Detection of microregional fluctuations in erythrocyte flow using laser Doppler microprobes.
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Animals↗
Biomedical subjects
D J Chaplin
Publications and source records attributed to D J Chaplin.
Microregional fluctuations in perfusion within human tumours detected using laser Doppler flowmetry.
PURPOSE: Transient fluctations in erythrocyte flux consistent with perfusion driven hypoxia have been previously reported using experimental tumour models. The present study was designed to establish whether such changes are a common feature of human tumours. METHODS AND MATERIALS: A multi-channel laser Doppler system was used to monitor microregional changes in flow in human tumours. Eight individual tumours were investigated, two primary and one locally recurrent breast carcinoma, two metastatic skin deposits and three metastatic lymph nodes. Six custom designed microprobes (diameter of 300 microns), each monitoring a nominal sampling volume of approximately 10(-2) mm3 were inserted into the tumour and perfusion monitored over a period of 60 min. RESULTS: The results show that in 54% of the regions monitored there was a change in microregional blood flow by a factor of 1.5 or more. Over the whole 60-min period, 19% of the changes were reversed, with a time course of 4-44 min. CONCLUSIONS: This finding demonstrates that microregional fluctuations in perfusion occur frequently in human tumours. Furthermore, the observation that 19% of the changes were reversed implies that at least some of the cells are subject to transient acute hypoxia.
Aged↗
Transient perfusion and radiosensitizing effect after nicotinamide, carbogen, and perflubron emulsion administration.
In order to improve the effect of radiation on tumour response, nicotinamide, perflubron emulsion and carbogen were administered which act on both diffusion limited hypoxia and intermittent perfusion limited hypoxia. These treatments were used in different combinations. The maximal radiosensitizing effect was found with the combination of the three treatments. The aim of this study was to use a double staining method (Hoechst 33342 and DiOC7(3) to evaluate the influence of nicotinamide, perflubron emulsion and carbogen on transient perfusion in three tumour cell lines transplanted onto nude mice: one rodent (EMT6), two human (HRT18, a rectal adenocarcinoma; and Na11+, a melanoma). For untreated groups, the percentage of closed and mismatched vessels depended on the tumour cell line. Carbogen alone or carbogen plus perflubron emulsion decreased the number of mismatched and closed vessels only for the two human cell lines. Nicotinamide was effective in decreasing the percentage of mismatched and closed vessels only for the melanoma cell line. The combination of nicotinamide, carbogen and perflubron emulsion was the most effective at decreasing both percentage of mismatched and closed vessels in all three tumours studies. This combination was also the most effective at enhancing the radiation response in all three tumours.
Adenocarcinoma↗
Effect of carbogen breathing on tumour microregional blood flow in humans.
BACKGROUND AND PURPOSE: Carbogen is currently being re-evaluated as a radiosensitiser. It acts primarily by increasing tissue pO2, although there is evidence to suggest that enhanced tumour blood flow may also be a component of its action. MATERIALS AND METHODS: Ten tumours in eight patients with advanced malignant disease were studied. Up to six microprobes, each with an estimated sampling volume of 10(-2) mm3, were inserted into the tumours. Ten min of baseline readings were taken prior to a 10 min carbogen (95% O2/5% CO2) breathing period, measurements were continued for a further 10 min. RESULTS: The results show that in 34 microregions analysed no overall change in tumour perfusion was seen with carbogen breathing. Individual tumour analysis demonstrated variation in response between patients to carbogen-after 6 min of carbogen four tumours showed an increase in blood flow by more than 10% of the pre-breathing value, two a decrease and four no change. The magnitude of change was small, with only two tumours fluctuating by more than 25%. CONCLUSIONS: These findings confirm the presence of transient fluctuations in microregional blood flow in human tumours but suggest that the radiosensitising action of carbogen lies primarily in its effect on increasing the oxygen capacity of blood. This supports the addition of agents such as nicotinamide with carbogen in order to overcome both diffusion and perfusion limited hypoxia.
Administration, Inhalation↗
DNA damage and repair in tumour and non-tumour tissues of mice induced by nicotinamide.
In vivo DNA damage and repair was induced by nicotinamide (NAM) in adenotype 12 virus-induced mouse sarcoma A12B3 and sarcoma F inoculated into CBA mice. DNA damage, NAM and NAD concentrations were measured after in vivo exposure to NAM, in tumours and spleens by alkaline elution and by HPLC analysis. Our results indicate that NAM between 100-1000 mg kg-1 causes a high level of in vivo DNA strand breaks in tumours and normal tissues in mice bearing the immunogenic sarcoma A12B3 but not in the non-immunogenic sarcoma F. The repair process was also delayed by the NAM treatment probably owing to inhibition of the DNA repair enzyme, poly(ADP-ribose)polymerase, as evidenced by accumulation of NAM and NAD. These data are consistent with NAM having a mechanism of action as a radiosensitiser at least in part by DNA repair inhibition. In addition, it should also be considered that high doses of NAM might cause considerable complications to normal tissue in tumour-bearing individuals.
Animals↗
Reduced capacity of tumour blood vessels to produce endothelium-derived relaxing factor: significance for blood flow modification.
The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.
Acetylcholine↗
Inhibition of intracellular pH control and relationship to cytotoxicity of chlorambucil and vinblastine.
The uptake and cytotoxicity of weakly acidic or basic chemotherapeutic agents is determined in part by passive diffusion along the pH gradient between the intracellular and extracellular compartments. In vivo it is known that tumour extracellular pH is more acidic than intracellular pH. Using CaNT murine tumour cells in vitro, we found the cytotoxicity of chlorambucil (a weak acid) increased as the extracellular pH of the culture medium (pHmed) was acidified. The cytotoxicity of vinblastine shows a reverse pH relationship with reduced cytotoxicity as pHmed was acidified. Chlorambucil cytotoxicity increased at acidic pHmed because the weak acidic function is ionised to a lesser extent at acidic pH and, therefore, favours drug uptake into the relatively neutral intracellular compartment. Vinblastine cytotoxicity decreased at acidic pHmed because the weak basic function is ionised to a greater extent at acidic pH and therefore does not favour drug uptake into the relatively neutral intracellular compartment. Using a combination of an inhibitor of the cell membrane proton pump, amiloride, and the ionophore, nigericin, the intracellular compartment can be acidified. This results in a time-dependent increase in sensitivity of the cells to low pHmed with significant cytotoxicity after 6 h exposure to pHmed = 6.2 and suggests that there is potential for direct tumour cytotoxicity in vivo if the tumour extracellular pH were equally acidic. An indirect effect of intracellular acidification is to alter the distribution of drugs between the extra- and intracellular compartment by reducing the pH gradient across the cell membrane. In response to intracellular acidification, the cytotoxicity of chlorambucil was reduced and that for vinblastine was increased. Inhibition of cellular pH control may result in direct cytotoxicity by acidification due to inhibition of proton efflux or indirectly by resulting in differential uptake of chemotherapeutic agents with weak acidic or basic functions.
Amiloride↗
Antivascular approaches to solid tumour therapy: evaluation of tubulin binding agents.
We have assessed the vascular effects of vinblastine and four other tubulin binding agents (dolastatin 10, dolastatin 15, combretastatin A1 and combretastatin A4), which are awaiting clinical evaluation. All five agents induce a reduction in tumour blood flow as measured by uptake of RbCI 24 h post drug administration. The degree of reduction ranged from 50% with combretastatin A1 to 90% with dolastatin 10. These reductions were similar to that seen with flavone acetic acid (FAA) and indicate that antivascular effects are a common feature of tubulin binding agents. We subsequently evaluated whether the blood flow reductions, induced by FAA and vinblastine, could be used to enhance the activity of the bioreductive drug tirapazamine. Since the kinetics and extent of blood flow reductions induced by the agents is comparable, similar therapeutic response was expected. Potentiation was only evident with FAA, indicating that this effect is not directly related to killing of hypoxic tumour cells induced as a consequence of blood flow reduction.
Animals↗
Immune reaction induced by X-rays and pions and its stimulation by schizophyllan (SPG).
Female C57BL/6 mice aged 6-8 weeks with transplanted Lewis lung cancer cells were used to investigate the anti-tumour effects and immune reactions in tumour tissue induced by X-ray and pion irradiation and their modification by schizophyllan (SPG). The effect of SPG on the rate of lung metastasis and the survival time of the mice was also studied using the same tumour system. These studies showed that in this tumour system the "practical' relative biological effectiveness (RBE) of pions was 1.33 in the dose ranges used (3 Gy x 4 = P3; 6 Gy x 4 = P6). SPG increased the suppression of tumour growth associated with moderate doses of radiation: X-rays (4 Gy x 4 = X4) or P3. SPG also decreased the number of lung metastases and prolonged the life span of the mice, these effects being independent of radiation. The addition of SPG to radiation increased both the macrophage infiltration and T-lymphocyte infiltration in the local tumour and the lung nodules. There did not appear to be any major differential effect of SPG on the pion-treated mice compared with those treated with X-rays.
Adjuvants, Immunologic↗
The influence of oxygen and carbon dioxide tension on the production of TNF alpha by activated macrophages.
The influence of oxygen tension and carbon dioxide levels on human TNF alpha (hTNF alpha) production by the monocyte/macrophage cell line U937 has been examined. The cells were stimulated under different oxygen and carbon dioxide concentrations in the presence and absence of the known TNF alpha inducer phorbol 12-myristate 13-acetate (PMA). The results indicated that U937 cells stimulated with PMA produced up to 10 times more TNF alpha when incubated under tumour-relevant oxygen tensions of 1% rather than under aerobic conditions, i.e. 21% O2. Increasing the carbon dioxide levels from 5% to 7% however decreased the amount of PMA-stimulated hTNF alpha produced. No hTNF alpha was produced in the unstimulated U937 cells, indicating that low oxygen tensions (hypoxia) alone did not induce the production of TNF alpha, in this case.
Carbon Dioxide↗
Tumour blood flow modification by endothelin-related peptides in the rat HSN fibrosarcoma.
Modification of tissue blood flow and tissue vascular resistance was examined in the female CBH rat, bearing a HSN fibrosarcoma, following bolus intravenous administration of 1 nM kg-1 endothelin-1 (ET-1) or 1 nM kg-1 sarafotoxin S6c (SX6c), selective agonists for endothelin A (ETA) and B (ETB) receptors respectively. Blood flow was measured 15 min after drug administration by the tissue uptake of 125I-labelled-iodoantipyrine. ET-1 and SX6c produced increases in mean arterial blood pressure (MABP) of 52 mmHg and 42 mmHg respectively. Blood flow to the tumour was unaffected by ET-1 treatment, whereas blood flow to normal tissues was reduced, the exception being the heart and the brain in which flow was increased. In contrast, tumour blood flow following SX6c was significantly increased, whereas blood flow in normal tissues was either unaltered or reduced. Vascular resistance was increased in all tissues and the tumour by ET-1 demonstrating that the tumour vasculature was constricting via ETA receptor activation. SX6c however, did not modify tumour vascular resistance, whereas it increased vascular resistance in all normal tissues, suggesting that the tumour lacks a functional population of ETB receptors. This discrepancy may provide a means for selectively modifying tumour blood flow.
Animals↗
Changes in coagulation and permeability properties of human endothelial cells in vitro induced by TNF-alpha or 5,6 MeXAA.
5,6 dimethyl xanthenone acetic acid (5,6 MeXAA), an analogue of flavone acetic acid (FAA), has been shown to be more active against murine tumours than FAA. As both drugs have a vascular component in their mechanism of action similar to that observed for TNF-alpha, we have studied the effects of 5,6 MeXAA alone and in combination with TNF-alpha on endothelial function in vitro. The changes induced by the drugs on procoagulant activity and permeability were determined under tumour-simulated conditions of low oxygen tension and the presence of tumour-secreted factors. Procoagulant activity was assayed by measuring the time taken for human umbilical vein endothelial cells (HUVECs) to clot normal human plasma, increased activity resulting in reduced clotting times. HUVECs incubated under aerobic conditions were more sensitive to TNF-alpha than cells incubated at < or = 0.2% oxygen. Culture medium conditioned by the human breast adenocarcinoma cell line MDA-MB-231 strongly upregulated procoagulant activity under both aerobic and hypoxic conditions; clotting times were further reduced by TNF-alpha. Both 5,6 MeXAA and FAA potentiated the effect of TNF-alpha on normal hypoxic endothelial cells; however, under all other conditions, neither drug in combination with TNF-alpha upregulated clotting activity. The presence of tumour-secreted factors had a far greater effect on upregulating procoagulant activity than did oxygen tension. In contrast to procoagulant activity, permeability was insensitive to TNF-alpha and low concentrations of 5,6 MeXAA also caused no change in permeability.
Antineoplastic Agents↗
Effect of nitro-L-arginine on blood flow, oxygenation and the activity of hypoxic cell cytotoxins in murine tumours.
This study was an investigation into the ability of nitro-L-arginine to change blood flow, oxygenation status and the activity of hypoxic cell cytotoxic agents in two different transplanted murine tumours. The tumour models were the C3H mammary carcinoma grown in the feet of female CDF1 mice and the SaF grown on the backs of CBA mice. Treatments were carried out in restrained non-anaesthetised animals when tumours were about 100 to 200 mm3 in size. Blood flow was monitored using laser Doppler flowmetry; oxygen partial pressure (pO2) distributions were obtained with an Eppendorf oxygen electrode; and response to treatment with hyperthermia (43.5 degrees C; 30 min) and RB6145 (250 mg kg-1;i.p.) assessed using a tumour growth delay assay. Nitro-L-arginine (10 mg kg-1; i.v.) significantly reduced blood flow by around 40-60% within 15 min after injection in C3H tumour and by 30 min in the SaF. However, nitro-L-arginine had absolutely no effect on tumour pO2 measured at the time of maximal blood flow reduction in both tumour types. It also failed to enhance the response of the C3H tumour to heat, but did produce a small yet significant increase in the response of the SaF tumour to RB6145.
Animals↗
Microregional blood flow in murine and human tumours assessed using laser Doppler microprobes.
A multichannel laser Doppler system has been used to measure microregional fluctuations in perfusion in the HT29 human tumour xenograft and in patients with advanced malignant disease. A comparison is made with previously obtained data for the SaF, a transplantable murine tumour. The 300 microns diameter probes recorded fluctuations in erythrocyte flux in tumour microregions with an estimated volume of 10(-2) mm3. Of the 66 human tumour microregions sampled, 26% showed a change in erythrocyte flux by a factor of 2 or more over the 60 min measurement period, compared with 37% of HT29 and 48% of SaF microregions. In each of the studies more than 50% of changes were completed within 20 min, although slower changes were more common in the human tumours than in the experimental systems. Within the 1 h monitoring period at least 30% of the changes were reversed (human tumours 30%, HT29 45%, SaF 31%). These findings demonstrate that microregional changes in erythrocyte flux, consistent with transient, perfusion-driven changes in oxygenation, are a feature of human malignancies as well as experimental transplanted tumours.
Aged↗
Early skin response to fractionated doses of pions for determining therapeutic gain factors.
This study concerns the biological effectiveness of fractionated doses of pions on early skin reactions near the implanted tumor, to evaluate the therapeutic gain factors of pions. The C3H mouse limbs bearing microscopical SCCVII tumors were irradiated with pions (9.6-38.4 Gy) or x-rays (14.4-50.4 Gy) in 2-7 fractions. Nicotinamide (500 mg/kg) and carbogen (a mixture of 95% O2 + 5% CO2), as hypoxic radiosensitizers, were administered prior to the x-rays, to confirm the presence of hypoxic cells in the skin. The mean skin scores and number of damaged nails assessed. The ratios of x-ray/pion doses needed for giving comparable skin reactions were 1.3-1.5. Nicotinamide and carbogen enhanced the skin reactions. When the ratios were compared with those of tumor cure, the pions showed no therapeutic gains. One of the possible causes was that the presence of hypoxic cells in the skin may have reduced the therapeutic gain.
Animals↗
Ischemia reperfusion injury in tumors: the role of oxygen radicals and nitric oxide.
Oxidative stress is a key process involved in the action of several therapeutic modalities used in cancer treatment. Ischemia reperfusion insult provides a model system for investigating the processes involved in determining the sensitivity of tumor tissue to oxidative stress. We have investigated the response of the murine CaNT tumor to ischemia reperfusion injury and the role that oxygen radicals and nitric oxide may play in this phenomenon. Our results show that little or no cell kill is detected in tumors exposed to up to 3 h of ischemia if the tumors are excised immediately before reperfusion. However, if reperfusion is permitted, then extensive cell kill is evident 24 h later. i.v. administration of superoxide dismutase or catalase, at the time when vascular reperfusion occurred, resulted in a significant protection against tumor cell kill, suggesting that the damage was mediated by oxygen radicals. Conversely, administration of an inhibitor of nitric oxide synthase, N omega-nitro-L-arginine, resulted in potentiation of tumor cell damage. Administration of a nitric oxide (NO) donor, diethylamine NO, at the time when vascular reperfusion occurred resulted in significant protection against tumor damage. These results suggest that nitric oxide is a potent mediator in determining tumor damage after ischemia reperfusion injury. The role of intrinsic NO production by murine tumors was investigated by measuring the accumulation of nitrate in the medium of tumor explants cultured in vitro in two tumors with differing sensitivity to ischemia reperfusion damage. The clamp-insensitive tumor SaS showed a greater nitrate accumulation than the clamp-sensitive tumor CaNT, which may confer a greater capacity for preventing tumor and endothelial cell damage after oxidative stress.
Adenocarcinoma↗
Anti-vascular approaches to solid tumour therapy: evaluation of vinblastine and flavone acetic acid.
Several agents have now been identified which exert their anti-tumour effects in large part via the tumour vasculature; these include TNF alpha and flavone acetic acid (FAA). More recently, Vincristine and Vinblastine have also been shown to cause a prolonged and selective decrease in tumour perfusion. Vinblastine, unlike, FAA, causes no increase in plasma TNF alpha levels in mice bearing the CaNT tumour, suggesting 2 distinct mechanisms of anti-vascular activity for these structurally diverse agents. Since FAA and Vinblastine also show quite different normal tissue toxicities, which are separately dose-limiting, we have examined the strategy of combining these 2 agents. When Vinblastine preceded FAA by 24 hr or less, tumour growth delay was significantly enhanced without a concomitant increase in toxicity. The level of enhancement was not significantly reduced by a 5-fold decrease in Vinblastine dose, though any reduction in the dose of FAA caused a rapid reduction in treatment effectiveness. Investigation of the functional vasculature of treated tumours suggested that increased anti-vascular effects may contribute to the enhanced growth inhibition of the combined treatment. Our results demonstrate the potential benefit of combining 2 different classes of anti-vascular agent, using Vinblastine and FAA (or 5,6-MeXAA) as prototype drugs.
Adenocarcinoma↗
Temporal heterogeneity in microregional erythrocyte flux in experimental solid tumours.
Using a multichannel laser Doppler system equipped with custom-developed microprobes, we have evaluated real-time fluctuations in microregional blood flow in two experimental murine tumour systems. The results show that in both the sarcoma F and the carcinoma NT over 50% of the microregions evaluated show a fluctuation in red blood cell flux by at least a factor of 2 over a 60 min time period. Approximately 20% of the regions monitored demonstrated a change in flow by a factor of 5 or more. Within the 1 h monitoring period, approximately 20% of the changes were reversed (SaF, 21%; CaNT, 19%). The duration of change for these regions ranged from 6 to 45 min. Similar temporal fluctuations in flow were seen in anaesthetised and unanaesthetised animals, indicating that artifacts due to probe movement were minimal. These findings clearly demonstrate that fluctuations in microregional erythrocyte flux are a common feature of the experimental tumours studied.
Adenocarcinoma↗