翌圣生物 Yeasen｜生命科学试剂与诊断原料一站式平台

Abstract

The progression of hepatocellular carcinoma (HCC) was highly dependent on pathological angiogenesis, with tumor-associated macrophages (TAMs) serving as key mediators regulating angiogenesis within the tumor microenvironment. The role of protein tyrosine phosphatase 2 (SHP2), a multi-signaling pathway regulator containing an SH2 domain, in TAM-mediated angiogenesis remained unclear. This study aimed to elucidate the molecular mechanism by which SHP2 regulates HCC angiogenesis through the ROS/Src pathway in TAMs under hypoxic conditions. We employed a comprehensive approach. THP-1 macrophage cells were transfected with SHP2 shRNA lentivirus and co-cultured with HCC cell line Huh-7 cells. The co-cultured cells were then subcutaneously transplanted into nude mice. Tumor size was assessed via HE staining in nude mouse tumor formation assays. We engineered luciferase-transfected Huh-7 cells to establish an orthotopic HCC implantation model and measured tumor area via in vivo fluorescence imaging. THP-1 cells were respectively cultured in media supplemented with antioxidants, pro-oxidants, and Src inhibitors, followed by transfection with SHP2 shRNA lentivirus and SHP2 mock lentivirus. These cells were then co-cultured with Huh-7 cells and Hep3B cells, respectively. The proportion of CD68-positive cells detected by immunofluorescence staining was used to ensure that the observed effects in the study were primarily derived from tumor-associated macrophages. Western blot analysis was employed to detect expression levels of SHP2, ROS/Src pathway-related proteins, tumor migration-related proteins, and angiogenesis-related proteins in the cells. Relative fluorescence intensity of intracellular ROS in THP-1 cells was visualized using the fluorescent probe DCFH-DA combined with fluorescence microscopy. Cell migration and invasion capabilities were assessed via wound healing assays and transwell assays. Immunofluorescence staining further validated SHP2 expression levels in THP-