Zajdela Ascitic Hepatoma As a Continuum for Tumor Cells in a Transitional Stateстатья
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Аннотация:AbstractPreviously, from the adhesive cell line of Zajdela rat ascitic hepatoma, we obtained daughter sublines of two types (holoclonal and meroclonal), the cells of which differed in morphology (fibroblast- and epithelium-like, respectively), clonogenicity in tests in vitro, and tumorigenicity in vivo. To identify the potential role of Zajdela hepatoma adherent cells in processes of epithelial–mesenchymal transition (EMT) and metastasis, we compared all its sublines with monolayer lines of other hepatocellular tumors by the parameters that determine invasive and migratory properties of the cells. We have established that the cells of the holoclonal sublines are characterized by an increased activity of matrix metalloproteinase (MMP) 1, individual type of migration, and high motility rate as compared with the cells of other lines. The cells of all monolayer lines of Zajdela hepatoma secreted active forms of MMP-9 and translocated the intracellular domain of epithelial cell adhesion molecule (EpCAM) to their nucleus, which indicates the acquisition of an invasive phenotype by the cells and activation of Wnt/β-catenin signaling pathway. Our results indicate that the clonal sublines that we obtained from Zajdela ascitic hepatoma are consisted of the cells in transitional states between the epithelial and mesenchymal phenotypes.INTRODUCTIONThe process of metastasis includes a number of sequential stages called the “metastatic cascade,” during which cells detach from the primary tumor, migrate into the circulation, and with blood/lymph flow reach the target organs, where they initiate the growth of secondary tumors. It is considered that this process is performed by a special subpopulation of tumor stem cells (TSCs)—metastatic tumor-initiating cells or metastatic TSCs (Baccelli and Trumpp, 2012) possessing stem properties and abilities to invade underlying tissues, migrate, and form secondary tumors.The phenotype of migratory TSCs of the primary tumor can be formed in the zone of invasive growth as a result of the EMT (Mani et al., 2008). In the process of EMT, the cells lose their epithelioid form, polarity and the organization of the actin cytoskeleton, their dense intercellular contacts are destroyed due to the suppression of E-cadherin gene expression, so the cells become fibroblast-like, express mesenchymal markers, including N-cadherin, vimentin, fibronectin, MMPs and acquire mobility,The primary Zajdela solid hepatoma was induced in rats with 4-dimethylaminoazo-benzene, while the ascitic tumor was then formed from it spontaneously in the process of tumor progression (Zajdela, 1963). A high frequency of metastasis in paratracheal lymph nodes is a peculiarity of this tumor (Kiseleva and Panikarovskii, 1964). Current view on the process of tumor progression allows one to suggest that the cells of Zajdela solid hepatoma in the zone of invasive growth did undergo to EMT.To study the cellular organization of Zajdela ascitic hepatoma and determine the role of its individual subpopulations in metastasis, we transferred the cells of multicellular islets into the culture in vitro and divided the initial cell population by long-term selection into two lines (monolayer and suspension) differing from each other in adhesiveness to the substrate and tumorigenicity (Teryukova et al., 2013). As a result of subsequent cloning of monolayer line cells using a limiting dilution method, we obtained two types of clonal sublines (Teryukova et al., 2016), including holoclonal ones with the traits of TSCs and meroclonal ones with the traits of tumor progenitor cells (TPCs). The cells of holoclonal (3H, 5F, and 6H) sublines have a predominantly fibroblast-like shape, produce mostly holoclones during recloning, and initiate ascitic hepatoma growth in vivo experiments. Cells of meroloclonal (1E and 9C) sublines have a predominantly epythelium-like shape, produce mostly meroclones during recloning, and do not initiate ascitic hepatoma growth.Previously, when studying tumor-associated antigens among the proteins of the outer membranes of Zajdela ascitic hepatoma cells, we identified basigin (known to be an MMP inductor) and EpCAM (involved in the pathway helping cells to maintain a low-differentiated status) (Teryukova et al., 2010). In the present work, we compared the cells of the obtained clonal sublines of Zajdela hepatoma with each other, as well as with the cells of permanent lines of other hepatocellular tumors, by several parameters associated with metastasis: MMP secretion and activity, motility rate, EpCAM expression, and EpICD localization.