Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy

Teriple-negative breast cancer (TNBC) is a heterogeneous tumor, and there is a lack of effective therapies. Immune checkpoint inhibitors (ICIs) therapy has been widely used to treat a variety of human cancers including TNBC. Despite the FDA's approval of therapies like Atezolizumab (anti- PD-L1) and Pembrolizumab (anti-PD-1) for certain TNBC cases, more than half of all TNBC patients, especially those with low PD-L1 levels and in advanced stages, remain unresponsive. Therefore, there is urgent clinical need to find other innovative combination therapy strategy for these TNBC patients. In this study, we examined the efficacy of sulindac to enhance the response of TNBC to anti-PD-L1 immunotherapy. We utilized a 4T1 syngeneic mouse tumor model to compare the inhibitory effects of PD-L1 antibody, sulindac, and their combination on 4T1 tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of activated T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. We also established a PBMC humanized mouse model to further confirmed that combination therapy could significantly reduce the tumor size of TNBC patient-derived xenografts (56S) and the combination group were found to have the most infiltrating activated human T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. Immunofluorescent staining of organoids also confirmed that organoids from combination group have more CD8+ T cell and the results of organoid cell viability assay in vitro indicated that combination of sulindac with PD-L1 antibody together with activated human PBMC significantly reduce 56S organoid cell viability, which is consistent with in vivo study. When investigating the mechanism of action, first we found sulindac could downregulate exosomal PD-L1 by decreasing expression of nsMase2 which is a major regulator in production of exosomal PD-L1. Second, we demonstrated that sulindac could downregulate PD-L1 by blocking Stat3 signaling and enhancing the expression of miR-570-3p which can potentially target PD-L1, which in turn led to a further decrease in exosomal PD-L1. Previous study showed that PD-L1 antibody could be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating exosomal PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent in combination with anti-PD-L1 therapy for the treatment of TNBC.