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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

Evaluation of chloride intracellular channel 1(CLIC1) role on glioblastoma development. A) Kaplan–Meier survival curve of mice intracranially transplanted with 105 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; χ2 = 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: χ2 = 17.5; P < .0001; hGBM#18: χ2 = 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 105 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 IgG1 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; χ2= 6.36; df=1.
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Figure 7: Evaluation of chloride intracellular channel 1(CLIC1) role on glioblastoma development. A) Kaplan–Meier survival curve of mice intracranially transplanted with 105 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; χ2 = 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: χ2 = 17.5; P < .0001; hGBM#18: χ2 = 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 105 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 IgG1 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; χ2= 6.36; df=1.

Mentions: To determine the in vivo relevance of CLIC1 silencing, we performed an orthotopic transplantation assay. We stereotaxically implanted dissociated neurospheres infected with a lentivirus expressing either NT or shRNA specific for CLIC1 (sh) into the nucleus caudatus of immunodeficient mice. We monitored tumor formation and growth until the appearance of neurological signs. Survival of mice injected with CLIC1-silenced cells was prolonged in comparison with NT control mice (χ2 = 6.21; df = 1; P < .05) (Figure 7A; Supplementary Figure 7, available online). Both control and CLIC1-silenced mice eventually developed GBMs according to WHO classification. When we killed the transplanted mice at the same time (ie, at the appearance of the neurological signs in control mice), CLIC1-silenced mice (sh) were still presymptomatic (pre), and their tumors were statistically significantly smaller than those of the control mice (NT); however, when we analyzed CLIC1-silenced symptomatic (sym) mice, their tumors reached the size of control tumors (Figure 7, B and C) (ANOVA with Tamhane multiple comparison tests: NT vs pre: P < .05; pre vs sym: P < .05; NT vs sym: P = not significant). CLIC1-silenced xenografts lacked CLIC1 expression as detected by immunohistochemistry at the early time point, whereas CLIC1 expression level became comparable between CLIC1-silenced (sh) tumors and control tumors (NT) at the late time point (Figure 7B). Interestingly, when lower numbers of cells (102 and 10 for GBM#10 and 103, 102, and 10 for GBM#18) were injected into mice, none of the mice that received CLIC1-silenced cells developed tumors (Figure 7D). The calculated stem cell frequency by the extreme limiting dilution assay (ELDA)algorithm was statistically significantly lower in CLIC1-silenced cells (hGBM#10: χ2 = 17.5, df = 1, P < .0001; hGBM#18: χ2 = 34.2, df = 1, P < .0001) and was underestimated because of the observed CLIC1 re-expression in all formed tumors. Thus CLIC1 appears to be relevant for the formation of tumors in 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)

Evaluation of chloride intracellular channel 1(CLIC1) role on glioblastoma development. A) Kaplan–Meier survival curve of mice intracranially transplanted with 105 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; χ2 = 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: χ2 = 17.5; P < .0001; hGBM#18: χ2 = 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 105 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 IgG1 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; χ2= 6.36; df=1.
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Figure 7: Evaluation of chloride intracellular channel 1(CLIC1) role on glioblastoma development. A) Kaplan–Meier survival curve of mice intracranially transplanted with 105 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; χ2 = 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: χ2 = 17.5; P < .0001; hGBM#18: χ2 = 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 105 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 IgG1 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; χ2= 6.36; df=1.
Mentions: To determine the in vivo relevance of CLIC1 silencing, we performed an orthotopic transplantation assay. We stereotaxically implanted dissociated neurospheres infected with a lentivirus expressing either NT or shRNA specific for CLIC1 (sh) into the nucleus caudatus of immunodeficient mice. We monitored tumor formation and growth until the appearance of neurological signs. Survival of mice injected with CLIC1-silenced cells was prolonged in comparison with NT control mice (χ2 = 6.21; df = 1; P < .05) (Figure 7A; Supplementary Figure 7, available online). Both control and CLIC1-silenced mice eventually developed GBMs according to WHO classification. When we killed the transplanted mice at the same time (ie, at the appearance of the neurological signs in control mice), CLIC1-silenced mice (sh) were still presymptomatic (pre), and their tumors were statistically significantly smaller than those of the control mice (NT); however, when we analyzed CLIC1-silenced symptomatic (sym) mice, their tumors reached the size of control tumors (Figure 7, B and C) (ANOVA with Tamhane multiple comparison tests: NT vs pre: P < .05; pre vs sym: P < .05; NT vs sym: P = not significant). CLIC1-silenced xenografts lacked CLIC1 expression as detected by immunohistochemistry at the early time point, whereas CLIC1 expression level became comparable between CLIC1-silenced (sh) tumors and control tumors (NT) at the late time point (Figure 7B). Interestingly, when lower numbers of cells (102 and 10 for GBM#10 and 103, 102, and 10 for GBM#18) were injected into mice, none of the mice that received CLIC1-silenced cells developed tumors (Figure 7D). The calculated stem cell frequency by the extreme limiting dilution assay (ELDA)algorithm was statistically significantly lower in CLIC1-silenced cells (hGBM#10: χ2 = 17.5, df = 1, P < .0001; hGBM#18: χ2 = 34.2, df = 1, P < .0001) and was underestimated because of the observed CLIC1 re-expression in all formed tumors. Thus CLIC1 appears to be relevant for the formation of tumors in 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