Cancer stem cells from human glioma cell line are resistant to Fas-induced apoptosis

Abstract

Abstract. Glioblastoma is the most common primary brain tumor, characterized by its resistance to treatments. To define efficient therapy, the origin of tumor-forming cells needs to be elucidated in order to search for new therapeutic pathways. The objective of this study was to determine the different cell populations constituting a human glioblastoma cell line, U-87 MG and their sensitivity to apoptosis induced through the activation of Fas, a membranous death receptor. By a cell sorting method, the sedimentation field flow fractionation, two major cell subpopulations were identified, a most differentiated cell fraction, containing large and adherent cells, sensitive to Fas-induced apoptosis and another one, characterized by small cells forming aggregates, expressing CD133, a marker of stem cells and more resistant to Fas-activated apoptosis. By using a selective method of culture, adapted for neural stem cell cultures, we have verified that the U-87 MG cell line contained cancer stem cells similar to the immature ones obtained by the cell sorting method. Interestingly, while these tumor stem cells, expressing CD133, were resistant to Fas-induced apoptosis, monomeric form of Fas protein was detected predominantly in these cells. In contrast, the most mature cells, responsive to Fas-activated apoptosis, collected in another cell fraction, contained oligomeric aggregates of Fas protein, a pre-signalling form of the Fas receptor, essential for the initiation of apoptosis through its activation. These results suggest that these immature stem cells in glioma could be an important factor of resistance to chemotherapy requiring apoptosis through Fas signalling system. Indeed, future strategies of treatment, inducing differentiation of these stem cells need to be considered to enhance therapeutic efficiency.

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells

“…We wondered whether the biophysical changes revealed by SdFFF analysis could help us to distinguish among sub-cellular populations, especially CSCs. As expected, and as already published [ 28 ], the F3 U87pLKO cell fraction exhibited more CSCs markers as compared with cells of the F1 fraction (data not shown). In the cell line in which autophagy was down-regulated (U87shBeclin1), analysis of CSCs markers confirmed a weak enrichment of the F3 fraction, based on enhancement of LGR5 and POU5F1 expressions (*p<0.05 and **p<0.01 respectively, Figure 6A ).…”

“…Thus, larger and less dense cells generating more hydrodynamic lift forces within the mobile phase and not very sensitive to centrifugal force (blue cells on Figure 5A ), will be eluted before smaller and denser cells (red cells, Figure 5A ) generating less hydrodynamic forces and more sensitive to the multigravitational field. As for a number of other cell models [ 27 , 29 , 31 ], we found that under the elution conditions, U87 cells were eluted under the hyperlayer elution mode [ 28 , 30 ] schematized in Figure 5A .…”

“…The counterpart is the dilution of the analytical cell sample limiting the number of cells in the sorted sub-populations forcing to increase the number of runs. Based on previous results of our group [ 28 ], we expected CSCs enrichment of the F3 fraction. Obtained data with sorted cells confirmed that the F3 fraction preferentially contained cells with characteristics of CSCs, as shown by the enhancement of LGR5 and POU5F1 expression, especially with the U87shBeclin1 cell line.…”

“…In each case, we observed the classical two-peak profile ( Figure 5B ); the first peak corresponds to the void peak (VP, t 0 /t 0 = R obs ≈ 1) containing unretained species (cell debris, proteins, cell aggregates…), and the second corresponds to the cell peak (CP, t 0 /t r = R obs < 1). These broad cell peaks ( Figure 5B ) correspond to very polydisperse populations, reflecting the heterogeneity of these GB cell lines containing a cell continuum, from fully differentiated cells to cancer stem cells [ 28 , 30 , 35 ].…”

“…Over the past decade, applications of SdFFF cell sorting have been developed in many fields, including oncology. A pioneering study of CSCs sorting from within a GB cell line [ 28 ] made it possible to sort other cancer cells [ 29 ] and to improve cell culture conditions ahead of cell sorting and CSCs detection [ 30 ]. Moreover, these studies allowed an opening towards the field of stem cell biology [ 31 ].…”