Uncovering the mechanism of cancer-associated fibroblasts induced immune evasion of hepatocellular carcinoma cells via in situ fluorescence imaging

Tumor microenvironment (TME) promotes the immune evasion hindering the hepatocellular carcinoma (HCC) immunotherapy. Cancer-associated fibroblasts (CAFs) as dominant stroma cells in the TME play critical roles in the immune evasion of HCC. However, the underlying molecular mechanism remains unclear. Herein, we construct an in-situ fluorescence imaging strategy to monitor the dynamic activation and distribution of CAFs, which is associated with a near-infrared (NIR) fluorescence probe Cy-FAP. This probe consists of cyanine derivative and valine-proline dipeptide responsive toward fibroblast activation protein-α (FAP), a biomarker of CAFs. Based on intramolecular charge transfer (ICT), after FAP cutting off the dipeptide specifically, fluorescence intensity at 705 nm enhances significantly. Through in vivo imaging with Cy-FAP, we revealed that the FAP expression was positively related to the immune evasion of HCC. Subsequently, we found that the JAK2 of CAFs was oxidized by the excessively generated ROS, resulting in the down-regulation of TNF-α and INF-γ and up-regulation of PD-L1. These led to exhaustion of CD8+ T cells and further enhanced the immune evasion of HCC. In conclusion, the real-time and in-situ monitoring of CAFs established here provides a powerful tool for exploring the roles of CAFs in the development of HCC.