Statistical Analysis of Ultrasonic Scattered Echoes Enables the Non-invasive Measurement of Temperature Elevations inside Tumor Tissue during Oncological Hyperthermia.

Non-invasive monitoring of temperature elevations inside tumor tissue is imperative for the oncological thermotherapy known as hyperthermia. In the present study, two cancer patients, one with a developing right renal cell carcinoma and the other with pseudomyxoma peritonei, underwent hyperthermia. The two patients were irradiated with radiofrequency current for 40 min during hyperthermia. We report the results of our clinical trial study in which the temperature increases inside the tumor tissues of patients with right renal cell carcinoma and pseudomyxoma peritonei induced by radiofrequency current irradiation for 40 min could be detected by statistical analysis of ultrasonic scattered echoes. The Nakagami shape parameter m varies depending on the temperature of the medium. We calculated the Nakagami shape parameter m by statistical analysis of the ultrasonic echoes scattered from the tumor tissues. The temperature elevations inside the tumor tissues were expressed as increases in brightness on 2-D hot-scale maps of the specific parameter αmod, indicating the absolute values of the percentage changes in m values. In the αmod map for each tumor tissue, the brightness clearly increased with treatment time. In quantitative analysis, the mean values of αmod were calculated. The mean value of αmod for the right renal cell carcinoma increased to 1.35 dB with increasing treatment time, and the mean value of αmod for pseudomyxoma peritonei increased to 1.74 with treatment time. The increase in both αmod brightness and the mean value of αmod implied temperature elevations inside the tumor tissues induced by the radiofrequency current; thus, the acoustic method is promising for monitoring temperature elevations inside tumor tissues during hyperthermia.

Temperature elevation in tissue detected in vivo based on statistical analysis of ultrasonic scattered echoes.

It is demanded to monitor temperature in tissue during oncological hyperthermia therapy. In the present study, we non-invasively measured the temperature elevation inside the abdominal cavity and tumour tissue of a living rat induced by capacitive-coupled radiofrequency heating. In the analysis of ultrasound scattered echoes, the Nakagami shape parameter m in each region of interest was estimated at each temperature. The Nakagami shape parameter m has temperature dependence; hence, the temperature increase inside tissue specimens can be detected with the m values. By carrying out in vivo experiments, we visualized the temperature increase inside the abdominal cavity and tumour tissue of living rats using two-dimensional hot-scale images indicating the absolute values of the ratio changes of the m values. In both the abdominal cavity and tumour tissue, the brightness in the hot-scale images clearly increased with increasing temperature. The increases in brightness in the hot-scale images imply the temperature elevations inside the abdominal cavity and tumour tissue of the living rats. The study results prove that the acoustic method we proposed is a promising method for monitoring changes in the internal temperature of the human body under hyperthermia treatment.

In vitro comparison of conventional hyperthermia and modulated electro-hyperthermia.

Radiofrequency-induced hyperthermia (HT) treatments for cancer include conventional capacitive coupling hyperthermia (cCHT) and modulated electro-hyperthermia (mEHT). In this study, we directly compared these methods with regard to in vitro cytotoxicity and mechanisms of action under isothermal conditions. Hepatoma (HepG2) cells were exposed to HT treatment (42°C for 30 min) using mEHT, cCHT or a water bath. mEHT produced a much higher apoptosis rate (43.1% ± 5.8%) than cCHT (10.0% ± 0.6%), the water bath (8.4% ± 1.7%) or a 37°C control (6.6% ± 1.1%). The apoptosis-inducing effect of mEHT at 42°C was similar to that achieved with a water bath at 46°C. mEHT also increased expression of caspase-3, 8 and 9. All three hyperthermia methods increased intracellular heat shock protein 70 (Hsp70) levels, but only mEHT greatly increased the release of Hsp70 from cells. Calreticulin and E-cadherin levels in the cell membrane also increased after mEHT treatment, but not after cCHT or water bath. These results suggest that mEHT selectively deposits energy on the cell membrane and may be a useful treatment modality that targets cancer cell membranes.

Effects of nitrogen on the apoptosis of and changes in gene expression in human lymphoma U937 cells exposed to argon-based cold atmospheric pressure plasma.

Cold atmospheric pressure plasma (CAP) is known as a source of biologically active agents, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present study, we examined the effects of nitrogen (N2) on the apoptosis of and changes in gene expression in human lymphoma U937 cells exposed to argon (Ar)-CAP. Enormous amounts of hydroxyl (·OH) radicals in aqueous solution were produced using Ar­CAP generated using a 20 kHz low frequency at 18 kV with a flow rate of 2 l/min. The increase in the levels of ·OH radicals was significantly attenuated by the addition of N2 to Ar gas. On the other hand, the level of total nitrate/nitrite in the supernatant was significantly elevated in the Ar + N2-CAP­exposed U937 cells. When the cells were exposed to Ar­CAP, a significant increase in apoptosis was observed, whereas apoptosis was markedly decreased in the cells exposed to Ar + N2-CAP. Microarray and pathway analyses revealed that a newly identified gene network containing a number of heat shock proteins (HSPs), anti-apoptotic genes, was mainly associated with the biological function of the prevention of apoptosis. Quantitative PCR revealed that the expression levels of HSPs were significantly elevated in the cells exposed to Ar + N2-CAP than those exposed to Ar­CAP. These results indicate that N2 gas in Ar­CAP modifies the ratio of ROS to RNS, and suppresses the apoptosis induced by Ar­CAP. The modulation of gaseous conditions in CAP may thus prove to be useful for future clinical applications, such as for switching from a sterilizing mode to cytocidal effect for cancer cells.

Evaluation of ¹77Lu-EDTMP in Dogs with Spontaneous Tumor Involving Bone: Pharmacokinetics, Dosimetry and Therapeutic Efficacy.

177Lu-EDTMP is currently being investigated as a potential agent for providing palliative care to the patients suffering from bone pain due to metastatic skeletal carcinoma. The present article describes the evaluation of 177Lu-EDTMP complex in four different canine patients with different types of primary and metastatic skeletal lesions with respect to its pharmacokinetic properties, dosimetry and therapeutic efficacy. The dogs were treated with a dose of ~44.4 MBq (1.2 mCi) per kg body weight of 177Lu-EDTMP, synthesized in-house with high radiochemical purity (98.8 ± 0.4 %) and excellent in vitro stability. The radiopharmaceutical showed favourable pharmacokinetic properties, such as, preferential accumulation at skeletal lesion sites and fast clearance from blood and other non-target organs through urinary route. The administered dose of the radiopharmacutical showed excellent therapeutic efficacy in case of a dog suffering from skeletal metastasis originating from primary tumor elsewhere. On the other hand, two of the remaining three patients with primary bone cancer showed stable disease intially with palliative effect. The fourth patient having metal implant induced osteosarcoma with severe limb oedema did not show any response to the treatment.

Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy.

BACKGROUND: The treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs. METHODS: The CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied. RESULTS: mEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity. CONCLUSIONS: This study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy.

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.

Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia.

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.

Modulated electro-hyperthermia enhances dendritic cell therapy through an abscopal effect in mice.

The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3+ and CD8+ cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3+ and CD8+ cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Strong synergy of heat and modulated electromagnetic field in tumor cell killing.

BACKGROUND AND PURPOSE: Hyperthermia is an emerging complementary method in radiooncology. Despite many positive studies and comprehensive reviews, the method is not widely accepted as a combination to radiotherapy. Modulated electrohyperthermia (mEHT; capacitive, electric field modulated, 13.56 MHz) has been used in clinical practice for almost 2 decades in Germany, Austria and Hungary. This in vivo study in nude mice xenograft tumors compares mEHT with "classic" radiative hyperthermia (radHT). MATERIAL AND METHODS: Nude mice were xenografted with HT29 human colorectal carcinoma cells. 28 mice in four groups with seven animals each and two tumors per animal (totally 56 tumors) were included in the present study: group 1 as untreated control; group 2 treated with radHT at 42 degrees C; group 3 treated with mEHT at identical 42 degrees C; group 4 treated with mEHT at 38 degrees C (by intensively cooling down the tumor). 24 h after treatment, animals were sacrificed and the tumor cross sections studied by precise morphological methods for the respective relative amount of "dead" tumor cells. RESULTS: The effect of mEHT established a double effect as a synergy between the purely thermal (temperature-dependent) and nonthermal (not directly temperature-dependent) effects. The solely thermal enhancement ratio (TER) of cell killing was shown to be 2.9. The field enhancement ratio (FER) at a constant temperature of 42 degrees C was measured as 3.2. Their complex application significantly increased the therapeutic enhancement to 9.4. CONCLUSION: mEHT had a remarkable cancer cell-killing effect in a nude mice xenograft model.

Usefulness of (99m)Tc(V)-dimercaptosuccinic acid scintigraphy in the assessment of response to external radiation therapy in soft tissue sarcoma in Giant Snauzer dog.

A nine-year-old male black Giant Schnauzer dog was referred for the scintigraphic evaluation with a history of malignant fibrosarcoma with a rapidly growing non painful mass on the left shoulder region quite near to the site of an operation performed four months ago. We carried out oncological scintigraphy using pentavalent (99m)Technetium labelled dimercaptosuccinic acid [(99m)Tc(V)-DMSA], a tumour localising radiopharmaceutical agent. The study was performed to assess the margins, vascularity of the tumour and response to the cancer therapy. Uniform intense radiopharmaceutical uptake was observed in the lesion indicating its margins, vascularity and malignant nature. The dog was subjected to external radiation therapy to control the growth of the cancer and to bring the tumour mass to an operable size. The dog was followed up with (99m)Tc(V)-DMSA scintigraphy pre-irradiation and post-irradiation. Immediately after the post-irradiation scintigraphy, the dog was operated on. During the surgery, resection of the tumour margins was performed carefully using a hand held gamma probe to assure that no tumour tissue was left inside. In conclusion, the authors would like to state that (99m)Tc(V)-DMSA oncoscintigraphy is valuable in the assessment and evaluation of therapy in canine soft tissue cancer.

Preliminary studies with 188rhenium-tin colloid for radiation synovectomy: preparation, size determination, in-vivo distribution, effect and dosimetry studies.

BACKGROUND: Generator-produced beta-emitting radionuclides such as (188)Re are gaining in importance for radiosynoviorthesis because of their availability on a regular basis. MATERIAL AND METHODS: We prepared a (188)Re-tin colloid in a reaction carried out either at 100 degrees C or at room temperature (RT). The size of the colloid particles was measured with a laser lightscattering method, and their biodistribution, dosimetric aspects and therapeutic effects were studied in an antigen-induced arthritis (AIA) model in rabbits. (188)Re-tin colloid solution was injected intra-articularly into the knee joints of rabbits with AIA and imaging studies were performed. Blood samples were collected post injection for estimation of the blood residence time. We also injected 2 intact rabbits in the same manner with (188)Re perrhenate solution in order to observe its effects and distribution in the body. All the treated rabbit knees were subjected to histopathology. RESULTS: The colloid particle size distribution was different after preparation at the different reaction temperatures, with a more suitable mean of 4.53 micro m in the RT preparation. The dose delivered to the synovial surface was between 3.51 and 4.21 Gy and that to the bone surface was between 0.70 and 0.84 Gy. Histopathologic examination revealed the development of fibrous connective tissue in the AIA knees 4 weeks after treatment, but not in the control group. CONCLUSIONS: The (188)Re-tin colloid preparation used in this study was suitable for radiation synovectomy application. It requires modifications in the preparation protocol so as to increase the labeling efficiency in correlation with an appropriate particle size.

Sentinel lymph node detection in canine oncological patients.

BACKGROUND: Sentinel lymph node detection was investigated in dogs with spontaneously occurring tumours. MATERIAL AND METHODS: In this pilot study, 24 client-owned spontaneously tumorous dogs presented for sentinel lymph node detection. A multiple method was used with a nuclear medicine technique (injection of 99mTc human serum albumin colloid) with scintigraphy and intraoperative guidance, and blue dye injection. RESULTS: Of the 35 lymph nodes histologically demonstrated to contain metastases, 34 (97%) were found by radioguided surgery, which means that one would have been missed in the intraoperative localisation process; 31 nodes (89%) were clearly visualised in the gamma camera images; only 27 (77%) were blue-stained by vital dye; a mere 8 lymph nodes (23%) were enlarged and therefore easily detectable by palpation. CONCLUSIONS: Data obtained from the harmless application of the sentinel node concept are useful for the radiopharmaceutist. The sentinel lymph node concept is well applicable in the veterinary clinic.