Fig 2: HBO facilitated teniposide to induce cGAS-STING signaling in mouse HCC tumor model. (A) Schematic illustration of the tumor microenvironment survey experiments in mouse HCC tumor model. Hepa1-6 liver orthotopically tumor-bearing mice were intraperitoneally injected with DMSO (Ctrl), oxaliplatin (10 mg/kg) and teniposide (10 mg/kg) twice at indicated time points. HBO therapy was administrated alone or after chemotherapy for a total of five times (n=3 per group). Tumors were resected and weighed after the final HBO administration. (B–C) HIF-1a expression of mice tumor tissues was determined by immunohistochemical staining (B) and immunoblotting (C) after HBO administration. Scale bars: 50 µm. (D) p-IRF3 expression of mice tumor tissues from different treatment groups was determined by immunohistochemical staining. Scale bars: 50 µm. (E) Ifn-b, Ccl5 and Cxcl10 mRNA expression of mice tumor tissues from different treatment groups was detected by RT-qPCR. (F) Representative images and quantitative analysis of p-IRF3low (n=60) and p-IRF3high (n=60) staining in tumor tissues from 120 patients with HCC. Scale bars: 50 µm (upper) and 25 µm (lower). (G) Disease-free survival (left) and overall survival (right) analyses of 120 patients with HCC showed a positive correlation between high p-IRF3 expression level and favorable prognosis. The prognostic significance was assessed by Cox regression analysis. (H–I) Kaplan–Meier curves were generated from the public microarray databases of human patients using an online software (kmplot.com/analysis/). Low expression levels of HIF1A (H) and high expression levels of IFN-B1, CCL5 and CXCL10 (I) were positively correlated with better survival of patients with HCC. Data in (C) are representative of three independent experiments. Data in (E) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; Ctrl, control; HBO, hyperbaric oxygen; DMSO, Dimethyl sulfoxide; HCC, hepatocellular carcinoma; HIF-1a, hypoxia inducible factor 1a; IRF3, interferon regulatory factor 3; Oxa, oxaliplatin; Rel. expression, relative expression; RT-qPCR, real time quantitative PCR; Teni, teniposide.

Fig 3: Teniposide triggered type ? interferon and NF-?B signaling through the cGAS-STING activation in human HCC cells. (A–C) Hep3B and Huh7 cells were treated with oxaliplatin or teniposide at each IC50 for 24 hours and the supernatant IFN-ß was then measured by ELISA (A) and the mRNA expression of IFIT-1 and IFIT-2 (B) and CCL5 and CXCL10 (C) was measured by RT-qPCR. (D) Hep3B and Huh7 cells transduced with cGAS-shRNA or scramble-shRNA were treated with teniposide at each IC50 for 24 hours and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting; ß-actin was used as a loading control. (E) Hep3B and Huh7 cells transduced with STING-shRNA or scramble-shRNA were treated as in (D) and the cellular protein expression of p-IRF3 and p-P65 was then detected by immunoblotting. Data in (D) and (E) are representative of three independent experiments. Data in (A), (B) and (C) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. cGAS-STING, cyclic GMP-AMP synthase-stimulator of interferon genes; Ctrl, control; HCC, hepatocellular carcinoma; IC50, 50% inhibitory concentration; IFN-ß, interferon ß; IRF3, interferon regulatory factor 3; NF-?B, nuclear factor kappa-B; Oxa, oxaliplatin; Rel. expression, relative expression;RT-qPCR, real time quantitative PCR; sh-SCR, short hairpin RNA-scrambled; sh-cGAS, short hairpin RNA-cGAS; Teni, teniposide.

Fig 4: Combined HBO with teniposide chemotherapy sensitized tumor response to PD-1 antibody. (A) Schematic illustration of anti-PD-1 sensitization experiments in vivo. Mice with subcutaneously established tumors were intraperitoneally injected with DMSO (Ctrl) and teniposide (10 mg/kg) twice at indicated time points, and HBO therapy was administrated after teniposide chemotherapy in the HBO+teniposide+PD-1 combination group for a total of five times. PD-1 antibody (200 µg per mouse) was intraperitoneally injected every 3 days at indicated time points (n=5 per group). (B–D) Tumor growth curves of Hepa1-6 (B), MC38 (C) and B16 (D) bearing mice during anti-PD-1 sensitization experiments. (E) Schematic diagram of the combination therapy based on HBO and teniposide. *P<0.05, **P<0.01, ***P<0.001. cGAS, cyclic GMP-AMP synthase; CTL, cytotoxic T lymphocyte; Ctrl, control; DC, dendritic cell; DMSO, Dimethyl sulfoxide; HBO, hyperbaric oxygen; IFN-I, interferon type I; IFN-?, interferon ?; IRF3, interferon regulatory factor 3; NF-?B, nuclear factor kappa-B; PD1, programmed cell death protein-1; STING, stimulator of interferon genes; Teni, teniposide; TNF-a, tumor necrosis factor-a.

Fig 5: HBO helped teniposide to activate the antitumor response in mouse HCC tumor model. (A) Multiplex immunofluorescence staining of p-IRF3 (green), CD11c (red), CD3 (yellow), CD8 (purple) and DAPI (blue) in p-IRF3 high expression regions and adjacent p-IRF3 low expression regions of human HCC tissues. Scale bars: 50 µm. (B) Tumor-infiltrating macrophages, M1-like macrophages (CD11b+F4/80+/CD86+) and M2-like macrophages (CD11b+F4/80+/CD206+) of each treatment group were identified by FACS. (C) Tumor-infiltrating CD11c+ cells of each treatment group were detected by immunohistochemistry. Scale bar: 50 µm. (D–E) Expression of CD86, MHC-I and MHC-II on the surface of CD11c+ cells in the liver orthotopic tumor from each treatment group was detected by FACS. (F) Tumor-infiltrating CD8+ cells of each treatment group were detected by immunohistochemistry. Scale bar: 50 µm. (G) Total infiltrating CD8+ cells and the intracellular expression of IFN-?, TNF-a, GZMB and Ki67 of CD8+ T cells in the liver orthotopic tumor from each treatment group were detected by FACS. Data in (B), (E) and (G) are shown as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. Ctrl, control; DAPI, 4',6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; GZMB, granzyme B; HBO, hyperbaric oxygen; HCC, hepatocellular carcinoma; IFN-?, interferon ?; IRF3, interferon regulatory factor 3; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; Oxa, oxaliplatin; Teni, teniposide; TNF-a, tumor necrosis factor-a.