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Localization patterns of cathepsins K and X and their predictive value in glioblastoma

Barbara Breznik
Barbara Breznik
, 
Clara Limback
Clara Limback
, 
Andrej Porcnik
Andrej Porcnik
, 
Andrej Blejec
Andrej Blejec
, 
Miha Koprivnikar Krajnc
Miha Koprivnikar Krajnc
, 
Roman Bosnjak
Roman Bosnjak
, 
Janko Kos
Janko Kos
, 
Cornelis J.F. Van Noorden
Cornelis J.F. Van Noorden
 and 
Tamara T. Lah
Tamara T. Lah
   | Oct 18, 2018
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Article Category: Research Article
Published Online: Oct 18, 2018
Page range: 433 - 442
Received: May 23, 2018
Accepted: Sep 11, 2018
DOI: https://doi.org/10.2478/raon-2018-0040
© 2018 Barbara Breznik, Clara Limback, Andrej Porcnik, Andrej Blejec, Miha Koprivnikar Krajnc, Roman Bosnjak, Janko Kos, Cornelis J.F. Van Noorden, Tamara T. Lah, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

mRNA expression of cathepsins K and X in gliomas of different grades and GBM subtypes. Public transcriptomic datasets were used as described in Materials and methods. Higher cathepsin K and X mRNA expression was found in GBM (grade IV glioma) versus grade II and III glioma (A, D), in GBM versus normal brain (B, E), and in the mesenchymal subtype of GBM versus the classical, proneural and neural subtypes, as classified according to Verhaak et al.39(C, F). Boxplots show the distribution of mRNA expression (log2) in glioma grade II and III and GBM. Data were retrieved from GlioVis portal.37 The significance was set at p < 0.01 (**), p < 0.001 (***).
mRNA expression of cathepsins K and X in gliomas of different grades and GBM subtypes. Public transcriptomic datasets were used as described in Materials and methods. Higher cathepsin K and X mRNA expression was found in GBM (grade IV glioma) versus grade II and III glioma (A, D), in GBM versus normal brain (B, E), and in the mesenchymal subtype of GBM versus the classical, proneural and neural subtypes, as classified according to Verhaak et al.39(C, F). Boxplots show the distribution of mRNA expression (log2) in glioma grade II and III and GBM. Data were retrieved from GlioVis portal.37 The significance was set at p < 0.01 (**), p < 0.001 (***).

Figure 2

Immunohistochemical staining of cathepsins K and X in serial paraffin-embedded GBM sections. The expression of cathepsins K and X in 21 GBM samples, quantified as percentage (%) of immunostained areas and box plots show the distribution of cathepsin expression in GBMs (A). Heterogeneous imunohistochemical staining was found in different parts of GBM samples for cathepsin K (B, D) and for cathepsin X (C, E). Inserts present high magnification images of GBM cells containing cathepsin K and X protein. Cathepsin K (F) and cathepsin X (G) expression was present in specific cells in normal brain tissue of GBM patients. These cells were not further identified. Immunohistochemical labelling of cathepsins was performed with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/purple). Scale bar = 50 μm.
Immunohistochemical staining of cathepsins K and X in serial paraffin-embedded GBM sections. The expression of cathepsins K and X in 21 GBM samples, quantified as percentage (%) of immunostained areas and box plots show the distribution of cathepsin expression in GBMs (A). Heterogeneous imunohistochemical staining was found in different parts of GBM samples for cathepsin K (B, D) and for cathepsin X (C, E). Inserts present high magnification images of GBM cells containing cathepsin K and X protein. Cathepsin K (F) and cathepsin X (G) expression was present in specific cells in normal brain tissue of GBM patients. These cells were not further identified. Immunohistochemical labelling of cathepsins was performed with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/purple). Scale bar = 50 μm.

Figure 3

Kaplan-Meier survival curves of overall survival in relation to cathepsin K and X mRNA expression. Cathepsin K mRNA expression did not correlate with survival of all GBM patients (A), whereas patients with tumors expressing high cathepsin X mRNA levels exhibited poorer survival than patients with low cathepsin X mRNA expression (B) When we stratified GBMs in the different subtypes, cathepsin X mRNA expression had predictive value in the classical GBM subtype only (p = 0.049 by log-rank and p = 0.0084 by Wilcoxons’ test) (C). Data for the Kaplan-Maier survival curves were obtained from GlioVis data portal.36,37 GBM tumors were stratified into two groups, tumors with high and with low cathepsin K or X mRNA expression, using median values as cutoff.
Kaplan-Meier survival curves of overall survival in relation to cathepsin K and X mRNA expression. Cathepsin K mRNA expression did not correlate with survival of all GBM patients (A), whereas patients with tumors expressing high cathepsin X mRNA levels exhibited poorer survival than patients with low cathepsin X mRNA expression (B) When we stratified GBMs in the different subtypes, cathepsin X mRNA expression had predictive value in the classical GBM subtype only (p = 0.049 by log-rank and p = 0.0084 by Wilcoxons’ test) (C). Data for the Kaplan-Maier survival curves were obtained from GlioVis data portal.36,37 GBM tumors were stratified into two groups, tumors with high and with low cathepsin K or X mRNA expression, using median values as cutoff.

Figure 4

Serial GBM sections immunohistochemically stained for peri-arteriolar GSC niche markers and cathepsins K and X. SMA-positive smooth muscle cells were present in the tunica media of the arteriolar wall as indicated by black arrows (A) CD133- and SDF-1α-positive cells (B, C) were present in the cellular layers adjacent to the tunica adventitia of the arteriole. CD68-positive macrophages and microglia were found in peri-arteriolar regions as indicated by black arrows (D) Cathepsins K (E) and X (F) were expressed in a CD133-, SDF-1α- and CD68-positive areas around the arteriole and their expression overlapped that of CD133-positive cells as indicated by white arrows (B, E, F). Cathepsins and SDF-1α were present in the endothelial cells of arterioles as indicated by black arrows (C, E, F). Immunohistochemical labelling of SMA (A) was performed with AEC as substrate (red color) and of the other proteins (B-F) with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/ purple). a, lumen of arteriole; ta, tunica adventitia; tm, tunica media. Scale bar = 100 μm. A B C D
Serial GBM sections immunohistochemically stained for peri-arteriolar GSC niche markers and cathepsins K and X. SMA-positive smooth muscle cells were present in the tunica media of the arteriolar wall as indicated by black arrows (A) CD133- and SDF-1α-positive cells (B, C) were present in the cellular layers adjacent to the tunica adventitia of the arteriole. CD68-positive macrophages and microglia were found in peri-arteriolar regions as indicated by black arrows (D) Cathepsins K (E) and X (F) were expressed in a CD133-, SDF-1α- and CD68-positive areas around the arteriole and their expression overlapped that of CD133-positive cells as indicated by white arrows (B, E, F). Cathepsins and SDF-1α were present in the endothelial cells of arterioles as indicated by black arrows (C, E, F). Immunohistochemical labelling of SMA (A) was performed with AEC as substrate (red color) and of the other proteins (B-F) with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/ purple). a, lumen of arteriole; ta, tunica adventitia; tm, tunica media. Scale bar = 100 μm. A B C D

Figure 5

High-magnification images of serial GBM sections labelled for CD133, SDF-1α and cathepsins K and X. The region around the arteriole as shown in upper left corner of the Figure was magnified. CD133- (A), SDF-1α- (B), cathepsin K- (C) and cathepsin X- (D) positive cells were adjacent to the tunica adventitia of the arteriole. Immunohistochemical labelling of proteins was performed with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/purple). a, lumen of arteriole; ta, tunica adventitia; tm, tunica media. The interrupted black line indicates the outer border of the tunica adventitia. Scale bar = 20 μm. around arterioles using the macrophage and microglia marker CD68 (Figure 4A-D).
High-magnification images of serial GBM sections labelled for CD133, SDF-1α and cathepsins K and X. The region around the arteriole as shown in upper left corner of the Figure was magnified. CD133- (A), SDF-1α- (B), cathepsin K- (C) and cathepsin X- (D) positive cells were adjacent to the tunica adventitia of the arteriole. Immunohistochemical labelling of proteins was performed with DAB as substrate (brown color). Cell nuclei were stained using hematoxylin (blue/purple). a, lumen of arteriole; ta, tunica adventitia; tm, tunica media. The interrupted black line indicates the outer border of the tunica adventitia. Scale bar = 20 μm. around arterioles using the macrophage and microglia marker CD68 (Figure 4A-D).

Figure 6

Correlation of microarray-based gene expression levels of cathepsin K and GSC niche markers. Cathepsin K did not correlate with GSC marker CD133/ PROM1 (A), but negatively correlated with GSC marker SOX2 (B) and positively correlated with GSC niche markers CD31/PECAM1 (C), α-smooth muscle actin SMA/ ACTA2 (D), chemotactic cytokine SDF-1α/CXCL12 (E) and its receptor CXCR4 (F), as well as with macrophage/microglia markers CD68 (G) and Iba1/AIF1 (H). Trend lines indicate linear regression estimates. Log2-transformed mRNA expression data were obtained via GlioVis portal.
Correlation of microarray-based gene expression levels of cathepsin K and GSC niche markers. Cathepsin K did not correlate with GSC marker CD133/ PROM1 (A), but negatively correlated with GSC marker SOX2 (B) and positively correlated with GSC niche markers CD31/PECAM1 (C), α-smooth muscle actin SMA/ ACTA2 (D), chemotactic cytokine SDF-1α/CXCL12 (E) and its receptor CXCR4 (F), as well as with macrophage/microglia markers CD68 (G) and Iba1/AIF1 (H). Trend lines indicate linear regression estimates. Log2-transformed mRNA expression data were obtained via GlioVis portal.

Figure 7

Correlation of microarray-based gene expression levels of cathepsin X and GSC niche markers. Cathepsin X negatively correlated with GSC markers CD133/PROM1 (A) and SOX2 (B) and positively correlated with GSC niche markers CD31/PECAM1 (C), α-smooth muscle actin SMA/ACTA2 (D), chemotactic cytokine SDF-1α/CXCL12 (E) and its receptor CXCR4 (F), as well as with macrophage/microglia markers CD68 (G) and Iba1/AIF1 (H). Trend lines indicate linear regression estimates. Log2-transformed mRNA expression data were obtained via GlioVis portal.
Correlation of microarray-based gene expression levels of cathepsin X and GSC niche markers. Cathepsin X negatively correlated with GSC markers CD133/PROM1 (A) and SOX2 (B) and positively correlated with GSC niche markers CD31/PECAM1 (C), α-smooth muscle actin SMA/ACTA2 (D), chemotactic cytokine SDF-1α/CXCL12 (E) and its receptor CXCR4 (F), as well as with macrophage/microglia markers CD68 (G) and Iba1/AIF1 (H). Trend lines indicate linear regression estimates. Log2-transformed mRNA expression data were obtained via GlioVis portal.
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