Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Chloride intracellular channel 1 (CLIC1) expression level in human gliomas. A ) Box-plot showing CLIC1 mRNA levels in control (nontumor) brain tissues, astrocytomas (World Health Organization [WHO] grades II–III), and glioblastomas (GBM; WHO grade IV) derived from the National Cancer Institute’s Repository for Molecular Brain Neoplasia Data (REMBRANDT) database. B and C ) Association of CLIC1 mRNA expression with patient prognosis. B ) Kaplan–Meier survival plot based on patient data from REMBRANDT database. C ) Kaplan–Meier survival plot based on subgroup from REMBRANDT database comprising only GBM patients. In each graph, patient samples have been divided into CLIC1 low-expressing tumors (CLIC1 low , blue ) and CLIC1 high-expressing tumors (CLIC1 high , red ) based on whether the tumors had CLIC1 mRNA levels that were less than or greater than median levels. D and E ) Association of CLIC1 mRNA levels with GBM subtypes (proneural [PN], proliferative [Prolif], neural [N], classical [CL], and mesenchymal [MES]): microarray data set from Phillips et al. (26) ( D ) and from Verhaak et al. (27) ( E ) works were examined. In panels A , D , and E , the solid lines within the boxes represent the median value; the boxes show the 25th and 75th percentile range of CLIC1 mRNA levels; maximum and minimum values are depicted as horizontal bars ; circles represent outliers. ** P < .001 and *** P < .0001 of differences between means of indicated pairs calculated by analysis of variance with Tamhane multiple comparison test; NS = not significant.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Expressing, Control, Derivative Assay, Microarray, Comparison

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Chloride intracellular channel 1 (CLIC1) expression in astrocytic tumors and in patient-derived glioblastoma (GBM) neurospheres. A ) CLIC1 expression levels by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in normal brain specimens (n = 20) and in astrocytic tumors of different grades (n = 13 for World Health Organization [WHO] grade II, n = 28 for WHO grade III, and n = 20 for WHO grade IV). The solid lines represent the mean value; Error bars represent 95% confidence intervals. *** P < .0001 of differences between means of indicated pairs calculated by analysis of variance with Tamhane multiple comparison test; NS = not significant. B ) CLIC1 expression levels by qRT-PCR in different patient-derived GBM neurospheres. Experiments were performed in triplicate; error bars represent 95% confidence intervals. C ) Representative images of CLIC1 immunostaining in GBM-derived neurospheres. Dissociated neurospheres were fixed and processed for immunofluorescence (CLIC1: red ; Sox2, Nestin, and glial fibrillary acidic protein [GFAP]: green ; 4’,6-diamidino-2-phenylindole [DAPI]: blue ; merge: yellow ). Scale bar = 50 μm. D ) Statistical measures of inter-rater agreement of immunoreactive cells in ( C ) evaluated by Cohen’s kappa index. Cohen’s kappa is close to 1 for highly associated markers and close to 0 for unrelated markers. The statistical significance of observed kappa values has been evaluated by means of the χ 2 test. Immunostained cells were counted at 20× magnification, five fields for each sample (mean cell number per field was 150). Three independent experiments were performed.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Expressing, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Immunostaining, Immunofluorescence

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Chloride intracellular channel 1 (CLIC1) subcellular localization in normal and tumoral human neurospheres. A ) Representative images of CLIC1 immunostaining in nonpermeabilized normal and tumoral human neurospheres. Dissociated neurospheres were fixed and processed for immunofluorescence (CLIC1: red ; 4’,6-diamidino-2-phenylindole [DAPI]: blue ). Cells were analyzed using confocal laser scanning microscopy, and a single optical x-y plane section is shown. Scale bar = 10 µm. hGBM#7 = neurospheres isolated from GBM patient 7; NPC = normal human progenitor cells. B ) Western blotting analysis of CLIC1 expression levels in whole cell lysates ( left panel ) and plasma membrane and cytoplasm-containing fractions ( right panel ) derived from normal (NPC) and tumoral neurospheres (hGBM#7). Sodium–potassium pump and GAPDH expression were examined to assess the purity of plasma membrane and cytoplasmic fractions, respectively. Reversible Ponceau staining was used as a control for equal protein loading. C ) CLIC1-mediated chloride (Cl − ) currents measured by perforated patch clamp technique in normal (NPC) and tumoral (hGBM#7) neurospheres. Cl − currents mediated by CLIC1 (I IAA94 ) were isolated using the specific CLIC1 inhibitor indanyloxyacetic acid–94 (IAA94) and normalized to the total cell current (I Tot ) (I IAA94 /I Tot %). Mean values derived from five independent experiments were represented. Error bars represent 95% confidence intervals. Generalized linear model test of between-subjects effects: F for “potential” = 1.44, degrees of freedom (df) = 4, P = .24 (not significant); F for “cell type” = 36.17, df = 1, P = .001; F for variables interaction = 1.30, df = 8, P = .27 (not significant). No statistical significance for interaction means a similar pattern of I IAA94 / I Tot change for different cell types at different membrane potential values, even if mean I IAA94 / I Tot values are different between different cell types.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Immunostaining, Immunofluorescence, Confocal Laser Scanning Microscopy, Isolation, Western Blot, Expressing, Clinical Proteomics, Membrane, Derivative Assay, Staining, Control, Patch Clamp

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Effect of chloride intracellular channel 1 (CLIC1) silencing in glioblastoma (GBM) neurospheres. A ) Western blotting analysis showing the efficiency of CLIC1 silencing in GBM neurospheres isolated from four patient samples. Dissociated neurospheres were transduced with lentivirus carrying either nontargeting short-hairpin RNA (shRNA) (NT) or CLIC1 shRNA (sh). Mouse embryonic fibroblasts derived from CLIC1 knockout mice (MEF CLIC1-KO) were used as negative controls. Vinculin was used as loading control. B and D ) Representative current traces (total, indanyloxyacetic acid 94 [IAA94]–sensitive, and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid [DIDS]–sensitive currents) from NT ( B ) and CLIC1-silenced (sh) ( D ) cells derived from GBM patient 10 (hGBM#10) NS and elicited by different potential steps (from −60 mV to 60 mV). C and E ) The current–voltage relationships for the corresponding experiments in B and D . F ) CLIC1-sensitive currents (I IAA94 ) were isolated using the specific CLIC1 inhibitor IAA94, and normalized to the total cell current (I Tot ) (I IAA94 /I Tot %). G ) The other chloride (Cl − ) currents in the cells were evaluated by the inhibitor DIDS (I DIDS ) and normalized to the total cell current (I Tot ) (I DIDS /I Tot %). Mean values derived from four independent experiments are represented. Error bars represent 95% confidence intervals. GLM test of between-subjects effects on I IAA94 /I Tot values: F for “potential” = 0.108, d. f. = 4, p = 0.979 (n. s.); F for “cell type” = 50.038, d. f. = 1, p < 0.001; F for variables interaction = 0.058, d. f. = 4, p = 0.993 n. s. No significance for interaction means a similar pattern of I IAA94 /I Tot change for different cell types at different membrane potential values, even if mean I IAA94 /I Tot values are different between different cell types. Generalized linear model test of between-subjects effects on I DIDS / I Tot values: F for “potential” = 4.031, degrees of freedom (df) = 4, P = .01; F for “cell type” = 3.590, df = 1, P = .07; F for variables interaction = 0.085, df = 4, P = .99. No statistical significance for interaction means a similar pattern of I DIDS /I Tot change for different cell types at different membrane potential values.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Western Blot, Isolation, Transduction, shRNA, Derivative Assay, Knock-Out, Control, Membrane

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Effects of chloride intracellular channel 1 (CLIC1) silencing on clonogenicity and proliferation of glioblastoma (GBM) stem/progenitor cells. A ) Representative microphotographs of control (NT) and CLIC1-silenced (sh) neurospheres formed in methilcellulose-containing medium after 15 days in culture. Scale bar = 300 µm. B ) Neurosphere formation assay. The clonogenic capacity of control (NT) and CLIC1-silenced (sh) cells was evaluated by plating cells in methylcellulose-containing medium. After 15 days, each plate was examined under a light microscope, and the total number of neurospheres was determined. C ) Quantification of the maximal diameters of control (NT) and CLIC1-silenced (sh) neurospheres from GBM patient 7 (hGBM#7). Ten neurospheres for each sample were analyzed. D ) Quantification of hGBM#7 neurosphere cell number. Ten neurospheres for each sample were picked and dissociated, and the cell number was determined. E ) The growth of control (NT) and CLIC1-silenced (sh) cells isolated from three patient samples was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Three independent experiments were performed; error bars represent 95% confidence intervals; ** P < .001. Generalized linear model tests of between-subjects effects showed statistically significant difference in all patients for the relative cell growth according to the time, the interference, and the interaction between those two variables. F ) Control (NT) and CLIC1-silenced (sh) neurospheres isolated from three patient samples were subjected to BrdU incorporation assay: BrdU-positive cells were quantified by immunofluorescence. Immunostained cells were counted at 20× magnification, five fields for each sample (mean cell number per field was 150). For results in panels B , C , D , and F ) an unpaired two-sided Student t test was used. Three independent experiments were performed; error bars represent 95% confidence intervals; * P < .05, ** P < .001, *** P < .0001.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Control, Tube Formation Assay, Light Microscopy, Isolation, MTT Assay, BrdU Incorporation Assay, Immunofluorescence

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Effects of chloride intracellular channel 1 (CLIC1) antibody treatment on glioblastoma (GBM) neurospheres. A and B ) The effect of CLIC1 antibody on CLIC1 currents was assessed by perforated patch clamp technique. A ) Representative whole cell current traces recorded in the perforated patch configuration at 50 mV from control (NT) and CLIC1-silenced (sh) cells derived from cells from GBM patient 10 (hGBM#10) are shown. IAA94 = indanyloxyacetic acid 94; IgG = immunoglobulin G. B ) Quantification of the different treatments as in ( A ) on whole cell current traces. Mean values derived from four independent experiments were represented. The statistical significance of the differences in relative total current after Tahmane test for dishomogeneous variances is shown: *** P < .0001; NS = not significant. C ) Effect of CLIC1 antibody on GBM neurospheres derived from hGBM#7 and hGBM#10 patients. GBM neurospheres were treated with increasing concentrations of CLIC1 antibody (1, 5, and 10 µg/mL) for 72 hours, and cell viability was monitored by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay; error bars represent 95% confidence intervals; three independent experiments were performed. The difference between cell viability at different antibody concentrations and reference mean viability in control conditions was evaluated by Bonferroni test. The statistical significance of the differences is shown: * P < .05, *** P < .0001. OD = optical density. D ) Effect of CLIC1 antibody treatment on BrdU incorporation ( left panel ) and caspase 3 activation ( right panel ) in hGBM#7 neurospheres. BrdU- or cleaved caspase 3–positive cells were counted at 20× magnification, five fields for each sample (mean cell number per field was 150). Three independent experiments were performed. An unpaired two-sided Student t test was used: * P < .05; NS = not significant.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Patch Clamp, Control, Derivative Assay, MTT Assay, BrdU Incorporation Assay, Activation Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

doi: 10.1093/jnci/djt278

Figure Lengend Snippet: Evaluation of chloride intracellular channel 1(CLIC1) role on glioblastoma development. A ) Kaplan–Meier survival curve of mice intracranially transplanted with 10 5 control (NT) and CLIC1-silenced (sh) cells. Number of mice at risk expressed as weeks (number of mice at risk): 0 (5), 9.8 (5), 10.57 (4), 14 (3), 14.5 (2), 16.5 (1) for NT; 0 (5), 14.5 (5), 18.57 (4), 19.3 (3), 22.8 (2), and 23.8 (1) for sh group. Data are from one experiment with five mice per group. P value was calculated with log rank test: * P < .05; χ 2 = 6.27; degrees of freedom (df) = 1. B ) Representative brain images from mice intracranially injected with control (NT) and CLIC1-silenced (sh) cells stained with hematoxylin and eosin (HE) ( top row ; scale bar = 3mm) or CLIC1 ( bottom row ; scale bar = 300 µm). C ) Tumor volume quantification, as indicated. Experiment was carried out using three mice per group. Error bars represent 95% confidence intervals; * P < .05. One-way analysis of variance with Bonferroni correction was used. Pre = presymptomatic; Sym = symptomatic. D ) Table representing the incidence of tumor formation of tumor bearing mice and the cancer stem cell frequency calculated in the glioblastoma (GBM) neurospheres (estimate). hGBM#10: χ 2 = 17.5; P < .0001; hGBM#18: χ 2 = 34.2; P < .0001. E ) Representative hematoxylin and eosin–stained histological images from mice intracranially injected with hGBM#7 cells treated with CLIC1 antibody (Ab) or isotype control antibody (scale bar = 3mm). Mice were killed at the second, third, and fourth week, as shown. IgG = immunoglobulin G. F ) Kaplan–Meier survival analysis of mice intracranially implanted with 10 5 hGBM#7 cells treated with CLIC1 antibody or isotype control antibody. Number of mice at risk expressed as weeks (number of mice at risk): 0 (6), 6.4 (6), 96.5 (5), 6.6 (4), 6.8 (2), 6.9 (1) for IgG 1 group; 0 (6), 6.6 (6), 7.0 (5), 12.6 (3), 13.3 (2), 16.7 (1) for CLIC1 group. Data are from one experiment with six mice per group. P value was calculated with log rank test: * P = .01; χ 2 = 6.36; df=1.

Article Snippet: For CLIC1 antibody studies, cells were incubated with CLIC1 antibody (mouse monoclonal, 5 μg/mL, clone 356.1; Santa Cruz Biotechnology) or isotype control antibody for 72 hours before implantation (10 5 cells) into the brain of immunodeficient mice.

Techniques: Control, Injection, Staining