File S1 - Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

This file contains Figures S1–S7 and Tables S1–S3. Figure S1. A scheme of the TH-302 (A) or pimonidazole (B) reductive activation pathway. One-electron reduction of nitroimidazole in each compound produces a radical anion intermediate, which can undergo futile redox cycling under normoxic conditions to generate superoxide, or fragmentation or further reduction under hypoxic conditions. Figure S2. Cell viability following a 2 h treatment of SCCVII and HT29 cells with varying concentrations of pyruvate under aerobic (21% oxygen) conditions. A, B, Cell viability of SCCVII (A) or HT29 (B) cells following a 2 h treatment with varying concentrations of pyruvate at 21% O2. Data are from 6 replicates; error bars represent the SE. Figure S3. Three-dimensional oxygen image in a SCCVII tumor using EPRI and MRI before and after pyruvate/TH-302 injection. A, T2-weighted anatomical image and pO2 maps measured before and 30 min after pyruvate injection in a representative SCCVII tumor-bearing mouse 7 days after tumor implantation. The T2 map was obtained before and 1 day after treatment with TH-302. B, T2-weighted anatomical image and pO2 maps measured before and after three consecutive days (days 7, 8 and 9) of TH-302 monotherapy. T2 map was obtained before and after three times TH-302 treatment. Figure S4. Percentage body weight change of model mice in each treatment group. A, C3H/Hen mice bearing murine SCCVII tumors (n = 5). B, Athymic NCr-nu/nu nude mice bearing human HT29 tumors. Figure S5. Immunoblotting of histone H2AX and caspase-3. A, Immunoblotting of phosphorylated S139 (pSer139) in histone H2AX and cleaved caspase-3 from SCCVII tumors at indicated times (h) after pyruvate/TH-302 treatment on day 7 (n = 3). B, Time-dependent increases in phosphorylation of H2AX (red) and cleavage of caspase-3 (green). C, TH-302 dose-dependent increases in phosphorylation of H2AX (red) and cleavage of caspase-3 (green). Figure S6. A, B, T2-weighted anatomical images and T2 maps scanned before (A) and 1 day after (B) pyruvate/TH-302 treatment in a representative SCCVII tumor bearing mouse on the indicated days after tumor implantation. Figure S7. Noninvasive detection of treatment response by MRI. A–B, T2-weighted anatomical, ADC and T2 maps measured before (A) and 1 day after (B) pyruvate/TH-302 treatment on a representative SCCVII tumor-bearing mouse 7 days after tumor implantation. C, Relative ADC changes with (Test) or without (Cont) pyruvate/TH-302 treatment. ΔADC (mm−3/s) = ADC (day8) – ADC (day7). Data are means ± SE of 4 experiments. *, PP2 value before (0 min) and 30 min after pyruvate treatment on SCCVII and HT29 tumors. Table S2. Percentage of hypoxic fraction (HF) of (DOCX)