Mesospheres form tumours in serial transplantations.

<p>(A) Ist-Mes-2 sphere cells (generation 1, G1) were subcutaneously grafted into Balb-c/nude mice at 10⁶ cells per animal. When tumours reached about 2,000 mm³, the mice were sacrificed, tumours excised and malignant cells grew <i>in vitro</i> as a cell line. The adherent cells were converted into spheres (G1) and these were grafted into Balb-c/nude mice to form tumours that were used for generation 2 (G2) spheres. This procedure was repeated two more times to derive G3 and G4 spheres. The inset in panel A shows the lag to tumour appearance following cell grafting for individual sphere cell generations. Cells of individual generations were evaluated for stemness markers as shown using qPCR (B) and WB (panel C shows the blots, panel D their densitometric evaluations) and for CS activity (E). (F) Tumours derived from adherent cells and spheres of individual generations were excised, paraffin-embedded and stained for the MM marker mesothelin (upper images show staining with the exclusion of the primary IgG) and with H&E. Data shown in panel A are derived from 5 animals and are mean values ± S.E.M, data in panels B and E are mean values from three independent experiments ± S.D. Images in panel C are representative of two individual experiments and their densitometric evaluations in panel D represent mean values with differences lower than 10%. Images in panel F were obtained using one tumour for each condition (generation). The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images are representative of three different tumours.</p

Mesospheres derived from different tumour generations show different mitochondrial features.

<p>Sphere cells representing individual generations were evaluated for respiration using the routine protocol (A) and the protocol for permeabilised cells (B) by means of the Oxygraph 2k high resolution respirometer. The symbols GM_L, GM_P, GMS_P, GMS_E and S(Rot)_E represent routine respiration, respiration via complex I, respiration via complex I+II, uncoupled respiration and uncoupled respiration via CII, respectively. (C) Mitochondrial mass was evaluated using MitoTracker Green as detailed in the Methods section. Superoxide was evaluated using the fluorescent probe MitoSOX (D), glucose uptake using the fluorescent glucose analogue 2-NBDG (E), ATP levels by a luciferase-based assay (F), ΔΨ_m using the fluorescent probe TMRM (G), lactate levels using a commercial kit (H), for SDH (I) and SQR (J) activities using enzymatic assays, and hand-held cells counter for cell size (K). (L) TEM was performed on individual generation sphere cells as detailed in the Methods section. Data in panels A-K are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel L are representative of three independent experiments. The white bar in panel K in the upper images represents 500 nm, in the lower images 200 nm.</p

IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.

<p>^aNumbers in bold indicate significantly different data (<i>p</i><0.05) from adherent CD24^-and parental cells.</p><p>The data are mean values ± S.D. from three independent experiments.</p><p>IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.</p

Sphere cells derived from MM cell lines show increased levels of ‘stemness’ markers.

<p>(A) Ist-Mes-2, Meso-2 and MM-BI cells were allowed to form spheres, after which these were placed in the complete medium to cause differentiation and re-adhesion of the cells. Relative levels <i>CD24</i>, <i>ABCG2</i> and <i>OCT4</i> mRNAs were assessed in the original adherent cells (set as 1), the spheres and the re-adherent cells. (B) Ist-Mes-2, Meso-2 and MM-BI cells spheres were tested for the level of <i>SOX4</i>, <i>C-MYC</i>, <i>ABCB5</i> and <i>KLF4</i> mRNA and their level expressed relative to that of adherent cells. The data are mean values ± S.D. derived from three independent experiments, the symbol ‘*’ stands significantly different data with <i>p</i><0.05, ‘**’ for those with <i>p</i><0.005.</p

CD24 supports initiation and progression of mesotheliomas.

<p>(A) Mock-transfected and CD24^- Ist-Mes-2 cells (10⁶ per animal) were grafted subcutaneously into NOD/SCID mice and tumour formation followed using USI. (B) CD24^- and mock-transfected Ist-Mes-2 cell-derived tumours were evaluated for CD24, CD47, EpCAM and Oct3/4 by western blotting using anti-actin IgG as a loading control, with panel C documenting densitometric evaluation of the blots. (D) A representative image of a mouse with CD24^- cell-derived tumour and parental cell-derived tumour is shown. (E) Representative USI images of a tumour derived from mock-transfected and CD24^- cells acquired on different days are shown, with the yellow arrows indicating the position of the tumours. Data in panel A are mean values ±S.E.M., and are derived from four animals. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel B are representative of two independent experiments with the densitometric evaluation showing average data with differences less than 10%.</p

Effect of CD24 knock-down on mitochondrial function.

<p>Parental and CD24^- adherent and sphere Ist-Mes-2, non-permeabilised cells were evaluated for routine respiration using the Oxygraph instrument (A). Panel B shows respiration related to the maximum respiratory capacity of the cells (ETC). The symbols in panel A stand for: R, routine, L, leak, E, ETC, netR, R-L, ROX, residual respiration. Parental, mock-transfected, CD24^- adherent and sphere Ist-Mes-2 cells were evaluated for ΔΨ_m using TMRM (C), superoxide generation using MitoSOX (D), lactate production using a commercial kit (E), citrate synthase (CS) activity (F), relative SDH (G) and SQR activity (H), glucose uptake (I) and ATP level (J). Panel K documents the level of <i>PCG1α</i>mRNA in parental, mock-transfected and CD24^- adherent and sphere Ist-Mes-2 cells. Data shown are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05.</p