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This study investigated long-term microenvironmental responses (oxygenation, perfusion, metabolic status, proliferation, vascular endothelial growth factor (VEGF) expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O(2)) for the whole period of tumour growth (chronic hypoxia). A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO(2)) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO(2) was significantly improved (median pO(2)=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels (maybe the result of a simultaneous reduction in arterial blood pressure). In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion.

The effects of combined and simultaneously applied localised 43 degrees C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg(-1) i.v.), (iii) localised HT, (iv) Bchl-ser-PDT+HT. The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied.

The possibility of enhancing aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) by simultaneous application of localised hyperthermia (HT) was evaluated. Treatments of rat DS-sarcomas included: (i) control, (ii) ALA administration (375 mg kg(-1), i.p.), no illumination, (iii) 'nonthermal' illumination, (iv) ALA-PDT: that is, ALA administration, 'nonthermal' illumination, (v) localised HT, 43 degrees C, 60 min (vi) ALA-PDT+HT: ALA administration with full spectrum irradiation resulting in ALA-PDT and HT. Tumour volume was monitored for 90 days or until a target volume (3.5 ml) was reached. No differences were seen between the first three groups, with all tumours reaching the target volume by 8-11 days. A total of 13 and 15% of tumours did not reach the target volume by day 90 following HT or ALA-PDT treatment, respectively. ALA-PDT+HT showed the greatest antitumour effect (P=0.0001), with 61% of the tumours not reaching the target volume. Viability and in vitro growth were also assessed in cells from tumours excised after treatment. ALA-PDT+HT reduced the fraction of viable tumour cells by 85%, and in vitro culture showed pronounced growth delay compared to control cells. These results demonstrate an enhanced antitumour effect upon ALA+HT, which appears to involve direct cell toxicity rather than solely vascular damage.

Isolated limb perfusion allows the direct application of therapeutic agents to a tumour-bearing extremity. The present study investigated whether the dihydropyridine-type Ca(2+)-channel blocker nifedipine could improve blood flow and oxygenation status of experimental tumours during isolated limb perfusion. Perfusion was performed by cannulation of the femoral artery and vein in rats bearing DS-sarcoma on the hind foot dorsum. Perfusion rate was adjusted to maintain a perfusion pressure of 100-140 mmHg throughout the experiment. Following equilibration, nifedipine was continuously infused for 30 min (8.3 microg min(-1) kg(-1) BW). During constant-pressure isolated limb perfusion, nifedipine can significantly increase perfusion rate (+100%) and RBC flux (+60%) through experimental leg tumours. "Steal phenomena" in favour of the surrounding normal tissue and oedema formation were not observed. Despite the increased oxygen availability (+63%) seen upon application of this calcium channel blocker, nifedipine does not result in a substantial reduction of tumour hypoxia, most probably due to an increase in O(2) uptake with rising O(2) supply to the tumour-bearing hind limb. Nifedipine application during isolated limb perfusion can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled isolated limb perfusion., (Copyright 2002 Cancer Research UK)

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT-PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia., (Copyright 2002 Cancer Research UK)

Perfusion and oxygenation of experimental tumours were studied during angiotensin II (AT II) administration whereby the rate of the continuous AT II infusion was chosen to increase the mean arterial blood pressure (MABP) by 50-70 mmHg. In subcutaneous DS-sarcomas the red blood cell (RBC) flux was assessed using the laser Doppler technique and the mean tumour oxygen partial pressure (pO2) was measured polarographically using O2-sensitive catheter and needle electrodes. Changes in RBC flux with increasing MABP depended mainly on tumour size. In small tumours, RBC flux decreased with rising MABP whereas in larger tumours RBC flux increased parallel to the MABP. As a result of these volume-dependent effects on tumour blood flow, the impact of AT II on tumour pO2 was also mainly tumour volume-related. In small tumours oxygenation decreased with increasing MABP during AT II infusion, whereas in large tumours a positive relationship between blood pressure and O2 status was found. This disparate behaviour might be the result of the co-existence of two functionally distinct populations of tumour vessels. In small tumours, perfusion decreases presumably due to vasoconstriction of pre-existing host vessels feeding the tumour. In larger malignancies, newly formed tumour vessels predominate and seem not to have this vasoresponsive capability (lack of smooth muscle cells and/or AT receptors), resulting in an improvement of perfusion which is not tumour-related per se, but is due to the increased perfusion pressure.

The radiosensitivity of solid tumours in anaemic rats treated with recombinant human erythropoietin (rhEPO, epoetin beta) was studied. Anaemia was induced by a single dose of carboplatin (45 mg kg(-1) i.v.), resulting in a reduction in the haemoglobin concentration by 30%. In a second group, the development of anaemia was prevented by rhEPO (1000 IU kg(-1)) administered s.c. three times per week starting 6 days before the carboplatin application. Three days after carboplatin treatment, DS-sarcomas were implanted subcutaneously onto the hind foot dorsum. Neither carboplatin nor rhEPO treatment influenced tumour growth rate. Five days after implantation, tumours were irradiated with a single non-curative dose (10 Gy), resulting in a growth delay with a subsequent regrowth of the tumours. In the rhEPO-treated group, the growth delay lasted significantly longer (9.5 days vs. 4.5 days) and the regrowth was slower (6.0 days vs. 4.1 days) compared with the anaemic group. These data suggest that the correction of chemotherapy-induced anaemia by rhEPO (epoetin beta) treatment increases tumour radiosensitivity, presumably as a result of an improved oxygen supply to tumour tissue.

The effect of localized hypothermia on microcirculatory and metabolic parameters in s.c. DS sarcomas on the hind foot dorsum of Sprague-Dawley rats was investigated. Tumours were cooled by superfusion of the tumour surface with cooled saline solution to 25 degrees C or 15 degrees C. Control tumours remained at 35 degrees C. These temperatures were maintained for 30 min. In tumour oxygenation measurements, hypothermia at 25 degrees C and 15 degrees C caused progressive decreases in the size of the fraction of pO2 measurements between 0 and 2.5 mmHg together with a reduction in pO2 variability. No significant changes in median or mean pO2 or in the fraction of pO2 measurements between 0 and 5 mmHg, and 0 and 10 mmHg were observed. Using laser Doppler flowmetry, red blood cell flux was found to decrease significantly upon 25 degrees C or 15 degrees C hypothermia treatment to 67% and 37% of starting values respectively, whereas no significant changes were seen in control tumours over the whole observation period. Viscosity was measured in blood and plasma samples over a range of temperatures and was found to increase with decreasing temperature. Assessment of tumour glucose levels showed an increased concentration of glucose following 15 degrees C hypothermia, an observation consistent with a 'slowing down' of glycolysis. No changes in lactate or adenylate phosphate levels were observed. As a way of improving tumour oxygenation, localized hypothermia may therefore be a useful means of radiosensitization.