EMT6 Tumors Research Articles

P16 Biological profiles of two ERBB2-amplified human breast cancer xenografts diversely sensitive to Trastuzumab

Activated T lymphocytes are known to kill tumor cells by triggering cytolytic mechanisms; however, their ability to enhance radiation responses remains unclear. This study examined the radiosensitizing potential of mouse CD8+ T cells, obtained by T-cell–tailored expansion and immunomagnetic purification. Activated CD8+ T cells displayed an interferon (IFN)-γ+ phenotype and enhanced by 1.8-fold the radiosensitivity of EMT-6 tumor cells in 1% oxygen, which modeled tumor-relevant hypoxia. Radiosensitization was counteracted by neutralizing IFN-γ or by blocking the inducible isoform of nitric oxide synthase, thus delineating the immune–tumor cell interaction through the IFN-γ secretion pathway. Reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter data in agreement detected downregulation of the IFN-γ gene by hypoxia, which caused IFN-γ deficiency next to radioresistance. Therefore, immune and radiation responses are likely to be allied in the hypoxic tumor microenvironment, and CD8+ T cells may bridge immunostimulatory and radiosensitizing strategies.

P38 Radioimmunotherapy (RIT) of refractory or relapsed Hodgkin's lymphoma (HL) with 90Yttrium-labelled antiferritin antibody

Purpose: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-γ (IFN-γ) and thereby radiosensitize tumor cells.Methods and Materials: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03–3 μg/mL), and afterward analyzed for the expression and secretion of IFN-γ by reverse transcriptase–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-γ to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS).Results: OM-174 activated the production of IFN-γ at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase–polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-γ. The same mechanism of radiosensitization through the IFN-γ secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-γ responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-γ gene while upregulating iNOS at transcriptional level.Conclusion: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-γ secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174.

Aqueous gold nanosols stabilized by electrostatic protection generated by X-ray irradiation assisted radical reduction

Reductant, stabilizer-free colloidal gold solutions were fabricated by a new room-temperature synchrotron X-ray irradiation method. The influence of process parameters such as the pH value and the exposure time on the structure of gold nanoparticles was investigated. The mechanisms underlying the X-ray-triggered reduction of gold ions and the formation of gold clusters are discussed in detail. The X-ray irradiation derived highly concentrated gold nanoparticles are readily to be re-dispersed and possess suitable colloidal stability within cellular environment. The characterization included a study of the possible cytotoxicity for the EMT-6 tumor cell line: the negative results indicate that the gold clusters produced with our approach are biocompatible.

Fluorine-18-Labeled Benzamide Analogues for Imaging the σ2 Receptor Status of Solid Tumors with Positron Emission Tomography

A series of fluorine-containing benzamide analogs was synthesized and evaluated as candidate ligands for positron emission tomography (PET) imaging of the sigma-2 (sigma2) receptor status of solid tumors. Four compounds having a moderate to high affinity for sigma2 receptors and a moderate to low affinity for sigma-1 (sigma1) receptors were radiolabeled with fluorine-18 via displacement of the corresponding mesylate precursor with [18F]fluoride. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four F-18-labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection. An analysis of the chemistry and biodistribution data suggested that N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-methylbenzamide ([18F]3c) and N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-iodo-3-methoxybenzamide ([18F]3f) are acceptable compounds for imaging the sigma2 receptor status of solid tumors.

Activation of Heat Shock Protein 70 Promoter with meso-Tetrahydroxyphenyl Chlorin Photodynamic Therapy Reported by Green Fluorescent Protein In Vitro and In Vivo¶

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT–mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue–exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/GFP–transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.

The radiosensitizing effect of immunoadjuvant OM-174 requires cooperation between immune and tumor cells through interferon-gamma and inducible nitric oxide synthase

To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-gamma (IFN-gamma) and thereby radiosensitize tumor cells. Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 mug/mL), and afterward analyzed for the expression and secretion of IFN-gamma by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-gamma to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). OM-174 activated the production of IFN-gamma at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-gamma. The same mechanism of radiosensitization through the IFN-gamma secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-gamma responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-gamma gene while upregulating iNOS at transcriptional level. Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-gamma secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174.

In Vivo Tumor Imaging in Mice Using a State-of-the-Art Clinical PET/CT in Comparison with a Small Animal PET and a Small Animal CT

The aim of this prospective study was to assess the feasibility of a state-of-the-art clinical PET/CT scanner for in vivo tumor imaging in mice in comparison to a small animal PET scanner and a small animal CT scanner. Seventeen female NMRI nude mice bearing a subcutaneous transplanted murine mammary carcinoma (EMT6) on the right hind limb were examined using a clinical PET/CT scanner (biograph Sensation 16), a dedicated small animal PET scanner (MOSAIC system), and a dedicated small animal CT scanner (MicroCAT II). 18F-fluoroazomycin arabinoside (18F-FAZA) was used as PET tracer for imaging of regional tumor hypoxia. The examination of tumor-bearing mice using a state-of-the-art clinical PET/CT scanner, a dedicated small animal PET scanner and a dedicated small animal CT scanner revealed good image quality and allowed the evaluation and correlation of molecular/metabolic alterations of cancers with anatomical/morphological findings. The tumor lesions showed a 1.89 +/- 0.6 higher mean tumor-to-background ratio +/- SD for the dedicated small animal PET scanner than the PET from the clinical PET/CT scanner (P<0.001). The mean tumor-to-background ratio of the PET of the clinical PET/CT scanner showed a high correlation with the mean tumor-to-background ratio of the dedicated small animal PET scanner (r2 = 0.92). The tissue hypoxia in the subcutaneous transplanted EMT6 tumors did not correlate with the tumor volume. Clinical PET/CT scanners are widely available and could contribute not only to human clinical routine examinations, but also to tumor research in animals. The molecular/metabolic information is reduced when imaging small tumors of mice with clinical PET/CT scanners due to the low spatial resolution and sensitivity of the PET scanners. The examination of small tumors in mice should be reserved to research centers with small animal PET/CT scanners because they provide promising hope to achieve accurate measurement of activity concentration. For tumor research in humans and animals using clinical PET/CT scanners, PET scanners with a higher spatial resolution and a higher sensitivity will be highly promising, but will be in competition to PET/MRI.

Efaproxiral (RSR13) Plus Oxygen Breathing Increases the Therapeutic Ratio of Carboplatin in EMT6 Mouse Mammary Tumors

Carboplatin, a member of the platinum family of alkylating agents, is often used in combination with radiotherapy. Some studies, including a recent publication from our laboratory, have suggested that the cytotoxic effects of platinum compounds may be altered by changes in the post-treatment oxygenation. The study reported here assessed whether post-treatment changes in tumor oxygenation caused by oxygen breathing alone or in combination with efaproxiral (RSR13) altered the effects of carboplatin. Efaproxiral, which allosterically modifies hemoglobin-oxygen binding to increase tumor pO(2), has been shown to increase the effects of radiation in animal tumor models and is in a second, confirmatory phase III clinical trial as an adjuvant to radiotherapy. These studies with EMT6 tumors in BALB/c Rw mice used clonogenic assays to assess tumor cell survival and tumor growth studies to assess antineoplastic activity and treatment-related toxicity. Efaproxiral plus oxygen breathing for 5 hrs after carboplatin treatment significantly increased the antineoplastic effects of carboplatin. The increased antineoplastic effects of carboplatin produced by efaproxiral plus oxygen breathing occurred without a concomitant increase in host toxicity. These findings suggest that the increases in tumor oxygenation produced by Efaproxiral plus oxygen breathing increased the therapeutic ratio of carboplatin.

Characterization of a novel iodinated sigma-2 receptor ligand as a cell proliferation marker

Overexpression of sigma-2 receptors in human tumors, such as melanoma, breast cancer, small cell lung carcinoma and prostate cancer, has been reported. Furthermore, the expression of sigma-2 receptors parallels the proliferative status of breast tumors implanted in nude mice. Thus, radiolabeled probes with a high affinity and high selectivity targeting sigma-2 receptors may be useful as tumor imaging agents. A conformationally flexible benzamide derivative, 5-bromo-2,3-dimethoxy- N-[2-(6,7-dimethoxy-3,4-dihydro-1 H-isoquinolin-2-yl)-butyl]-benzamide, displayed greater than 1000-fold selectivity for sigma-2 receptors ( K i=8.2 and 12,900 nM for sigma-2 and sigma-1, respectively). The corresponding radioiodinated ligand, 5-iodo-2,3-dimethoxy- N-[2-(6,7-dimethoxy-3,4-dihydro-1 H-isoquinolin-2-yl)-butyl]-benzamide ([ 125I]/[ 123I] I), was successfully prepared via an iododestannylation reaction. Binding studies carried out in membrane homogenates prepared from the mouse mammary tumor cell line (EMT6) with [ 125I] I showed the desired high binding affinity and high capacity ( K d=0.68±0.06 nM, B max=1005±46 fmol/mg protein). The sigma-2-like pharmacological profile of [ 125I] I binding sites was confirmed by competition studies. Similar binding parameters were also found in EMT6 xenografts (Day 10 and Day 19 implants) from BALB/c mice ( K d=0.43–1.1 nM, B max=2025–4528 fmol/mg protein). It was determined by dissection and microSPECT imaging that [ 125I]/[ 123] I accumulated in EMT6 tumors established in BALB/c mice (Day 10 implants). Two hours after the tracer injection, the dissection ratio of EMT6 tumor to background (muscle) reached 6–7. However, microSPECT imaging could not clearly delineate the tumors, while the specific localization could be confirmed by ex vivo autoradiography. In summary, the novel iodinated ligand with high affinity and considerable selectivity for sigma-2 receptors may provide a useful tool to characterize sigma-2 receptors in vitro. Further modification of the ligand and the imaging parameters will be needed to improve the signal for in vivo detection.

Monitoring the effect of mild hyperthermia on tumour hypoxia by Cu-ATSM PET scanning

Purpose: Mild hyperthermia can improve tumour oxygenation and enhance radiosensitivity. Imaging the hypoxic fraction of a tumour can guide hyperthermia treatment planning and facilitate treatment optimization. 64Cu-ATSM (Copper-diacetyl-bis(N4-methylthiosemicarbazone)) is a positron emitting compound that has been demonstrated to have rapid uptake and selective retention in hypoxic cells and has been used for imaging human and animal tumours. The purpose of the present report is to establish methodology that will allow one to use Cu-ATSM PET scanning to detect the impact of hyperthermia on tumour physiology in as little time as possible.Materials and methods: EMT6 tumours (mouse mammary carcinoma) were implanted into the subcutaneous tissue of both thighs of 10 BALB/c mice (one heated, one control tumour per animal). The target thermal dose was 41.5°C × 45 min. Without interrupting heating, 64Cu-ATSM (mean activity 1.8 mCi) was then injected and serial PET scans were obtained. In a sub-group of four animals, a low administered activity (∼0.3 mCi) 64Cu-ATSM scan was also conducted before heating to permit a direct comparison of the effects of hyperthermia on the same tumours. In another sub-group of five animals, a low activity (∼0.3 mCi) 64Cu-PTSM (pyruvaldehyde-bis(N*-methylthiosemicarbazone)) scan was conducted before heating, to confirm a posited correlation between perfusion and early 64Cu-ATSM uptake.Results: This study corrected for perfusion differences by dividing tumour uptake by the average early (first minute) uptake (‘self-normalized uptake’). The 10 heated tumours showed a significantly (p = 0.007) lower self-normalized uptake than control tumours by 2 min. For the four mice with low activity Cu-ATSM scans performed before hyperthermia, the tumours to be heated demonstrated self-normalized uptake consistent with the unheated control tumours and which departed significantly (p ≤ 0.02) from their post-hyperthermia scans by 5 min. Comparisons between scans and needle electrode surveys were performed in an additional four animals with eight tumours. For technical reasons electrode surveys were done after the end of hyperthermia—and, therefore, these animals also had comparison scans taken after hyperthermia. Reduced self-normalized uptake on scans was associated with increased pO2 on electrode surveys. These data also suggested a substantial degradation of the effect on tumour hypoxia by ∼15–45 min after the end of mild hyperthermia.Conclusion: Short imaging times of ∼5 min with modest (∼4–10) numbers of mice can discriminate the effects of mild hyperthermia on tumour physiology. The long-term objective is to use this tool to identify as short and mild a hyperthermia session as possible.

The Distribution of the Anticancer Drug Doxorubicin in Relation to Blood Vessels in Solid Tumors

Anticancer drugs gain access to solid tumors via the circulatory system and must penetrate the tissue to kill cancer cells. Here, we study the distribution of doxorubicin in relation to blood vessels and regions of hypoxia in solid tumors of mice. The distribution of doxorubicin was quantified by immunofluorescence in relation to blood vessels (recognized by CD31) of murine 16C and EMT6 tumors and human prostate cancer PC-3 xenografts. Hypoxic regions were identified by injection of EF5. The concentration of doxorubicin decreases exponentially with distance from tumor blood vessels, decreasing to half its perivascular concentration at a distance of about 40 to 50 mum, The mean distance from blood vessels to regions of hypoxia is 90 to 140 microm in these tumors. Many viable tumor cells are not exposed to detectable concentrations of drug following a single injection. Limited distribution of doxorubicin in solid tumors is an important and neglected cause of clinical resistance that is amenable to modification. The technique described here can be adapted to studying the distribution of other drugs within solid tumors and the effect of strategies to modify their distribution.

1364 ORAL Predicting the radiotherapy service requirements in scotland in 2015

Activated T lymphocytes are known to kill tumor cells by triggering cytolytic mechanisms; however, their ability to enhance radiation responses remains unclear. This study examined the radiosensitizing potential of mouse CD8+ T cells, obtained by T-cell–tailored expansion and immunomagnetic purification. Activated CD8+ T cells displayed an interferon (IFN)-γ+ phenotype and enhanced by 1.8-fold the radiosensitivity of EMT-6 tumor cells in 1% oxygen, which modeled tumor-relevant hypoxia. Radiosensitization was counteracted by neutralizing IFN-γ or by blocking the inducible isoform of nitric oxide synthase, thus delineating the immune–tumor cell interaction through the IFN-γ secretion pathway. Reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter data in agreement detected downregulation of the IFN-γ gene by hypoxia, which caused IFN-γ deficiency next to radioresistance. Therefore, immune and radiation responses are likely to be allied in the hypoxic tumor microenvironment, and CD8+ T cells may bridge immunostimulatory and radiosensitizing strategies.

131I]Iodoazomycin arabinoside for low-dose-rate isotope radiotherapy: radiolabeling, stability, long-term whole-body clearance and radiation dosimetry estimates in mice

Background The preliminary characterization of [ 131I]iodoazomycin arabinoside ([ 131I]IAZA) as a potential radiotherapeutic radiopharmaceutical is described. Methods High-specific-activity [ 131I]IAZA was prepared in therapeutic doses (up to 3 GBq per batch) by isotope exchange in pivalic acid melt and was purified on Sep-Pak cartridges. Stability in 15% ethanol in saline at 4°C was determined by high-performance liquid chromatography. IAZA cytotoxicity (IC 50, ∼0.1 mM) against both murine (EMT-6) and human (143B, 143B-LTK) tumor cells determined by MTT test was in the range previously reported for EMT-6 cells using a clonogenic assay. Tissue radioactivity levels were measured in a murine tumor model for the 24- to 168-h postinjection period. Radiation dose estimates obtained from the tissue activity levels for this period were calculated from pharmacokinetic (WinNonlin) and dosimetry (MIRD and RAdiation Dose Assessment Resource) parameters. Results The radioiodination efficiency was >90%, but with systematic losses during Sep-Pak purification, the recovered yields of [ 131I]IAZA were ∼75%. The product (specific activity, 4.6–6.4 GBq/μmol) was stable for at least 2 weeks, with only ∼6% degradation over this storage period. Extended biodistribution studies in Balb/c mice bearing implanted EMT-6 tumors showed that the highest tumor/blood radioactivity ratio (T/B; 4.8) occurred 24 h after dosing; the T/B ratio was ∼1.5 at the end of the 7-day study. The 24- to 168-h tissue radioactivity data fit a one-compartment model except for liver data, which best fit a two-compartment model. Dosimetry estimates showed a tumor self-dose of 7.4 mGy/MBq, which is several-fold higher than for the liver or the kidney. Conclusions [ 131I]IAZA can be efficiently radiolabeled at high specific activity, purified by a simple Sep-Pak technique and stored with little radiolysis or chemical decomposition at these specific activities. Based on measured radioactivity burdens during the week following injection and on published animal ([ 125I]IAZA) and clinical ([ 123I]IAZA) dosimetry data, the current dose estimates point to selective tumor irradiation at low dose rates.

Radiosynthesis, in vitro cellular uptake and in vivo biodistribution of 3′- O-(3-benzenesulfonylfuroxan-4-yl)-5-[ 125I]iodo-2′-deoxyuridine, a nucleoside-based nitric oxide donor

Introduction 3′- O-(3-Benzenesulfonylfuroxan-4-yl)-5-iodo-2′-deoxyuridine ( 1) is a cytotoxic nitric oxide (NO) donor-nucleoside dual action prodrug designed to exploit both NO and an antimetabolite nucleoside for cancer therapy. Methods 1 was radiolabeled by radioiodide exchange and purified by HPLC in 16% overall radiochemical yield. The specific activity of [ 125I] 1 was 31.8 μCi/μg (680 MBq/μM). Protein binding, deiodination, cellular uptake and incorporation of 1 into cellular nucleic acids were measured by standard methods, and its in vivo biodistribution was determined in Balb/c mice bearing implanted EMT-6 tumors following intravenous injection. Results [ 125I] 1 degraded rapidly during the in vitro tests, thus impeding unequivocal assessment but indicating that it was only weakly protein bound and that it was resistant to deiodination under test conditions. Uptake of [ 125I] 1 by murine tumor cells (KBALB and KBALB-STK) in vitro was low (∼17 fmol/μg protein over 2 h) with only ∼0.3% (0.04–0.06 fmol/μg protein) of total uptake present in the DNA fraction. In the murine tumor model, liver, kidney, intestine and tumor showed the greatest uptake, with liver, intestine and blood all containing >5 injected dose per gram of tissue (%ID/g) during the 15-min to 2-h postinjection period. Maximum tissue/blood level ratios were 3.6 (2 h) for tumor and 6.4 (24 h) for liver. Low uptake in thyroid and stomach was indicative of minimal in vivo deiodination. Conclusions [ 125I] 1 undergoes only minimal deoiodination under both in vitro and in vivo conditions. Under conditions of the in vitro NO release assay, 1 reacts to produce a single, major, unstable adduct that decomposes upon workup. Protein binding of [ 125I] 1 could not be assessed because of similar chemical reaction with albumin. Incorporation of radioactivity into the cellular nucleic acid fraction was low, and in vivo distribution of [ 125I] 1 was consistent with nonspecific reactivity towards tissue nucleophiles. The chemical reactivity of [ 125I] 1 mitigates against its use as a NO donor and as a tracer for this class of compounds.

Radiobiologic effect of intermittent radiation exposure in murine tumors

In stereotactic irradiation using a linear accelerator, the effect of radiation may be reduced during intermittent exposures owing to recovery from sublethal damage in tumor cells. After our previous in vitro study suggesting this phenomenon, we investigated the issue in murine tumors. We used EMT6 and SCCVII tumors approximately 1 cm in diameter growing in the hind legs of syngeneic mice. Three schedules of intermittent radiation were investigated. First, 2 fractions of 10 Gy were given at an interval of 15-360 min to investigate the pattern of recovery from sublethal damage. Second, 5 fractions of 4 Gy were given with interfraction intervals of 2.5-15 min each. Third, 10 fractions of 2 Gy were given with interfraction intervals of 1-7 min each. Doses of 15-20 Gy were also given without interruption to estimate the dose-modifying factors. Tumors were excised 20 h later, and tumor cell survival was determined by an in vivo-in vitro assay. In the 2-fraction experiment, the increase in cell survival with elongation of the interval was much less than that observed in our previous in vitro study. In the 5- and 10-fraction experiments, no significant increase in cell survival was observed after the intermittent exposures. Moreover, cell survival decreased at most points of the 5-fraction experiments by interruption of radiation in both EMT6 and SCCVII tumors. In the 10-fraction experiment, cell survival also decreased when the interruption was 3 or 7 min in EMT6 tumors. The results of the present in vivo studies were different from those of our in vitro studies in which cell survival increased significantly when a few minutes or longer intervals were posed between fractions. This suggests that recovery from sublethal damage in vivo may be counterbalanced by other phenomena such as reoxygenation that sensitizes tumor cells to subsequent irradiation.

Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy

A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3–NTR neo, WiDr/WiDr–NTR neo), Chinese hamster (V79/V79–NTR puro) and murine (EMT6/EMT6–NTR puro) cell line pairs, and were compared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 μM) precluded the determination of IC 50 values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24 h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.

Investigation into 64Cu-labeled Bis(selenosemicarbazone) and Bis(thiosemicarbazone) complexes as hypoxia imaging agents

Background Cu-diacetyl-bis( N 4-methylthiosemicarbazone) [Cu-ATSM], although excellent for oncology applications, may not be suitable for delineating cardiovascular or neurological hypoxia. For this reason, new Cu hypoxia positron emission tomography (PET) imaging agents are being examined to search for a higher selectivity for hypoxic or ischemic tissue at higher oxygen concentrations found in these tissues. Two approaches are to increase alkylation or to replace the sulfur atoms with selenium, resulting in the formation of selenosemicarbazones. Methods Three 64Cu-labeled selenosemicarbazone complexes were synthesized and one was screened for hypoxia selectivity in vitro using EMT-6 mouse mammary carcinoma cells. Rodent biodistribution and small animal PET images were obtained from BALB/c mice implanted with EMT-6 tumors. One alkylated thiosemicarbazone was synthesized and examined. Results Of the three bis(selenosemicarbazone) ligands synthesized and examined, only 64Cu-diacetyl-bis(selenosemicarbazone) [ 64Cu-ASSM] was isolated in high-enough radiochemical purity to undertake cell uptake experiments where uptake was shown to be independent of oxygen concentration. The bis(thiosemicarbazone) complex synthesized, 64Cu-diacetyl-bis( N 4-ethylthiosemicarbazone) [ 64Cu-ATSE], showed hypoxia selectivity similar to 64Cu-ATSM although at a higher oxygen concentration. Biodistribution studies for 64Cu-ASSM and 64Cu-ATSE showed high tumor uptake at 20 min ( 64Cu-ASSM, 10.33±0.78% ID/g; 64Cu-ATSE, 7.71±0.46% ID/g). PET images of EMT-6 tumor-bearing mice visualized the tumor with 64Cu-ATSE and revealed hypoxia selectivity consistent with the in vitro data. Conclusion Of the compounds synthesized, only 64Cu-ASSM and 64Cu-ATSE could be examined in vitro and in vivo. Although the stability of bis(selenosemicarbazone) complexes increased upon addition of methyl groups to the diimine backbone, the fully alkylated species, 64Cu-ASSM, demonstrated no hypoxia selectivity. However, the additional alkylation present in Cu-ATSE modifies the hypoxia selectivity and in vivo properties when compared with Cu-ATSM.

An investigation into the potential of phenothiazinium-based photo-sensitisers to act as PDT agents

There is an urgent need for effective cancer treatments and increasingly, photo-dynamic therapy (PDT) is being used to fulfil this need as it offers a number of advantages over traditional cancer treatments. Here, the potential of a series of phenothiazinium-based photo-sensitisers (PhBPs) as PDT agents is tested. PhBPs were incubated with EMT-6 tumour cells and erythrocytes respectively under dark and light conditions (3.15Jcm(-2) over 30min). "Comet assay" and haemolytic assay were then used to assess cellular photo-damage induced by these PhBPs. Additionally, in vitro assays were used to determine light adsorption characteristics, singlet oxygen yields (ΦΔPhBP) and lipophilicity (logP) of these PhBPs. "Comet assay" showed EMT-6 incubation with PhBPs under light conditions to produce DNA "tails", which were circa 35μm long, indicating the presence of DNA photo-damage. Corresponding incubations under dark conditions led to no such damage. The majority of the PhBPs tested possessed significant singlet oxygen yields (ΦΔPhBP>0.7), suggesting the general use of type II mechanisms for photo-sensitization, and were generally lipophilic (logP>0). Incubation of erythrocytes with these PhBPs in the dark produced between 6% and 19% haemolysis. These levels were generally unaffected by illumination except in the case of DMMB, which showed haemolytic levels increasing from 11% to 61%. It is suggested that DNA may be the primary target for the photo-dynamic anti-tumour activity of the PhBPs tested with the exception of DMMB, which may potentially also target tumour cell membranes.

Macrophages enhance the radiosensitizing activity of lipid A: A novel role for immune cells in tumor cell radioresponse

This study examines whether activated macrophages may radiosensitize tumor cells through the release of proinflammatory mediators. RAW 264.7 macrophages were activated by lipid A, and the conditioned medium (CM) was analyzed for the secretion of cytokines and the production of nitric oxide (NO) through inducible nitric oxide synthase (iNOS). EMT-6 tumor cells were exposed to CM and analyzed for hypoxic cell radiosensitivity. The role of nuclear factor (NF)-kappaB in the transcriptional activation of iNOS was examined by luciferase reporter gene assay. Clinical immunomodulator lipid A, at a plasma-relevant concentration of 3 microg/mL, stimulated RAW 264.7 macrophages to release NO, tumor necrosis factor (TNF)-alpha, and other cytokines. This in turn activated iNOS-mediated NO production in EMT-6 tumor cells and drastically enhanced their radiosensitivity. Radiosensitization was abrogated by the iNOS inhibitor aminoguanidine but not by a neutralizing anti-TNF-alpha antibody. The mechanism of iNOS induction was linked to NF-kappaB but not to JAK/STAT signaling. Interferon-gamma further increased the NO production by macrophages to a level that caused radiosensitization of EMT-6 cells through the bystanding effect of diffused NO. We demonstrate for the first time that activated macrophages may radiosensitize tumor cells through the induction of NO synthesis, which occurs in both tumor and immune cells.

131 Pharmacodynamic analysis of apoptosis and anti-vascular activity in GIST patients treated with Imatinib

To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated.Tumor oxygenation was measured using an Eppendorf pO2 histograph when tumors, implanted intradermally in the rear dorsum, reached 150–200 mm3. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue.Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO2 6 hours after injection of MGd was 8.0 ± 2.4 mm Hg. The control value was 1.5 ± 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6–8 hours after i.v. injection of 40 μmol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 ± 4.3% <2.5 mm Hg; Control: 69.4 ± 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation.MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.