Selective tumor heating by shortwave radiofrequency (RF)

Experimental solid tumors were treated in vivo with external high frequency dielectric heating to observe any heat selectivity between the tumor mass on one hand, and the subcutaneous tissue, muscle, and systemic temperatures on the other. Methylcholantrene-induced sarcoma cells were inoculated into the subcutaneous tissue or muscles of the posterior thigh of isologous Fischer rats. When the tumor mass reached the desired size, dielectric heating with a fixed frequency of 13.56 megahertz (MHz) was applied locally to the tumor-bearing area. All the periods of treatment were kept constant at 1 hour. Temperature was measured with thermocouple probes inserted directly into the tumor mass and the tissues lying within the electromagnetic field. Systemic temperature was monitored via a clinical mercury thermometer inserted into the rectum. Temperature recordings were taken at 5-minute intervals during which time the power was turned off in order to avoid the RF interference and to allow thermal equilibrium between the probes and the tissue. The results showed a high selective temperature gradient for the tumor mass as compared to the subcutaneous and muscle tissues when tumor masses were greater than 1.0 cu cm. No selectivity was detected in small tumors or in nontumor-bearing controls. Systemic temperature did not rise by these treatments. No tumor regression was observed at this dosage. Burns were noted in those animals in which normal tissue temperature rose above 43 C.     

Combined treatment of inoperable carcinomas of the uterine cervix with radiotherapy and regional hyperthermia. Results of a phase II trial

AIM: The disappointing results for inoperable, advanced tumors of the uterine cervix after conventional radiotherapy alone necessitates improving of radiation therapy. Simultaneous chemotherapy or altered radiation fractionation, such as accelerated regimen, increase acute toxicity and treatment is often difficult to deliver in the planned manner. The purpose of this phase II study was to investigate the toxicity and effectiveness of a combined approach with radiotherapy and regional hyperthermia. PATIENTS AND METHODS: From January 1994 to October 1995 18 patients with advanced carcinomas of the uterine cervix were treated in combination with radiotherapy and hyperthermia. The patients were treated with 6 to 20 MV photons delivered by a linear accelerator in a 4-field-box technique to a total dose of 50.4 Gy in 28 fractions. In the first and fourth week 2 regional hyperthermia treatments were each applied with the Sigma-60 applicator from a BSD-2000 unit. After this a boost to the primary tumor was given with high-dose-rate iridium-192 brachytherapy by an afterloading technique with 4 x 5 Gy at point A to a total of 20 Gy and for the involved parametrium anterioposterior-posterioanterior to 9 Gy in 5 fractions. RESULTS: The acute toxicity was low and similar to an external radiotherapy alone treatment. No Grade III/IV acute toxicity was found. The median age was 47 years (range 34 to 67 years). In 16 of 18 patients a rapid tumor regression was observed during combined thermo-radiotherapy, which allowed the use of intracavitary high-dose-rate brachytherapy in these cases. Complete and partial remission were observed in 13 and 4 cases, respectively. One patient did not respond to the treatment. The median follow-up was 24 months (range 17 to 36 months). The local tumor control rate was 48% at 2 years. Median T20, T50 and T90 values were 41.7 degrees C (range 40.3 to 43.2 degrees C), 41.1 degrees C (range 39.2 to 42.5 degrees C) and 39.9 degrees C (range 37.7 to 41.9 degrees C), respectively. Cumulative minutes of T90 > 40 degrees C (Cum40T90) and cumulative minutes, which were isoeffective to 43 degrees C, were calculated (CEM43T90, CEM43T50, CEM43T20). CEM43T90 was found to be a significant parameter in terms of local tumor control for the 4 hyperthermia treatments (p = 0.019). CONCLUSIONS: This treatment modality has proved to be feasible and well tolerable. The rapid tumor shrinkage in the combined approach of radiotherapy with hyperthermia before beginning brachytherapy seems to be a good prerequisite for improving of the disappointing results in cure of advanced cancer of the uterine cervix.     

Enhancement by hyperthermia of the in vitro cytotoxicity of mitomycin C toward hypoxic tumor cells

Mitomycin C and hyperthermia are both toxic to chronically hypoxic EMT6 tumor cells. Combinations of this drug and heat were tested in vitro in normally aerated and chronically hypoxic EMT6 mouse mammary tumor cells to establish whether greater than additive cytotoxicity could be achieved by combined treatment. Cell survival was measured at four concentrations of mitomycin C (0.01, 0.1, 1.0, and 10 microM) at 37 degrees or at elevated temperatures (41, 42, and 43 degrees) for durations of 1, 2, 3, and 6 hr. At 42 degrees, exposure to mitomycin C for 3 and 6 hr produced a 2- to 3-fold increase in hypoxic tumor cell kill at all drug concentrations over that expected for strict additivity. A 15-fold enhancement in the kill of hypoxic tumor cells was obtained at 1.0 and 10 microM mitomycin C at 43 degrees for 6 hr of exposure. Under most conditions, additivity was observed for the antibiotic and heat in oxygenated cells, except at 43 degrees with 0.01 and 0.1 microM mitomycin C following 3 and 6 hr of treatment, conditions under which a 5- to 10-fold potentiation of tumor cell kill was obtained. The rate of formation of reactive metabolites from mitomycin C under anaerobic conditions in EMT6 cell-free preparations was measured. A 30 to 50% increase in alkylating activity was observed at elevated temperatures, suggesting that the enhanced cytotoxicity of mitomycin C with heat toward hypoxic cells may, in part, be due to an increase in activation of the drug.     

Preclinical assessment of hypoxic marker specificity and sensitivity

PURPOSE: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. MATERIALS AND METHODS: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. RESULTS: The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. CONCLUSIONS: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.     

Improvement of tumor oxygenation status by mild temperature hyperthermia alone or in combination with carbogen

The effects of mild temperature hyperthermia (MTH) on the oxygenation and radioresponse in rodent tumors were investigated. FSall tumors grown in the legs of C3H mice and R3230 AC tumors grown in the legs of Fischer rats were heated with a water bath and the partial pressure of oxygen (pO2) was determined using the microelectrode method. In FSall tumors, the pO2 measured immediately after heating at 41.5 degrees C for 1 hour was markedly higher than that in the control tumors, whereas heating at higher temperatures for 1 hour decreased the tumor oxygenation. In R3230 AC tumors, heating at 41.5 degrees C for 1 hour caused a moderate increase in the pO2 and heating at 42.5 degrees or 43.5 degrees C for 30 minutes markedly increased the pO2. However, heating at 42.5 degrees C or higher temperatures for 1 hour decreased the pO2 in the R3230 AC tumors. The improvement of oxygenation in FSall tumors by heating at 41.5 degrees C for 1 hour increased the radiation-induced cell death in these tumors. The combination of carbogen breathing with MTH was far more potent than carbogen breathing or MTH alone in increasing tumor oxygenation and potentiating the radiation effect in FSall tumors.     

Kinetic responses of murine sarcoma cells to radiation and hyperthermia in vivo and in vitro

Cells of a solid mouse mammary sarcoma that can be cultured in vitro and which, upon inoculation, grow in vivo into new tumors, were exposed either in vivo or in vitro to doses of 300 or 600 rads of X-rays and/or to a temperature of 43 degrees for 1 hr. DNA histograms obtained with flow cytofluorometry were sampled at regular time intervals after treatments in order to obtain information on the cells' postexposure kinetics. X-irradiation of exponentially growing cells induced the expected G2 block; heat exposure caused cells to accumulate in S and G2. The sequential treatment (300 rads followed by 1 hr of hyperthermia) resulted in a mitotic delay that was longer than the sum of the delays of the individual treatments. The proliferative behavior of cycling cells in the tumor treated with a dose of X-rays was qualitatively similar to that seen for exponentially growing cells in vitro; however, marked differences were seen after 43 degrees exposure. The heat treatment of tumors in vivo caused a significant decrease in the tumor cell density as compared to the X-ray treatment alone. Sequential X-ray and heat treatment induced a higher fraction of cycling cells than that found in control tumors. However, X-ray or heat treatment alone caused no significant recruitment of resting cells into cycle 1 day after treatment. A model that permits estimation of the fraction of resting cells in a tumor is described.     

Augmentation in chemosensitivity of intratumor quiescent cells by combined treatment with nicotinamide and mild hyperthermia

C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Nicotinamide was administered intraperitoneally before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. The tumors were then excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from tumors that had not been pretreated with BrdU labeling. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). In both tumor systems, the MN frequency in Q cells was lower than that in the total cell population. Nicotinamide treatment elevated the MN frequency in total SCC VII cells. Mild heating raised the MN frequency more markedly in Q cells than in total cells. The combination of nicotinamide and mild heat treatment increased the MN frequency more markedly than either treatment alone. In total SCC VII cells, nicotinamide increased 195mPt-cisplatin uptake. Mild heating elevated 195mPt-cisplatin uptake in total EMT6/KU cells. Cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. Nicotinamide sensitized tumor cells including a large acutely hypoxic fraction, such as those of SCC VII tumors, through inhibition of the fluctuations in tumor blood flow. Tumor cells including a large chronically hypoxic fraction such as Q cells were thought to be sensitized by mild heating through an increase in tumor blood flow.     

Optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells

Electrochemical treatment (ECT) of cancer utilizes direct current to produce chemical changes in tumors. ECT has been suggested as an effective alternative local cancer therapy. However, a methodology is not established, and mechanisms are not well studied. In vivo studies were conducted to evaluate the effectiveness of ECT on animal tumor models. Radiation-induced fibrosarcomas were implanted subcutaneously in 157 female C3H/HeJ mice. Larger rat fibrosarcomas were implanted on 34 female Fisher 344 rats. When the spheroidal tumors reached 10 mm in the mice, two to five platinum electrodes were inserted into the tumors at various spacings and orientations. Ten rats in a pilot group were treated when their ellipsoidal tumors were about 25 mm long; electrode insertion was similar to the later part of the mouse study, i.e., two at the base and two at the center. A second group of 24 rats was treated with six or seven electrodes when their tumors were about 20 mm long; all electrodes were inserted at the tumor base. Of the 24 rats, 12 of these were treated once, 10 were treated twice. and 2 were treated thrice. All treated tumors showed necrosis and regression for both mice and rats; however, later tumor recurrence reduced long-term survival. When multiple treatments were implemented, the best 3 month mouse tumor cure rate was 59.3%, and the best 6 month rat tumor cure rate was 75.0%. These preliminary results indicate that ECT is effective on the radiation-induced fibrosarcoma (RIF-1) mouse tumor and rat fibrosarcoma. The effectiveness is dependent on electrode placement and dosage.     

Characterization of organ-specific immunoliposomes for delivery of 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in a mouse lung-metastasis model

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. 111In-labeled 34A-liposomes containing monosialoganglioside (GM1) were prepared that included [3H]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [3H]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of 111In-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen. Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.     

Ultrasonic hyperthermia and drugs as therapy for human tumor xenografts

Ultrasonic energy can generate controlled, local hyperthermia (42 degrees C--43 degrees C), and ultrasonic hyperthermia results in increased cytotoxicity of chemotherapeutic drugs for the treatment of cancer. Human breast (MX-1) and lung (LX-1) tumors implanted subcutaneously in athymic nude mice were treated with a single application of ultrasonic (670 kHz, peak intensity of 5 watts/cm2) hyperthermia (43.0 degrees C +/- 0.5 degrees C) for 30 minutes in combination with suboptimal doses of cyclophosphamide, melphalan, or procarbazine. Delays in the tumor growth rate and temporary tumor regression were observed. Comparison of the tumor growth rate with single modalities, ie, drug or hyperthermia alone, shows evidence of synergistic effects of the combination of ultrasonic hyperthermia and melphalan on MX-1 and of hyperthermia and procarbazine on LX-1 tumors.     

Combined preoperative and postoperative immunotherapy for murine C1300 neuroblastoma

Preoperative treatment of murine C1300-neuroblastoma (C1300) with triple immunotherapy using low-dose cyclophosphamide (CY), retinyl palmitate (RP), and interleukin-2 (IL2), followed by tumor resection leads to significant initial tumor control and prolonged survival. However, because long-term tumor recurrence is 67%, the efficacy of continued postoperative immunotherapy is now evaluated. Thirty-two A/J mice with 1 cm subcutaneous C1300 tumors were treated for 13 days with CY-100 mg/kg, intraperitoneally (IP), on day 2 of treatment then 25 mg/kg on day 9, RP-2500 IU IP 2 x/week, and IL2 1.6 x 10(5) U IP BID on days 4 to 9 and 11 to 13. On day 14, mice were divided into five treatment groups: (1) OP (operated-tumor resection, n = 6); (2) OP+CY (resection and postoperative CY, n = 7); (3) OP+CY+RP (resection and postoperative CY+RP, n = 7); (4) OP+CY+RP+IL2 (resection and postoperative CY+RP+IL2, n = 7); and (5) CY+RP+IL2 (continued CY+RP+IL2 with no resection, n = 5). Survival and postoperative tumor recurrence were followed for 60 days. The cure rates were group 1 33% (2/6), group 2 43% (3/7), group 3 29% (2/7), group 4 71% (5/7), and group 5 20% (1/5). After surgery, tumors that recurred did so in 8 to 22 days, with no statistical difference noted between groups. MHC class I antigenic expression of tumors resected on day 14 and recurrent tumors was determined with monoclonal antibodies and flow cytometry. In tumors resected on day 14, class I expression measured by mean fluorescence, was 374.8 +/- 27.40.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microdialysis assessment of 5-fluorouracil release from thermosensitive magnetoliposomes induced by an electromagnetic field in tumor-bearing mice

The current study was designed to evaluate the properties of thermosensitive magnetoliposomes (TMs), a new drug carrier proposed by the authors, in an electromagnetic field pertaining to their selective heating and drug release under an in vivo condition. TMs containing 5-fluorouracil (5-FU) were prepared by reverse-phase evaporation, injected into the tumor mass of B 16-BL6 melanoma in mice, and selectively heated by a 500-kHz electromagnetic field. The release profile of 5-FU from TMs was examined by using a microdialysis technique. The temperature of TMs in the tumor was effectively elevated to 42 degrees C and maintained at this temperature, overcoming the "cooling effect" of blood flow and surrounding tissues. The release kinetics of 5-FU from TMs was successfully analyzed by physiological modeling, which allows the prediction of intratumor drug concentrations during electromagnetic field exposure under various conditions. In conclusion, this study first demonstrated an in vivo evidence for the electromagnetic field-induced thermosensitive release of a drug from TMs in a tumor with the use of microdialysis.     

Measurement of tumor vascular damage in mice with 99mTc-MIBI following photodynamic therapy

The clinical perfusion agent 99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin (PII)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg-1 PII. Twenty-four hours later, one tumor was illuminated (600-650 nm, 200 mW cm-2, 400 J cm-2) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of 99mTc-hexakismethoxyisobutylisonitrile (MIBI) (0.18 MBq g-1) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g-1) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of 99mTc-MIBI is a simple method to assess TVP and vascular damage induced by PDT.     

Correlation of very late activation integrin and CD44 expression with extrarenal invasion and metastasis of renal cell carcinomas

Cell adhesion molecules mediate cell-cell and cell-matrix interactions, and they are thought to play an important role in tumor invasion and metastasis. Altered expression of integrins and CD44 in renal cell carcinoma has been recently demonstrated, but an association with invasive or metastatic behavior has not been reported. We examined very late activation (VLA) integrin and CD44 expression in 37 renal cell carcinomas and correlated adhesion molecule expression with multiple histological and clinical parameters. Most tumors exhibited positive staining for VLA3 (81%). Approximately one third of the tumors stained positively for VLA6 and CD44, and fewer (27%) were positive for VLA2. Only a few tumors were positive for VLA4 (8%) and VLA5 (14%). Most of the tumors exhibiting positive staining showed a combination of membranous and cytoplasmic staining patterns. Low-grade tumors positive for VLA6 showed a tendency for basilar staining of the tumor cells, whereas high-grade tumors exhibited diffuse cytoplasmic staining. All tumors exhibiting weak or strong positive staining for VLA4 or VLA5 showed extrarenal invasion or were known to have developed metastases at the time of nephrectomy. All tumors strongly positive for VLA2 or CD44 showed invasion beyond the renal capsule or metastases. In contrast to a previous study, no association was observed between positive staining and tumor grade. Nor were tumor size, architectural pattern, cell type, or DNA ploidy found to be associated with particular staining patterns. Although many of the invasive tumors showed no difference in VLA integrin or CD44 expression compared with tumors confined to the kidney, increased expression in some of them suggests that these cell adhesion molecules may contribute to the invasive or metastatic phenotype.     

Antitumor activity of nigericin and 5-(N-ethyl-N-isopropyl)amiloride: an approach to therapy based on cellular acidification and the inhibition of regulation of intracellular pH

The extracellular pH (pHe) in solid tumors is frequently lower than the pHe in normal tissues, but the intracellular pH (pHi) is regulated to physiological levels. Cell killing can be achieved in an acidic environment in tissue culture by nigericin, which acidifies cells by transporting H+ from the extracellular space into the cytoplasm; this cell killing can be enhanced when used with 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of membrane-based Na+/H+ exchange, which plays a major role in the regulation of pHi (R. P. Maidorn; E. J. Cragoe; I. F. Tannock, Br. J. Cancer 67:297-303; 1993). We have therefore assessed the ability of nigericin and EIPA to kill cells in two murine solid tumors (the KHT fibrosarcoma and the EMT-6 sarcoma). Hydralazine, which reduces tumor blood flow, or glucose, which stimulates glycolysis leading to accumulation of lactate, were also administered to mice to lower pHe in the tumors. We observed only a small decrease in the surviving fractions of cells in the tumors when tolerated doses of nigericin and EIPA were given IP to tumor-bearing mice. When nigericin and EIPA were combined with administration of hydralazine, the surviving fraction of cells in both tumors was reduced by a factor of 0.01, but there were minimal effects on growth delay. Administration of glucose with nigericin and EIPA led to a smaller reduction in surviving fraction of the KHT tumor (by approximately 0.1), although glucose was more effective than hydralazine in lowering the mean tumor pHe. When KHT tumors were treated with 15 Gy X-rays followed immediately by nigericin, EIPA, and hydralazine, a reduced surviving fraction as well as an increase in tumor growth delay was observed compared to radiation alone; however, there was little evidence to suggest that these agents were selectively toxic to the cells that survived radiation. Nigericin and EIPA, with or without hydralazine, had minimal effects on normal tissues, as assessed by changes in body weight, number of leukocytes, and serum creatinine levels. We conclude that pharmacological effects to acidify cells and to prevent regulation of pHi under the acidic conditions that exist in solid tumors can lead to moderate levels of cell killing, if additional strategies are used to lower tumor pHe.     

Hydralazine at thermoradiotherapy: tumor size and blood flow effects

PURPOSE: This study was aimed to assess the dependence on tumor size and blood flow of the efficacy of a vasoactive drug hydralazine with thermoradiotherapy. METHODS AND MATERIALS: Experiments were performed on mice bearing SCC-VII tumors with volumes of about 85 and 340 mm3 (7-8 or 11-12 days after transplantation, respectively). Local hyperthermia (water bath, 43 degrees C, 0.5 h) was started 3 h after irradiation of tumors. Hydralazine (2.5 mg/kg, IP) was given 0.5 h before heating. Tumor blood flow was evaluated by laser Doppler flowmetry before, during and up to 2 days after the treatments. RESULTS: It was shown that hydralazine and hyperthermia, even in combination with each other, had very weak anti-tumor effect, especially for 85 mm tumors. The agents also insignificantly enhanced the efficacy of radiotherapy excluding the case of polyradiomodification for 340 mm3 tumors when a dose modifying factor of about 2.0 was achieved. Thermometry showed only a small improvement by HDZ in heating patterns of tumors of both sizes. Meanwhile, the therapeutic efficacy of hydralazine and heat was correlated with the changes in tumor blood flow, first of all with the delayed effects. The radiomodifiers induced only minor and transient suppression of perfusion in the smaller tumors, and more markedly and for longer time decreased blood flow in the larger tumors. In the latter case, the inhibiting effect of the drug plus hyperthermia remained for at least 48 h after the treatment. CONCLUSION: (a) The combined use of hydralazine and heat seems to be advisable only at radiotherapy of rather large advanced tumors; (b) the efficacy of such radiomodification is correlated with prolonged inhibition of tumor blood flow by these agents; and (c) hydralazine and hyperthermia are likely to kill selectively both acutely and chronically hypoxic radioresistant cancer cells.     

Posttraumatic hypothermia in the treatment of axonal damage in an animal model of traumatic axonal injury

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)2D3 was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)2D3, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)2D3-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)2D3 alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)2D3 pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)2D3 can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)2D3 was shown, either alone or in combination with CTX, to inhibit tumor growth.     

Expression of keratinocyte growth factor mRNA in ex vivo and in vitro specimens of human cornea and conjunctiva

Treatment of HIV and related malignancies with pharmacologic and biologic agents has not appreciably modified the course of disease. Immunologic impairment remains the critical factor in response. We report the long-term results of a single session of low-flow (0.3 L/min) extracorporeal perfusion hyperthermia on 29 men and 2 women with disseminated Kaposi's sarcoma and profound immunologic impairment. Any antiretroviral drug employed by the patient was stopped 72 hours prior to treatment and withheld during the period of follow-up. Core temperature was raised to 42 degrees C and held for 1 hour with extracorporeal perfusion and ex vivo blood heating to 49 degrees C as the means of temperature control. Of 31 patients, 2 died of complications secondary to treatment (cardiac arrhythmia; CNS bleed). There were two cases of intravascular coagulopathy. Pressure point skin damage may occur despite adequate cushioning. At 30 days posttreatment complete or partial regressions were seen in 20/29 of those treated, with regressions persisting in 14/29 of those treated by 120 days posttreatment. At 360 days, 4/29 maintain tumor regressions with 1 in complete remission (at 26 months). The patient in complete remission remains culture-negative and PCR-negative for HIV. CD4+ counts rose from around 250 to, and remain around, 800 in this man. Selected healed lesions were biopsied to demonstrate tumor absence. Patients were selected for treatment if pretreatment testing of the tumor showed regression in vitro with heat exposure. Analysis of the early and midterm failures showed little sustained rise of the CD4+ cells if presenting total CD4+ counts were below 50 and had been at such low levels for extended periods, although other surrogate markers of HIV activity declined (semiquantitative PCR) during this period and is felt to support the hypothesis of apoptosis proposed in this illness. Analysis of the tumors of the few men not responding demonstrated elevated levels of IL-6 as compared to responders (12 vs < 1 pg/ml). At 120 days 29/31 patients remained alive (expected, 20). At 360 days, 21/31 remained alive (expected, 11). In no patient was HIV activity stimulated with heat exposure. CMV retinitis did clear in some patients treated (both techniques), but treatment alone did not prevent later development of retinopathy. EBV parameters were markedly altered in the short term with heat exposure in some patients. Few patients showed herpes simplex activation. Varicella-zoster virus remitted in some patients. There is utility in the use of systemic hyperthermia to control HIV and related malignancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.