immunity. To date, (Programmed Death 1) PD-1, cytotoxic T-lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin mucin-3 (TIM-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and Lymphocyte-activation gene 3 (LAG-3) have been identified as markers of exhausted T cells or NK cells [19‐21]. To examine the potential role for preventing CD8⁺ T and NK cell exhaustion by the STAT3-blocked HCC vaccine, we first examined the expression of these exhaustion markers on T cells and NK cells from mice bearing H22 cells. We observed that the number of PD-1⁺CD8⁺ T cells and TIGIT⁺CD8⁺ T cells was lower in H22-bearing mice immunized with the STAT3-blocked HCC vaccine than in the other groups, while LAG-3 and TIM-3 expression did not show significant differences (Fig. 6a). We then examined the production of Tumor Necrosis Factor α (TNF-α) and Interferon γ (IFN-γ) to determine whether CD8⁺ T cells exhibited an exhausted phenotype. We found that the CD8⁺ T cells displayed the most profound impairment in production of IL-2 (data not shown); however, the STAT3-blocked HCC vaccine improved the production of TNF-α and IFN-γ by CD8⁺T cells (Fig. 6b), as well as the percentage of perforin-expressing CD8⁺T cells. A similar trend was observed with Granzyme B, although this did not reach statistical significance (Fig. 6c). Meanwhile, as shown in Fig. 6d , LAG-3 and TIGIT, but not PD-1, LAG-3, or CTLA-4, were lower on NK cells from mice immunized with the STAT3-blocked HCC vaccine. Finally, the levels of IL-10 and TGF-β in mice immunized with the STAT3-blocked HCC vaccine were lower than those in the other groups (Fig. 6e). These results indicated that the STAT3-blocked HCC vaccine could prevent CD8⁺ T cells and NK cells from HCC-induced exhaustion.