Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

GFAP+NNP, isolated from the cerebrum of human fetuses, were purchased from Lonza, formerly Cambrex (CC-2565, NHA-Normal Human Astrocytes; Walkersville, MD). GFAP+NNP at passage 0 (or rarely passage 1) were grown for 8 hours in expansion medium (supplemented with rhEGF, insulin, AA with 3% fetal bovine serum: AGM Bullet Kit Media, Lonza), then for 24 hours in serum-starvation medium; DMEM/F12 medium (Gibco) supplemented with N2 (10×) (Gibco), insulin (10 ng/mL; Invitrogen), and epidermal growth factor (EGF) (10 ng/mL; Invitrogen). Then, the medium was changed to neural differentiation medium: DMEM/F12 supplemented with N2 (10×). The cells were grown in the differentiation medium for 2–20 days.

Tissue samples were obtained from patients with glioblastoma treated in the Department of Neurosurgery, Polish Mother Memorial Hospital Research Institute of Lodz and Department of Neurosurgery Medical University of Lodz, Poland. All samples were collected under protocols approved by Medical University of Lodz. The tumor cells were dispersed by means of collagenase type IV (20 U/mL, 37°C). Subsequently, the cells were for 12 hours in expansion medium. Twelve hours later, the medium was changed to the serum-starvation medium, and aggregates were isolated after 1–4 days of incubation. For each tumor 20–40 aggregates were tested.

The aggregates were isolated and transferred into cell culture dishes covered with Matrigel (Growth Factor-reduced; BD Discovery Labware, Bedford, MA) and cultured in neural differentiation medium: DMEM/F12 supplemented with N2 (10×). After 12–24 hours of incubation, it was observed that cells were released from the aggregates. The aggregates were then gently removed by means of a 1-mL pipette and the cells which migrated out of the aggregates were left on the dish for further experiments. The aggregate-derived cells were immunocytochemically stained after 12–24 hours and at 5, 10, 15, and 20 days of growth.

The aggregates could be propagated for at least 10 months, incubated in neural differentiation medium and transferred every 5–20 days. The experiments presented in this paper were performed after three to six transfers of the aggregates into new medium (no longer than five weeks of propagation).

Glioblastoma cells forming aggregates, non-selected glioblastoma cells and undifferentiated GFAP+NNP were cultured in alpha-MEM media containing 10% FBS (fetal bovine serum).

The protein extracts from cell cultures were obtained with the use of NucleoSpin (Macherey-Nagel). The commercially available Brain Tissue Lysate was used as a positive control in Western blot analysis (Abcam). Equal amounts of protein extracts, 25 μg per line, were separated by 5% or 10% SDS-PAGE followed by transfer onto Immobilon™-P polyvinylidene difluoride membranes (Sigma-Aldrich). After blocking of nonspecific binding, the membranes were incubated for 1 hour at room temperature with primary antibodies (Table 1). After extensive washing in TBST buffer, the membranes were incubated for 1 hour at room temperature with species-specific secondary antibody (Santa Cruz Biotechnology, Inc.), diluted 1:2000. Membranes were then washed in TBST buffer and finally in TBS buffer (TRIS Buffered Saline; pH 7.5); antigen-antibody complexes were detected by enhanced chemiluminescence, using Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Inc.) and visualized with the use of BIO-RAD camera and Quantity One software.

Immunocytochemistry assays for double- or triple-immunofluorescent labeling were performed. For immunofluorescence studies, cells were grown on tissue-culture chamber slides or for single cell assay experiment, in 16-well chamber slides (Nunc). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature and blocked with 2% donkey serum in PBS for 1 hour at room temperature. For double or triple immunolabeling, fixed cells were subsequently incubated with appropriate primary antibodies (Table 1) for 1 hour at room temperature. Double- or triple-labeling was achieved by simultaneous incubation with a combination of species-specific fluorochrome-conjugated secondary antibodies (1 hour, room temperature). For double immunolabeling, a mixture of donkey anti-rabbit AlexaFluor^®488 (dilution 1:250) and donkey anti-mouse AlexaFluor^®594 (dilution 1:250) antibodies (Molecular Probes) were applied. For triple labeling, the following combination of antibodies was used: donkey anti-rabbit AlexaFluor^®488 (dilution 1:250), donkey anti-mouse AlexaFluor^®594 (dilution 1:250), donkey anti-goat AlexaFluor^®350 (dilution 1:250); Molecular Probes. After a final rinse with PBS, the slides were mounted using ProLong^® Gold Antifade Reagent (Molecular Probes). For nuclei staining, the ProLong^® Gold Antifade Reagent with DAPI (Molecular Probes) was used. The slides were coverslipped and examined using an Olympus BX-41 fluorescence microscope. Semi-quantitative analysis based on measurement of fluorescence intensity was performed with the use of WCIF Image J software (Wright Cell Imaging Facility, Toronto Western Research Institute). MAP2+^high signal was defined based on current measurements and results published by Witusik et al [6]. Cells showing intensity higher than 120 units/pixel were defined as MAP2+ ^high. For immunocytochemical BrdU staining, the vendor protocol was applied (Sigma).

Multiplex PCR was performed for evaluation of EGFR amplification with superoxide dismutase 1 (SOD1) used as a reference gene. EGFR and SOD1 were amplified using the following primers: 5'-ctactagaagttgatggctt-3' and 5'-ggtccatgaaaaagcagatg-3' (110 bp); 5'-ttaagaagacttggtggtccatgaaaaagcagatg-3' and 5'-aaaaaagcttggaatgtttattgggcgatcc-3' (163 bp). PCR products were separated by electrophoresis in 2% agarose gel, visualized using a Bio-Rad Gel Doc 1000 and analyzed with Molecular Analyst software as described before [9].

DNA was isolated from the cells obtained from the aggregates and from immunostained cells, original tumor cultures and blood isolated from the patients, by means of Macherey-Nagel DNA/RNA/Protein purification kit. LOH and MSI analyses were performed using paired tumor specimens and corresponding peripheral blood samples. The following LOH markers were used: D1S2734, D1S197, D1S162 D1S156, D9S319, D9S162, D10S587, D10S1267, D17S1828. MSI markers have already been described [10]. Forward primers were 5'-end fluorescence-labeled. PCR was performed in thermocycling conditions individually established for each pair of primers. PCR products were denatured and gel electrophoresis in LiCor automatic sequencer system was applied to the separation and analysis of PCR-generated alleles.

Four genomic regions of P53 gene (exons 5–8) were amplified by PCR using sets of primers encompassing each exon [11]. Sequencing was performed as described before [11] using the dideoxy termination method, SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies) and LiCor automated sequencer.

To assess the mitotic activity of glioblastoma cells, 10 μM BrdU (Sigma, St. Louis, MO), a marker of DNA synthesis, was added to the cells cultured in expansion media for 48–72 hours. To assess the mitotic activity of cells derived from glioblastomas, 10 μM BrdU was added to DMEM/F12 media supplemented with N2 on days 1 and 15 of culture. Similarly, BrdU was added to the glioblastoma cells passaged in 10% alpha-MEM. BrdU incorporation was assessed after 5, 10, 15 and 20 passages. After 48–72 hours of incorporation, the cultures were fixed with 4% paraformaldehyde for 15 minutes and processed for immunocytochemistry.

Glioblastoma cells showing CD44+, Vimentin+, Fibronectin+, and neural markers positive, phenotype were plated in 6-well tissue culture dishes, and allowed to reach 80% confluence. Triplicate wells were incubated for 3 weeks in adipogenic medium, containing 0.5 μM hydrocortisone (Sigma), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma) and 60 μM indomethacin (Sigma). The medium was replaced every 3 to 4 days. To assess lipid deposition, the cultured cells were washed with PBS and fixed for 1 hour with 10% formalin, then stained for 15 minutes with a diluted Oil Red-O solution (Sigma) with shaking at room temperature, then washed three times with dH₂O, and subsequently examined under a light microscope. Negative controls were glioblastoma cells showing mesenchymal phenotype but not exposed to adipogenic medium; positive controls consisted of marrow stromal cells exposed to adipogenic medium and stained with Oil Red-O.

Glioblastoma cells obtained after dislodging the aggregates were used for single cell assay: one cell was suspended in the medium directly into one conical well of a 96-well or 16-well immunocytochemistry plate. Then, the cells were grown in several conditions: serum-starvation medium, neural differentiation medium, and expansion medium, as described under the GFAP+NNP growth and differentiation section. The single cell assay was also started at passage 0, just after the beginning of the tumor cell cultures. The cells were stained, photographed and subsequently DNA was isolated and LOH analysis was performed to check if clones were obtained from the tumor cells.

Cells isolated from eight glioblastomas, as well as GFAP+NNP, were grown in the same culture conditions: first in expansion medium, then in serum-starvation medium, and finally in neural differentiation medium, as described in Materials and methods. All undifferentiated GFAP+NNP presented the multilineage phenotype, defined as co-expression of GFAP, CD44, Beta III-tubulin, MAP2 and Nestin, SOX-2, Vimentin [5‐7]. GFAP+NNP were CD133 negative (Fig. 1a). In initial monolayer cultures isolated from eight glioblastomas, 10% (GBM6) to 95% (GBM1) of cells presented an evident multilineage phenotype (Table 2). All glioblastoma cells with multilineage phenotype were also SOX-2 positive (Fig. 1b). In addition to the population of cells with multilineage phenotype, cells with one or more of the following phenotypes were also observed in different glioblastoma cultures: MAP2+^high/GFAP+/CD44-; CD44+/GFAP-/MAP2-; GFAP+/CD44+/MAP2-; MAP2+^high/GFAP-/CD44-; CD133+/CD44+/MAP2+/GFAP+ and CD133+/MAP2-/GFAP- (Table 2).

Between 24–48 hours of incubation in the serum-starvation medium, glioblastoma cells growing initially in a monolayer, began forming aggregates (Fig. 1c). A spontaneous ability to form aggregates consisting of multilineage tumor cells was observed in five of eight glioblastomas (GBM1, GBM2, GBM3, GBM4, GBM8), which presented more than 40% of cells with multilineage phenotype. The aggregates were very stable and able to be transferred and micro-surgically manipulated. To confirm that the aggregates were formed by a pure population of tumor cells with the multilineage phenotype, every aggregate was gently cut into two parts. One part was removed from the culture for DNA/protein isolation and immunocytochemical staining, and the second part was further propagated.

Glioblastoma cells forming aggregates, as well as undifferentiated GFAP+NNP, co-expressed SOX-2, Nestin, Beta III-tubulin, MAP2, GFAP, Vimentin, Fibronectin and CD44, when cultured under serum-starvation media. The expression of these markers was detected by immunocytochemistry (Fig. 1a–f) [5‐7], and Western blotting (Fig. 2a). Under these conditions, GFAP+NNP did not form aggregates and were maintained in monolayer. Because the aggregates were formed by homogenous tumor cells, in terms of the multilineage phenotype, aggregate formation allowed for separation of these cells from other tumor cells in glioblastoma cultures. Aggregates, but not monolayer of glioblastoma cells, were used for a majority of further experiments.

The phenotypical discrepancies of eight glioblastomas presented here prompted us to perform basic molecular testing. The molecular alterations in glioblastoma are variable, however, two predominant genetic pathways typical for primary (EGFR amplifications) and secondary glioblastomas (mutations of P53) have been described [12, 13]. Therefore, we performed sequencing of P53 gene and EGFR amplification analysis by multiplex PCR in DNA isolated directly from the tumors and from the respective cell cultures. EGFR amplification was identified in GBM1, GBM3 and GBM5, whereas GBM2, GBM6 and GBM8 showed P53 gene mutations (Table 2, Fig. 2b–c). None of these alterations were found in GBM4 and GBM7 (Table 2). The statistical analysis did not reveal any important association between the molecular background and cellular phenotype.

Non-neoplastic stem cells have been previously described to infiltrate tumor tissue; we verified whether the population of the aggregate-forming cells was composed purely of tumor cells. We showed that DNA isolated from frozen tumors was heterogeneous, most likely as a result of contamination by non-tumor cells: both normal DNA and allele with chromosomal loss was found in the tumor samples. However, DNA isolated from the aggregates showed no retention of the normal allele in LOH analysis, indicating that these aggregates consisted of only tumor cells (GBM1-GBM4) (Fig. 2d).

Next, we replaced the serum-starvation medium with neural differentiation medium in GFAP+NNP cultures as well as glioblastoma cell aggregates. Under these conditions, GFAP+NNP rarely formed aggregates. If aggregates appeared, they were unstable and completely dispersed after one or two transfers due to massive release of differentiating cells. The dispersed cells showed features of differentiation and grew in monolayer (Fig. 3a,b). Using triple immunocytochemical staining, we showed that at early stages of differentiation (after 2–3 days of incubation in the neural differentiation medium), in 25–35% of the GFAP+NNP derivatives, MAP2+^high/GFAP+^low/CD44- phenotype could be induced. Cells with upregulated MAP2 and Beta III-tubulin expression, and downregulated GFAP expression, express TH (tyrosine hydroxylase), an enzyme required for catecholamines synthesis, which further confirms their neuronal characteristics (Fig. 4a–h). The morphology of these cells, lack of CD44 and high expression of MAP2 and Beta III-tubulin, suggested that they were potential neuronal intermediates (Fig. 5a). Moreover, their percentage was almost as high as the percentage of neuronal cells observed two days later. Among cells showing GFAP and MAP2 co-expression, most (about 70%) presented only the remnants of GFAP, which constitutes additional evidence that the neuronal cells originated from the cells initially showing a multilineage phenotype. The existence of MAP2+, GFAP+, CD44- cells has already been presented by our group [7]. We showed that the percentage of those cells after 5–7 days of neural differentiation did not exceed 5% [7].

When cultured under serum-starvation conditions, in contrast to GFAP+NNP, glioblastoma cells formed stable aggregates, and aggregate cells maintained the multilineage phenotype for several months (as currently observed for 8 months). The differences between aggregates of glioblastomas and GFAP+NNP cultures are shown in Fig. 3a,b. When culture conditions were changed to neural differentiation conditions after 12–24 hours, tumor cells migrated from aggregates and formed a monolayer (Fig. 1d,e). After their appearance in monolayer, we continued with two parallel cultures. The monolayer of cells was left in culture for further propagation, and the aggregates were transferred to new dishes.

The monolayer cells released from glioblastoma aggregates presented the multilineage phenotype. In these cells a co-expression of CD44, GFAP, and MAP2 was observed after 24 hours and remained after 5 days of exposure to neural differentiation medium (Fig. 1b,c,d,e). In comparison, after five days of culture in the neural differentiation medium, nearly all GFAP+NNP differentiated into neuronal and astrocytic cells (Fig. 5a,b,c).

After 5–20 days of culture in the neural differentiation medium, the phenotype of the cells released from glioblastoma aggregates was characterized by immunocytochemistry. The results revealed differences in the differentiation patterns and the capacity between cells obtained from examined tumors. GBM2 cells released from aggregates showed the highest resistance to neural differentiation conditions: predominantly, cells sustaining the multilineage phenotype (CD44+/GFAP+/MAP2+) were observed (Table 3; Fig. 5d). GBM1 and GBM3 cells released from aggregates, in the same culture conditions, showed three phenotypes: multilineage (CD44+/GFAP+/MAP2+), non-neural (CD44+/GFAP-/MAP2-) and neuronal intermediate (MAP2+^high/GFAP+/CD44-) (Table 3; Fig. 5e,f). Comparative analysis showed that the neuronal intermediate phenotype was also observed as a result of GFAP+NNP differentiation (Fig. 5a). However, in GFAP+NNP this phenotype was observed only temporarily prior to differentiation into neural cells, whereas it was very stable in glioblastoma cells. These results suggest that it was possible to trigger the neural phenotype in GBM1 and GBM3 cells, although it was arrested at early stages of neural differentiation. GBM4 cells released from aggregates contained a higher percentage of cells with glial phenotype (greater than 25%) after neural differentiation than GBM1 and GBM3 (Table 3), whereas GBM8 cells contained greater than 40% of glial cells (Table 3; Fig. 5g). In addition to glial, neural differentiation of GBM8 cells yielded a subpopulation resembling neuronal cells (Fig. 5g). In all of these cases (GBM1, GBM2, GBM3, GBM4, GBM8), during the prolonged neural differentiation (15 days), Nestin expression was not eliminated as it was in neuronal derivatives of GFAP+NNP (Fig. 5h) [5]. In parallel, the aggregates were cultured and the phenotype was monitored. After 3–4 months, the aggregates cultured in neural differentiation medium lost the ability to attach to tissue culture surface and to release cells into a monolayer. However, the cells within the aggregates remained viable and displayed the multilineage phenotype (data not shown). These results suggest that in contrast to GFAP+NNP, induction of neural phenotype in the glioblastoma aggregate-derived cell was significantly inhibited.

To observe the mesenchymal differentiation of GFAP+NNP and glioblastoma aggregate-derived cells, the cells were transferred to the alpha-MEM medium supplemented with 10% FBS. When cultured under these conditions, GFAP+NNP generated a population of cells with robust expression of Fibronectin and maintenance of CD44 expression (Fig. 6a,b). We previously defined this population as mesenchymal [8]. We show that similarly to GFAP+NNP, the aggregate-derived cells from GBM1, GBM2, GBM3, GBM4 and GBM8 exposed to alpha-MEM with 10% FBS for a number of passages, were induced to non-neural, mesenchymal phenotype (CD44+/GFAP-/MAP2-) and robustly expressed Fibronectin (Fig. 6c,d). The same effect was observed when heterogeneous glioblastoma cells isolated from GBM1, GBM2, GBM3, GBM4, GBM5 and GBM8, were exposed to alpha-MEM with 10% FBS (Fig. 6e). However, GBM2 and GBM4 cells required more passages (above 15) than GBM1, GBM3, GBM5, GBM8 (above 10), to present purely non-neural, mesenchymal phenotype. Interestingly, in GBM1 and GBM3 aggregate-derived cells, non-neural differentiation was also observed after exposure to neural differentiation medium, as described in the previous paragraph (Fig. 6d). Moreover, glioblastoma-derived mesenchymal cells exposed to adipogenic medium, showed features of adipogenesis (Fig. 6f). Considering our data, and recent literature demonstrating mesenchymal differentiation of glioblastoma cells [1], we defined the CD44+, GFAP-, MAP2- population of glioblastoma cells as mesenchymal.

These results show that two of the analyzed glioblastomas (GBM1 and GBM3) exhibited non-neural differentiation under both neural- and non-neural differentiation conditions.

Molecular analyses (LOH and/or P53 sequencing) confirmed that the population of cells with non-neural phenotype consisted entirely of glioblastoma cells and were not contaminated with normal cells as LOH, without contamination with the normal allele, nor were P53 mutations identified (Fig. 2c,d).

In addition, our results show that one out of eight glioblastomas (GBM7) did not form aggregates and did not differentiate in accordance with the GFAP+NNP differentiation model (Table 2).

CD133 positive cells were observed in the primary cultures isolated from six out of eight glioblastomas (Table 2; Fig. 1f). All these glioblastomas after exposure as a monolayer culture to the alpha-MEM with 10% FBS, showed a complete loss of CD133-positive cells. Both aggregates of glioblastoma cells and the cells released from the aggregates were negative for CD133 (Fig. 7a,b). Assay of BrdU incorporation revealed that acquisition of the mesenchymal phenotype correlated with very low mitotic activity when compared to the original population of glioblastoma cells isolated from GBM1, GBM 3, GBM 4, GBM 5, GBM 6, and GBM 8 (Fig. 7c,d). Only GBM2 did not show this regression of mitotic activity.

Glioblastoma cells with the multilineage phenotype obtained from the aggregates and the dispersed cells of all eight tumors were grown in cloning conditions in different media (see Methods). We were able to clone only cells isolated from GBM2, GBM4, and GBM6. GBM2 cells maintained the multilineage phenotype when exposed to serum-starvation medium (Fig. 8a). However, GBM4 and GBM6 acquired mesenchymal phenotype in all tested single cell assay conditions (Fig. 8b). These results showed that single cell assay can be applied only for some glioblastomas. Moreover, single cell assay altered the cellular phenotype in two out of three cloned cell lines. LOH and/or MSI analysis confirmed the neoplastic origin of the cloned cells.