In vivo evaluation of [18F]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography.

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [(18)F]fluoroetanidazole ([(18)F]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [(18)F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [(18)F]FETA, with an octanol-water partition coefficient of 0.16+/-0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60-120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO(2) values <5 mmHg (r=0.805, P=0.027) and carbogen breathing decreased [(18)F]FETA-derived radioactivity levels (P=0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30-60 min. Higher fractional retention of [(18)F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P=0.05) reflected higher percentage of pO(2) values <1 mmHg (P=0.023), lower vessel density (P=0.026), and higher radiobiological hypoxic fraction (P=0.008) of the HT1080/1-3C tumours. In conclusion, [(18)F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.

In vitro selectivity, in vivo biodistribution and tumour uptake of annexin V radiolabelled with a positron emitting radioisotope.

The availability of a noninvasive method to detect and quantify apoptosis in tumours will enable tumour response to several cancer therapies to be assessed. We have synthesised two radiotracers, annexin V and the N-succinimidyl-3-iodobenzoic acid (SIB) derivative of annexin V, labelled with radio-iodine ((124)I and (125)I) and provided proof of the concept by assessing specific binding and biodistribution of these probes to apoptotic cells and tumours. We have also assessed the tumour uptake of [(124)I]annexin V in a mouse model of apoptosis. RIF-1 cells induced to undergo apoptosis in vitro showed a drug concentration-dependent increased binding of [(125)I]annexin V and [(125)I]SIB-annexin V. In the same model system, there was an increase in terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL)-positive cells and a decrease in clonogenic survival. Radiotracer binding was completely inhibited by preincubation with unlabelled annexin V. In RIF-1 tumour-bearing mice, rapid distribution of [(125)I]SIB-annexin V-derived radioactivity to kidneys was observed and the radiotracer accumulated in urine. The binding of [(125)I]SIB-annexin V to RIF-1 tumours increased by 2.3-fold at 48 h after a single intraperitoneal injection of 5-fluorouracil (165 mg kg(-1) body weight), compared to a 4.4-fold increase in TUNEL-positive cells measured by immunostaining. Positron emission tomography images with both radiotracers demonstrated intense localisation in the kidneys and bladder. Unlike [(124)I]SIB-annexin V, [(124)I]annexin V also showed localisation in the thyroid region presumably due to deiodination of the radiolabel. [(124)I]SIB-annexin V is an attractive candidate for in vivo imaging of apoptosis by PET.

Proportion of infiltrating IgG-binding immune cells predict for tumour hypoxia.

Macrophages can account for up to 50% of tumour mass and secrete many angiogenic factors. Furthermore, tumour hypoxia is thought to play a major role in the activation of macrophages and the regulation of angiogenesis. In this paper, we demonstrate a strong correlation between hypoxia and the recruitment of immune cells binding to IgG in 8 experimental tumours. We provide evidence that IgG binding immune cells in 3 tumour lines are predominately composed of macrophages. Reduced oxygenation may act as a stimulus for recruitment of immune cells to the tumour mass, and the detection of either IgG-positive host cells or macrophages may offer an alternative method for monitoring tumour hypoxia.

Pentoxifylline improves the oxygenation and radiation response of BA1112 rat rhabdomyosarcomas and EMT6 mouse mammary carcinomas.

Tumor hypoxia can significantly impact the efficacy of cancer therapy. Pentoxifylline, a methylxanthine derivative, can improve oxygen delivery to tissues and is widely used in the treatment of peripheral vascular disease and various cerebrovascular disorders. In this article, we show that pentoxifylline, combined with oxygen breathing, significantly improves the radiation response of two experimental tumors in vivo through improved tumor oxygenation. We also demonstrate that pentoxifylline does not directly radiosensitize EMT6 cells in vitro and does not modify the tumor radiation response when administered postirradiation to solid EMT6 tumors. Our findings confirm that preirradiation administration of pentoxifylline can improve radiation efficacy, but suggest that its role as a postirradiation modifier of treatment response, reported by others, may be tumor-specific.

Tumour response to hypercapnia and hyperoxia monitored by FLOOD magnetic resonance imaging.

Flow and oxygenation dependent (FLOOD) MR images of GH3 prolactinomas display large intensity increases in response to carbogen (5% CO2/95% O2) breathing. To assess the relative contributions of carbon dioxide and oxygen to this response and the tumour oxygenation state, the response of GH3 prolactinomas to 5% CO2/95% air, carbogen and 100% O2 was monitored by FLOOD MRI and PO2 histography. A 10-30% image intensity increase was observed during 5% CO2/95% air breathing, consistent with an increase in tumour blood flow, as a result of CO2-induced vasodilation, reducing the concentration of deoxyhaemoglobin in the blood. Carbogen caused a further 40-50% signal enhancement, suggesting an additional improvement due to increase blood oxygenation. A small 5-10% increase was observed in response to 100% O2, highlighting the dominance of CO2-induced vasodilation in the carbogen response. Despite the large FLOOD response, non-significant increases in tumour pO2 were observed in response to the three gases. Tissue pO2 is determined by the balance of oxygen supply and demand, hence increased blood flow/oxygenation may not necessarily produce a large increase in tissue PO2. The FLOOD response is determined by the level of deoxygenation of blood, the size of this response relating to vascular density and the potential of high-oxygen content gases to improve the oxygen supply to tumour tissue.

Improvement in human tumour oxygenation with carbogen of varying carbon dioxide concentrations.

BACKGROUND AND PURPOSE: Carbogen (95%O2, 5%CO2) is being used in clinical trials as a hypoxic radiosensitiser. Tolerance to carbogen can be a problem, this study compares tumour oxygenation during inhalation of hyperoxic gas containing either 2% or 5% CO2. MATERIALS AND METHODS: Tumour pO2 was measured in 16 patients using the Eppendorf pO2 histograph. RESULTS: After breathing gas containing either 5% or 2% CO2 an increase in median pO2 was measured in every tumour, the frequency of low pO2 values ( < or = 10 mmHg) fell from 47% to 29% in the 5% group and from 55% to 17% in the 2% group. CONCLUSIONS: This study confirms that breathing 2% CO2 and 98% O2 is well tolerated and effective in increasing tumour oxygenation.

Polarographic measurements of oxygen tension in human glioma and surrounding peritumoural brain tissue.

This study quantifies the spatial distribution of pO2 in glioma and in the surrounding brain tissue. Both glioma and peritumoural brain contain regions at oxygen tensions less than 2.5 mmHg. Modalities targeting hypoxia to improve the efficacy of therapy may have an important role in the management of this disease.

The effects of host carbogen (95% oxygen/5% carbon dioxide) breathing on metabolic characteristics of Morris hepatoma 9618a.

Characteristics of the tumour metabolic profile play a role in both the tumour-host interaction and in resistance to treatment. Because carbogen (95% oxygen/5% carbon dioxide) breathing can both increase sensitivity to radiation and improve chemotherapeutic efficacy, we have studied its effects on the metabolic characteristics of Morris hepatoma 9618a. Host carbogen breathing increased both arterial blood pCO2 and pO2, but decreased blood pH. A fourfold increase in tumour pO2 (measured polarographically) and a twofold increase in image intensity [measured by gradient recalled echo magnetic resonance (MR) imaging sensitive to changes in oxy/deoxyhaemoglobin] were observed. No changes were seen in blood flow measured by laser Doppler flowmetry. Tumour intracellular pH remained neutral, whereas extracellular pH decreased significantly (P < 0.01). Nucleoside triphosphate/inorganic phosphate (NTP/Pi), tissue and plasma glucose increased twofold and lactate decreased in both intra- and extracellular compartments, suggesting a change to a more oxidative metabolism. The improvement in energy status of the tumour was reflected in changes in tissue ions, including Na+, through ionic equilibria. The findings suggest that the metabolic profile of hepatoma 9618a is defined partly by intrinsic tumour properties caused by transformation and partly by tissue hypoxia, but that it can respond to environmental changes induced by carbogen with implications for improvements in therapeutic efficacy.

Preclinical evaluation of the novel hypoxic marker 99mTc-HL91 (Prognox) in murine and xenograft systems in vivo.

PURPOSE: The 99mTc-labelled amine oxime 99mTc-HL91 (Prognox) is under investigation as a potential noninvasive clinical marker of tumour hypoxia whose uptake can be monitored by gamma camera imaging. The aim was to assess its retention in 3 tumours under control and enhanced oxygenation conditions. MATERIALS AND METHODS: The SaF murine sarcoma, grown subcutaneously in CBA mice, and human colon carcinoma HT29 and lung adenocarcinoma A549, grown as xenografts in SCID mice, were used at 6-8 mm diameter. Oxygenation status was enhanced by giving 500 mg/kg nicotinamide i.p. and breathing carbogen until the point of assay. Oxygenation/hypoxia was measured using the Eppendorf pO2 histograph (KIMOC 6650) with at least 5 tracks and at least 70 values, and expressing pO2 values as % < 2.5 mmHg. 99mTc-HL91 (0.8 or 8 MBq per mouse) was injected i.v. immediately before nicotinamide or saline, and animals were killed 2 h after injection. Tumour, skin, muscle, and blood samples were counted and isotope retention was expressed as % injected dose per gram. 14C-labelled uncomplexed HL91 was used similarly (0.2-0.4 MBq per mouse) and samples were solubilised and decolourised before counting. RESULTS: Nicotinamide and carbogen treatment reduced 99mTc-HL91 retention in all tumours to 54%-64% of control; it also reduced the proportion of pO2 values < 2.5 mmHg in all tumours. The mean proportion of pO2 values < 2.5 mmHg correlated very well with the mean ratio of tumour to blood retention at 2 h for all tumours, both unperturbed and oxygen-enhanced (r = 0.996, p < 0.001). Retention of 14C-HL91 in SaF tumour was unchanged by nicotinamide and carbogen, confirming that 99mTc complexation of the ligand is required for hypoxia specificity. CONCLUSION: There is excellent correlation between 99mTc-HL91 retention and hypoxia, as measured by the Eppendorf histograph, over the range of 50%-90% of values < 2.5 mmHg in 3 different tumour models, including 2 human xenografts. 99mTc complexation of the ligand is required for hypoxia specificity. 99mTc-HL91 (Prognox) shows good potential as a clinical marker for hypoxia and warrants further development.

Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity.

PURPOSE: To measure the effects of breathing high-oxygen-content gases, with a CO2 fraction of between 0 and 10%, on tumour radiosensitivity, blood flow and oxygenation. METHODS AND MATERIALS: The murine sarcoma F was used, implanted subcutaneously (s.c.) in syngeneic CBA mice. We assessed the induced changes in tumour microregional blood flow and oxygenation using laser Doppler flowmetry, and pO2 histography, respectively. Radiation response was determined using an in vivo-in vitro clonogenic assay 18-20 h post treatment. RESULTS: The results show that the level of radiosensitization achieved is dependent on both the CO2 content of the inspired gas and the duration of gas breathing. No radiosensitization was evident following inhalation of 90% O2 + 10% CO2. All other gases elicited radiosensitization; however, that achieved with 100% O2 disappeared at the extended preirradiation breathing time of 45 min. At this time, radiosensitization was maintained for gases containing 1%, 2.5%, or 5% CO2. Changes in oxygenation, as measured by PO2 electrodes, did indicate improved oxygenation status during inhalation of the gases. However, the time-course and extent of the changes did not mirror accurately the changes in radiosensitization. All the gases with a CO2 content of 2.5% or greater induced a 10-20% reduction in microregional blood flow, with no change evident following inhalation of 100% O2 or 99% O2 + 1% CO2. CONCLUSIONS: The data imply that the decreased radiosensitization seen at extended breathing times of oxygen is unrelated to blood flow changes. The fact that radiosensitization is seen with extended breathing times of gases containing 2.5% and 5% CO2, despite blood flow decreases, is indicative of other overriding physiological changes, perhaps related to oxygen utilisation. The studies overall indicate that, at least in the tumour investigated, radiosensitization is not affected if the CO2 content of the inspired gas is reduced from 5% to 2.5%, or even 1%. Further evaluation of the radiosensitizing effects of such gas mixtures is now warranted. In addition, comparison with recent studies of other tumour types, where carbogen has been shown to improve tumour blood flow, suggests that this may be a tumour-specific phenomenon. Based on these data, further effort is required to elucidate the physiological mechanisms that determine these blood flow changes.

Measurement of tumor oxygenation: a comparison between polarographic needle electrodes and a time-resolved luminescence-based optical sensor.

A novel oxygen sensor which does not rely on electrochemical reduction has been used to measure the oxygenation of the murine sarcoma F in a comparative study with an existing polarographic electrode that is available commercially. The prototype luminescence sensor yielded an oxygen distribution comparable with readings made using a pO2 histograph. The percentage of regions detected that had a pO2 less than 5 mm Hg was 79 and 75 using the Eppendorf pO2 histograph and the luminescence fiber optic sensor, respectively. These values were compatible with a measured radiobiologically hypoxic fraction of 67% in this tumor. The polarographic method detected more regions with a pO2 of 2.5 mm Hg or less (69%) compared with the optical sensor (50%) (P < 0.05). This could reflect differences in the oxygen use of the sensing devices. This initial assessment indicates the potential of a fiber-optic-based oxygen-monitoring system. Such a system should have several advantages including monitoring temporal oxygen changes in a given microregion and use with NMR procedures.