Experience in creating primary cultures of endometrial cancer and studying cells carrying phenotype of cancer stem cells

Purpose of the study. Was to investigate the possibility of applying the method of spheroid formation in culture for assessment of the endometrial cancer (EC) tumor stem cells (TSC) content in complex samples containing various tumor cells and microenvironment.

Materials and methods. Primary cultures were obtained from fragments of tumors removed during surgery as a first stage of treatment at the Department of Gynecological Oncology, the National Medical Research Center for Oncology. After enzymatic disaggregation of tissue, cell suspension was passaged in DMEM medium containing 10 % fetal bovine serum and 1 % gentamicin to obtain primary two-dimensional cultures. To study the ability of cells to form spheroids, the primary culture was removed from the culture plate and passaged with 2.0 × 10⁴ cells per well of a six-well plate (n = 6) in DMEM medium containing 0.35 % agarose and growth factors EGF (20 ng/ml) and FGF (20 ng/ml). After two weeks of cultivation, the average size, number of formed spheroids, and frequency of spheroid formation were determined. For those cultures that had formed spheroids, immunofluorescent staining of the two-dimensional culture for the marker CD133 was performed, after which the frequency of CD133⁺ cells was determined.

Results. A total of nine primary cultures of EC were obtained, five of which formed spheroids within two weeks of cultivation under non-adhesive conditions. In these cultures, small polygonal CD133⁺ cells showed the strongest association with spheroid formation, which were associated with the largest spheroids (98–110 μm in diameter).

Conclusion. There is a large number of microenvironmental cells in mixed cultures of CSC, some of which may express CD133, including healthy stem cells that also form spheroids in soft agar. A more detailed study of CSC subpopulations compared to normal endometrium is required to establish a link between the observed diversity of cells in culture and their ability to form spheroids and other characteristics of tumor stem cells.

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