Figure 1.
Cytotoxicity of sorafenib on E.G7 cells.
E.G7 cells were treated with various concentrations (0–25 µM) of sorafenib for 24 hours. AlamarBlue assay was performed to determine the cell viability. The IC50 of sorafenib is 15 µM on E.G7 cells.
Figure 2.
Restoration of immunosuppressive factors in E.G7 cells by sorafenib.
(A) (Left panel) Expressions of immunosuppressive factors were assayed by Western blotting, and (right panel) their quantitative results. IDO, VEGF, IL-10 and TGF-βwere decreased in a dose-dependent manner in E.G7 cells treated with 5–15 µM sorafenib for 24 hours. In addition, expressions of pSTAT3 and CCL2/MCP-1 also were decreased. (B) Expression of IDO in E.G7 cells treated with exogenous IFN-γto mimic the tumor-bearing animal model. Sorafenib decreases both endogenous and exogenous IDO expressions. (C) Expression of TGF-βreceptor I was assessed by flow cytometry. TGF-βreceptor I was significant decreased after 2–15 µM sorafenib treatments. (D) Survival of CD8+ T cells would affect the therapeutic outcome of ACT, and apoptosis of CD8+ T cells is mainly through Fas/Fas ligand (FasL) pathway. Thus, expression of FasL was assayed by flow cytometry, and the expression of FasL was suppressed in E.G7 cells by sorafenib. The experiments were repeated more than three times, and one of the representative was shown here. (* as compared with that of vehicle, *p<0.05, **p<0.01, ***p<0.001; # as compared with that of 2 µM sorafenib treatment, #p<0.05, ##p<0.01)
Figure 3.
Sorafenib enhances functions and migrations of CD8+ T cells.
(A, B) Activities of CD8+ T cells were evaluated from expressions of tomato fluorescent protein and intracellular IFN-γby flow cytometry. Both were elevated after co-cultured with sorafenib-treated E.G7 cells. The experiments were repeated three times, and one of the representative was shown here. (C) Cytotoxicities of CD8+ T cells were enhanced when co-cultured with sorafenib-treated E.G7 cells at different effector-to-target (E:T) ratio. (D) Migratory capability of CD8+ T cells was determined by transwell assay. Sorafenib significantly increased migrations of CD8+ T cells. The experiments were repeated twice. (* p<0.05, ** p<0.01, **** p<0.0001)
Figure 4.
WP1066, an STAT3-specific inhibitor, was used to clarify the role of STAT3 in immunosuppressive microenvironment.
(A) (Left panel) Expressions of IDO, VEGF, IL-10, and CCL2/MCP-1 were assayed with Western blotting, and (right panel) their quantitative results. All proteins were suppressed after treated with WP1066. (B, C) Expressions of tomato fluorescent protein and intracellular IFN-γwere assayed by flow cytometry. Significantly higher tomato fluorescent protein and IFN-γsignals were found in CD8+ T cells co-cultured with sorafenib and WP1066-treated E.G7 cells, respectively. (D) Effect of WP1066 on migration of CD8+ T cells was evaluated with transwell assay. More migratory CD8+ T cells were found in WP1066-treated E.G7 cells. The experiments were repeated three times, and one of the representative was shown here. (* p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001)
Figure 5.
Serial low doses of sorafenib augment recruitment and activation of transferred CD8+ T cells, and exhibit better tumor responses of ACT.
(A) The experimental design in vivo. (B) Bioluminescent imaging was used to monitor the activation of CD8+ T cells from day 0 post ACT. Transferred CD8+ T cells survived longer in tumor lesions in 2T+sora group compared with that of CD8+ T cells alone group. (C) Quantification of BLI signals from tumors. No significant difference was found between 2T+ sora and 5T groups. Though no significant difference was found between 2T+ sora and 2T groups, about two folds higher average radiance was shown in 2T+ sora group as compared with that of 2T group. (D) Tumor growth was tracked by caliper measurement. Significant tumor shrinkage was observed in 5T and 2T+ sora groups, and no significant difference was found between these two groups. Shrinkage of tumors initiated from day 3 after ACT. Notably, day 3 was the time point with peak BLI signals. The experiments were repeated five times, and one of the representative was shown here. (*as compared with that of the control group, * p<0.05, ** p<0.01, **** p<0.0001; # as compared with that of the sorafenib group, #p<0.05, ##p<0.01; †as compared with that of the 2T group, †p<0.05)
Figure 6.
Sorafenib ameliorates therapeutic efficacy of ACT via reversing the unfavorable microenvironment.
(A) (Upper panel) Immunosuppressive factors in tumors treated with or without sorafenib were evaluated with ex vivo Western blotting, and (lower panel) the results were quantified. Immunosuppressive factors were downregulated after sorafenib treatment via STAT3 and TGF-β inhibitions. (B, C) Percentages of Tregs in tumor drainage lymph node (TDLN) and MDSCs in the spleen, respectively, were decreased after serial low doses (7.5 mg/kg/day) of sorafenib treatment. (D, E) Percentages of host CD4+ and CD8+ T cells remained unchanged after serial low doses of sorafenib treatments.
Figure 7.
Sorafenib augments the therapeutic efficacy of ACT by inhibiting STAT3 expression and downregulating immunosuppressive factors in tumor cells and tumor microenvironment.
Downregulation of immunosuppressive factors, including TGF-β, IL-10, CCL2/MCP-1 and VEGF, lowers the populations of both Tregs and MDSCs. Transferred CD8+ T cells show better activation and killing effects due to declined Tregs and MDSCs. Furthermore, more transferred CD8+ T cells accumulate in tumors and survive longer, so that enhanced therapeutic responses can be achieved.