Saikosaponin D and paeoniflorin improve the HCC immune microenvironment via CLCF1/PD-L1 mediated crosstalk between CAFs and tumor cells

Radix Bupleuri-Radix Paeoniae Alba (RB-RPA) has the potential to ameliorate the immune microenvironment in hepatocellular carcinoma (HCC). The interactions between various cells influence the immune microenvironment. However, the effects of the active components (saikosaponin D (SSD) and paeoniflorin) of RB-RPA on HCC and the cell interactions within its microenvironment warrant further investigations. We established orthotopic mouse models of HCC using Hepa 1–6 cells (a hepatoma cell line) to investigate the infiltration of cancer-associated fibroblasts (CAFs). Following this, we knocked down CLCF1 in the CAFs to explore their interactions with Hepa 1–6 cells. We subsequently treated CAFs with SSD-paeoniflorin. In T-cell activation assays, we overexpressed PD-L1 in Hepa 1–6 cells and co-cultured these cells with T cells. Additionally, we treated HCC mice with SSD-paeoniflorin and induced the overexpression of CLCF1 in vivo. In mice with HCC, tumors were characterized by substantial infiltration of CAFs, which displayed elevated levels of the protein CLCF1. These CAFs play a role in suppressing the HCC immune microenvironment and elevating PD-L1 levels through the production of CLCF1. At the cellular level, SSD-paeoniflorin inhibited the growth of CAFs and the expression of CLCF1 and also suppressed the PD-L1 level in Hepa 1–6 cells through CAF-derived CLCF1. Mechanistically, CLCF1 may promote PD-L1 expression via the JAK/STAT3 signaling pathway. Furthermore, the overexpression of PD-L1 in Hepa 1–6 cells suppressed T cell proliferation and activation. In animal studies, SSD-paeoniflorin improved the HCC immune microenvironment by enhancing the interaction between CAFs and Hepa 1–6 cells through CLCF1. Collectively, SSD-paeoniflorin inhibits CLCF1 secretion in CAFs to downregulate PD-L1 expression in Hepa 1–6 cells and may reactivate the anti-tumor immune microenvironment in HCC.