Evaluation of the effects of Stattic (a STAT3 inhibitor) on improvement of immunogenic chemotherapy in B16 melanoma cells

Abstract

Background: Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. However, immunosuppressive microenvironment is the main obstacle in cancer immunotherapy. STAT3 is a pathway involved in induction immunosuppression found constitutively active in many cancers and plays a major role in cancer progression. Stattic is known as a selective inhibitor of SH2 domain of STAT3. Aim: To evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3. Methods: The growth inhibitory effects of stattic and chemotherapeutic agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box 1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA. Results: Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion were found in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, no significant increase was observed in the level of ICD in CT26 cells and IL-12 secretion by DCs, when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups. Conclusion: These findings indicate that stattic as an STAT3 inhibitor can increase ICD induced by DOX.