Mechanistic studies of tyrosine phosphatase SHP2 in hypoxic environment to promote angiogenesis in HCC by modulating the ROS/Src pathway

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-1 cells. Additionally, we examined the effects of SHP2 on HCC angiogenesis using tube formation assays, Western Blot analysis, HE staining, and immunohistochemical staining for CD31. The impact of SHP2 on HCC cell proliferation was investigated through colony formation assays and cell cycle analysis by flow cytometry. Following co-culture of SHP2 shRNA-infected THP-1 cells with Huh-7 cells, nude mouse tumor formation assays and in vivo fluorescence imaging revealed significantly reduced tumor volume and area. Following antioxidant supplementation in THP-1 cell culture medium, western blot analysis revealed markedly reduced expression levels of ROS/Src pathway-associated proteins, tumor migration proteins, and angiogenesis-related proteins. Fluorescent probe-based fluorescence microscopy demonstrated decreased relative fluorescence intensity of ROS within tumor cells. Furthermore, Western blot and immunofluorescence staining experiments jointly demonstrated that SHP2 in TAMs can regulate the ROS/Src pathway. Wound healing assays, transwell assays, and tube formation assays demonstrated that SHP2 in TAMs enhanced HCC cell migration, invasion, and angiogenesis capabilities. Furthermore, HE staining and immunohistochemical staining for CD31 indicated that inhibiting ROS/Src pathway suppressed HCC angiogenesis. Colony formation assay and flow cytometry analysis revealed that SHP2 in TAMs promotes HCC proliferation and cell cycle progression via the ROS/Src pathway. Under hypoxic conditions, tyrosine phosphatase SHP2 in TAMs regulates angiogenesis in HCC through the modulation of the ROS/Src pathway.