Davina Jean Honess

Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer

Its All in the Delivery Pancreatic cancer is almost universally associated with a poor prognosis, in part because the tumors are resistant to chemotherapeutic drugs. Working with a mouse tumor model that displays many features of the human disease, Olive et al. (p. 1457, published online 21 May; see the Perspective by Olson and Hanahan) found that the tumors were poorly vascularized, a factor likely to impede drug delivery. Treatment of the mice with the chemotherapeutic drug gemcitabine in combination with a drug that depletes tumor-associated stromal tissue led to an increase in tumor vasculature, enhanced delivery of gemcitabine, and a delay in disease progression. Thus, drugs targeting the tumor stroma may merit investigation as a way to enhance the efficacy of conventional chemotherapy for pancreatic cancer. Pancreatic tumors are unresponsive to chemotherapy because their limited vasculature precludes efficient drug delivery. Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

Oxygen-sensitive enzyme-prodrug gene therapy for the eradication of radiation-resistant solid tumours.

Overwhelming clinical and experimental data demonstrate that tumour hypoxia is associated with aggressive disease and poor treatment outcome as hypoxic cells are refractive to radiotherapy and some forms of chemotherapy. However, hypoxia is rare in physiologically normal tissues representing a tumour-specific condition. To selectively target this therapeutically refractive cell population, we have combined bioreductive chemotherapy with hypoxia-directed gene therapy. We have transfected the human fibrosarcoma cell line, HT1080, with a hypoxia-regulated expression vector encoding the human flavoprotein cytochrome c P450 reductase (HRE-P450R). This conferred hypoxia-dependent sensitivity to the alkylating nitroimidazole prodrug RSU1069 in vitro, with a greater than 30-fold increase in oxic/hypoxic cytotoxicity ratio compared with controls. Xenografts of both the HRE-P450R and empty vector transfectants had comparable hypoxic fractions and were refractive to single dose radiotherapy of up to 15 Gy. However, combining a prodrug of RSU1069 with a reduced radiotherapy dose of 10 Gy represents a curative regimen (50% tumour-free survival; day 100) in the HRE-P450R xenografts. In complete contrast, 100% mortality was apparent by day 44 in the empty vector control xenografts treated in the same way. Thus, an oxygen-sensitive gene-directed enzyme prodrug therapy approach may have utility when incorporated into conventional radiotherapy and/or chemotherapy protocols for loco-regional disease in any tissue where hypoxia is a contra-indication to treatment success.

Interaction of hyperthermia and the hypoxic cell sensitizer Ro-07-0582 on the EMT6 mouse tumour.

The combination of hyperthermia and the hypoxic cell radiosensitizer Ro-07-0582 has been investigated on the EMT6 tumour implanted into the legs of BALB/c mice. Treatments at a drug dose of 1 mg/g over a range of waterbath temperatures from 37 to 45 degrees C are described. The surviving clonogenic fraction following treatment was assayed in vitro. Measurements of intra-tumour temperature have been made, and shown to be better correlated with the cytocidal effect on the tumour than the waterbath temperature. No significant effect of Ro-07-0582 was observed at 37 degrees C. However, marked cytotoxicity due to the drug was seen at intra-tumour temperatures above 42-5 degrees C for 1 h. These were in addition to the cytocidal effect of the hyperthermia. The results are discussed in relation to the distribution of temperatures and hypoxic cell populations throughout the tumour.

The effect of systemic hyperthermia on melphalan pharmacokinetics in mice.

The effect of 45 min systemic heating at 41 degrees C on plasma and RIF-1 tumour pharmacokinetics of intraperitoneally administered melphalan (MEL) was studied in C3H mice. This heat dose causes greater potentiation of MEL in tumour than in marrow cells, resulting in a therapeutic gain for the combined therapy (Honess &Bleehen, 1985). MEL (7.5 mg kg-1) was administered at the start of heating and concentrations assayed from 20-90 min by high-performance liquid chromatography (HPLC). With or without heat peak concentrations were achieved by 20 min and were 3 to 4 micrograms ml-1 in plasma and 1-3 micrograms g-1 in tumour. Higher MEL concentrations in both plasma and tumour were found in heated animals at times after 20 min from injection, but the effect was greater in plasma (2.5-4 fold) than in tumour (1.5-2 fold) where differences were not always significant. At 40 min after a dose of 7.5 mg kg-1, plasma and tumour concentrations in heated animals were equivalent to those after 12.5 mg kg-1 and 8.5 mg kg-1, respectively, without heating. Tumour/plasma ratios were usually lower in heated than in unheated animals where they often exceeded 100%. The apparent plasma elimination half-life (t1/2) was 17.5-25 min in unheated and 24-44 min in heated animals. The area under the curve (AUC) was increased by a factor of 1.2-1.5 in heated animals, at least partly due to a decrease in volume of distribution. The heat induced increase in MEL exposure may be involved in the enhanced response to the drug, but does not appear to explain the therapeutic gain compaired to MEL alone.

The interaction of thermal tolerance with drug cytotoxicity in vitro

The effect of preheating EMT6 cells in vitro on their response to cytotoxic agents of either 43 degrees C or 37 degrees C has been investigated. Preheating for 3 h at 40 degrees C produced measurable protection (thermal tolerance) to subsequent treatment for 1 h at 43 degrees C. This preheat treatment was further found to reduce cell killing by BLM and BCNU (drug tolerance) present during 1 h at 43 degrees C. In contrast, no such heat-induced drug tolerance was seen with ADR. An additional effect with ADR was the apparent elimination of heat-induced thermal tolerance at toxic drug doses. However, preheating under these conditions had no effect on the subsequent cytotoxicity of any of these drugs at 37 degrees C. Also, preheating for 1 h at 43 degrees C was found to sensitize cells to BLM and BCNU toxicity at 37 degrees C but to protect against ADR toxicity. The results are discussed in relation to known mechanisms of cell killing by heat and of thermal tolerance.

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3′-Deoxy-3′-18F-Fluorothymidine PET and Diffusion-Weighted MR Imaging

Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. Methods: Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by 18F-FDG and 18F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2′-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography–mass spectrometry. Results: Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. 18F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in 18F-FDG uptake were only marginal. No clear relationship between 18F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence 18F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. Conclusion: A direct relationship of 18F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.

Effects of local hyperthermia on the pharmacokinetics of misonidazole in the anaesthetized mouse.

The effects of sodium pentobarbitone anaesthesia, the presence of a tumour, and local hyperthermia to a tumour-bearing leg, on the pharmacokinetics of MISO in the mouse are reported. Analysis of MISO and its metabolite Ro 05-9963 was by high-performance liquid chromatography. The plasma kinetics of MISO were largely unaffected by any of these treatments, but hyperthermia substantially reduced tumour concentrations of the drug. The effects of tumour site and size on unheated-tumour drug concentrations were also studied, and an increase in tumour size was shown to decrease tumour MISO levels, but to different degrees according to whether implanted in the leg or flank. Uniformity of MISO distribution throughout heated and unheated tumours was examined, and levels were found to be constant within tumours. The presence of a temperature detector in heated tumours did not affect their drug concentration.

Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases

The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans.

Thymidine Metabolism as Confounding Factor of 3’-Deoxy-3’-[18F]Fluorothymidine Uptake after Therapy in a Colorectal Cancer Model

Noninvasive monitoring of tumor therapy response helps in developing personalized treatment strategies. Here, we performed sequential PET and diffusion-weighted MRI to evaluate changes induced by a FOLFOX-like combination chemotherapy in colorectal cancer xenografts, to identify the cellular and molecular determinants of these imaging biomarkers. Methods: Tumor-bearing CD1 nude mice, engrafted with FOLFOX-sensitive Colo205 colorectal cancer xenografts, were treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) weekly. On days 1, 2, 6, 9, and 13 of therapy, tumors were assessed by in vivo imaging and ex vivo analyses. In addition, HCT116 xenografts, which did not respond to the FOLFOX treatment, were imaged on day 1 of therapy. Results: In Colo205 xenografts, FOLFOX induced a profound increase in uptake of the proliferation PET tracer 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) accompanied by increases in markers for proliferation (Ki-67, thymidine kinase 1) and for activated DNA damage response (γH2AX), whereas the effect on cell death was minimal. Because tracer uptake was unaltered in the HCT116 model, these changes appear to be specific for tumor response. Conclusion: We demonstrated that 18F-FLT PET can noninvasively monitor cancer treatment–induced molecular alterations, including thymidine metabolism and DNA damage response. The cellular or imaging changes may not, however, be directly related to therapy response as assessed by volumetric measurements.