Improvement of STING-mediated cancer immunotherapy using immune checkpoint inhibitors as a game-changer

Abstract

Various cancer therapies, such as surgery, radiotherapy, chemotherapy, and immunotherapy, have been used to treat cancer. Among cancer immunotherapies, stimulators of interferon genes (STING) activate various immune cells and induce them to attack cancer cells. However, the secretion of type I interferon (IFN α and β) increases after stimulation of the immune cell as a side effect of STING agonist, thereby increasing the expression of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Therefore, it is necessary to reduce the side effects of STING agonists and maximize cancer treatment by administering combination therapy. Tumor-bearing mice were treated with cisplatin, tumor-specific peptide, neoantigen, DMXAA (STING agonist), and immune checkpoint inhibitor (ICI). The combination vaccine group showed a reduction in tumor mass, an increased survival rate, and IFN-γ⁺ (interferon gamma) CD8⁺ (cluster of differentiation 8) T cells in the spleen and TME. The distribution of immune cells in the spleen and TME was confirmed, and the number of active immune cells increased, whereas that of immunosuppressive cells decreased. When measuring cytokine levels in the tumor and serum, the levels of pro-inflammatory cytokines increased and anti-inflammatory cytokines decreased. This study demonstrated that when various cancer therapies are combined to treat cancer, it can lead to an anticancer immune synergistic effect by increasing the immune response and reducing side effects.

Keywords: CD8+ T cell; Cancer immunotherapy; Immune checkpoint inhibitor; STING; Tumor microenvironment; Type I IFN.

Fig. 1

Tumor treatment effects of tumor-specific antigen and STING agonist co-administration in cisplatin-treated mouse model. C57BL/6 mice were subcutaneously injected with 2 × 10⁵ TC-1 cells/mouse on day 0. Mice were then treated intraperitoneally with 5 mg/kg cisplatin on days 15 and 18 and intratumorally with 20 µg/mouse E7 long peptide and 100 µg/mouse DMXAA on days 16 and 19. a Schedule flowchart. b Tumor mass was measured until the mice died or the tumor diameter was > 2 cm (n = 5). c Mouse survival was observed for 60 days (n = 5). d One week after the last vaccination, the tumor tissues and spleens of TC-1 tumor-bearing mice were harvested and re-stimulated with E7 short peptide and then analyzed for IFN-γ⁺ CD8⁺ T cells by flow cytometry (n = 5). IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Characterization after the co-administration of tumor-specific antigen and STING agonist in cisplatin-treated mouse model. In the in vivo experiments, the groups were as follows: cisplatin treatment with E7 long peptide vaccination (DMSO) and cisplatin treatment with E7 long peptide and DMXAA vaccination (DMXAA). a Tumor mass was measured until the mice died or the tumor diameter reached > 2 cm (n = 5). b Mouse survival was observed for 60 days (n = 5). c One week after the last vaccination, the tumor tissues and spleens of TC-1 tumor-bearing mice were harvested and re-stimulated with E7 short peptide and then analyzed for IFN-γ⁺ CD8⁺ T cells by flow cytometry (n = 5). d, e Tumor tissues and spleens of the mice were harvested on day 22. Bar graphs depict the presence of CD4⁺ T cells, CD8⁺ T cells, MDSCs, and Treg cells and the M1 and M2 distribution percentages of CD11b⁺ F4/80⁺ macrophages, as evaluated by flow cytometry analysis (n = 5). f, g One week after the last vaccination, the tumor tissues and serum from the same mice in c were harvested. Bar graphs represent the levels of cytokines in the tumor tissue and serum of mice as measured by ELISA (n = 5). IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

PD-L1 expression by STING activation in vivo and in vitro. On day 20, the tumor tissues of mice were harvested. a Schedule flowchart. b Bar graphs depict the expression of PD-L1 in CD45⁻ and CD45⁺ immune cells by flow cytometry (n = 5). c Photomicrograph of tumor tissue obtained by H&E staining and PD-L1 IHC staining (n = 3). d Intravenous injection of 100 µg/mouse Cy-5 labeled-PD-L1 antibody into tumor-bearing mice, and 18 h later analyzed by IVIS spectrum imaging system. The bar graphs show the fluorescence radiance of the Cy-5 labeled-PD-L1 antibody in tumor tissues (n = 5). e–h Bar graphs depict the in vitro expression of PD-L1 in TC-1 tumor cells determined by flow cytometry. e BMDCs and pDCs isolated and differentiated from C57BL/6 mice were treated with or without 10 μg/ml DMXAA, and the supernatant was treated with TC-1 cells overnight. f TC-1 cells were treated with 100 ng/ml recombinant protein. g, h TC-1 cells were treated with 10 ng/ml blocking antibody for 30 min, washed, and then treated overnight with the supernatant of DMXAA-treated BMDCs or pDCs. IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Tumor treatment effect of combination therapy using immune checkpoint inhibitor in large tumors. C57BL/6 mice were subcutaneously injected with 2 × 10⁵ TC-1 cells/mouse on day 0. Mice were then treated intraperitoneally with 5 mg/kg cisplatin on days 18 and 21 and intratumorally with 20 µg/mouse E7 long peptide and/or with 100 µg/mouse DMXAA on days 19 and 22. The cells were then treated and/or intraperitoneally with 100 µg/mouse PD-1/PD-L1 antibody on days 19, 21, 23, 25, and 27. a Schedule flowchart. b Tumor mass was measured until the mice died or the tumor diameter reached > 2 cm (n = 5). c Mouse survival observed for 60 days (n = 5). d One week after the last vaccination, the tumor tissues and spleens of TC-1 tumor-bearing mice were harvested and re-stimulated with E7 short peptide and then analyzed for IFN-γ⁺ CD8⁺ T cells by flow cytometry (n = 5). IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Suppression of tumor treatment effect due to STING deficiency. In the in vivo experiments, C57BL/6 (wild-type; WT) mice and STING KO (knockout) mice were used. a Schedule flowchart. b Tumor mass was measured until the mice died or the tumor diameter was > 2 cm (n = 5). c Mouse survival observed after 60 days (n = 5). d One week after the last vaccination, the tumor tissues and spleens of TC-1 tumor-bearing mice were harvested and re-stimulated with E7 short peptide and then analyzed for IFN-γ⁺ CD8⁺ T cells by flow cytometry (n = 5). e Bar graphs depicting the in vitro expression of PD-L1 in TC-1 tumor cells determined by flow cytometry. BMDCs and pDCs isolated and differentiated from C57BL/6 and STING KO mice were treated with or without 10 μg/ml DMXAA and the supernatant was treated with TC-1 cells overnight. IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Tumor treatment effects of neoantigen and combination therapy in CT26 murine colon carcinoma tumor model in vivo and in vitro. a–d BALB/c mice were injected with 2 × 10⁵ CT26 cell/mouse subcutaneously on day 0. Mice were then treated intraperitoneally with 5 mg/kg cisplatin on days 12 and 15 and/or intratumorally with neoantigen WT (wild-type) or MT (mutant) peptide (20 µg/mouse) on days 13, 16, 19, 22, and 25. Next, mice were treated intratumorally with DMXAA (100 µg/mouse) on days 13 and 16 and/or intraperitoneally with PD-1/PD-L1 antibody (100 µg/mouse) on days 13, 15, 17 and 19. a Schedule flowchart. b Tumor mass was measured until the mice died or the tumor diameter reached > 2 cm (n = 5). c Mouse survival was observed for 60 days (n = 5). d One week after the last vaccination, splenocytes were isolated and re-stimulated with neoantigen WT/MT peptide for 2 days, and the levels of IFN-γ were estimated by ELISA (n = 5). e–h Bar graphs depict the in vitro expression of PD-L1 in CT26 tumor cells determined by flow cytometry. e BMDCs and pDCs isolated and differentiated from BALB/c mice were treated with or without 10 μg/ml DMXAA, and the supernatant was treated with CT26 cells overnight. f CT26 cells were treated with 100 ng/ml recombinant proteins. g–h CT26 cells were treated with 10 ng/ml blocking antibody for 30 min, washed, and then incubated overnight with supernatants of DMXAA-treated BMDCs or pDCs. IBM SPSS Statistics Base 22.0 was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001