Figure 1.
Stem cell-related properties, migration, and invasion ability of rhabdospheres.
(A) Phase contrast pictures of rhabdospheres derived from RD grown in anchorage-independent condition, in serum-starved medium supplemented with bFGF and EGF. Representative image, scale bar 100 µm. (B) Sphere-forming efficiency of rhabdospheres over three serial passages. The graph shows the amount of the primary, secondary (generated from dissociated primary spheres), and tertiary (generated from dissociated secondary spheres) spheres from 2000 cells. *p<0.05 vs primary spheres. (C) mRNA levels for the stem cell markers OCT3/4 and NANOG in rhabdospheres compared to native RD by Real Time PCR. *p<0.05. (D) Western blotting for OCT3/4 and NANOG in rhabdospheres compared to RD native cells (left, representative images) and densitometric analysis (right; *p<0.05). (E) Differentiation assays of rhabdospheres after incubation with appropriate differentiating stimuli. Left: osteogenic differentiation evaluated by Alizarin Red S staining, scale bar 100 µm; middle: adipogenic differentiation evaluated by Oil-Red-O lipid staining, scale bar 10 µm; right: chondrogenic differentiation evaluated by Alcian Blue staining, scale bar 50 µm. Representative images. (F) Transwell chemotaxis assay of rhabdospheres vs native RD. The graph shows the number of migrated cells in five X20 fields after 8 h. *p<0.05. (G) mRNA levels for MMP9 in rhabdospheres vs native RD by Real Time PCR. *p<0.05. (H) MMPs activity in the supernatant of rhabdospheres vs RD native cells by gelatin quenching assay. *p<0.05. (I) mRNA levels for CXCR4 in rhabdospheres compared to native RD by Real Time PCR. *p<0.05. (L) Cytofluorimetric analysis of CXCR4-positive cell fraction in rhabdospheres and native RD. Representative intensity plots for rhabdospheres and native RD (left) and percentage of CXCR4-positive cells (right). **p<0.001.
Figure 2.
Analysis of chemoresistance of rhabdospheres.
(A) Percentage of cell viability inhibition of rhabdospheres compared to native RD after treatment with different doses of cisplatin, calculated vs untreated cells (B) Percentage of cell growth inhibition of rhabdospheres compared to native RD after treatment with different doses of DXR, calculated vs untreated cells *p<0.05. (C) DXR nuclear uptake by confocal microscopy (left, representative images, scale bar 50 µm) and quantification of the intensity level of nuclear signal by image analysis (right; *p<0.05). (D) mRNA levels for MDR1 by Real Time PCR (*p<0.05) and (E) Western blotting for MDR1 protein expression (representative image). Rhabdospheres vs native RD, MG63-DXR100 multidrug resistant cells as positive control. (F) ATP intracellular content evaluated in rhabdospheres in comparison to native RD cells.
Figure 3.
Increased lysosome acidity and V-ATPase expression in rhabdospheres.
(A) AO uptake in rhabdospheres vs native RD by confocal microscopy. Red staining is associated with acidic vesicles, whereas green staining is associated with high pH. Representative images of AO staining (left; scale bar 50 µm) and emission spectra graphs (right) of lysosomes within the cells indicated in the left panel by the white arrows. X-axis, wavelength (λ); Y-axis, intensity index (max = 1). (B) Total number of lysosomes (top) and quantification of the red band contribution (R%) (bottom; ****p<0.0001) after AO staining in rhabdospheres and native RD. (C) Quantitative analysis of pHc through carboxy-SNARF-1. *p<0.05. (D) mRNA levels for ATPase V0c by Real Time PCR (p<0.05). (E) Western blotting for ATPase V0c (top, representative image) and densitometric analysis (bottom, *p<0.05). (F) Confocal analysis of rhabdosphere cells after immunofluorescence staining of ATPase V0a1 subunit localization (green) in the vesicular compartment (cytoskeleton marked by phalloidin-TRITC, middle) or in the cytoplasmic membrane (arrows in the bright field, left). The squared detail of plasmatic membrane localization is enlarged (right). Nuclei were counterstained with Hoechst 33258. Representative images of an xy field, scale bar 20 µm.
Figure 4.
Effects of strategies targeting V-ATPase in rhabdospheres.
(A) Percentage of cell growth inhibition of rhabdospheres and RD cells after treatment with AO at different concentrations evaluated by viable cell counting with respect to untreated cells. (B) Percentage of cell viability inhibition after treatment with OME at different concentrations with respect to untreated cells, evaluated by the acid phosphatase indirect assay (*p<0.05 spheres vs RD and #p<0.05 spheres or RD vs untreated). (C) Cell number evaluated by dye exclusion assay after OME treatment. *p<0.05. (D) Hoechst 33258 staining to evaluate apoptosis of rhabdosphere cells after OME treatment. Representative pictures. Scale bar 50 µm. (E) Cell cycle distribution of rhabdosphere cells by flow cytometry after OME treatment. Left, representative images of double stained cells indicating the total content of DNA (Propidium Iodide, X-axis) and Bromodeoxyuridine (BrdU) incorporation into newly synthesized DNA by proliferating cells during S-phase (BrdU-FITC, Y-axis). Right, graph of the percentages (*p = <0.05).
Figure 5.
Analysis of V-ATPase expression and OME effectiveness in other CSC models.
(A) Real Time PCR analysis of mRNA levels for the stem cell markers OCT3/4 and NANOG in spheres obtained from A-673, SK-ES-1 (ES, left; *p<0.05, **p<0.01), NB-100, and CHP-212 (NB, right; *p<0.05) cell lines in comparison to native cells. *p<0.05. (B) Western blotting for OCT3/4 and NANOG in ES spheres compared to native cells (left, representative images) and densitometric analysis (right; *p<0.05). (C) mRNA levels for ATPase V0c by Real Time PCR in ES and NB spheres compared to native cells (*p<0.05). (D) Percentage of inhibition of cell viability after 72 h of treatment of ES spheres with OME, evaluated by the acid phosphatase indirect assay with respect to untreated cells (*p<0.05).
Figure 6.
Effects of OME on chemoresistance of rhabdospheres.
(A) Percentage of growth inhibition of rhabdospheres pre-treated with OME and incubated with DXR (left; *p<0.05 vs DXR alone-treated cells) or cisplatin (right) with respect to untreated cells, evaluated by the acid phosphatase indirect assay. (B) Confocal microscope analysis and quantification of DXR uptake in rhabdospheres after OME pre-treatment. Nuclear DXR fluorescence intensity indicated with colour and height in surface intensity plots (representative images, top), and quantification of nuclear signal by bar graph (bottom; *p<0.05 for 10 µM and ****p<0.0001 for 20 µM). (C) mRNA analysis for ATPase V0c after rhabdosphere electroporation with a specific siRNA against the V0c subunit. (D) Cell survival of rhabdospheres electroporated with the specific siRNA against ATPase V0c and treated with 25 ng/mL of DXR, determined by the acid phosphatase indirect assay (*p<0.05 vs no siRNA).
Figure 7.
Effects of OME on invasiveness of rhabdospheres.
(A) Transwell chemotaxis assay of rhabdospheres after pre-treatment with OME. *p<0.05. (B) MMPs activity in the supernatant of rhabdospheres after pre-treatment with OME by gelatin quenching assay. *p<0.05. (C) Cytofluorimetric analysis of CXCR4-positive cell fraction in rhabdospheres after treatment with OME. Left, representative intensity plots. Right, graph of the percentages. *p<0.05.
Figure 8.
Targeting lysosomal acidity in rhabdomyosarcoma CSC.
Lysosome acidification in RMS CSC is mediated by V-ATPase, and plays an important role for growth, chemoresistance, and invasiveness of these cells (A). The anti-V-ATPase OME is an effective drug and it can be proposed as a valuable strategy to affect RMS CSC (B).