Abstract A34: Combined focal radiation and anti-mCTLA-4 antibody therapy modulates myeloid subset phospho-STAT3 levels in the 4T1 tumor microenvironment and results in tumor growth inhibition
Authors: Draper David , Evens Hillary , Wong Alden , Wise Scott , Franklin Maryland R. 
Published: June 2021

Abstract
Tumor-promoting effects of STAT3 signaling within myeloid subsets are being uncovered by ongoing research. Thus, selective targeting of STAT3 in myeloid cells has become an attractive therapeutic approach as it can prevent adverse effects that may otherwise be triggered by broad inhibition of the pathway. Using a murine breast cancer model, we provide evidence that CTLA-4 checkpoint blockade combined with radiation therapy selectively reduces the levels of phosphorylated STAT3 in myeloid-derived suppressor cell subsets (MDSCs). To this end, we treated 4T1 tumor-bearing mice with focal radiation (SARRP, Xstrahl), an anti-mCTLA-4 checkpoint inhibitor, or the combination of both. Tumor burden was monitored volumetrically. In the radiation-only group, a modest tumor growth inhibition occurred (34% TGI), while anti-mCTLA-4 therapy alone had no effect. However, radiation in combination with anti-mCTLA-4 therapy further enhanced tumor growth inhibition over radiation alone (71% TGI). On day 22 post-implant, tumors were harvested for analysis. To examine the effects that in vivo therapy had on STAT3 signaling, we used a recently developed method to quantify phospho-protein levels in multiple distinct cell subsets simultaneously. Here, we measured phospho-STAT3 levels in tumor-derived MDSCs, tumor-associated macrophage subsets (TAMs), as well as nonhematopoietic cells (CD45−). Immune subset infiltration into the tumor was also examined. Direct ex vivo phospho-flow analysis revealed that treatment-induced effects on phospho-STAT3 levels differed depending on the therapy administered and the subset analyzed. Notably, CTLA-4 blockade reduced phospho-STAT3 levels in granulocytic-MDSCs, the most prevalent myeloid subset in the 4T1 tumor. This was not further enhanced when combined with radiation at the timepoint analyzed. Reduced phospho-STAT3 levels were also observed in monocytic-MDSCs, but combined therapy was required to trigger this effect. Interestingly, phospho-STAT3 levels increased in M1 and M2 TAMs and were unchanged in CD45− cells following combined therapy. To examine the effects on immune cell infiltration, eleven subsets were profiled. A dramatic reduction in B cells (&gt;95%) was observed in each tumor in the combined therapy group when compared to the untreated group. A less extensive but similar reduction was observed in regulatory T cells (Tregs). Notably, B-cell reductions that exceeded 90% were also observed in some animals within the isotype control group. Furthermore, we examined the CD8+ T-cell phenotype. Combination therapy, but not single agents, increased the expression of both CD69 and PD-1 on CD8+ T cells, suggesting an enhancement of antitumor cytotoxicity in these cells. Taken together, our data suggest that anti-mCTLA-4 and radiation selectively disrupt STAT3 signaling in MDSCs and combine to enhance CD8+ T-cell activity, which may play a role in the 4T1 tumor growth inhibition observed.
Citation Format: David Draper, Hillary Evens, Alden Wong, Scott Wise, Maryland R. Franklin. Combined focal radiation and anti-mCTLA-4 antibody therapy modulates myeloid subset phospho-STAT3 levels in the 4T1 tumor microenvironment and results in tumor growth inhibition [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A34.