Induction of senescence like macrophages by hepatocellular carcinoma cells in vitro

Cellular senescence has been regarded as a tumor-suppressive mechanism when occurring in cancer cells. However, when senescence occurs in immune cells, where it can exert opposing effects. Macrophages, one of crucial component of the immune cell have been shown to undergo senescence in mouse models of glioma and lung cancer. Yet, evidence for macrophage senescence in hepatocellular carcinoma (HCC) remains limited. To investigate the senescent state of macrophage in HCC we established an in vitro co-culture model of murine hepatocellular carcinoma cell line Hepa1-6 and macrophages-like transformed murine cell line RAW264.7. SA-β-Gal staining, real-time quantitative PCR, bulk RNA-seq, immunofluorescence were employed to demonstrate macrophage senescence which co-culture with hepatocellular carcinoma. Real-time qPCR, phagocytosis assays, flow cytometry, and transwell invasion experiments revealed the function of macrophages. RAW 264.7 cells exhibited morphological changes consistent with senescence-like change, along with elevated staining of the senescence-associated marker SA-β-Gal and higher proportion of G1 phase in Cell cycle analysis. Co-cultured RAW264.7 cells exhibited upregulated expression of senescence-related genes while downregulating proliferation and cell cycle-related genes. Additionally, senescent macrophages displayed impaired function and adopted an M2-like phenotype. In summary, our findings indicate that macrophages co-cultured with hepatocellular carcinoma cells acquire senescence-like phenotype and the function has also changed.