Article Figures & Data
Figures
- Figure 1.
miRNA and pluripotency gene expression profiles in primary ESFT spheres and adherent cells in vitro. A, expression of NANOG and OCT-4, as measured by RT-PCR, expressed as fold changes in spheres and adherent cells from the four primary ESFT. B, fold change of expression of selected miRNAs as measured by RT-PCR in the same spheres and adherent cells as in A. Results were analyzed by one-way ANOVA, and mean values with SD are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 2.
Differential response to doxorubicin and enoxacin of CSC-enriched primary ESFT spheres, adherent cells, and cell lines. Effect of 96-hour primary sphere and adherent cell treatment with increasing doses of doxorubicin as assessed by MTS. A, proportion of living cells relative to control treated cells. Data were analyzed by two-way ANOVA. B and C, effect on spheres, adherent cells, and cell lines of 96-hour 10 μg/mL enoxacin treatment as assessed by MTS. The proportion of living cells relative to control-treated cells is shown (B, tumor cell line and hpMSC compared with primary sphere response; C, primary sphere compared to corresponding adherent cell response). D, left, ratio of living cells to control-treated cells after 24, 48, and 72 hours of 10 μg/mL of enoxacin treatment of sh control or shTARBP2-infected A673 cells. D, right, Western blotting and TARBP2 quantification in A673 cells bearing sh control or shTARBP2. Results from growth curves analyzed by two-way ANOVA and significance of enoxacin-treated relative to control-treated cells are shown. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.
- Figure 3.
Induction of apoptosis selectively in CD133+ cells by enoxacin. A, induction of apoptosis in enoxacin-treated relative to control-treated CD133+ and CD133− cells, as measured by Annexin PI staining of cells harvested at subconfluence. Dots and triangles, induction of apoptosis in CD133+ and CD133− populations, respectively, from the four tumors as indicated (left). FACS image of Annexin V and PI staining of ESFT-1 cells gated on the CD133+ and CD133− subpopulations (right). Graphs are representative of three independent experiments. B, sphere formation by DMSO- and enoxacin-treated cells. Graphs, sphere number. Data were analyzed by the Student t test. Mean values ± SD are shown. *, P < 0.05; ****, P < 0.0001.
- Figure 4.
Enoxacin alone depletes the CD133+ compartment but is not sufficient to inhibit primary ESFT xenograft growth. 5,000 ESFT-1 and 10,000 ESFT-3 cells were injected into the subcapsular kidney compartment of NGS mice and treatment was started 10 days later. A, left, tumor weight after 5 weeks of 5 day/week treatment with vehicle or 50 mg/kg of enoxacin alone; right, CD133+ subpopulation fractions in residual tumors at the end of the 5-week treatment. The Student t test was performed on the two groups. n.s., nonsignificant. B, gross anatomy and histology of two representative control- and enoxacin-treated ESFT-3. C, CD99 staining of ESFT 1 and ESFT-3 xenografts. *, P < 0.05.
- Figure 5.
Synergistic antitumor activity of doxorubicin and enoxacin on primary ESFT xenografts. A, representative 3D reconstruction of ultrasound images of ESFT-3 tumors after 4 weeks of the indicated treatment. B, tumor weight of ESFT-3 after 5 weeks of treatment with vehicle, enoxacin alone (50 mg/kg 5 days/week), doxorubicin alone (single weekly 0.5 mg/kg dose), or a combination of the two drugs at the same doses. The Student t test was performed for comparison of doxorubicin only– and doxorubicin/enoxacin-treated groups. C, CD133+ cell fraction in residual ESFT-3 following treatment. Results were analyzed by one-way ANOVA, and mean values with distribution are shown. *, P < 0.05.
- Figure 6.
Enoxacin in combination with doxorubicin induces extensive tumor necrosis in vivo, increases miRNA expression, and depletes tumor-initiating capacity. A, H&E staining of residual tumors (ESFT-1) 5 weeks after the indicated treatment. B, number of living cells × 106/gram of dissociated tumor following treatment. Results were analyzed by one-way ANOVA. C, relative fold change of miRNA expression in residual tumors as assessed by RT-PCR. Mean values and distributions are shown. D, 5000 residual viable cells from treated tumors (ESFT-1) were reinjected into healthy recipient mice and tumor development was scored 6 weeks later. Data were analyzed by the χ2 test. E, percentage of CD133+ cells in residual tumors following treatment (before reinjection) and in secondary xenografts. Mean values and distribution are indicated. *, P < 0.05; ****, P < 0.0001.
- Figure 7.
Onecut2, a candidate predictor marker of ESFT response to enoxacin whose expression may correlate with prognosis. A, Kaplan–Meier curve of the clustering of 111 targets of at least three out of five miRNAs (let-7f, mir-26a, mir-100, mir-145, and mir-181a). B, clustering of 37 individual significantly prognostic targets of three out of five miRNAs. C, Kaplan–Meier curve of survival probability of patients, according to ONECUT2 expression above or below the median. D, expression of ONECUT2 in the CD133+ fraction of the four ESFT samples relative to that of ESFT-1. Results analyzed by one-way ANOVA. Mean values ± SD are shown. **, P < 0.01; ****, P < 0.0001.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplemental Table S1 - Supplemental Table S1. Target genes of 5 TARBP2-dependent miRNAs.
- Supplemental Figure S1 - Supplemental Figure S1. Tumor origins and specifications and primary cell characterization.
- Supplemental Figure S2 - Supplemental Figure S2. ESFT-1 response to enoxacin in vivo.
- Supplemental Figure Legends - Supplemental Figure Legends. Legends for Supplemental Figures S1-S2.
Volume 74, Issue 22
