Clonal Evolution of Glioblastoma under Therapy methods
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Cell culture, lentivirus production and cell growth analysis
After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
METHODS
The specimens in cohort INCB originate from the Besta Brain Tumor Biobank, which is partly funded by the Italian Minister of Health. All patients signed an informed consent for the use of their biological material for research purposes. One case (R012) from this cohort had a history of lower grade glioma prior to the first GBM. All patients were treated by standard Stupp treatment with surgery followed by radiotherapy plus concomitant and adjuvant TMZ.
Reconstruction of the main routes of GBM evolution
Estimates of divergence time suggest that the recurrent clone diverged from the untreated clone many years before disease was detected ( ). The median among the 45 well-fitting patients had a divergence time of 12.6 years (range 2.3-50.5, IQR 7.2-22.6). Since the remaining 47 non-fitting patients may fail the model's assumption that untreated and recurrent tumors be evolutionarily distinct (i.e., monophyletic), we caution that divergence time for these patients be interpreted only as a heuristic measure of genetic difference between the two tumor samples. In general, uncertainty in the divergence time was large, with the median well-fitting patient showing a 95% CI 24 years wide. Still, even the bottom of the 95% CI exceeded three years for a majority of patients.
Cell culture, lentivirus production and cell growth analysis
Evaluation of cell growth was performed using the MTT assay. Cells were plated at density of 2.5 × 10 3 cells/well into 96 well plates in 6 replicates and allowed to adhere for 24 h. Viability was assessed daily by adding MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium, Sigma 5mg/ml in PBS). Following 4h incubation period, medium was removed and formazan crystal were solubilized with acidic isopropanol (0.1 N HCl in absolute isopropanol. The absorbance at 550 nm was measured with a plate reader.
Western Blot
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulphate, 1.5 mM Na 3 VO 4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerolphosphate and EDTA-free protease inhibitor cocktail (Roche)). Lysates were cleared by centrifugation at 15,000 r.p.m. for 15 min at 4 °C. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in TBS with 5% non-fat milk and 0.1% Tween20, and probed with primary antibodies. Antibodies and working concentrations are: LTBP4 (1:200, sc-393666) obtained from Santa-Cruz Biotechnology; P-SMAD7 (1:1000, #3101) and SMAD7 (1:1000, #5339) obtained from Cell Signaling Technology; β-actin (1:2,000 dilution; A5441) obtained from Sigma.
Gene Fusion Validation
For validation of fusion transcripts and RT-PCR assays were performed. Total RNA was extracted from the tissues by AllPrep DNA/RNA Mini kit according to the manufacturer's instructions (Qiagen). The total RNA (0.5 µg) was reverse transcribed to synthesize template cDNA by a random primer using the SuperScriptIII First-Strand System(Life Technologies), and 20 µl synthesized cDNA was diluted 10 times with DW. For RT-PCR, EzWay Taq PCR MasterMix (Komabiotech, KOREA) and 5 µl synthesized cDNA as template were used. Thermal cycling was carried out under the following conditions: 1 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C. The primer pairs used in this experiment were designed to make the amplification product including the breakpoints of the fusion genes. PCR products were analyzed by agarose gel electropho...
Measurement outputs
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Protein expression by western blot
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Quantify the primary readouts for this experiment: Protein expression by western blot.
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Evidence quotes (6)
After infection cells were selected with Puromycin (Sigma) at concentration of 2 mg/ml for 48 h. Cells were analysed by western blot, qRT-PCR and growth assay 3 days later.
The specimens in cohort INCB originate from the Besta Brain Tumor Biobank, which is partly funded by the Italian Minister of Health. All patients signed an informed consent for the use of their biological material for research purposes. One case (R012) from this cohort had a history of lower grade glioma prior to the first GBM. All patients were treated by standard Stupp treatment with surgery followed by radiotherapy plus concomitant and adjuvant TMZ.
Estimates of divergence time suggest that the recurrent clone diverged from the untreated clone many years before disease was detected ( ). The median among the 45 well-fitting patients had a divergence time of 12.6 years (range 2.3-50.5, IQR 7.2-22.6). Since the remaining 47 non-fitting patients may fail the model's assumption that untreated and recurrent tumors be evolutionarily distinct (i.e., monophyletic), we caution that divergence time for these patients be interpreted only as a heuristic measure of genetic difference between the two tumor samples. In general, uncertainty in the divergence time was large, with the median well-fitting patient showing a 95% CI 24 years wide. Still, even the bottom of the 95% CI exceeded three years for a majority of patients.
Evaluation of cell growth was performed using the MTT assay. Cells were plated at density of 2.5 × 10 3 cells/well into 96 well plates in 6 replicates and allowed to adhere for 24 h. Viability was assessed daily by adding MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium, Sigma 5mg/ml in PBS). Following 4h incubation period, medium was removed and formazan crystal were solubilized with acidic isopropanol (0.1 N HCl in absolute isopropanol. The absorbance at 550 nm was measured with a plate reader.
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulphate, 1.5 mM Na 3 VO 4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerolphosphate and EDTA-free protease inhibitor cocktail (Roche)). Lysates were cleared by centrifugation at 15,000 r.p.m. for 15 min at 4 °C. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in TBS with 5% non-fat milk and 0.1% Tween20, and probed with primary antibodies. Antibodies and working concentrations are: LTBP4 (1:200, sc-393666) obtained from Santa-Cruz Biotechnology; P-SMAD7 (1:1000, #3101) and SMAD7 (1:1000, #5339) obtained from Cell Signaling Technology; β-actin (1:2,000 dilution; A5441) obtained from Sigma.
For validation of fusion transcripts and RT-PCR assays were performed. Total RNA was extracted from the tissues by AllPrep DNA/RNA Mini kit according to the manufacturer's instructions (Qiagen). The total RNA (0.5 µg) was reverse transcribed to synthesize template cDNA by a random primer using the SuperScriptIII First-Strand System(Life Technologies), and 20 µl synthesized cDNA was diluted 10 times with DW. For RT-PCR, EzWay Taq PCR MasterMix (Komabiotech, KOREA) and 5 µl synthesized cDNA as template were used. Thermal cycling was carried out under the following conditions: 1 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C. The primer pairs used in this experiment were designed to make the amplification product including the breakpoints of the fusion genes. PCR products were analyzed by agarose gel electrophoresis. The primers were summarized in.
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