Optimizing the factors which modify thermal enhancement of melphalan in a spontaneous murine tumor

Background: Hyperthermia enhances the cytotoxicity of some chemotherapeutic agents. Both clinical and laboratory studies suggest melphalan may be an important drug when hyperthermia is added to chemotherapy treatments. Factors that may modify the thermal enhancement of melphalan were studied to optimize its clinical use with hyperthermia. Methods: The tumor studied was an early-generation isotransplant of a spontaneous C3Hf/Sed mouse fibrosarcoma, Fsa-II. All studies were performed under supervision of the Animal Care and Use Committee. Hyperthermia was administered by immersing the tumor-bearing foot into a constant temperature water bath. Four factors were studied: duration of hyperthermia, sequencing of hyperthermia and melphalan, intensity of hyperthermia, and tumor size. To study duration of hyperthermia tumors were treated at 41.5°C for 30 or 90 min immediately after intraperitoneal administration of melphalan. For sequencing of hyperthermia and melphalan, animals received hyperthermia treatment of tumors for 30 min at 41.5°C immediately after drug administration, both immediately and 3 h after administration of drug or only at 3 h after administration of drug. Intensity of hyperthermia was studied using heat treatment of tumors for 30 min at 41.5 or 43.5°C immediately following drug administration. Effect of tumor size was studied by delaying experiments until three times the tumor volume (113 mm³) was observed. Treatment of tumors was for 30 min at 41.5°C immediately following drug administration. Tumor response was studied by the mean tumor growth time. Results: Hyperthermia in the absence of melphalan had a small but significant effect on tumor growth time at 43.5°C but not at 41.5°C. Hyperthermia at 41.5°C immediately after melphalan administration doubled mean tumor growth time at 30 min and caused a threefold increase at 90 min (P=0.0002) when compared to tumors treated with melphalan alone at room temperature. Application of hyperthermia for one-half hour immediately following drug administration was the most effective in delaying tumor growth. No significant difference in mean tumor growth time was observed with an increase in temperature from 41.5 to 43.5°C. For large tumors heat alone and melphalan alone caused a moderate increase in tumor growth delay. These effects in large tumors were greatly increased by a combination of chemotherapy and hyperthermia. Conclusions: From our data it would appear that the administration of intraperitoneal melphalan immediately prior to 90 min of heat at 41.5°C may optimize anti-neoplastic activity. These data may be useful in formulating clinical protocols in which melphalan and heat are combined.     

Dissecting the role of hyperthermia in natural killer cell mediated anti-tumor responses

The effects of hyperthermia on natural killer (NK) cell cytotoxicity against tumor cell targets are not yet fully understood. A more complete understanding of these effects could be important for maximizing the clinical benefits obtained by using hyperthermia for cancer therapy. Here, we summarize results in the literature regarding the effects of elevated temperatures on NK cells and our own recent data on the effects of fever-range temperatures. At treatment temperatures above 40°C, (which is above the physiological body temperatures normally achieved during fever or exercise), both enhancing and inhibitory effects on cytotoxic activity of NK cells against tumor cells have been reported. Our own results have shown that fever-range thermal stress (using a temperature of 39.5°C) enhances human NK cell cytotoxicity against tumor target cells. This effect requires function of the NKG2D receptor of NK cells, and is maximal when both NK and tumor cell targets are heated. Reported heat sensitive cellular targets affected by hyperthermia on tumor cells include heat shock proteins, MICA and MHC Class I. In NK cells, plasma membrane reorganization may occur after mild heat stress. We conclude this review by listing several unresolved questions that should be addressed for a more complete understanding of the molecular mechanisms which underlie the effects of thermal stress on the function of NK cells. Altogether, the available data indicate a strong potential for heat-induced enhancement of NK cell activity in mediating, at least in part, the improved clinical responses seen when hyperthermia is used in combination with other therapies.     

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer

Even with current promising antitumor antibodies, their antitumor effects on stroma‐rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa‐2; moderate, BxPC‐3; and abundant, Capan‐1 and Ope‐xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra‐abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa‐2 model, moderate (1063) in the BxPC‐3 model, and negative in the Capan‐1 and Ope‐xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8‐fold (2980, 3015) in the BxPC‐3 model, 2.5‐ or 4.8‐fold (1881, 3615) in the Capan‐1 model, and 3.2‐ or 4.2‐fold (1469, 1922) in the Ope‐xeno model, respectively. Cetuximab was effective in treating even stroma‐rich and k‐ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.     

Ultrasound-induced hyperthermia for the treatment of human superficial tumors

Ultrasonic systems were developed for the treatment of superficial human tumors, generating local tumor hyperthermia at tumor center temperatures ranging from 43°to 50°. Twenty-eight patients with disease of varying histology were evaluable for response, and demonstrated an overall response rate of 57 % with a complete response rate of 18 %. In 11 patients who had received definitive radiation therapy to the heat treated area, the response rate was 81 %; there were no toxicities other than those noted for the overall study. As temperature was escalated a marked increase was observed both in response rates (from 53% (43–44°C) to 83% (48–50°C) and in duration of response (from 29 to 250 days for the same temperatures). Toxicities were minimal, consisting of superficial blistering in four patients and pain in six patients. Pain was usually associated with tumor involvement in the periostium.     

Radio frequency hyperthermia of advanced human sarcomas

Hyperthermia ? 42°C is tumoricidal in vitro and in many animal models, although such temperatures have only recently been achieved experimentally in some human cancers. A recently developed radio frequency device that provides safe hyperthermia to any depth without surface tissue injury now permits evaluation of the effects of hyperthermia on advanced human sarcomas. Twelve patients with large sarcomas located intraabdominally [7], in the chest wall [2], proximal extremity [2], and the neck [1], were evaluated in this study. Tumor types include liposarcoma [3], rhabdomyosarcoma [2], leiomyosarcoma [2], neurofibrosarcoma [2], and one each malignant mesothelioma, undifferentiated sarcoma, and osteosarcoma. Intratumor temperatures ?42°C were observed in all tumors, with virtually no normal tissue injury. Selective tumor heatin ?45°C occurred in 9/12 (75%) and ?50°C in 6/12 (50%). One to five weekly treatments ?50°C and ten daily treatments ?45°C resulted in significant tumor necrosis and pain relief in some patients. Hyperthermia of advanced sarcomas is possible with little host toxicity and may be of potential therapeutic benefit.     

Implications of increased tumor blood flow and oxygenation caused by mild temperature hyperthermia in tumor treatment

In many past clinical studies in which hyperthermia enhanced the efficacy of radiotherapy, the tumor temperatures could be raised only to 40–42°C range in most cases. The heat-induced cell death, cellular radiosensitization, and vascular damage induced by such mild temperature hyperthermia (MTH) are likely to be insignificant despite the increased response of tumors to radiotherapy. Heating rodent tumors at 40–42°C was found to cause an enduring increase in blood flow and oxygenation in the tumors. Recent studies with canine soft tissue sarcoma and human tumor clinical studies also demonstrated that MTH improves tumor oxygenation, and enhances response of the tumors to radiotherapy or chemoradiotherapy. The increased blood flow and vascular permeability caused by MTH may also improve the delivery of various therapeutic agents such as chemotherapy drugs, immunotherapeutic agents and genetic constructs for gene therapy to tumor cells. MTH as a means to potentiate the efficacy of radiotherapy and others warrants further investigation.     

Quality assurance guidelines for interstitial hyperthermia

Quality assurance (QA) guidelines are essential to provide uniform execution of clinical hyperthermia treatments and trials. This document outlines the clinical and technical consequences of the specific properties of interstitial heat delivery and specifies recommendations for hyperthermia administration with interstitial techniques. Interstitial hyperthermia aims at tumor temperatures in the 40–44?°C range as an adjunct to radiation or chemotherapy. The clinical part of this document imparts specific clinical experience of interstitial heat delivery to various tumor sites as well as recommended interstitial hyperthermia workflow and procedures. The second part describes technical requirements for quality assurance of current interstitial heating equipment including electromagnetic (radiative and capacitive) and ultrasound heating techniques. Detailed instructions are provided on characterization and documentation of the performance of interstitial hyperthermia applicators to achieve reproducible hyperthermia treatments of uniform high quality. Output power and consequent temperature rise are the key parameters for characterization of applicator performance in these QA guidelines. These characteristics determine the specific maximum tumor size and depth that can be heated adequately. The guidelines were developed by the ESHO Technical Committee with participation of senior STM members and members of the Atzelsberg Circle.     

Effects of intraoperative hyperthermia on canine sciatic nerve: histopathologic and morphometric studies

Failure to achieve local control in the treatment of pelvic and retroperitoneal tumours results in a high rate of recurrences. The objective of intraoperative hyperthermia (IOHT) is to enhance the effect of intraoperative radiation therapy and to increase local tumour control. The tolerance of peripheral nerves to heat may limit the heat dose that can be applied to tumours. Histopathologic and histomorphometric changes of canine sciatic nerve after 60-min IOHT were studied in three groups of five dogs each for temperatures of 43, 44 and 45°C. IOHT was performed using a water-circulating hyperthermia device with a multichannel thermometry system on surgically exposed sciatic nerve. Histopathologic and histomorphometric studies were done immediately, 3 weeks and 12 months after IOHT. Histologic changes observed immediately after treatment were minimal but at 3 weeks following 60-min 45°C IOHT both axon and myelin loss and an increase in endoneurial fibrous tissue were observed. Twelve months after treatment a statistically significant decrease in axon, myelin and small vessel percentages as well as an increase in endoneurial and epineural connective tissue were observed for dog treated to 45°C. Dog treated to 44°C for 60 min had similar statistically significant but less severe changes. Twelve months after 43°C IOHT for 60 min, nerve fibres appeared normal and endoneurial connective tissue was only increased mildly around small and medium-sized vessels. These results suggest that temperatures to the peripheral nerve >44°C for 60 min are likely to cause significant histopathologic changes that can be found 12 months after treatment. A hypothesis of the mechanism of heat injury to peripheral nerves was developed.     

Localized current field hyperthermia: Effect on normal ocular tissue

Using a 500 kHz radiofrequency electromagnetic heating system, the effects of localized current field hyperthermia in normal rabbit eyes were examined. A specially designed scleral plaque placed on normal rabbit eyes was heated to temperatures of 43°C, 45°C, and 47°C for a period of 45 min. The effects of hyperthermia were monitored by clinical examination, fluorescein angiography, electroretinography and histopathology. A graded effect with increasing temperature was found at the lower temperature, and it was confined to the treatment field. At 47^CC the electroretinogram was extinguished due to diffuse photoreceptor damage outside the treatment field, as demonstrated by histo-pathology and electron microscopy. This study indicates that hyperthermia at 45°C for 45 min is me maximum allowable temperature without causing diffuse retinal damage in the normal rabbit eye.     

Sensitizing for cis-diamminedichloroplatinum(II) action by hyperthermia in resistant cells

cDDP-resistant Ehrlich ascites tumour (EAT) cells (ER cells) were tested for cellular content of total glutathione, heat sensitivity, cDDP sensitivity and synergistic effects of a combined treatment of heat and chemotherapy. In comparison with the non-resistant EAT cells (EN) the ER cells had an elevated level of glutathione. Treatment with d,l-buthionine-(S,R)-sulphoximine SO), resulting in almost complete depletion of cellular glutathione, did not cause drug sensitization. The ER cells were somewhat less heat sensitive compared with the EN cells. Heat chemosensitization was observed for the EN cells as well as for the ER cells. At 43°C (but not at 42°C) the thermal enhancement ratio (TER) for cDDP toxicity was significantly higher in the ER cells. The total number of cells killed by the combined treatment was less in the ER cells than in the EN cells. After analysing existing literature, combined with the current results, it is concluded that although cDDP-resistant cells can often considerably be chemosensitized by hyperthermia, in most cases the difference in cDDP sensitivity cannot be overcome totally. In those situations where cDDP-resistant cells are more sensitive to heat and also show a high TER, especially at clinically relevant temperatures, hyperthermia as added modality is indicated for clinical treatment.     

Clinical experiences with local microwave hyperthermia

At the Claire Zellerbach Saroni Tumor Institute, Mount Zion Hospital and Medical Center, 38 patients who failed definitive radiotherapy and chemotherapy were treated with 915 megahertz and 2450 megahertz microwave hyperthermia to observe normal tissue tolerance and therapeutic responses. Superficial and measurable lesions were selected. Thirty-seven courses were given with radiation and eleven courses were given alone. When hyperthermia was combined with radiation, complete clinical regression occurred in 41 % ($\text{15}\text{37}$) of patients and partial regression in 37% ($\text{14}\text{37}$); however with hyperthermia alone, complete regression occurred in 18% ($\text{2}\text{11}$) of patients and partial regression in 18 % ($\text{2}\text{11}$). Thus, moderate local tumor hyperthermia (42.5°C) following low dose irradiation (1800–2700 rad) has resulted in significant responses in recurrent tumors in previously irradiated areas. The maximum temperature achieved during a course of treatment appeared to correlate with tumor responses. Also a relationship existed between radiation dose and tumor response. There was no relationship between radiation dose and thermal side effects. Thermal dosimetry remained an outstanding problem for clinical hyperthermia, in part because of inadequacy of heat delivery and measurement systems, and in part because of patient variations in terms of tolerance to beat and tumor physiological changes with fractions of hyperthermia. Side effects of thermal blistering and burns were correlated with maximum temperatures attained during heat treatments. They were tolerable by patients, and can be decreased by appropriate skin cooling in some patients. Further protocol studies are needed to determine the optimal temperature/radiation dosage, treatment schedules and sequences, and treatment techniques.     

Effects of hyperthermic temperatures and the synthesis of heat-shock proteins on the lateral diffusion of H-2Kk

The effect of hyperthermia, fractionated heat and the synthesis of heat shock proteins on the lateral diffusion of H-2K^k was examined in RDM-4, Ch-1 and mouse L cells. Cells in suspension were examined immediately after heating, adaptation to growth at 40°C for 72 h or 6 h after exposure to sodium arsenite (5 μM), iodoacetamide (10 μM) or a 20 min heat shock at 45°C. No heat shock protein synthesis could be measured in CH-1 cells in response to either heat or chemical stress. Synthesis of these proteins was observed in both RDM-4 and mouse L cells in response to heat or chemical treatments, and the kinetics of synthesis were similar to those reported in the literature for other eukaryotic cell lines. The diffusion of fluorescein isothiocyanate-labelled monoclonal anti-H-2K^k bound to heat (or chemically) treated cells was measured by the technique of fluorescence recovery after pattern photobleaching. After a 30 min exposure to temperatures between 41°C and 45°C, a temperature-dependent decrease in recovery was observed. At 45°C, 75–100% of the surface antigen was found to be immobile. The diffusion coefficients of the mobile fractions were not significantly changed at all the temperatures examined. Heat effects on the recovery patterns of all the three cell lines were qualitatively similar and unaffected by the presence or absence of heat or chemically induced heat shock proteins. After 48–60 h following a heat shock, the recovery patterns were similar to those of unheated control cells.     

Change in the in vivo hyperthermic response resulting from the metabolic effects of temporary vascular occlusion

Previous workers have reported that clamping of animal tumors in vivo enhanced the effect of hyperthermia; the enhancement has been attributed to pH and nutritional effects of vascular occlusion. It has not been clear, however, the degree to which improved heating patterns or effects on the tumor cells and vasculature from the clamping procedure itself might have contributed to the observed effect. In the experiments herein reported, care was taken to insure comparable heating of C3H mouse mammary tumors transplanted on the flank whether clamped or unclamped. Clamping for one hour with hyperthermia during the final 30 minutes caused a marked thermosensitization as measured by tumor control. The temperature at 30 minutes heating to control 50% of the tumors for 120 days (TCT 50–120) was reduced from 46.8°C in controls to 43.5°C in clamped tumors, a difference of 3.3 ± 0.09°C. No cytotoxicity from the clamping alone was evident by assessment of subsequent tumor growth and no lasting vascular effects could be detected by ¹³³Xe washout and tumor growth. Since the techniques used produced essentially identical heating patterns, we conclude that the striking enhancement in hyperthermic response in clamped tumors can be attributed to the metabolic consequences of temporary vascular occlusion.     

Temperature-dependent changes in physiologic parameters of spontaneous canine soft tissue sarcomas after combined radiotherapy and hyperthermia treatment

PURPOSE: The objectives of this study were to evaluate effects of hyperthermia on tumor oxygenation, extracellular pH (pHe), and blood flow in 13 dogs with spontaneous soft tissue sarcomas prior to and after local hyperthermia. METHODS AND MATERIALS: Tumor pO2 was measured using an Eppendorf polarographic device, pHe using interstitial electrodes, and blood flow using contrast-enhanced magnetic resonance imaging (MRI). RESULTS: There was an overall improvement in tumor oxygenation observed as an increase in median pO2 and decrease in hypoxic fraction (% of pO2 measurements <5 mm Hg) at 24-h post hyperthermia. These changes were most pronounced when the median temperature (T50) during hyperthermia treatment was less than 44 degrees C. Tumors with T50 > 44 degrees C were characterized by a decrease in median PO2 and an increase in hypoxic fraction. Similar thermal dose-related changes were observed in tumor perfusion. Perfusion was significantly higher after hyperthermia. Increases in perfusion were most evident in tumors with T50 < 44 degrees C. With T50 > 44 degrees C, there was no change in perfusion after hyperthermia. On average, pHe values declined in all animals after hyperthermia, with the greatest reduction seen for larger T50 values. CONCLUSION: This study suggests that hyperthermia has biphasic effects on tumor physiologic parameters. Lower temperatures tend to favor improved perfusion and oxygenation, whereas higher temperatures are more likely to cause vascular damage, thus leading to greater hypoxia. While it has long been recognized that such effects occur in rodent tumors, this is the first report to tie such changes to temperatures achieved during hyperthermia in the clinical setting. Furthermore, it suggests that the thermal threshold for vascular damage is higher in spontaneous tumors than in more rapidly growing rodent tumors.     

BIOLOGICAL EFFECTS OF TWEEN－80 IN COMBINATION WITH HYPERTHERMIA ON HUMAN STOMACH CANCER CELL LINE BGC－823

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The pharmacologic manipulation of blood flow in hyperthermia therapy

Many human tumors treated by hyperthermia do not reach therapeutic temperatures (42°C). The explanation for this difference may be that some tumors react to thermal stress in a manner similar to normal tissues; ie, they increase blood flow during hyperthermia in order to dissipate the heat. Higher temperatures might be achieved in these heat-resistant tumors by administering vasoconstrictive agents in an effort to reduce blood flow. In this preliminary study, we determined the extent to which pharmacologic inhibition of local blood flow might allow higher temperatures to develop in normal muscles exposed to localized radiofrequency hyperthermia. We found that the local muscle temperature rise could be increased by at least 90% in two dogs and six rabbits with the use of a local vasoconstrictive drug.     

A comparison of temperatures in canine solid tumours during local and whole-body hyperthermia administered alone and simultaneously

Temperature measurements were made in canine solid tumours during whole-body hyperthermia (WBH) alone, local hyperthermia alone and local hyperthermia given simultaneously with WBH. During the plateau phase of WBH alone, mean intratumoral temperature ranged from 41.3± 0.2°C to 41.7 ± 0.1°C and was statistically lower (P=0.0028) and more variable than rectal temperature, which ranged from 42.0 ± 0.02°C to 42.1 ± 0.03°C. The temperature distribution in solid tumours during WBH is more uniform than during local hyperthermia. The simultaneous administration of whole-body and local hyperthermia in five dogs resulted in increased tumour temperatures in comparison to WBH and in more uniformly increased tumour temperatures in comparison to local hyperthermia alone. Median intratumoral temperatures (± 95 % confidence intervals) resulting from local hyperthermia alone and local hyperthermia given simultaneously with WBH were 39.9°C (39.7–40.1) and 42.9°C (42.6–43.1), respectively, and were statistically different (P=0.0012). Local applied power requirements to meet predetermined intratumoral temperature limits were decreased by 50% (P=0.011) in dogs undergoing combined local/whole-body hyperthermia versus local hyperthermia alone. Dogs tolerated the combination of local and WBH without complication.     

Dissecting the role of hyperthermia in natural killer cell mediated anti-tumor responses.

The effects of hyperthermia on natural killer (NK) cell cytotoxicity against tumor cell targets are not yet fully understood. A more complete understanding of these effects could be important for maximizing the clinical benefits obtained by using hyperthermia for cancer therapy. Here, we summarize results in the literature regarding the effects of elevated temperatures on NK cells and our own recent data on the effects of fever-range temperatures. At treatment temperatures above 40 degrees C, (which is above the physiological body temperatures normally achieved during fever or exercise), both enhancing and inhibitory effects on cytotoxic activity of NK cells against tumor cells have been reported. Our own results have shown that fever-range thermal stress (using a temperature of 39.5 degrees C) enhances human NK cell cytotoxicity against tumor target cells. This effect requires function of the NKG2D receptor of NK cells, and is maximal when both NK and tumor cell targets are heated. Reported heat sensitive cellular targets affected by hyperthermia on tumor cells include heat shock proteins, MICA and MHC Class I. In NK cells, plasma membrane reorganization may occur after mild heat stress. We conclude this review by listing several unresolved questions that should be addressed for a more complete understanding of the molecular mechanisms which underlie the effects of thermal stress on the function of NK cells. Altogether, the available data indicate a strong potential for heat-induced enhancement of NK cell activity in mediating, at least in part, the improved clinical responses seen when hyperthermia is used in combination with other therapies.     

Effects of heat stress on the level of heat shock protein 70 on the surface of hepatocellular carcinoma Hep G2 cells: implications for the treatment of tumors

The ability to distinguish tumor cells from normal cells is vital to allow the immune system to selectively destroy tumor cells. In order to find an effective marker, we used enzyme-linked immunosorbent assay, immunocytochemistry, immunofluorescence, and flow cytometry to investigate the effects of heat stress on the amount of heat shock protein 70 on the surface of tumor cells (Hep G2 cells). Heat shock protein 70 is the major stress-induced heat shock protein found on the surface of tumor cells. Our results indicate that the percentage of Hep G2 cells with a detectable level of heat shock protein 70 on their cell surface increased significantly (P?<?0.05) following heat stress at 42 °C for 2 h (up to 1.92 times the level before heat treatment). The detectable level of heat shock protein 70 on the surface of Hep G2 cells reached its peak 12 h after treatment. However, the fluorescent intensity of stressed and unstressed Hep G2 cells was not significantly different (P?>?0.05). The increase in the level of heat shock protein 70 on the surface of tumor cells following heat stress could provide a basis for finding novel immunotoxins as targets for drug action and may have application to be used in conjunction with hyperthermia in the treatment of tumors.