Fig 1.
Serum induces mainly astrocytic differentiation in neurospheres.
a. Pictures of neurosphere cultured and differentiated adherent monolayers of six different cell lines, GSC23, GG1, GG9, GG12, GG14, GG16 (magnification x10). b. Immunostaining for astrocytic (GFAP) and neuronal (β3 Tubulin) differentiation markers in differentiated GSC23, GG1 and GG9 (magnification x20; GFAP and β3 Tubulin in red and DAPI in blue). c. Western blot detecting the expression of stem cell marker SOX2 and differentiation markers GFAP and β3 Tubulin in differentiated and undifferentiated GSC23, GG1, GG9, GG14 and GG16.
Fig 2.
Serum-induced differentiation enhances migration and invasion in vitro.
a. GSC23 whole neurosphere differentiation on poly-l-lysine coated cover slips in media containing serum, along with co-immunostaining with SOX2 (green) and GFAP (red; DAPI in blue; magnification x10). b. Inverted microscope images of GSC23 neurospheres and undifferentiated monolayer GSC23 cells cultured in serum free medium on laminin-coated plates (magnification x10). c. Western blot showing the expression of Nestin and the stem cell marker SOX2 and differentiation marker GFAP in the neurospheres and undifferentiated laminin-coated GSC23 monolayer cells. d. Wound healing assay performed with differentiated monolayer and undifferentiated laminin-coated monolayer GSC23 cells showing differences in wound closure capacity (magnification x5), quantification of wound closure of 3 independent experiments are shown in (e) (**** p<0.0001). f. Representative pictures (magnification x5) of transwell membranes comparing the invading capacity of differentiated and undifferentiated GG16 cells; quantification shown in g. The bars represent the mean of three independent experiments measured in triplicate ± SEM (*P < 0.05, ***P < 0.001).
Fig 3.
Serum-differentiated GBM cells show enhanced infiltration in vivo.
a. Immunohistochemical staining with Nestin on xenografts derived from differentiated (GG16D) and undifferentiated (GG16 Nsp) at day 7 and day 21 post intracranial injection; overview (magnification x5) and enlarged picture (magnification x20) are being shown. b. Mann-Whitney U tests showing significant differences in the number of infiltrating cells between implanted neurosphere and differentiated cells, both at 7 days (p = 0,001) and 21 days (p = 0,004). The average number of infiltrating cells per section and standard deviations are indicated. c. Immunohistochemical staining with GFAP.
Fig 4.
Serum-differentiation leads to enhanced MMP9 expression.
a. Western blot showing MMP9 expression in differentiated and undifferentiated GSC23, GG1, GSC14 and GG16 cells. The numbers (SD between parentheses) indicate the intensity of the quantified bands corrected for loading (β-actin) and the level in neurospheres was set at 1. b. Immunohistochemical staining for MMP9 on xenografts derived from differentiated and undifferentiated GG16 at day 7 and day 21 post intracranial injection showing elevated MMP9 expression in xenografts derived from the differentiated cells in comparison to the undifferentiated cells; overview (magnification x5) and enlarged picture (magnification x20). c. Western blot showing a decrease in MMP9 expression in differentiated GG16 cells following the administration of CP 471474, a chemical inhibitor of MMP9. d. Representative pictures (magnification x5) of transwell membranes showing a reduction of the invading capacity of differentiated GG16 cells following pre-treatment with the MMP9 inhibitor; quantification is shown in e. Data represent the means ± SEM of 3 independent experiments where 30 neurospheres were included in each experiment (****P < 0.0001).
Fig 5.
Serum-differentiated GBM cells can revert back to an undifferentiated state and re-acquire stemness.
a. Serum-differentiated GSC23, GG1, GG9, GG14 and GG16 (primary) neurospheres (Nsp) reverted back to (secondary) neurospheres following exposure to neural stem cell medium (NSM). b. Quantification of the size of primary and secondary GG9 and GG16 neurospheres. (***P<0.001). c. Western blot showing the expression level of GFAP in undifferentiated (primary Nsp), differentiated and de-differentiated (secondary Nsp) GG9 and GG16 cells. d. Limiting dilution assay showing the reduction in the neurosphere formation potential in GSC23, GG9, GG14 and GG16 following differentiation. Data represent the means ± SEM of 3 independent experiments and neurospheres were counted from 96 wells in each experiment (**P<0.01, ***P < 0.001).
Fig 6.
De-differentiation of serum-differentiated GG16 cells reduces their invasive potential.
a. Heatmap based on fold change of the indicated stem cell and differentiation markers generated by qRT-PCR analyses showing partial gain of stem cell and loss of differentiation markers following dedifferentiation of GG16 cells. b. Representative transwell membranes (magnification x5) showing reduced numbers of invading cells after dedifferentiation of GG16 cells (secondary Nsp) that is almost equal to that of primary neurosphere cells; quantification in c. showing mean ± SEM of 3 independent experiments (*P<0.05). d. Western blot showing the absence of MMP9 expression in primary and secondary GG16 neurosphere cells in comparison to the differentiated GG16 cells.
Fig 7.
Model depicting the proposed effect of differentiation on GBM progression.
a. Differentiation enhances the migration and invasive potential of GBM cells. The differentiated cells can (partially) revert back to an undifferentiated/ stem cell state involving signals derived from the microenvironment. b. During GBM progression differentiated cells emanating from the primary tumor have enhanced infiltrative capacity and penetrate deeper in to the normal brain parenchyma. The early differentiated cells can revert back to an undifferentiated/stem cell state with unlimited proliferative potential and produce a secondary lesion away from the primary tumor. Alternatively an additional possibility is that the more invasive differentiated GBM cells pave the way for GSCs by generating a passage and suitable conditions for GSCs to follow their track.