Tumor-associated macrophages promoting PD-L1 expression in infiltrating B cells through the CXCL12/CXCR4 axis in human hepatocellular carcinoma

The inflammation-related tumor microenvironment (TME) is one of the major driving forces of hepatocarcinogenesis. We aimed to investigate cell-to-cell communication among Hepatocellular Carcinoma (HCC) through re-analyzing HCC single-cell RNA-seq data, and to confirm such cellular interaction through in vitro and in vivo study. We found a subset of Regulatory B cells with PD-L1 expression (PD-L1⁺ Bregs), mainly located in adjacent HCC tissues. In co-localization with PD-L1⁺ Bregs, a subset of Tumor Associated Macrophages with high expression of CXCL12 (CXCL12⁺ TAMs) was also mainly located in adjacent HCC tissues. Moreover, CXCL12⁺ TAMs can be stimulated in vitro using an HCC conditional medium. Using CellChat analysis and Multiplex Immunohistochemistry staining (mIHC), CXCL12⁺ TAMs were found to be first recruited by Cancer-Associated Fibroblasts (CAFs) through a CD74/macrophage migration inhibitory factor (MIF) pattern, and further differentiated into TGF-β-enriched tissues. Furthermore, CXCL12⁺ TAMs recruited PD-L1⁺ Bregs via the CXCL12/CXCR4 axis, and CXCR4 expression was significantly positively correlated to PD-L1 expression in PD-L1⁺ Bregs. At last, we confirmed the communications among CAFs, Macrophages and B cells and their tumor-promoting effects by using an orthotopic mouse model of HCC. Immunosuppressive HCC TME involving cell-to-cell communications comprised MIF-secreting CAFs, CXCL12-secreting TAMs, and PD-L1-producing Bregs, and their regulation could be promising therapeutic targets in future immunotherapy for human HCC.