Drug Resistance and Cellular Adaptation to Tumor Acidic pH Microenvironment

Despite advances in developing novel therapeutic strategies, a major factor underlying cancer related death remains resistance to therapy. In addition to <i>biochemical</i> resistance, mediated by xenobiotic transporters or binding site mutations, resistance can be <i>physiological</i>, emerging as a consequence of the tumor’s physical microenvironment. This review focuses on extracellular acidosis, an end result of high glycolytic flux and poor vascular perfusion. Low extracellular pH, pHe, forms a physiological drug barrier described by an “ion trapping” phenomenon. We describe how the acid-outside plasmalemmal pH gradient negatively impacts drug efficacy of weak base chemotherapies but is better suited for weakly acidic therapeutics. We will also explore the physiologic changes tumor cells undergo in response to extracellular acidosis which contribute to drug resistance including reduced apoptotic potential, genetic alterations, and elevated activity of a multidrug transporter, p-glycoprotein, pGP. Since low pHe is a hallmark of solid tumors, therapeutic strategies designed to overcome or exploit this condition can be developed

ADC maps and histograms of DW-MRI from representative animals at different time points pre- and post-treatment initiation.

<p>A dramatically increased ADCs observed in HS766T, but not in the other two tumor types.</p

Time-course imaging result of DCE-MRI and DW-MRI.

<p>A) Time-courses of normalized mean K^trans values. The mean values of normalized K^trans decreased 69% for TH-302 treated mice in Hs766t tumors, decreased 46% for Mia PaCa-2 tumors and increased 5% in SU.86.86 tumors. B) Changes in normalized mean tumor ADC values over time. A substantial increase in relative mean ADCs was observed for the TH-302 treated group at post- 24 and 48 hours (29% increasing for 24h, p<0.01; 17% increasing for 48h, p<0.01). MIA PaCa-2 is not statistically significant different between conducted groups (8% increasing for 24h, <i>P</i>>0.05; 4% increasing for 48h, p>0.05). For SU.86.86, no significant change was detected by DW-MRI for both TH-302 and control group (3% decreasing for 24h, p>0.05; 0.5% increasing for 48h, p>0.05). The normalization was calculated by the average of the difference of pre- and post-treatment in Th302 group relative to average of the difference of pre- and post-treatment in control group. Error bars stand for standard deviation.</p

Representative K^trans maps for HS766t, Mia PaCa-2 and Su.86.86 tumor type from DCE-MRI (L-R).

<p>As demonstrated in the histogram, K^trans generated from HS766t and Mia-PaCa-2 tumors were dramatically decreased 48 hours after TH-302 treatment compared to pre-treatment values. There were no significant changes observed for a SU.86.86 mouse at the same time point.</p

pH electrode results indicate that MIA PaCa-2 tumors exhibit “steal” Effect in response to hydralazine.

<p>Mice bearing subcutaneous SU.86.86 (n = 7), Hs766t (n = 3) or MIA PaCa-2 (n = 4) flank tumors were anesthetized and tumor pH measurements were obtained. A reference electrode was inserted under the skin of the mouse in a non-tumor site while the pH electrode was inserted up to 1.3 cm into the center of each tumor. Two measurements were taken at each time point and averaged. Following initial pH measurements, mice were administered 10 mg/kg hydralazine via ip injection. Tumor pH was measured for 3 hours following treatment. Data reported represents the average maximum change in each cohort, the average pH change for each cohort and the single animal maximum pH change for each cohort. Data is reported as mean change in pH ± SEM. * <i>P</i><0.05; **<i>P</i><0.007.</p

Combination therapy dosing regimen optimization increases TH-302 efficacy in MIA PaCa-2 tumors.

<p>Mice bearing MIA PaCa-2 tumors were pair-matched into 3 cohorts (n = 10 per cohort): TH-302 monotherapy, TH-302 30 minutes after hydralazine, and simultaneous administration of TH-302 and hydralazine. TH-302 (50 mg/kg) and hydralazine (10 mg/kg) were administered ip, and treatment consisted of 2 cycles of 5 continuous treatments with 2 days off. A) Tumor volume measurements of MIA PaCa-2 tumors. B) Kaplan-Meier curve showing time until MIA PaCa-2 tumors reached 1000 mm³. Tumor volumes are presented as mean tumor volume ± SEM. * <i>p</i><0.05; ↑  =  dose administered.</p

MIA PaCa-2 tumors lack tonal mature vasculature.

<p>Hs766t, MIA PaCa-2 and SU.86.86 tumors were fixed and embedded in paraffin in preparation for IHC staining for tumor vasculature markers, CD31 and SMA. A) Representative images of tumors stained with CD31 and SMA. Scale bars represent 100 µm. Positive pixel analysis of B) CD31 and C) SMA staining across the whole area of a tumor. Data is presented as % Positivity [(Positive pixels/total pixels) × 100] ± SEM. * <i>P</i><0.05; ** <i>P</i><0.005.</p

Hydralazine treatment results in a reduction in tumor blood flow within 15 minutes.

<p>Mice bearing MIA PaCa-2 tumors were analyzed by Doppler ultrasound to quantify tumor blood flow. A) <i>Left Panel:</i> Tumors were scanned using color Doppler imaging to identify major tumor vasculature prior to treatment. Red represents blood flow through tumor vasculature. White arrow marks vasculature chosen for analysis. <i>Right Panel:</i> Following hydralazine administration, pulsed wave (PW) Doppler was used every 5 minutes for 30 minutes to quantify blood flow through tumor vasculature. Representative diagrams showing a decrease in tumor blood flow following hydralazine treatment. B) Quantification of tumor blood flow changes in MIA PaCa-2 subcutaneous tumors (n = 4) following hydralazine treatment. Data are reported as mean velocity (mm/sec) ± SEM. C) Quantification of tumor blood flow changes in Mia PaCa-2 orthotopic pancreatic tumors (n = 3) following hydralazine treatment. Data are reported as mean velocity (mm/sec) ± SEM. D) Tumor oxygenation was measured pre- and post-hydralazine using OxyLite (oxygen) needle electrodes in subcutaneous Mia PaCa-2 tumors (N = 5). Measurements are collected over time at a single site within the tumor. Complete experimental details can be found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113586#s2" target="_blank">methods</a> section. Data are presented as pO2 (mmHg). The arrow at T = 10 minutes designates I.P. injection of hydralazine.</p

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

<div><p>Background</p><p>TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.</p><p>Methodology/Results</p><p>The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and <i>in</i><i>vivo</i> tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. <i>In vivo</i> EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.</p><p>Conclusions/Significance</p><p>Our <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.</p></div

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.

<p><b>A, B,</b> Cell proliferation of SCCVII (<b>A</b>) or HT29 (<b>B</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.</p