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Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells.

Setti M, Savalli N, Osti D, Richichi C, Angelini M, Brescia P, Fornasari L, Carro MS, Mazzanti M, Pelicci G - J. Natl. Cancer Inst. (2013)

Bottom Line: CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment.The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

View Article: PubMed Central - PubMed

Affiliation: Affiliations of authors: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy (MS, DO, CR, PB, LF, GP); Department of Bioscience, University of Milan, Milan, Italy (NS, MA, MM); Department of Anesthesiology, Division of Molecular Medicine, University of California-Los Angeles, Los Angeles, CA (NS); Department of Neurosurgery, Neurocenter, University of Freiburg, Freiburg, Germany (MSC).

ABSTRACT

Background: Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis.

Methods: We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane's multiple comparison test. Kaplan-Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided.

Results: CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1(low) vs CLIC1(high) survival: χ(2) = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.

Conclusions: Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

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Related in: MedlinePlus

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.
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Figure 5: 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.

Mentions: We next investigated the role of CLIC1 in regulating the maintenance and the growth of GBM neurospheres. In vitro self-renewal capacity of CLIC1-silenced and control cells was evaluated by methylcellulose assay. Single cells were plated in semisolid medium, single clones were counted after 15 days, and the clonogenic cells were calculated as the percentage of the total number of seeded cells. CLIC1-silenced cells formed statistically significantly fewer (hGBM#7 NT: 11.63±5.23%, sh: 3.83±1.75%; hGBM#9 NT: 14.86±3.37%, sh: 3.20±0.71%; hGBM#10 NT: 14.00±3.23%, sh: 3.35±0.98%; P < .01 in all experiments) (Figure 5, A and B; Supplementary Figure 5A, available online) and smaller colonies (NT: 502.5±56.85 µm; sh: 264.0±13.50 µm; P < .01; n = 5) (Figure 5C), with a lower cellular content compared with control cells (NT: 830.00±119.22 cells; sh: 483.33±85.68 cells; P < .01) (Figure 5D). When spheres generated at the first plating were dissociated and single cells were seeded on methyl-cellulose, control cells formed spheres with statistically significantly high efficiency, whereas CLIC1-silenced cells generated only a few small spheres, suggesting reduced self-renewal capacity (Supplementary Figure 5B, available online). Interestingly, there was no difference in clonogenic capacity between CLIC1-silenced and control cells at the third replating when CLIC1-silenced cells re-expressed the protein (Supplementary Figure 5, B and C, available online). Furthermore, CLIC1 silencing strongly reduced cellular growth kinetics in all patient-derived GBM neurospheres analyzed, as shown by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (hGBM#7: F = 233.5, df = 1, P < .001; hGBM#9: F=208.5, df = 1, P < .001; hGBM#10: F = 62.8, df = 1, P < .001) (Figure 5E; Supplementary Figure 5D, available online). Consistent with cell proliferation data, CLIC1 silencing strongly reduced the percentage of BrdU-positive cells in GBM-derived neurospheres (hGBM#7 NT: 64.32±3.93%, sh: 12.09±2.25%; hGBM#9 NT: 28.58±2.58%, sh: 14.48±0.18%; hGBM#10 NT: 41.39±0.80%, sh: 18.61±1.32%; P < .05 in all hGBM analyzed) (Figure 5F; Supplementary Figure 5E, available online). However, cell cycle analysis showed no alteration in cell cycle progression (Supplementary Figure 6A, available online). Moreover, no difference in the percentage of apoptotic cells between CLIC1-silenced cells and the control cells was detected (Supplementary Figure 6, B and C, available online). Together, these data indicate that CLIC1 downregulation affects the ability to steadily propagate GBM neurospheres.


Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells.

Setti M, Savalli N, Osti D, Richichi C, Angelini M, Brescia P, Fornasari L, Carro MS, Mazzanti M, Pelicci G - J. Natl. Cancer Inst. (2013)

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.
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Figure 5: 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.
Mentions: We next investigated the role of CLIC1 in regulating the maintenance and the growth of GBM neurospheres. In vitro self-renewal capacity of CLIC1-silenced and control cells was evaluated by methylcellulose assay. Single cells were plated in semisolid medium, single clones were counted after 15 days, and the clonogenic cells were calculated as the percentage of the total number of seeded cells. CLIC1-silenced cells formed statistically significantly fewer (hGBM#7 NT: 11.63±5.23%, sh: 3.83±1.75%; hGBM#9 NT: 14.86±3.37%, sh: 3.20±0.71%; hGBM#10 NT: 14.00±3.23%, sh: 3.35±0.98%; P < .01 in all experiments) (Figure 5, A and B; Supplementary Figure 5A, available online) and smaller colonies (NT: 502.5±56.85 µm; sh: 264.0±13.50 µm; P < .01; n = 5) (Figure 5C), with a lower cellular content compared with control cells (NT: 830.00±119.22 cells; sh: 483.33±85.68 cells; P < .01) (Figure 5D). When spheres generated at the first plating were dissociated and single cells were seeded on methyl-cellulose, control cells formed spheres with statistically significantly high efficiency, whereas CLIC1-silenced cells generated only a few small spheres, suggesting reduced self-renewal capacity (Supplementary Figure 5B, available online). Interestingly, there was no difference in clonogenic capacity between CLIC1-silenced and control cells at the third replating when CLIC1-silenced cells re-expressed the protein (Supplementary Figure 5, B and C, available online). Furthermore, CLIC1 silencing strongly reduced cellular growth kinetics in all patient-derived GBM neurospheres analyzed, as shown by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (hGBM#7: F = 233.5, df = 1, P < .001; hGBM#9: F=208.5, df = 1, P < .001; hGBM#10: F = 62.8, df = 1, P < .001) (Figure 5E; Supplementary Figure 5D, available online). Consistent with cell proliferation data, CLIC1 silencing strongly reduced the percentage of BrdU-positive cells in GBM-derived neurospheres (hGBM#7 NT: 64.32±3.93%, sh: 12.09±2.25%; hGBM#9 NT: 28.58±2.58%, sh: 14.48±0.18%; hGBM#10 NT: 41.39±0.80%, sh: 18.61±1.32%; P < .05 in all hGBM analyzed) (Figure 5F; Supplementary Figure 5E, available online). However, cell cycle analysis showed no alteration in cell cycle progression (Supplementary Figure 6A, available online). Moreover, no difference in the percentage of apoptotic cells between CLIC1-silenced cells and the control cells was detected (Supplementary Figure 6, B and C, available online). Together, these data indicate that CLIC1 downregulation affects the ability to steadily propagate GBM neurospheres.

Bottom Line: CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment.The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

View Article: PubMed Central - PubMed

Affiliation: Affiliations of authors: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy (MS, DO, CR, PB, LF, GP); Department of Bioscience, University of Milan, Milan, Italy (NS, MA, MM); Department of Anesthesiology, Division of Molecular Medicine, University of California-Los Angeles, Los Angeles, CA (NS); Department of Neurosurgery, Neurocenter, University of Freiburg, Freiburg, Germany (MSC).

ABSTRACT

Background: Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis.

Methods: We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane's multiple comparison test. Kaplan-Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided.

Results: CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1(low) vs CLIC1(high) survival: χ(2) = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.

Conclusions: Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

Show MeSH
Related in: MedlinePlus