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Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

Podergajs N, Brekka N, Radlwimmer B, Herold-Mende C, Talasila KM, Tiemann K, Rajcevic U, Lah TT, Bjerkvig R, Miletic H - Radiol Oncol (2013)

Bottom Line: In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF.Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect.We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.

Materials and methods: [corrected] To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.

Results: Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

Conclusions: We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

No MeSH data available.


Related in: MedlinePlus

bFGF increases EGFR and stem cell marker expression in GSCs. (A, B) Flow cytometry analysis of EGFR expression in GSCs, grown in the presence of EGF and/or bFGF for 48 hours. bFGF alone increased the fraction of EGFR expressing cells in the NCH644 cell line, but not in the NCH421k, compared to medium without growth factors. Data shown are mean ± SD of three independent experiments, ***: p<0,001. (C) Western blots of GSCs cultured under different conditions. Presence of bFGF in culturing medium for 14 days upregulated the expression of EGFR in NCH644, but not in NCH421k. (D) Western blots of GSCs. Presence of bFGF in culturing medium for 14 days increased the expression of stem cell markers CD133 and nestin in both cell lines compared to unstimulated cells whereas the expression of SOX2 was increased only in NCH644 cells. EGF had no effect. (E) Quantification of western blots confirmed the significant higher expression of CD133 and nestin in both cell lines and a significant higher expression of SOX2 only in NCH644 cell line under bFGF stimulation. Data shown are mean ± SD of three independent experiments, *: p<0.05; **: p<0.01; ***: p<0.001. Labels: NB=Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF.
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f2-rado-47-04-330: bFGF increases EGFR and stem cell marker expression in GSCs. (A, B) Flow cytometry analysis of EGFR expression in GSCs, grown in the presence of EGF and/or bFGF for 48 hours. bFGF alone increased the fraction of EGFR expressing cells in the NCH644 cell line, but not in the NCH421k, compared to medium without growth factors. Data shown are mean ± SD of three independent experiments, ***: p<0,001. (C) Western blots of GSCs cultured under different conditions. Presence of bFGF in culturing medium for 14 days upregulated the expression of EGFR in NCH644, but not in NCH421k. (D) Western blots of GSCs. Presence of bFGF in culturing medium for 14 days increased the expression of stem cell markers CD133 and nestin in both cell lines compared to unstimulated cells whereas the expression of SOX2 was increased only in NCH644 cells. EGF had no effect. (E) Quantification of western blots confirmed the significant higher expression of CD133 and nestin in both cell lines and a significant higher expression of SOX2 only in NCH644 cell line under bFGF stimulation. Data shown are mean ± SD of three independent experiments, *: p<0.05; **: p<0.01; ***: p<0.001. Labels: NB=Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF.

Mentions: In this study, we used two different, heterogeneous GSC lines, NCH421k and NCH644. These cell lines were derived from primary GBMs that showed typical GBM aberrations (e.g. loss of chromosome 10 and/or gain of chromosome 7) as indicated by DNA copy-number profiling by array-CGH. NCH421k additionally carried amplifications of the PDGFRA and CDK4 gene loci. These copy number aberrations were well preserved in the cell lines (Figure 1). Both primary tumors as well as the NCH421k and NCH644 cell lines derived from them lack amplification of the EGFR gene locus (position indicated by arrows in Figure 1). Both cell lines belong to the proneural category according to the classification by Verhaak et al.20 (data not shown). Recently it has been shown that the presence of EGF in the culture medium leads to reduced EGFR expression in tumor cells with EGFR amplification.13 However, cells without EGFR amplification might also express EGFR. Therefore, we first determined the expression level of EGFR by flow cytometry in both cell lines under different growth conditions. Before the experiment, cells were cultured in NB medium without growth factors for 10 days and then divided into different groups for growth factor treatment as indicated in Figure 2. FACS analysis for EGFR revealed that 0.4 ± 0.14% of NCH644 cells and 0.35 ± 0.35% of NCH421k cells stimulated by both EGF and bFGF (complete NB medium) were positive. However, while stimulation with EGF alone, bFGF alone or medium without growth factors did not alter the expression of EGFR in NCH421k cells, there was a change in NCH644 cells under the defined conditions as shown in Figure 2A and B. bFGF stimulation for 48 hours substantially increased the fraction of EGFR expressing NCH644 cells to 16.85 ± 1.48% (p<0.001), while the absence of growth factors or EGF stimulation alone revealed only 6.00 ± 0.28% (p<0.001) or 1.95 ± 0.64% (p<0.001) EGFR expressing cells, respectively. Thus, bFGF stimulation alone had the strongest positive effect on EGFR expression, while complete medium containing both growth factors and EGF had a negative effect. The upregulation of EGFR in NCH644 was verified by western blot, where cells were stimulated with bFGF for 14 days (Figure 2C). The difference in response to bFGF between the two cell lines might be explained by the variable levels of EGFR that are initially present in GBMs without EGFR amplification. Only a fraction of GBMs without EGFR amplification naturally expresses EGFR.21


Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

Podergajs N, Brekka N, Radlwimmer B, Herold-Mende C, Talasila KM, Tiemann K, Rajcevic U, Lah TT, Bjerkvig R, Miletic H - Radiol Oncol (2013)

bFGF increases EGFR and stem cell marker expression in GSCs. (A, B) Flow cytometry analysis of EGFR expression in GSCs, grown in the presence of EGF and/or bFGF for 48 hours. bFGF alone increased the fraction of EGFR expressing cells in the NCH644 cell line, but not in the NCH421k, compared to medium without growth factors. Data shown are mean ± SD of three independent experiments, ***: p<0,001. (C) Western blots of GSCs cultured under different conditions. Presence of bFGF in culturing medium for 14 days upregulated the expression of EGFR in NCH644, but not in NCH421k. (D) Western blots of GSCs. Presence of bFGF in culturing medium for 14 days increased the expression of stem cell markers CD133 and nestin in both cell lines compared to unstimulated cells whereas the expression of SOX2 was increased only in NCH644 cells. EGF had no effect. (E) Quantification of western blots confirmed the significant higher expression of CD133 and nestin in both cell lines and a significant higher expression of SOX2 only in NCH644 cell line under bFGF stimulation. Data shown are mean ± SD of three independent experiments, *: p<0.05; **: p<0.01; ***: p<0.001. Labels: NB=Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3814277&req=5

f2-rado-47-04-330: bFGF increases EGFR and stem cell marker expression in GSCs. (A, B) Flow cytometry analysis of EGFR expression in GSCs, grown in the presence of EGF and/or bFGF for 48 hours. bFGF alone increased the fraction of EGFR expressing cells in the NCH644 cell line, but not in the NCH421k, compared to medium without growth factors. Data shown are mean ± SD of three independent experiments, ***: p<0,001. (C) Western blots of GSCs cultured under different conditions. Presence of bFGF in culturing medium for 14 days upregulated the expression of EGFR in NCH644, but not in NCH421k. (D) Western blots of GSCs. Presence of bFGF in culturing medium for 14 days increased the expression of stem cell markers CD133 and nestin in both cell lines compared to unstimulated cells whereas the expression of SOX2 was increased only in NCH644 cells. EGF had no effect. (E) Quantification of western blots confirmed the significant higher expression of CD133 and nestin in both cell lines and a significant higher expression of SOX2 only in NCH644 cell line under bFGF stimulation. Data shown are mean ± SD of three independent experiments, *: p<0.05; **: p<0.01; ***: p<0.001. Labels: NB=Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF.
Mentions: In this study, we used two different, heterogeneous GSC lines, NCH421k and NCH644. These cell lines were derived from primary GBMs that showed typical GBM aberrations (e.g. loss of chromosome 10 and/or gain of chromosome 7) as indicated by DNA copy-number profiling by array-CGH. NCH421k additionally carried amplifications of the PDGFRA and CDK4 gene loci. These copy number aberrations were well preserved in the cell lines (Figure 1). Both primary tumors as well as the NCH421k and NCH644 cell lines derived from them lack amplification of the EGFR gene locus (position indicated by arrows in Figure 1). Both cell lines belong to the proneural category according to the classification by Verhaak et al.20 (data not shown). Recently it has been shown that the presence of EGF in the culture medium leads to reduced EGFR expression in tumor cells with EGFR amplification.13 However, cells without EGFR amplification might also express EGFR. Therefore, we first determined the expression level of EGFR by flow cytometry in both cell lines under different growth conditions. Before the experiment, cells were cultured in NB medium without growth factors for 10 days and then divided into different groups for growth factor treatment as indicated in Figure 2. FACS analysis for EGFR revealed that 0.4 ± 0.14% of NCH644 cells and 0.35 ± 0.35% of NCH421k cells stimulated by both EGF and bFGF (complete NB medium) were positive. However, while stimulation with EGF alone, bFGF alone or medium without growth factors did not alter the expression of EGFR in NCH421k cells, there was a change in NCH644 cells under the defined conditions as shown in Figure 2A and B. bFGF stimulation for 48 hours substantially increased the fraction of EGFR expressing NCH644 cells to 16.85 ± 1.48% (p<0.001), while the absence of growth factors or EGF stimulation alone revealed only 6.00 ± 0.28% (p<0.001) or 1.95 ± 0.64% (p<0.001) EGFR expressing cells, respectively. Thus, bFGF stimulation alone had the strongest positive effect on EGFR expression, while complete medium containing both growth factors and EGF had a negative effect. The upregulation of EGFR in NCH644 was verified by western blot, where cells were stimulated with bFGF for 14 days (Figure 2C). The difference in response to bFGF between the two cell lines might be explained by the variable levels of EGFR that are initially present in GBMs without EGFR amplification. Only a fraction of GBMs without EGFR amplification naturally expresses EGFR.21

Bottom Line: In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF.Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect.We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.

Materials and methods: [corrected] To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.

Results: Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

Conclusions: We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

No MeSH data available.


Related in: MedlinePlus