Immunosuppressive myeloid-derived suppressor cells (MDSC) subvert antitumor immunity and limit the efficacy of chimeric antigen receptor T cells (CAR-T). Previously, we reported that the GM-CSF/JAK2/STAT3 axis drives liver-associated MDSC (L-MDSC) proliferation and blockade of this axis rescued antitumor immunity. We extended these findings in our murine liver metastasis (LM) model, by treating tumor-bearing mice with STAT3 inhibitors (STATTIC or BBI608) to further our understanding of how STAT3 drives L-MDSC suppressive function. STAT3 inhibition caused significant reduction of tumor burden as well as L-MDSC frequencies due to decrease in pSTAT3 levels. L-MDSC isolated from STATTIC or BBI608-treated mice had significantly reduced suppressive function. STAT3 inhibition of L-MDSC was associated with enhanced antitumor activity of CAR-T. Further investigation demonstrated activation of apoptotic signaling pathways in L-MDSC following STAT3 inhibition as evidenced by an upregulation of the pro-apoptotic proteins Bax, cleaved caspase-3, and downregulation of the anti-apoptotic protein Bcl-2. Accordingly, there was also a decrease of pro-survival markers, pErk and pAkt, and an increase in pro-death marker, Fas, with activation of downstream JNK and p38 MAPK. These findings represent a previously unrecognized link between STAT3 inhibition and Fas-induced apoptosis of MDSCs. Our findings suggest that inhibiting STAT3 has potential clinical application for enhancing the efficacy of CAR-T cells in LM through modulation of L-MDSC.

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Liver is a tolerogenic organ and has variety of immune cells resulting in a profoundly immunosuppressive space. We recently reported that GM-CSF/JAK2/STAT3 axis drives liver myeloid suppressor cell (L-MDSC) proliferation and STAT3 inhibition causes activation of apoptosis signaling via Bax up-regulation. Herein, we explore liver specific programming events that promote L-MDSC suppressive conditioning. Bone marrow derived MDSC (BM-MDSC) were expanded in CD45.1+ mice in response to intraperitoneal MC38 tumors. CD45.1+ BM-MDSC were adoptively transferred into CD45.2+ recipient mice via tail vein (TV) or portal vein (PV). CD11b+ cells were harvested 48 hrs later from recipient liver and lungs. Liver from PV and lung from TV CD45.1+ MDSC recipients were compared. There was increased expansion of CD45.1+ MDSC (CD11b+Gr1+) in PV-liver as compared to TV-lung group (Liver-PV 45±3% vs. Lung-TV 15±2% vs. Tumor 43±3, p<0.001, n=5) with increased numbers of the more immunosuppressive monocytic MDSC (M-MDSC) subtype in CD45.1+ transferred cells in liver as compared to lung (Liver-PV 61±4%, Lung-TV 40±3%, Tumor 58±3 p<0.005, n=5). Enhanced pSTAT3 expression (mediator of MDSC expansion) in CD45.1+CD11b+Gr1+ cells in liver was observed as compared to lung (pSTAT3: Liver-PV 54±4, Lung-TV 30±5, p<0.05 n=5). Quantitative PCR of MDSCs isolated from Liver-PV showed significantly decreased levels (8.5 fold, p<0.05, n=4) of pro-apoptotic Bax protein as compared to Lung-TV indicating MDSC apoptosis resistance following conditioning in the liver. These data indicate that L-MDSCs are directed towards suppressive programming and blocking STAT3 may have clinical application for enhancing the efficacy of immunotherapy for liver tumors.

Myeloid derived suppressor cells (MDSC) subvert anti-tumor immunity. Previously we reported that the GM-CSF/ JAK2/STAT3 axis drives liver MDSC (L-MDSC) proliferation and CAR-T suppression. We hypothesized that STAT3 supports L-MDSC survival and suppressive function by inhibiting apoptosis. We treated liver metastasis (LM) in mice with STAT3 inhibitors (STATTIC or BBI) or with vehicle control. STAT3 inhibition caused a significant reduction in tumor burden (p<0.05) and L-MDSC frequency (DMSO 41±3% vs. STATTIC 29±3%/BBI 20±3%, p<0.0001, n=10) in association with lower pSTAT3 levels (DMSO 33±4% vs. STATTIC 11±3%/BBI 9±2%, p<0.0001, n=10). L-MDSC isolated from STATTIC or BBI treated mice were co-cultured with CAR-Ts and corresponding target tumor cells at 1:1:1 ratio. There was a significant decrease in tumor cell density (DMSO 100% vs. STATTIC 71±5%/BBI 20±3%, p<0.05, n=5) and enhancement of tumor cell killing (DMSO 28±4% vs. STATTIC 54±4%/BBI 52±7%, p<0.01, n=5). Rescue of CAR-T tumor killing function correlated with enhanced L-MDSC apoptosis signaling. We detected upregulation of pro-apoptotic proteins Bax, caspase 3, and Fas. Signaling molecules downstream of Fas, JNK and p38 MAPK (p<0.05), were also activated in L-MDSC. In contrast, L-MDSC pro-survival Bcl2, pErk, and pAkt (p<0.05) were downregulated in response to STAT3 inhibition. Microarray results confirmed the STAT3-induced changes in apoptotic and survival gene expression, which was validated by RT-PCR (p<0.05). Within LM, STAT3 inhibition drove L-MDSC apoptosis via the Fas/Fas- L pathway with downstream pro-apoptotic signaling through p38 MAPK. Blocking STAT3 may have clinical application for enhancing immunotherapy for LM.