Delicaflavone reactivates anti-tumor immune responses by abrogating monocytic myeloid cell-mediated immunosuppression

Myeloid cell-mediated immunosuppression is a major obstacle to checkpoint blockade immunotherapy. We previously reported that total biflavonoids extract from Selaginella doederleinii (TBESD) and a flavone monomer isolated from TBESD, named Delicaflavone, have favorable anti-tumor activity. However, whether TBESD and Delicaflavone could affect the tumor microenvironment (TME) remains unclear.In this study, we focused on the TME to determine whether TBESD and Delicaflavone could restore anti-tumor immune response.4T1 tumor-bearing immunocompetent BALB/c mice and T cell-deficient nude mice were used to examine the effect of TBESD on T cell-mediated immunity in vivo. Multi-parameter flow cytometry was conducted to evaluate the impacts of TBESD on TME. Primary cells, including murine CD8+ T cells, tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) were prepared to investigate the modulatory activities of TBESD on immune cells. It was further determined whether Delicaflavone or Amentoflavone, two typical functional biflavones from TBESD, mediated those effects of TBESD. Finally, the impacts of TBESD and Delicaflavone on Jak1/STAT6 signaling pathway were explored via western blot.We found that TBESD significantly reduced 4T1 tumor growth in immunocompetent BALB/c mice, but not in nude mice. This effect was associated with the regulation of TME, shown as an increase in functional T cells and M1 phenotype TAMs (M1-TAMs), and a decrease in M2 phenotype TAMs (M2-TAMs), monocytic-MDSCs (M-MDSCs) and regulatory T cells (Tregs) in TBESD-treated BALB/c mouse 4T1 tumors. It was found ex vivo that TBESD restrained the viability and immunosuppressive properties of M2-TAMs and M-MDSCs, especially for the loss of arginase-1 expression. Additionally, TBESD re-educated M2-TAMs to an M1 like phenotype. Further investigations determined that Delicaflavone predominantly mediated the immuno-modulatory activities of TBESD both ex vivo and in vivo. Finally, Delicaflavone and TBESD blocked Jak1/STAT6 signaling pathway in M2-TAMs and MDSCs.The present study suggests Delicaflavone as a potent natural inhibitor of M2-TAMs and MDSCs, which fills the gap in knowledge on the immuno-modulatory effects of TBESD and Delicaflavone, and could have translational implications to improve the efficacy of cancer immunotherapy.