Abstract P3-05-19: Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells
Authors: Liu C-Y , Lau K-Y , Huang T-T , Chen J-L , Chu P-Y , Huang C-T , Wang W-L , Tseng L-M 
Published: February 2018

Background

Metastatic triple-negative breast cancer (TNBC), despite being chemo-sensitive, remains poor prognostic. Rising data have suggested some anti-cancer agents exert immunostimulatory activities through inducing immunogenic cell death (ICD). ICD can be detected by presence of damage-associated molecular patterns (DAMPs) such as cell surface exposure of calreticulin (CRT), secretion of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells. Signal transducer and activator of transcription 3 (STAT3) activity is activated in TNBC and regorafenib has been shown to suppress p-STAT3 signal. We tested whether regorafenib induces ICD in TNBC.

Methods

Mice TNBC cell line 4T1 cells were treated with regorafenib and cell survival was examined by MTT assay and flow cytometric analysis. DAMPs were examined by western blot analysis, immunofluorescence microscopy and luminescent assay. The correlation between regorafenib-mediated ICD and STAT3 inhibition were validated in ectopic STAT3 transfected 4T1 cells. Moreover, we investigated the combination strategies of regorafenib with immune checkpoint blockade in syngeneic 4T1 tumor bearing mice (TNBC animal model) with treatment of regorafenib or mPD1 treatment alone/ combination of mPD1 and regorafenib.

Results

The results demonstrated that regorafenib reduced cell survival and induced cell apoptosis in a dose-dependent pattern in 4T1 cell line. Regorafenib induced ICD, as evidenced by it triggered the release of HMGB1 and ATP, as well as the exposure of CRT on the cell surface. Moreover, regorafenib-induced ICD was attenuated by ectopic expression of STAT3 (thus increased phosphorylation of STAT3) in STAT3-overexpressed 4T1 cells. Last but not the least, we observed 4T1 tumor bearing mice with treatment of regorafenib alone, or anti-PD1 monoclonal antibody (mAb) alone, or in combination of regorafenib and anti–PD1 mAb resulted in reduced size of primary tumors, increased survival and fewer lung metastases. Flow cytometric analysis revealed regorafenib treatment increased activated CD8+ T cells, increased antigen-presenting dendritic cells, and suppressed regulatory T cells (Tregs) in mice spleen.

Conclusions

Regorafenib induced ICD in 4T1 cells via the inhibition of p-STAT3. Regorafenib also promoted CD8+ T cells activation and antigen presenting ability of dendritic cells, and suppressed Tregs. Our study demonstrated regorafenib as an ICD-inducer and immunomodulator and its potential combination with immune checkpoint blockade in TNBC.

Citation Format: Liu C-Y, Lau K-Y, Huang T-T, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Tseng L-M. Regorafenib induces immunogenic cell death via p-stat3 inhibition in triple negative breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-19.