P. Guha1, J. C. Gardell1, M. Cunetta1, M. Lima1, J. Darpolor1, S. Ferree1,2, N. Espat1,2, S. C. Katz1,2 1Roger Williams Medical Center,Surgical Oncology,Providence, RI, USA 2Boston University,Boston, MA, USA
Introduction: Myeloid derived suppressor cells (MDSC) mediate intrahepatic immune suppression in liver metastasis (LM) patients. MDSCs are a heterogeneous group of immature myeloid cells that limit the efficacy of genetically engineered chimeric antigen receptor T cells (CAR-T). We seek mechanistic targets to limit MDSC expansion or induce death to enhance CAR-T efficacy for LM.
Methods: Tumor burden of murine LM model treated with DMSO or STATTIC or BBI was measured using bioluminescence. Flow cytometry, western blot, qPCR and immunofluorescence was performed on MDSC isolated from tumors. LDH assay and CFSE was used to measure MDSC induced suppression of CAR-T.
Results:Previously, we have shown that the GM-CSF/Jak2/STAT3 axis drives liver MDSC (L-MDSC) expansion and suppressive function in murine LM model. We hypothesized that blocking STAT3 activation would cause decreased L-MDSC viability in the tumor microenvironment. STATTIC and BBI target STAT3 by inhibiting its activation, dimerization and nuclear translocation. Both STATTIC and BBI caused a reduction in tumor burden in our LM model with decrease in L-MDSC and pSTAT3 levels as compared to the vehicle (DMSO) control mice. When L-MDSC isolated from STATTIC or BBI treated tumor bearing mice were co-cultured with CAR-T cells, there was a significant decrease in immunosuppression as evidenced by enhanced tumor cell cytotoxicity (29+4% DMSO,54+4% STATTIC p=0.02, 52+7% BBI p=0.01) and CAR-T expansion (22+2% DMSO,36+3% STATTIC p=0.001, 38+4% BBI p=0.001). There was significant increase in apoptosis of L-MDSCs (table) isolated from STATTIC or BBI treated tumor bearing mice as compared to DMSO control. Investigation of apoptosis pathway signaling molecules showed that there was an up-regulation of pro-apoptotic Bax (STATTIC-3.6 fold and BBI-2.7 fold over DMSO, p<0.05) and down-regulation of anti-apoptotic Bcl2 (STATTIC-0.8 fold and BBI-0.5 fold over DMSO, p<0.05) protein in L-MDSCs. This was also associated with significant decrease in proliferation markers such as phosphorylated Erk (pErk) (STATTIC-0.3 fold and BBI-0.5 fold over DMSO, p<0.05), phosphorylated Akt (pAkt) (STATTIC-0.3 fold and BBI-0.4 fold over DMSO, p<0.05). Interestingly, there was an increase in phosphorylated p38 (pp38) MAPK activity (STATTIC-2.7 fold and BBI-3.2 fold over DMSO, p<0.05), Fas (table) and TNFα (STATTIC-3.4 fold and BBI-2.8 fold over DMSO, p<0.05) expression in MDSCs.
Conclusion: Our findings indicate that STAT3 inhibition caused an increase in L-MDSC apoptosis through up-regulation of Fas/Fas-L with down-stream pro-apoptotic signaling via p38MAPK. Thus, blockade of STAT3 in tumor bearing mice may enable us to rescue the anti-tumor immunity for LM through induction of L-MDSC death.
