Hyperthermic Enhancement of Tumor Radiosensitization Strategies

Local hyperthermia of living tissue can cause significant increases in blood flow and oxygenation depending on time-temperature history. Increases in perfusion of the abnormal and insufficient vasculature found in solid tumors may increase tumor oxygenation, thereby increasing the radiation sensitivity of the tumor. We hypothesized that local heating of tumor would increase the oxygenation of the tumor tissue and allow other oxygenating agents to further modify tumor oxygenation and radiation response. In the present study the effect of moderate temperature hyperthermia (MTH) at 41.5–42.5°C for 30–60 min, 250 mg/kg nicotinamide, or carbogen breathing (95% O₂/5% CO₂) on the radiation sensitivity of FSaII murine fibrosarcomas or R3230 AC rat adenocarcinomas was studied. Individually, these treatments increased the tumor cell sensitivity to single dose 10–15 Gy X-irradiation by 1–5 fold on average, as measured by the in vivo/in vitro tumor excision assay. The combination of tumor MTH with nicotinamide or carbogen breathing increased the radiation sensitivity by 3–5 fold in FSaII tumors and 10–30 fold in R3230 tumors with varying levels of statistical significance. Finally, the triple combination of adjuvant MTH, nicotinamide and carbogen breathing increased the radiation-induced cell death in FSaII tumors to a similar extent as the dual combinations of MTH, nicotinamide or heat, carbogen breathing. However, in R3230 AC tumors the triple adjuvant combination significantly increased radiation-induced cell killing compared to all other dual adjuvant treatments (p < 0.04). To interrogate the mechanism by which heating alters tumor physiology, nitric oxide production in tumor and endothelial cells in culture and tumor tissue after heating was studied. Heating caused an increase in nitric oxide production over a 24 h period after treatment. Subsequently, inhibiting the enzymatic production of NO with L-NAME was found to increase heat-induced growth delay of FSaII tumors. The cause and effect of increased nitric oxide production and the response of the tumor vasculature to heat are discussed in the context of the tumor radiosensitization achieved by heating, carbogen breathing and nicotinamide.     

Changes in tumor hypoxia measured with a double hypoxic marker technique

Purpose: Development of a double hypoxic cell marker assay, using the bioreductive nitroimidazole derivatives CCI-103F and pimonidazole, to study changes in tumor hypoxia after treatments that modify tumor oxygenation.

Methods and Materials: Both hypoxic markers were visualized by immunohistochemical techniques to detect changes in hypoxic fraction induced by carbogen breathing (95% O₂ and 5% CO₂) or hydralazine injection. The protocol was tested in a human laryngeal squamous cell carcinoma xenograft line. Quantitative measurements were derived from consecutive tissue sections that were analyzed by a semiautomatic image analysis system. Qualitative analysis was obtained by double staining of the two hypoxic markers on the same tissue section.

Results: A significant correlation between the hypoxic fractions for the two markers, CCI-103F and pimonidazole, was found in air breathing animals. After carbogen breathing, the hypoxic fraction decreased significantly from 0.07 to 0.03, and after hydralazine treatment, the hypoxic fraction increased significantly. Reduction of hypoxia after carbogen breathing was most pronounced close to well-perfused tumor regions.

Conclusions: With this method, employing two consecutively injected bioreductive markers, changes in tumor hypoxia can be studied. A significant reduction in hypoxia after carbogen breathing and a significant increase in hypoxia after hydralazine administration was demonstrated.     

Survival of fish upon removal of cyanide from water

The effects of potassium cyanide and the removal of cyanide from water in vivo on the survival of fish were investigated. This research was initiated because of the catastrophe that took place at the end of January 2000 in the Carpathian basin, when an enormous amount of cyanide pollution swept through the Samos and Tisza rivers, and then to the Danube. Since nothing was done against the disaster, we have suggested a chemical solution to remove cyanide from waterways (Chem. Innovat. 30 (2000b) 53). Based on experiments, we describe that the most effective and harmless way to remove cyanide and to save the lives of fish from 40 to 160 x the lethal doses of cyanide is to use carbogen gas containing 5% carbon dioxide and 95% oxygen followed by aeration with air.     

Steroids, carbogen or placebo for sudden hearing loss: a prospective double-blind study

There is no consensus regarding treatment modalities for idiopathic sudden sensorineural hearing loss (SNHL). In order to evaluate the effectiveness of steroid or carbogen inhalation therapies, a prospective double-blind placebo controlled study was designed. All 41 patients enrolled in the study had unilateral SNHL with no prior history of SNHL, otological pathological history or otoscopic findings. Patients were assigned to four treatment groups: prednisone tablets, placebo tablets, carbogen inhalation or room air inhalation. All were treated for 5 days. The audiometric data at admission was compared to that at day 6 and to data collected at follow-up (average 33 days). Results revealed no significant difference between the groups for early or late audiometric outcome. Age, time from onset of symptoms to initiation of treatment, tinnitus, audiogram configuration, and the presence of vertigo at onset did not significantly affect the outcome. The discrimination scores that were poor in all patient groups on admission improved within days in all groups. These findings suggest that steroids or carbogen inhalation have no therapeutic advantage over placebo. Also, regardless of treatment modality, hearing continued to improve for at least a month after treatment was stopped. Received: 6 April 2001 / Accepted: 9 May 2001     

Increased radiosensitivity of tumors by perfluorochemicals and carbogen

The efficacy of an emulsion of perfluorochemicals (Fluosol-DA) to sensitize RIF-1 tumors of C3H mice to radiation was studied. An i.v. injection of 12 ml/kg of Fluosol-DA (20%) to tumor-bearing animals and keeping the animals in an atmosphere of a mixture of O2 (95%) and CO2 (5%) (carbogen) for 1 hr before and during irradiation of s.c. tumors in the thigh significantly enhanced the tumoricidal effect of radiation as measured with growth delay of the treated tumors. A lesser but significant enhancement of radiation damage in the tumors was also observed when the animals were kept in carbogen for 1 hr without injection of Fluosol-DA. It was concluded that Fluosol-DA increased the delivery of oxygen to hypoxic areas in the tumors and enhanced the response of hypoxic cells to radiation.     

Investigations in vivo of the effects of carbogen breathing on 5-fluorouracil pharmacokinetics and physiology of solid rodent tumours

Purpose We have shown previously that carbogen (95% 0₂, 5% CO₂) breathing by rodents can increase uptake of anticancer drugs into tumours. The aim of this study was to extend these observations to other rodent models using the anticancer drug 5-fluorouracil (5FU). 5FU pharmacokinetics in tumour and plasma and physiological effects on the tumour by carbogen were investigated to determine the locus of carbogen action on augmenting tumour uptake of 5FU.Methods Two different tumour models were used, rat GH3 prolactinomas xenografted s.c. into nude mice and rat H9618a hepatomas grown s.c. in syngeneic Buffalo rats. Uptake and metabolism of 5FU in both tumour models with or without host carbogen breathing was studied non-invasively using fluorine-19 magnetic resonance spectroscopy (¹⁹F-MRS), while plasma samples from Buffalo rats were used to construct a NONMEM pharmacokinetic model. Physiological effects of carbogen on tumours were studied using ³¹P-MRS for energy status (NTP/Pi) and pH, and gradient-recalled echo magnetic resonance imaging (GRE-MRI) for blood flow and oxygenation.Results In both tumour models, carbogan-induced GRE-MRI signal intensity increases of 60% consistent with an increase in tumour blood oxygenation and/or flow. In GH3 xenografts, ¹⁹F-MRS showed that carbogen had no significant effect on 5FU uptake and metabolism by the tumours, and ³¹P-MRS showed there was no change in the NTP/Pi ratio. In H9618a hepatomas, ¹⁹F-MRS showed that carbogen had no effect on tumour 5FU uptake but significantly (p=0.0003) increased 5FU elimination from the tumour (i.e. decreased the t_1/2) and significantly (p=0.029) increased (53%) the rate of metabolism to cytotoxic fluoronucleotides (FNuct). The pharmacokinetic analysis showed that carbogen increased the rate of tumour uptake of 5FU from the plasma but also increased the rate of removal. ³¹P-MRS showed there were significant (p0.02) increases in the hepatoma NTP/Pi ratio of 49% and transmembrane pH gradient of 0.11 units.Conclusions We suggest that carbogen can transiently increase tumour blood flow, but this effect alone may not increase uptake of anticancer drugs without a secondary mechanism operating. In the case of the hepatoma, the increase in tumour energy status and pH gradient may be sufficient to augment 5FU metabolism to cytotoxic FNuct, while in the GH3 xenografts this was not the case. Thus carbogen breathing does not universally lead to increased uptake of anticancer drugs.     

Necrosis predicts benefit from hypoxia-modifying therapy in patients with high risk bladder cancer enrolled in a phase III randomised trial

Background and purpose

Addition of carbogen and nicotinamide (hypoxia-modifying agents) to radiotherapy improves the survival of patients with high risk bladder cancer. The study investigated whether histopathological tumour features and putative hypoxia markers predicted benefit from hypoxia modification.

Materials and methods

Samples were available from 231 patients with high grade and invasive bladder carcinoma from the BCON phase III trial of radiotherapy (RT) alone or with carbogen and nicotinamide (RT + CON). Histopathological tumour features examined were: necrosis, growth pattern, growing margin, and tumour/stroma ratio. Hypoxia markers carbonic anhydrase-IX and glucose transporter-1 were examined using tissue microarrays.

Results

Necrosis was the only independent prognostic indicator (P = 0.04). Necrosis also predicted benefit from hypoxia modification. Five-year overall survival was 48% (RT) versus 39% (RT + CON) (P = 0.32) in patients without necrosis and 34% (RT) versus 56% (RT + CON) (P = 0.004) in patients with necrosis. There was a significant treatment by necrosis strata interaction (P = 0.001 adjusted). Necrosis was an independent predictor of benefit from RT + CON versus RT (hazard ratio [HR]: 0.43, 95% CI 0.25–0.73, P = 0.002). This trend was not observed when there was no necrosis (HR: 1.64, 95% CI 0.95–2.85, P = 0.08).

Conclusions

Necrosis predicts benefit from hypoxia modification in patients with high risk bladder cancer and should be used to select patients; it is simple to identify and easy to incorporate into routine histopathological examination.     

An attempt to enhance chemosensitivity of quiescent cell populations in solid tumors by combined treatment with nicotinamide and carbogen

cis-Diamminedichloroplatinum(II) (cisplatin) was intraperitoneally injected into mice bearing SCC VII or EMT6/KU tumors after ten administrations of 5-bromo-2-deoxyuridine (BrdU) to label all the proliferating tumor cells. The tumors were excised 1 h after the cisplatin injection, minced, and trypsinized. The tumor cell suspensions were then incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus frequency was determined, using immunofluorescence staining for BrdU. Cells that were not labeled with BrdU were regarded as quiescent. The micronucleus frequency in the total number of tumor cells was determined in tumors that had not been pretreated with BrdU. To modify the sensitivity to cisplatin, nicotinamide was intraperitoneally injected before the administration of cisplatin or mice were placed in a circulating carbogen (95% O₂, 5% CO₂) chamber for 30 min after cisplatin administration. In both tumor systems, the micronucleus frequency in quiescent cells was lower than that in the total cells. Nicotinamide pretreatment increased the micronucleus frequency in total and in quiescent cells in both tumor systems, and to a higher extent in total cells. The combination of nicotinamide and carbogen increased the micronucleus frequency more markedly than treatment with either nicotinamide or carbogen alone. In total cells of both tumors, the nicotinamide injection increased the uptake of [^195mPt]cisplatin. The combined treatment raised the uptake more markedly than did treatment with either agent alone. In total cells of the SCC VII tumor, these increases in micronucleus frequency and the [^195mPt]cisplatin uptake following nicotinamide or combined pretreatment were significant. In both tumors, carbogen breathing also elevated the micronucleus frequency to some degree in total and quiescent cells and the [^195mPt]cisplatin uptake in total cells. The combined nicotinamide and carbogen treatment was considered to be useful for sensitizing tumor cells to chemotherapy with cisplatin in vivo.Abbreviations Q cells quiescent cells - P cells proliferating cells     

Histological evaluation of cochlear blood flow using different fixation methods

Summary For an accurate histological evaluation of cochlear blood flow, it is essential to fix the cochlear vessels while maintaining their physiological state. In the present study, we administered 10% CO₂ to guinea pigs and then used phase-contrast microscopy to determine how two different methods of fixation influenced the cochlear vasculature. The first method of fixation employed perilymphatic perfusion in vivo, while the second one involved fixation after decapitation. Decapitation caused significant changes in the vessels of the stria vascularis, including constriction and sludging. In contrast, no sludging occurred in the perilymphatic perfusion method and erythrocyte morphology was preserved. However, dilatation of the strial blood vessels occurred after the inhalation of 10% CO₂ even in the decapitation method. The results demonstrate that particular attention must be paid to the fixation method used, especially when evaluating the blood flow of the stria vascularis.     

BOLD contrast fMRI of whole rodent tumour during air or carbogen breathing using echo-planar imaging at 1.5 T

The aim of this study was to evaluate the feasibility of functional MR imaging (fMRI) at 1.5 T, exploiting blood oxygenation level-dependent (BOLD) contrast, for detecting changes in whole-tumour oxygenation induced by carbogen (5% CO2+95% O2) inhalation of the host. Adult WAG/Rij rats with rhabdomyosarcomas growing subcutaneously in the lower flank were imaged when tumours reached sizes between 1 and 11 cm3 (n=12). Air and carbogen were alternatively supplied at 2 l/min using a snout mask. Imaging was done on a 1.5-T MR scanner using a T2*-weighted gradient-echo, echo-planar imaging (GE-EPI) sequence. Analysis of the whole-tumour EPI images was based on statistical parametric maps. Voxels with and without signal intensity changes (SIC) were recorded. Significance thresholds were set at p<0.05, corrected for multiple comparisons. In continuous air breathing condition, 3 of 12 tumours showed significant negative SIC and 1 tumour had a clear-cut positive SIC. The remaining tumours showed very little or no change. When switching to carbogen breathing, the SIC were significantly positive in 10 of 12 tumours. Negative SIC were present in 4 tumours, of which three were simultaneously characterised by positive SIC. The overall analysis indicated that 6 of the 12 tumours could be considered as strong positive responders to carbogen. Our research demonstrates the applicability of fMRI GE-EPI at 1.5 T to study whole-tumour oxygenation non-invasively. The observed negative SIC during air condition may reflect the presence of transient hypoxia during these measurements. Selection of tumours on the basis of their individual response to carbogen is possible, indicating a role of such non-invasive measurements for using tailor-made treatments.