Search

Cervical cancers exhibit substantial intra- and inter-tumour heterogeneities in blood flow prior to treatment, reflecting similar variability in vascularisation. When clinically relevant hyperthermia is applied as an adjuvant to established treatment modalities, blood flow may change in non-predictable directions, extents and durations, indicating subsequent variability in heat dissipation and in flow-associated parameters of the tumour microenvironment. Before heating, locally advanced cervical cancers are mostly hypoxic, acidic, exhibit substrate and energy deprivation and show lactate accumulation, which is spatially and temporally heterogeneous. Additionally a relatively homogeneous interstitial hypertension is observed. Most probably, metabolic parameters of the hostile microenvironment are able to greatly modulate the thermosensitivity of cancer cells. Adequate information concerning changes upon heat treatment is not available so far. Due to this lack of proven data for cervical cancers upon heat treatment, clinical studies are urgently needed in order to judge the possible impact of blood flow and the above-mentioned microenvironmental parameters.

Tumour blood flow before and during clinically relevant mild hyperthermia exhibits pronounced heterogeneity. Flow changes upon heating are not predictable and are both spatially and temporally highly variable. Flow increases may result in improved heat dissipation to the extent that therapeutically relevant tissue temperatures may not be achieved. This holds especially true for tumours or tumour regions in which flow rates are substantially higher than in the surrounding normal tissues. Changes in tumour oxygenation tend to reflect alterations in blood flow upon hyperthermia. An initial improvement in the oxygenation status, followed by a return to baseline levels (or even a drop to below baseline at high thermal doses) has been reported for some tumours, whereas a predictable and universal occurrence of sustained increases in O(2) tensions upon mild hyperthermia is questionable and still needs to be verified in the clinical setting. Clarification of the pathogenetic mechanisms behind possible sustained increases is mandatory. High-dose hyperthermia leads to a decrease in the extracellular and intracellular pH and a deterioration of the energy status, both of which are known to be parameters capable of acting as direct sensitisers and thus pivotal factors in hyperthermia treatment. The role of the tumour microcirculatory function, hypoxia, acidosis and energy status is complex and is further complicated by a pronounced heterogeneity. These latter aspects require additional critical evaluation in clinically relevant tumour models in order for their impact on the response to heat to be clarified.

It is generally accepted that tumor blood flow, microcirculation, oxygen and nutrient supply, tissue pH distribution, and the bioenergetic status-factors which are usually closely linked and which define the so-called metabolic microenvironment--can markedly influence the therapeutic response of malignant tumors to conventional irradiation, chemotherapy, other nonsurgical treatment modalities, and the cell proliferation activity within tumors. Currently available information on the parameters defining the metabolic micromilieu in human tumors is presented in this review. According to these data, significant variations in these relevant factors are likely to occur between different locations within a tumor, and between tumors of the same grade and clinical stage. The extent and mechanisms by which the microenvironmental factors influence cellular response to standard irradiation, chemotherapeutic drugs, immunotherapy and localized hyperthermia are described and "physiological" barriers to treatment are outlined.

The changes of microregional perfusion in a hamster cheek pouch membrane were investigated. The vessel network of the membrane was visualized by preparing a transparent chamber, which was heated with circulating water at 42 degree C. Blood perfusion was monitored by using a laser Doppler flowmeter (LDF), which was used either in a conventional way by positioning the probe stationary or in a novel way by constantly moving the probe over the surface of the chamber (scanning). When a segment of tissue was subjected to the LDF scanning, the profile of scanned LDF values was well correlated with the distribution of vessels. Therefore, this scanning technique was useful in localizing the probe in tissues with respect to vessels. Since the scanning can be repeated every other minute, this technique also offered continuous monitoring of tissue blood flow at multiple sites. Upon heating, different vessels individually responded to the first and second heatings followed by coolings, suggesting a heterogeneous heat response in the connective tissue of the hamster cheek pouch membrane. This scanning technique proved very useful in collecting information for the study of the heterogeneous nature of blood flow in normal and tumour tissues.

The effect of hyperthermia on microcirculatory and metabolic parameters in s.c. DS-sarcomas of different sizes on the hind foot dorsum of SD-rats was investigated. Hyperthermia was carried out using a novel water-filtered, infrared-A radiation technique. Heating was performed at a rate of 0.5 degrees C/min until 44 degrees C was achieved in the tumour centre, which was maintained for 60 min. Using a multichannel laser Doppler flowmeter, red blood cell flux could be assessed continuously and at several sites within the tumour tissue simultaneously. Substantial inter-site variations in laser Doppler flux (LDF) were observed during hyperthermia which were independent of tumour size, site of measurement, and temperature at the site of measurement, indicating that single site measurements of tumour LDF are poor predictors of the mean response of a tumour to hyperthermia. When mean LDF was considered, decreases in red blood cell fluxes occurred that were more pronounced the greater the tumour volume. In no case was vascular stasis observed. Hyperthermia did not affect tumour oxygenation substantially. Microregional and global assessment of lactate and ATP concentrations demonstrated increased lactate and decreased ATP levels following hyperthermia. Tumour glucose levels were increased following hyperthermia, possibly due to an enlarged distribution space resulting from development of interstitial oedema. Changes in lactate and ATP levels and the lack of changes in tumour oxygenation suggest a modification of energy metabolism following hyperthermia in the form of increased ATP hydrolysis, intensified glycolysis and impaired oxidative phosphorylation.

Tumor oxygenation has been measured polarographically in s.c. implanted DS-sarcomas on the dorsum of the hind foot of male Sprague-Dawley rats. pO2 was determined in all 3 spatial dimensions and 3-dimensional pO2 distributions as well as the mean extent of confluent areas with pO2 < 5 mmHg were calculated. Finally, the effect of elevating arterial pO2 (by carbogen breathing) as well as of increasing tumor blood flow (by angiotensin infusion) on the spatial pO2 distribution was analyzed. Depending on the tumor volume, the spatial pO2 distribution is more or less anisotropic. In smaller tumors, areas with physiological pO2 values are found adjacent to large hypoxic areas whereas larger tumors are almost completely hypoxic/anoxic. With carbogen breathing, the mean tissue pO2 is elevated although hypoxia is not eradicated in larger tumors. In small tumors, angiotensin leads to a vasoconstriction of tumor vessels followed by a worsening of tumor oxygenation whereas in large tumors the increased systemic perfusion pressure resulted in an improvement of oxygenation. Thus, carbogen predominantly affects pO2 diffusion by increasing the arterial pO2 whereas angiotensin influences tumor perfusion and leads to an increased oxygen supply to the tumor tissue.

The effect of cyclophosphamide (CP) injection (60 mg/kg i.p., single dose) on volume growth and tissue oxygenation (pO2 distribution) was investigated in rat DS-sarcomas. CP was administered 4 days after subcutaneous (s.c.) tumor implantation (volume approximately 0.35 ml). Polarographic pO2 measurements were performed in the subcutis at the hind foot dorsum and in tumors 72 h after CP administration. The oxygenation status of these tissues was compared with that of saline-treated controls. CP-injection caused a mean growth delay of 11 days in DS-sarcomas and had no impact on the oxygenation status of the subcutis. In contrast, in s.c. growing DS-sarcomas the pO2 distribution improved significantly when treated tumors (0.59 ml volume) were compared with their untreated counterparts (1.15 ml volume). Comparison of the oxygenation data of CP-treated tumors with size-matched controls revealed an identical oxygenation status in the experimental tumors used. Thus, when 'reoxygenation' is discussed, one should consider whether it is solely the result of tumor shrinkage or a volume-independent phenomenon.

Despite continued interest in the administration of nicotinamide (NA) as a tumor-specific radiosensitizer (an effect thought to be related to increases in tumor blood flow and oxygenation), little is known about the underlying mechanism(s) of this effect. The aim of this study was to investigate metabolic changes following NA application in both tumor and normal tissues. Increased concentrations of NAD+ were measured in DS-sarcomas, liver, and kidney tissue, with no changes in concentrations in resting skeletal muscle. Further investigations also examined the concentrations of glucose, lactate, ATP, ADP and AMP in tumor and resting skeletal muscle tissue following NA application. Here, the only change detected was an increase in lactate levels in tumor tissue. The changes in NAD+ concentrations described correlate well with reported changes in tissue blood flow measured following NA. On the basis of changes in tumor blood flow, oxygenation and metabolite concentrations found in this and other recent studies, possible mechanisms for tumor radiosensitization following nicotinamide administration are considered.

Purpose: Nicotinamide has been reported to preferentially radiosensitize tumor tissue, supposedly through a reduction in tumor hypoxia. This may occur as a result of nicotinamide-induced changes in tumor blood flow and therefore the present study was undertaken to evaluate the effect of nicotinamide on circulatory parameters in skeletal muscle and tumor tissue (subcutaneously-implanted DS-sarcomas) of the rat., Methods and Materials: Mean arterial blood pressure (measured in the common carotid artery using a pressure transducer) and red blood cell flux (as measured by laser Doppler flowmetry) were continuously monitored for 120 min following a single intraperitoneal application of nicotinamide (500 mg/kg). An arterial blood pressure/laser Doppler flux ratio was estimated for tumor and muscle tissue., Results: Nicotinamide significantly reduced the mean arterial blood pressure to a minimum value 25% below the pretreatment value 20 min after the commencement of drug administration, with partial recovery thereafter. Red blood cell flux through tumor tissue, following an initial rapid decrease, rose steadily to values 34% above those measured in control animals at t = 60 min, while the arterial blood pressure/laser Doppler flux ratio in tumor tissue fell to values 34% below those of control animals. In skeletal muscle similar trends were seen although the changes were not of the same extent as those seen in tumor tissue. Tumor pO2 was measured 60 min following i.p. application of nicotinamide using polarographic needle electrodes. Despite the significant increase in blood flow following nicotinamide, no significant difference was seen between pO2 histograms obtained in tumors in nicotinamide-treated and control animals., Conclusion: These findings suggest that nicotinamide preferentially improves tumor microcirculatory function and effectuates a decrease in the arterial blood pressure/laser Doppler flux ratio within tumor tissue, effects which reach their maximum approximately 60 min following nicotinamide administration.

The oxyhemoglobin saturation (HbO2) of single red blood cells within tumor microvessels (diameter: 3-12 micrometers) of DS-Carcinosarcoma was studied using a cryophotometric micromethod. In untreated control tumors (mean tissue temperature approx. 35 degrees C) the measured values scattered over the whole saturation range from zero to 100 sat. %, the mean being 51 sat. %. Upon heating at 40 degrees C for 30 min, the oxygenation of the tumor tissue significantly improved as compared with control conditions. After 40 degrees C-hyperthermia a mean oxyhemoglobin saturation of 66 sat. % was obtained. In contradistinction to this, after 43 degrees C-hyperthermia the tumor oxygenation was significantly lower and reached a mean HbO2 saturation value of 47 sat. %. A further temperature rise to 45 degrees C caused the oxygenation to drop drastically (mean oxyhemoglobin saturation value: 24 sat. %). This is due to a severe restriction of nutritive blood flow. The changes in tumor oxygenation after hyperthermia seem to be predominantly mediated through changes in tumor blood flow, including tumor microcirculation, which showed a similar temperature dependence. Metabolic effects probably play a minor role in the oxyhemoglobin saturation distribution within tumor microvessels.

Responses of tumor microcirculation (RBC flux) to i.p. glucose or mannitol injections were studied in early generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma (FSaII). RBC flux in superficial tumor microregions was assessed using laser Doppler flowmetry. After administration of glucose or mannitol (a nonmetabolized sugar alcohol), a dose-dependent reduction in laser Doppler flow, and a dose-dependent increase in systemic hematocrit occurred concurrently. Maximum flow reductions induced by i.p. glucose or mannitol were statistically indistinguishable for equal osmotic load. Maximum decreases in RBC flux for glucose or mannitol were 20 and 25% (1.25 mg/g i.p.), 42 and 48% (2.5 mg/g i.p.), 72 and 60% (5 mg/g i.p.), and 80 and 75% (10 mg/g i.p.), respectively. Maximum increases in systemic hematocrit ranged from 18% (1.25 mg/g glucose i.p.) to 33% (10 mg/g glucose i.p.). Examination of RBC count, blood hemoglobin concentration, and fluid accumulation in the abdominal cavity after glucose or mannitol administration were all compatible with a significant shift of intravascular/extracellular water into the abdominal cavity with resultant systemic hypovolemic hemoconcentration. RBC volume and mean hemoglobin content of RBC remained unchanged with glucose loading. The data suggest that reductions in laser Doppler flow are predominantly caused by hypovolemic hemoconcentration following i.p. administration of hyperosmolar sugar solutions. Changes in laser Doppler flow due to specific glucose-mediated or glucose-related phenomena are probably of minor importance in the murine tumor system investigated. Future studies on murine tumors, examining for specific effects of glucose on metabolism and/or therapy, should not use i.p. administration of hyperosmolar solutions.

Severe disturbances in microcirculation during advanced phases of tumor growth lead to restrictions of convective and diffusive transport. In addition, an inhomogeneous distribution of transport conditions develops, resulting in insufficient and heterogeneous substrate supply and an inadequate drainage of wastes. Polarographic measurements of the local tissue oxygen tension (PO2) using gold microelectrodes reveal that very low PO2 values are prevalent in C3H mouse mammary carcinomas. The tissue PO2 frequency distributions are shifted to low PO2 values and limited in variability. The mean PO2 value is 7 mm Hg. The median is 4 mm Hg, the modal class being 0 to 5 mm Hg. Within different microareas of the same tumor, pronounced heterogeneities exist. Due to an elevated rate of lactic acid production and its subsequent inadequate removal, a severe tissue acidosis is evidenced in malignant tumors. For C3H mouse mammary carcinomas, most of the measured pH values are in the range of 6.4 to 7.1, the modal class being 6.7 to 6.8 (mean pH, 6.73; median pH, 6.75). Within different microareas of the same tumor, clear heterogeneities in the pH distribution do occur. Very low pH values (5.8 to 6.3) have been observed in large ulcerated tumors. In extensively necrotic areas, pH values even higher than the arterial pH could be detected.

Tumor acidosis and energy deprivation enhance thermal sensitivity. We have used in vivo 31P nuclear magnetic resonance spectroscopy to noninvasively monitor changes in pH and energy metabolism in FSaII mouse tumors after i.p. administration of glucose. A dose of 5 g/kg glucose induced a pH drop of 0.31 units without any statistically significant change in energy status. The pH changes resolved within 2 h. In contrast, administration of 10 g/kg glucose resulted in a severe acidosis (mean nadir pH of 6.19 corresponding to a mean pH drop of 0.96 units) and loss of energy, the latter most probably being due to an acidosis-induced inhibition of glycolysis during ischemic hypoxia. The resulting acidosis and energy loss were more persistent and resolved in 5.5-28 h. In contrast, after an identical dose of mannitol (10 g/kg), a pH drop of approximately only 0.1 units over 72 min was noted. The data suggest that both cleavage of glucose to lactic acid and blood flow inhibition are involved in glucose induced tumor acidosis. In vivo 31P nuclear magnetic resonance spectroscopy may be useful clinically to monitor therapeutic attempts at enhancing thermal sensitivity.