Establishment and characterization of clonal lines with cancer stem- and progenitor-cell properties from monolayer Zajdela hepatoma

The aim of this study was to investigate the biology of cancer stem cells (CSC) from a metastatic tumor. Previously, we explanted the cells of rat ascites Zajdela hepatoma in vitro . We established a permanent monolayer cell line via selection of adhesive cells from multicellular floating islets. In the present work, we cloned these cells by a limiting-dilution method and established five novel clonal sublines of the hepatoma: three holoclonal sublines containing CSC and two meroclonal sublines. After a long-term cultivation (approximately 30 passages, freezing, and thawing), the cell of clonal sublines retained the features of CSC. They have a tumor-initiating potential and produce mainly holoclones upon recloning in the complete growth medium and large nonadhesive hepatospheres in the serum-free medium. Morphometric analysis showed that the cells of holoclonal and meroclonal sublines differed in the cell shape, area, nucleus size, and nuclear-cytoplasmic ratio. We have found for the first time that holoclonal cells of Zajdela hepatoma have a fibroblast-like morphology and form contacts with each other due to membrane protrusions. We suggest that the fibroblast-like morphology of CSC is an attribute of a metastatic tumor and demonstrates the capability of these cells for individual migration.