Cancer Biology

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, soft agar anchorage-independent assay were used to determine tumor transform activity of hepatoma cells with ISX over expression or knockdown in vitro.

This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters of progenitor bone cells growing under non-adherent conditions are called osteospheres. Such progenitor populations often possess characteristics of multipotency and can differentiate into multiple mesenchymal lineages. Cancer cells capable of growing in suspension have also been reported in osteosarcomas, tumors of the bone tissue. These spherical colonies formed from single cells (clonal) in non-adherent conditions are generally considered to represent self-renewing, stem-like cells and can be employed for other assays such as multipotency and limiting dilution analysis (LDA).