Table 1.
Primary and secondary antibodies used for immunocytochemical staining.
Figure 1.
Exemplary results of molecular analyses in glioblastoma cells.
(A) An exemplary FISH result presenting chromosome 7 polysomy (a single cell magnified in the rectangle); EGFR probe (red signals); CEP 7 control probe (green signals); magnification 1000x; (B) An exemplary TP53 sequencing result, the line marks the mutated nucleotide in codon 237; (C,D) Exemplary MLPA results showing (C) the genetic alterations (EGFR and MDM4 amplification) in an early passage of cell cultured under classical conditions and (D) their absence in a late passage of the cells from the same tumor.
Table 2.
Molecular characteristics of all analysed samples in the comparison to the population data and CCLE database data.
Figure 2.
Immunocytochemical identification of the cells overgrowing tumor cells.
(A) A late passage of classical monolayer cell culture. The majority of cells expressing αSMA is GFAP(−). A small population of GFAP(+)/α-SMA(−) cells is also visible; (B) The cells invaded radially from the spherical core and further grew as monolayer. Three populations were observed: GFAP-positive; αSMA-positive and double negative.
Figure 3.
The comparison of cell biology features of glioblastoma cell cultures.
The characteristics of cells in different culture conditions are depicted separately: neoplastic cells from cultures which provided a stable cell line (blue bars), neoplastic cells from cultures that did not provide a cell line (red bars) and normal cells from the latter cultures (green bars). (A) The differences in the tumor cell content in glioblastoma cell cultures; (B) The comparison of the percentage of the cells incorporating BrdU during a 7-day incubation; (C) The comparison of the percentage of senescent (positive for SA-β-Gal) cells; (D) The results of Mann-Whitney U test. Ser E – p0-p3 (1–3 weeks) under classical conditions; Ser L – p6-p13 (8–16 weeks) under classical conditions; SF E – p0-p1 (1–3 weeks) under serum-free monolayer conditions; SF L – p3-p5 (6–16 weeks) under serum-free monolayer conditions; Sph E – t0-t2 (up to 3 weeks) of spheroid culturing; Sph L – t4-t8 (3–5 months) of spheroid culturing; E – early passages/transfers; L – late passages/transfers; T – tumor cells, expressing TP53 or EGFR and GFAP; N – normal cells; s – cultures providing stable cell lines, u – cultures not providing stable cell lines.
Figure 4.
Exemplary results of the immunocytochemical BrdU co-staining in different GB-derived cell cultures.
(A,B) An early passage of cells cultured under the classical monolayer conditions after a 7-day incubation with BrdU. In both cases two populations of cells are detectable: (A) BrdU(+)/EGFR(−)/GFAP(−) and BrdU(−)/EGFR(+)/GFAP(+); (B) BrdU(+)/TP53(−)/GFAP(−) and BrdU(−)/TP53(+)/GFAP(+); (C) An early passage of cells cultured under serum-free conditions after 5-day incubation with BrdU, TP53(+)/GFAP(+) tumor cells are negative for BrdU; (D) A spheroid from the 2nd transfer after a 7-day incubation with BrdU, the majority of cells are BrdU-positive; (E) The cells which invaded radially from the spheroid core (2nd transfer) after a 48-hour incubation with BrdU. The majority of EGFR(+)/GFAP(+) cells incorporated BrdU; (F) The cells which invaded from spherical core after 2 months of culturing. Less than 10% of cells incorporated BrdU (10 days of incubation).
Figure 5.
Enzymocytochemical staining showing the senescence of glioblastoma cells cultured under various conditions.
SA-β-Gal-positive cells were observed in all tested conditions. (A) An early passage of GB cells under monolayer serum conditions; (B) A late passage of GB cells under monolayer serum conditions; (C) A late passage of GB cells under monolayer serum-free conditions; (D) The cells in an early spheroid; (E) The cells in a late spheroid; (F) The cells in a late floating spheroid.
Figure 6.
The senescence of neoplastic cells verified by the combination of immunocytochemistry and enzymocytochmistry.
(A,B) A late passage of cells cultured under classical conditions. Only GFAP-positive cells show SA-β-Gal activity; (C,D) A late passage of cells cultured under classical conditions. Only TP53(+)/GFAP(+) cell shows SA-β-Gal activity.
Figure 7.
The neoplastic cell senescence verified by two methods (BrdU incorporation assay and SA-β-Gal assay).
(A–C) Only two populations of cells are visible: BrdU(−)/SA-β-Gal(+)/TP53(+) and BrdU(+)/SA-β-Gal(−)/TP53(+).
Figure 8.
Real-time phase microscopy monitoring of glioblastoma cells.
During the 9-day observation (images taken in 36-hour periods) the cells grew and elongated, however, did not proliferate.
Figure 9.
The senescence of glioblastoma cells with stem cells characteristics verified by the combination of immunocytochemistry and enzymocytochemistry.
(A,B,C) An early passage of cells cultured under the classical conditions. SOX2-positive cells show SA-β-Gal activity and do not incorporate BrdU; (D, E, F) An early passage of cells cultured under the classical conditions. Nestin-positive cell shows SA-β-Gal activity.
Figure 10.
Examples of the additional mechanisms responsible for the glioblastoma cell lines stabilization failure.
(A,B) Neoplastic cells with the features of mitotic catastrophe (large planar cells with two nuclei): positive for GFAP (A) and for EGFR (B); (C,D) GFAP-positive cells with the features of apoptosis; (E,F) TP53-positive cell showing the features of neosis; nuclear budding visible, only a portion of the micronuclei is BrdU-positive.
Figure 11.
Invasion ability of glioblastoma cells invading from spheroid cultures.
(A, B) The GB cells form late transfers of spheroids which invaded thorough the Matrigel-coated filter to the lower surface of the filter of invasion chamber stained for SA-β-Gal activity (A) and with DAPI (B). (C) Box-whisker plots (mean, std. error and min/max values) showing percentage of invading SA-β-Gal (+) and SA-β-Gal (−) cells from early and late transfers of spheroids. The results of the statistical analysis (using Mann-Whitney U test) are presented for each case separately and for the cumulative group in the table below.