Tumor-Infiltrating Platelets Recruit Neutrophils to Promote Tumor Growth through the 5-HIAA–GPR35–ERK1/2 Axis in Hepatocellular Carcinoma

Tumor-associated neutrophils (TAN) promote tumor growth and metastasis in hepatocellular carcinoma (HCC). Platelets can activate neutrophils and contribute to inflammation and organ damage; however, the relationship between TANs and platelets in HCC remains unclear. We performed multiplex immunohistochemistry and found that tumor-infiltrating platelets and TANs exhibited similar spatial distributions in patient-derived HCC samples. Then, a transwell migration assay demonstrated that supernatants from platelets cocultured with HCC cells enhanced neutrophil migration in vitro compared with supernatants from platelets or HCC cells cultured alone. Subsequent metabolomics analysis and in vitro and in vivo validation revealed that platelet-derived 5-hydroxyindole acetic acid (5-HIAA) promoted neutrophil migration via G protein-coupled receptor 35 (GPR35). Furthermore, in Hepa1-6 mouse models of HCC, GPR35+ neutrophils were found to facilitate HCC growth. Analysis of human HCC single-cell RNA sequencing (RNA-seq) data and mouse neutrophil bulk RNA-seq data revealed significant upregulation of the MAPK signaling pathway and pathways associated with neutrophil migration and cytokine production in GPR35+ neutrophils. Finally, in vitro experiments demonstrated that 5-HIAA activated the ERK1/2 pathway, enhanced protumor cytokine production, and promoted HCC cell growth via GPR35, all of which were suppressed by the GPR35 inhibitor CID2745687. Together, these data indicate that tumor-infiltrating platelets recruit neutrophils to promote tumor growth through the 5-HIAA-GPR35-ERK1/2 axis in HCC.