Abstract 2470: Isolation and characterization of cancer stem cells from the human medulloblastoma cell line DAOY: adherent vs. suspension in vitro cell cultures

Abstract

Abstract Brain tumor stem cells (BTSC) in vitro cultures have been made possible by the formation of neurospheres in suspension conditions with the use of serum free medium supplemented with specific growth factors, as B27, EGF, or b-FGF. In this work, we tested a more recent alternative method for the culture of BTSC, using laminin pre-coated flasks and the same medium used for the neurosphere growth, which leads to BTSC expansion attached to a layer. BTSC cultures in adherence conditions by the use of laminin can afford a more homogeneous exposition to growth factors than sphere cultures do, which promotes a higher enrichment in stem-like cells. Also, it is known that the laminin interactions with integrines or other components of the cell matrix can lead to inhibition signals of cell differentiation. Moreover, the monolayer conditions can support a higher number of cells in the flask. The medulloblastoma cell line DAOY was cultured in different conditions of normoxia and hypoxia, and several cell populations were obtained for in vitro and molecular experiments. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have BTSC with a higher probability than the CD133- fraction. The colony formation assay presented similar results for the two cultured methods, but clonogenicity in soft agar was higher in the laminin culture cells. In the cell migration assay, medullospheres migrated similarly to the laminin cultured cells, since both cell populations lost their adherence capacity after being cultured with serum free medium. Gene expression of CD133, CD15 and EZH2 was compared as well, and significant differences were not detected between the two cell populations. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining BTSC in DAOY medulloblastoma cell line. Citation Format: Javier de la Rosa, Ander Sáenz Antoñanzas, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Isolation and characterization of cancer stem cells from the human medulloblastoma cell line DAOY: adherent vs. suspension in vitro cell cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2470.