Figure 1.
Cell proliferation following a 2 h treatment of SCCVII and HT29 cells with varying concentrations of TH-302 under aerobic (21% oxygen) or hypoxic (0.1% oxygen) conditions.
A, B, Cell proliferation of SCCVII (A) or HT29 (B) cells following a 2 h treatment with varying concentrations of TH-302 or pimonidazole (pimo) at different levels of oxygen. Data are from 3 experiments; error bars represent the SE.
Figure 2.
Monitoring the oxygen consumption rate (OCR) in SCCVII and HT29 cells using XF96 Analyzer (Seahorse Bioscience).
A, C, OCR trace before and after the treatment with 0.2, 0.5 or 2 mM of exogenous pyruvate. B, OCR increase ratio (%) after the treatment of SCCVII and HT29 cells with each concentration of pyruvate. C, D, OCR response by the treatment of SCCVII with 2 mM pyruvate before (C) and after (D) incubating the inhibitors (antimycin A and rotenone) of mitochondrial respiratory chain.
Figure 3.
Noninvasive monitoring and quantification of tumor median pO2 by EPRI and MRI before and after pyruvate injection in SCCVII and HT29 tumors.
A, B, T2-weighted anatomical image (left) and pO2 maps measured before (middle) and 30 min after (right) pyruvate (or vehicle for control group) injection into SCCVII (A) or HT29 (B) tumor-bearing mice at 7–12 and 10–16 days, respectively, after tumor implantation.
Figure 4.
Quantitation of median pO2 changes and proportional hypoxic regions.
A, B, Median pO2 profile before (0 min) and 30 min after pyruvate injection in tumors of 500–1500-mm3 size on SCCVII (A) and HT29 (B) tumors. C, D, Proportional hypoxic fraction (HF) at pO2<15.2 or <10 mmHg on SCCVII (C) and HT29 (D) tumors. Each value was measured across three 2-mm slices from the center of the 3D image. The values shown are the mean ± SE. *, P<0.05 as compared with the pO2 (30 min after pyruvate administration) on day 10. The number of animals at each time point is provided in Table S1 in File S1.
Figure 5.
Tumor doubling times after three treatments with pyruvate and/or TH-302.
Doubling time was measured as the increase from initial tumor size, which was the tumor volume prior to treatment. A, SCCVII tumor-bearing C3H/Hen mice. Mice were treated three times on days 7–9 (Day 7 start) or days 9–11 (Day 9 start). B, HT29 tumor-bearing athymic nude mice. Mice were treated three times on days 8/10/12 (Day 8 start) or days 14/16/18 (Day 14 start). Data are mean ± SE of 5 mice. **, P<0.01, ***, P<0.001; N.S., not significant.
Figure 6.
A, Representative images of hypoxic areas in tumors detected by pimonidazole binding (green) and the phosphorylation of S139 (pSer139) in histone H2AX (red). A bolus dose of pyruvate (1.15 mmol/kg) was intravenously injected, and pimonidazole (60 mg/kg, i.v.) and TH-302 (100 mg/kg, i.p.) were then administered 30 min later. B, Quantification of the percentage of pimonidazole-positive area from whole tumor sections with (Pyr +) or without (Pyr −) pyruvate injection. Data are mean ± SE of 5 and 3 tumors, respectively. **, P<0.01. C, Quantification of the percentage of γH2AX-positive area in pimonidazole-positive area with (Pyr +) or without (Pyr −) pyruvate injection. Data are means ± SE of 5 and 3 experiments, respectively. *, P<0.05. D, Representative images of the caspase activation (green) and the S139 phosphorylation of S139 (pSer139) in histone H2AX (red). Part of the nucleus is diminished by potential apoptotic cell death, forming the three-dimensional spaces. E, Immunoblotting of phosphorylated histone γH2AX and cleaved caspase-3 from SCCVII tumors 1 day after pyruvate/TH-302 treatment on each day indicated (n = 3). The data are shown as relative intensity to that on day 9. F, T2 intensity changes 1 day after pyruvate/TH-302 treatment on each day indicated (n = 3–5). Values shown represent means ± SE.