[Thermal therapy of prostate cancer using magnetic nanoparticles]. / Termoterapia en cáncer de próstata mediante el uso de nanopartículas magnéticas.

A novel method of interstitial heating using magnetic nanoparticles and a direct injection technique has been evaluated in human cancers in recent clinical trials. In prostate cancer, this approach was investigated in two separate phase-I-studies, employing magnetic nanoparticle thermotherapy alone and in combination with permanent seed brachytherapy. The feasibility and good tolerability was shown in both trials, using the first prototype of a magnetic field applicator. As with any other heating technique, this novel approach requires specific tools for planning, quality control and thermal monitoring, based on appropriate imaging and modelling techniques. In these first clinical trials, a newly developed method for planning and non-invasive calculations of the 3-dimensional temperature distribution based on computed tomography could be validated. Limiting factors of this approach at present are patient discomfort at high magnetic field strengths and suboptimal intratumoral distribution of nanoparticles. Until these limitations will be overcome and thermal ablation can safely be applied as a monotherapy, this treatment modality is being evaluated in combination with irradiation in patients with localized prostate cancer.

SV40 large T-antigen disturbs the formation of nuclear DNA-repair foci containing MRE11.

The accumulation of DNA repair proteins at the sites of DNA damage can be visualized in mutagenized cells at the single cell level as discrete nuclear foci by immunofluorescent staining. Formation of nuclear foci in irradiated human fibroblasts, as detected by antibodies directed against the DNA repair protein MRE11, is significantly disturbed by the presence of the viral oncogene, SV40 large T-antigen. The attenuation of foci formation was found in both T-antigen immortalized cells and in cells transiently expressing T-antigen, indicating that it is not attributable to secondary mutations but to T-antigen expression itself. ATM-mediated nibrin phosphorylation was not altered, thus the disturbance of MRE11 foci formation by T-antigen is independent of this event. The decrease in MRE11 foci was particularly pronounced in T-antigen immortalized cells from the Fanconi anaemia complementation group FA-D2. FA-D2 cells produce essentially no MRE11 DNA repair foci after ionizing irradiation and have a significantly increased cellular radiosensitivity at low radiation doses. The gene mutated in FA-D2 cells, FANCD2, codes for a protein which also locates to nuclear foci and may, therefore, be involved in MRE11 foci formation, at least in T-antigen immortalized cells. This finding possibly links Fanconi anaemia proteins to the frequently reported increased sensitivity of Fanconi anaemia cells to transformation by SV40. From a practical stand point these findings are particularly relevant to the many studies on DNA repair which exploit the advantages of SV40 immortalized cell lines. The interference of T-antigen with DNA repair processes, as demonstrated here, should be borne in mind when interpreting such studies.

Iodinated nitroimidazoles as radiosensitizers.

Four different nitroimidazole derivatives, with up to two iodine atoms on the imidazole ring, were investigated for their radiosensitizing potency under hypoxic conditions, in order to test whether the introduction of iodine atoms increases the radiosensitizing potency of nitroimidazoles. Misonidazole and metronidazole were used as controls. Human colonic adenocarcinoma cells were incubated with the drugs at different concentrations and for different time-periods. Photon energies of 50 kV, 60 kV and 20 MV and total radiation doses of up to 20 Gy were used. The introduction of additional iodine atoms into the nitroimidazole derivatives resulted in a strong increase in cytotoxicity of the compounds. In parallel, there were indications that the radiosensitizing potency was also increased.

Description and characterization of the novel hyperthermia- and thermoablation-system MFH 300F for clinical magnetic fluid hyperthermia.

Magnetic fluid hyperthermia (MFH) is a new approach to deposit heat power in deep tissues by overcoming limitations of conventional heat treatments. After infiltration of the target tissue with nanosized magnetic particles, the power of an alternating magnetic field is transformed into heat. The combination of the 100 kHz magnetic field applicator MFH 300F and the magnetofluid (MF), which both are designed for medical use, is investigated with respect to its dosage recommendations and clinical applicability. We found a magnetic field strength of up to 18 kA/m in a cylindrical treatment area of 20 cm diameter and aperture height up to 300 mm. The specific absorption rate (SAR) can be controlled directly by the magnetic field strength during the treatment. The relationship between magnetic field strength and the iron normalized SAR (SAR(Fe)) is only slightly depending on the concentration of the MF and can be used for planning the target SAR. The achievable energy absorption rates of the MF distributed in the tissue is sufficient for either hyperthermia or thermoablation. The fluid has a visible contrast in therapeutic concentrations on a CT scanner and can be detected down to 0.01 g/l Fe in the MRI. The system has proved its capability and practicability for heat treatment in deep regions of the human body.

Evaluation of magnetic fluid hyperthermia in a standard rat model of prostate cancer.

PURPOSE: To examine the feasibility and potential of magnetic fluid hyperthermia (MFH) as a minimally invasive method for hyperthermia treatment of prostate cancer. MATERIALS AND METHODS: Orthotopic Dunning R3327 prostate tumors were induced in 20 male Copenhagen rats. The animals either received MFH treatment following intratumoral administration of magnetic fluids or were used as either tumor growth controls for determination of iron distribution in selected organs or as histologic controls without MFH treatment. The MFH treatments were carried out at 45 degrees C or 50 degrees C using an AC magnetic field applicator system designed for small animals. RESULTS: Sequential treatments with MFH were possible following a single intratumoral injection of magnetic fluid. Intratumoral temperatures of 50 degrees C and more were obtained and were monitored online using fluoro-optic thermometry. Four days after MFH treatments, 79% of the injected dose of ferrites was still present in the prostate. CONCLUSIONS: The successful intraprostatic nanoparticle infiltration and stable steady-state intratumoral treatment temperatures demonstrate the feasibility of MFH in a prostate cancer model. Efficacy and survival benefit must be confirmed in further experiments.

A systematic proteomic study of irradiated DNA repair deficient Nbn-mice.

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress.

Thermotherapy of prostate cancer using magnetic nanoparticles: feasibility, imaging, and three-dimensional temperature distribution.

OBJECTIVES: To investigate the feasibility of thermotherapy using biocompatible superparamagnetic nanoparticles in patients with locally recurrent prostate cancer and to evaluate an imaging-based approach for noninvasive calculations of the three-dimensional temperature distribution. METHODS: Ten patients with locally recurrent prostate cancer following primary therapy with curative intent were entered into a prospective phase 1 trial. The magnetic fluid was injected transperineally into the prostates according to a preplan. Patients received six thermal therapies of 60-min duration at weekly intervals using an alternating magnetic field applicator. A method of three-dimensional thermal analysis based on computed tomography (CT) of the prostates was developed and correlated with invasive and intraluminal temperature measurements. The sensitivity of nanoparticle detection by means of CT was investigated in phantoms. RESULTS: The median detection rate of iron oxide nanoparticles in tissue specimens using CT was 89.5% (range: 70-98%). Maximum temperatures up to 55 degrees C were achieved in the prostates. Median temperatures in 20%, 50%, and 90% of the prostates were 41.1 degrees C (range: 40.0-47.4 degrees C), 40.8 degrees C (range: 39.5-45.4 degrees C), and 40.1 degrees C (range: 38.8-43.4 degrees C), respectively. Median urethral and rectal temperatures were 40.5 degrees C (range: 38.4-43.6 degrees C) and 39.8 degrees C (range: 38.2-43.4 degrees C). The median thermal dose was 7.8 (range: 3.5-136.4) cumulative equivalent minutes at 43 degrees C in 90% of the prostates. CONCLUSION: The heating technique using magnetic nanoparticles was feasible. Hyperthermic to thermoablative temperatures were achieved in the prostates at 25% of the available magnetic field strength, indicating a significant potential for higher temperatures. A noninvasive thermometry method specific for this approach could be developed, which may be used for thermal dosimetry in future studies.

Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme.

We aimed to evaluate the feasibility and tolerability of the newly developed thermotherapy using magnetic nanoparticles on recurrent glioblastoma multiforme. Fourteen patients received 3-dimensional image guided intratumoral injection of aminosilane coated iron oxide nanoparticles. The patients were then exposed to an alternating magnetic field to induce particle heating. The amount of fluid and the spatial distribution of the depots were planned in advance by means of a specially developed treatment planning software following magnetic resonance imaging (MRI). The actually achieved magnetic fluid distribution was measured by computed tomography (CT), which after matching to pre-operative MRI data enables the calculation of the expected heat distribution within the tumor in dependence of the magnetic field strength. Patients received 4-10 (median: 6) thermotherapy treatments following instillation of 0.1-0.7 ml (median: 0.2) of magnetic fluid per ml tumor volume and single fractions (2 Gy) of a radiotherapy series of 16-70 Gy (median: 30). Thermotherapy using magnetic nanoparticles was tolerated well by all patients with minor or no side effects. Median maximum intratumoral temperatures of 44.6 degrees C (42.4-49.5 degrees C) were measured and signs of local tumor control were observed. In conclusion, deep cranial thermotherapy using magnetic nanoparticles can be safely applied on glioblastoma multiforme patients.

Thermotherapy using magnetic nanoparticles combined with external radiation in an orthotopic rat model of prostate cancer.

BACKGROUND: We evaluated the effects of thermotherapy using magnetic nanoparticles, also referred to as magnetic fluid hyperthermia (MFH), combined with external radiation, in the Dunning model of prostate cancer. METHODS: Orthotopic tumors were induced in 96 male Copenhagen rats. Animals were randomly allocated to eight groups, including controls and groups for dose-finding studies of external radiation. Treatment groups received two serial thermotherapy treatments following a single intratumoral injection of magnetic fluid or thermotherapy followed by external radiation (10 Gy). On day 20, after tumor induction, tumor weights in the treatment and control groups were compared and iron measurements in selected organs were carried out. RESULTS: Mean maximal and minimal intratumoral temperatures obtained were 58.7 degrees C (centrally) and 42.7 degrees C (peripherally) during the first thermotherapy and 55.4 degrees C and 42.3 degrees C, respectively, during the second of two treatment sessions. Combined thermotherapy and radiation with 20 Gy was significantly more effective than radiation with 20 Gy alone and reduced tumor growth by 87.5-89.2% versus controls. Mean iron content in the prostates on day 20 was 87.5% of the injected dose of ferrites, whereas only 2.5% was found in the liver. CONCLUSIONS: An additive effect was demonstrated for the combined treatment at a radiation dose of 20 Gy, which was equally effective in inhibiting tumor growth as radiation alone with 60 Gy. Serial heat treatments were possible without repeated injection of magnetic fluid. The optimal treatment schedules of this combination regarding temperatures, radiation dose, and fractionation need to be defined in further experimental studies.

The effect of thermotherapy using magnetic nanoparticles on rat malignant glioma.

Thermotherapy using magnetic nanoparticles is a new technique for interstitial hyperthermia and thermoablation based on magnetic field-induced excitation of biocompatible superparamagnetic nanoparticles. To evaluate the potential of this technique for minimally invasive treatment, we carried out a systematic analysis of its effects on experimental glioblastoma multiforme in a rat tumor model. Tumors were induced by implantation of RG-2-cells into the brains of 120 male Fisher rats. Animals were randomly allocated to 10 groups of 12 rats each, including controls. Animals received two thermotherapy treatments following a single intratumoral injection of two different magnetic fluids (dextran- or aminosilane-coated iron-oxide nanoparticles). Treatment was carried out on days four and six after tumor induction using an alternating magnetic field applicator system operating at a frequency of 100 kHz and variable field strength of 0-18 kA/m. The effectiveness of treatment was determined by the survival time of the animals and histopathological examinations of the brain and the tumor.Thermotherapy with aminosilane-coated nanoparticles led up to 4.5-fold prolongation of survival over controls, while the dextran-coated particles did not indicate any advantage. Intratumoral deposition of the aminosilane-coated particles was found to be stable, allowing for serial thermotherapy treatments without repeated injection. Histological and immunohistochemical examinations after treatment revealed large necrotic areas close to particle deposits, a decreased proliferation rate and a reactive astrogliosis adjacent to the tumor.Thus, localized interstitial thermotherapy with magnetic nanoparticles has an antitumoral effect on malignant brain tumors. This method is suitable for clinical use and may be a novel strategy for treating malignant glioma, which cannot be treated successfully today. The optimal treatment schedules and potential combinations with other therapies need to be defined in further studies.

Selection and characterization of heat-resistant variants of multidrug-resistant human gastric carcinoma cell lines.

AIM: To generate heat resistant variants selected from established human gastric carcinoma cell lines exhibiting different types of multidrug resistance (MDR) phenotype, i.e. EPG85-257P, the drug sensitive parental cell-line, EPG85-257RDB, a classical MDR subline and EPG85-257RNOV, which is an atypical multidrug resistant subline. METHODS: Thermoresistance was induced by stepwise increase of the growth temperature from 37.0 to 39.4 degrees C. Thermoresistance was determined by change of population doubling time (PDT) and clonogenic survival after acute hyperthermia at 42, 43, 44 and 45 degrees C. RESULTS: Most of the cells exhibited necrosis at elevated culture temperature. The PDT of the surviving thermoresistant variants were increased two-fold (EPG85-257P-TR) and 1.2-fold (EPG85-257RNOV-TR), respectively. No PDT change was observed with the lowest thermoresistant subline EPG85-257RDB-TR. Dose response curves after acute hyperthermia indicated a stable increase of thermotolerance of the parental cell line and the atypical MDR subline (50-90-fold at 45 degrees C), but not of the classical MDR subline, which was only increased at 43 degrees C (3-4-fold). Acquired thermoresistance did not change after freezing/thawing procedures. CONCLUSION: All cell lines achieved chronically induced thermoresistance. Thermotolerance after acute hyperthermia was present in the drug sensitive parental cell line and the atypical MDR subline, but not in cells exhibiting a classical MDR phenotype.

Imaging of single human carcinoma cells in vitro using a clinical whole-body magnetic resonance scanner at 3.0 T.

The purpose of the present study was to examine whether single human carcinoma cells labeled with iron oxide nanoparticles could be detected by magnetic resonance (MR) imaging on a clinical 3-T scanner using a surface coil only. WiDr human colon carcinoma cells were loaded with two kinds of iron oxide nanoparticles differing by coating and size: aminosilan-coated (MagForce) and carboxy-dextran-coated particles (Resovist). The latter were preferred by the colon carcinoma cell line used here and taken up much faster (12 h) than the smaller carboxydextran-coated Resovist (48 h). Labeled single carcinoma cells, distributed in an agarose gel in a monodisperse layer as controlled by light microscopy, became detectable as punctuate signal extinctions when using a small circularly polarized surface coil in conjunction with a T(2)*-weighted GE sequence at 3 T. The threshold for the detectability of labeled colon carcinoma cells ranged at a load of 4-5 mug iron/10(6) cells. Obviating the need for special hardware additions, this study opens a new lane for single-cell tracking on clinical 3-T MR scanners amenable to patient studies.

Magnetic fluid hyperthermia (MFH)reduces prostate cancer growth in the orthotopic Dunning R3327 rat model.

BACKGROUND: Magnetic fluid hyperthermia (MFH) is a new technique for interstitial hyperthermia or thermoablation based on AC magnetic field-induced excitation of biocompatible superparamagnetic nanoparticles. Preliminary studies in the Dunning tumor model of prostate cancer have demonstrated the feasibility of MFH in vivo. To confirm these results and evaluate the potential of MFH as a minimally invasive treatment of prostate cancer we carried out a systematic analysis of the effects of MFH in the orthotopic Dunning R3327 tumor model of the rat. METHODS: Orthotopic tumors were induced by implantation of MatLyLu-cells into the prostates of 48 male Copenhagen rats. Animals were randomly allocated to 4 groups of 12 rats each, including controls. Treatment animals received two MFH treatments following a single intratumoral injection of a magnetic fluid. Treatments were carried out on days 10 and 12 after tumor induction using an AC magnetic field applicator system operating at a frequency of 100 kHz and a variable field strength (0--18 kA/m). On day 20, animals were sacrificed and tumor weights in the treatment and control groups were compared. In addition, tumor growth curves were generated and histological examinations and iron measurements in selected organs were carried out. RESULTS: Maximum intratumoral temperatures of over 70 degrees C could be obtained with MFH at an AC magnetic field strength of 18 kA/m. At a constant field strength of 12.6 kA/m, mean maximal and minimal intratumoral temperatures recorded were 54.8 degrees C (centrally) and 41.2 degrees C (peripherally). MFH led to an inhibition of tumor growth of 44%-51% over controls. Mean iron content in the prostates of treated and untreated (injection of magnetic fluids but no AC magnetic field exposure) animals was 82.5%, whereas only 5.3% of the injected dose was found in the liver, 1.0% in the lung, and 0.5% in the spleen. CONCLUSIONS: MFH led to a significant growth inhibition in this orthotopic model of the aggressive MatLyLu tumor variant. Intratumoral deposition of magnetic fluids was found to be stable, allowing for serial MFH treatments without repeated injection. The optimal treatment schedules and temperatures for MFH need to be defined in further studies.

Imatinib mesylate radiosensitizes human glioblastoma cells through inhibition of platelet-derived growth factor receptor.

Imatinib mesylate is a small molecule inhibitor of the c-Abl, platelet-derived growth factor (PDGF) receptor and c-Kit tyrosine kinases that is approved for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML) and gastrointestinal stromal tumors. Glioblastoma multiforme is a highly malignant primary brain tumor that is usually treated with surgery and/or radiotherapy. Previous studies implicate an autocrine loop caused by high expression of PDGF and its receptor, PDGFR, in the proliferation of some glioblastomas. Here, we demonstrate that pretreatment of a human glioblastoma cell line, RuSi RS1, with imatinib significantly enhanced the cytotoxic effect of ionizing radiation. This effect was not seen in human breast cancer (BT20) and colon cancer (WiDr) cell lines. Whereas c-Abl and c-Kit were expressed about equally in the three cell lines, RuSi RS1 cells showed significantly higher expression of PDGFR-beta protein in comparison to BT20 and WiDr. Imatinib treatment of RuSi RS1 cells decreased overall levels of cellular tyrosine phosphorylation and specifically inhibited phosphorylation of PDGFR-beta, while c-Abl was not prominently activated in these cells. These results suggest that imatinib may have clinical utility as a radiosensitizer in the treatment of human glioblastoma, possibly through disruption of an autocrine PDGF/PDGFR loop.

Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia.

The role of the Fanconi anaemia genes in DNA repair was examined by a quantitative analysis of nuclear DNA repair foci in FA primary fibroblasts after ionising irradiation using antibodies directed against RAD51, MRE11 and BRCA1 for visualisation. IR induced foci detected with anti-RAD51, but not those detected with anti-MRE11, are reduced in fibroblasts of all eight FA complementation groups in comparison to control cells. Correction of FA-A, FA-C and FA-G cells by retroviral cDNA transfer specifically corrected the RAD51-foci response but did not affect formation of foci containing BRCA1 or MRE11. Since all FA cells, except FA-D1, lack the monoubiquitinated FANCD2-L protein, this isoform is likely to be involved in the formation of nuclear foci containing RAD51 in diploid FA cells. FA-D1 cells show the same attenuation in RAD51 foci formation, suggesting that the unknown FANCD1 protein is similarly involved in RAD51 foci formation, either independently or as a subsequent step in the FANCD2 pathway. These findings indicate that Fanconi anaemia cells have an impairment in the RAD51-dependent homologous recombination pathway for DNA repair, explaining their chromosomal instability and extreme sensitivity to DNA cross-linking agents.