Tissue physiology and the response to heat

The most important physiological parameter influencing tissue response to heat is blood flow. At mild hyperthermia temperatures blood perfusion increases in many tumours and this effect is heating time-, temperature- and tumour-dependent. These flow increases can improve tumour oxygenation.

?When heating is terminated, perfusion and oxygenation commonly recover, although how quickly this occurs appears to be tumour-specific. While these effects are unlikely to have any anti-tumour activity they can be exploited to improve the combination of heat with other therapies. However, since similar physiological effects should occur in normal tissues, such combination therapies must be carefully applied. Heating tumours to higher temperatures typically causes a transient increase in perfusion during heating, followed by vascular collapse which if sufficient will increase tumour necrosis. The speed and degree of vascular collapse is dependent on heating time, temperature and tumour model used.

?Such vascular collapse generally occurs at temperatures that cause a substantial blood flow increase in certain normal tissues, thus preferential anti-tumour effects can be achieved. The tumour vascular supply can also be exploited to improve the response to heat. Decreasing blood flow, using transient physiological modifiers or longer acting vascular disrupting agents prior to the initiation of heating, can both increase the accumulation of physical heat in the tumour, as well as increase heat sensitivity by changing the tumour micro-environmental parameters, primarily an increase in tumour acidity.

?Such changes are generally not seen in normal tissues, thus resulting in a therapeutic benefit.     

Imaging tumour physiology and vasculature to predict and assess response to heat

The vascular supply of tumours and the tumour microenvironment both play an important role when tumours are treated with hyperthermia. Blood flow is one of the major vehicles by which heat is dissipated thus the vascular supply will influence the ability to heat the tumour. It also influences the type of microenvironment that exists within tumours, and it is now well-established that cells existing in areas of oxygen deficiency, nutrient deprivation and acidic conditions are more sensitive to the effect of hyperthermia. The vascular supply and microenvironment are also affected by hyperthermia. In general, mild heat temperatures transiently improve blood flow and oxygenation, while higher hyperthermia temperatures cause vascular collapse and so increase the adverse microenvironmental conditions. Being able to image these vascular and microenvironmental parameters both before and after heating will help in our ability to predict and assess response. Here we review the various techniques that can be applied to supply this information, especially using non-invasive imaging approaches.     

Role of HIF-1α in response of tumors to a combination of hyperthermia and radiation in vivo

Purpose: Mild temperature hyperthermia (MTH) increases blood flow and oxygenation in tumours. On the other hand, high-dose-per-fraction irradiation damages blood vessels, decreases blood flow and increases hypoxia in tumours. The radiation-induced hypoxia in tumours activates hypoxia-inducible factor-1α (HIF-1α) and its target genes, such as vascular endothelial growth factor (VEGF), promoting revascularization and recurrence. In the present study, we examined the hypothesis that MTH inhibits radiation-induced upregulation of HIF-1α and its target genes by increasing tumour oxygenation.

Materials and methods: FSaII fibrosarcoma tumours grown subcutaneously in the legs of C3H mice were used. Tumours were irradiated with 15?Gy using a ⁶⁰Co irradiator or heated at 41?°C for 30?min using an Oncothermia heating unit. Blood perfusion and hypoxia in tumours were assessed with Hoechst 33342 and pimonidazole staining, respectively. Expression levels of HIF-1α and VEGF were determined using immunohistochemical techniques. Apoptosis of tumour cells was quantitated via TUNEL staining and the effects of treatments on tumour growth rate were assessed by measuring tumour diameters.

Results: Irradiation of FSaII tumours with a single dose of 15?Gy led to significantly decreased blood perfusion, increased hypoxia and upregulation of HIF-1α and VEGF. On the other hand, MTH at 41?°C for 30?min increased blood perfusion and tumour oxygenation, thereby suppressing radiation-induced HIF-1α and VEGF in tumours, leading to enhanced apoptosis of tumour cells and tumour growth delay.

Conclusion: MTH enhances the anti-tumour effect of high-dose irradiation, at least partly by inhibiting radiation-induced upregulation of HIF-1α.     

Reply

Blood flow in tumours and healthy tissue determines the ability of obtaining satisfactory temperature distributions in clinical hyperthermia, as well as the success of hyperthermia and radiation treatment. During the hyperthermia treatment, diagnostic data related to tissue blood flow can be determined by analysing the relationship between the amount of power absorbed in the tissue and the resulting temperature rise. The interpretation of the perfusion data (PERF) is highly complicated by the lack of an adequate theory to describe the heat transport in vascularized tissues. In vascularized breast tissues about 10 times as much power is needed to maintain therapeutic temperatures as is necessary in a stationary breast phantom. This large difference in maintenance power levels indicates the extreme importance of blood flow in tissue heat transport.

PERF has been determined in 23 patients with advanced breast tumours. In this series (a) perfusion typically did not change during the stationary part of the individual hyperthermic sessions, (b) minimum tumour PERF was not related to tumour volume, and (c) there was no relation between tumour PERF and the ability to heat tumours. PERF can both increase and decrease after successful hyperthermia.     

Perfusion analyses in advanced breast carcinoma during hyperthermia

Blood flow in tumours and healthy tissue determines the ability of obtaining satisfactory temperature distributions in clinical hyperthermia, as well as the success of hyperthermia and radiation treatment. During the hyperthermia treatment, diagnostic data related to tissue blood flow can be determined by analysing the relationship between the amount of power absorbed in the tissue and the resulting temperature rise. The interpretation of the perfusion data (PERF) is highly complicated by the lack of an adequate theory to describe the heat transport in vascularized tissues. In vascularized breast tissues about 10 times as much power is needed to maintain therapeutic temperatures as is necessary in a stationary breast phantom. This large difference in maintenance power levels indicates the extreme importance of blood flow in tissue heat transport. PERF has been determined in 23 patients with advanced breast tumours. In this series (a) perfusion typically did not change during the stationary part of the individual hyperthermic sessions, (b) minimum tumour PERF was not related to tumour volume, and (c) there was no relation between tumour PERF and the ability to heat tumours. PERF can both increase and decrease after successful hyperthermia.     

Physiological mechanisms in hyperthermia: a review

In experimental animal systems, hyperthermia at therapeutic temperature (43-45 degrees C) causes a profound increase in blood flow in normal tissues while it induces only meager and temporal increases in blood flow in tumors. A severe vascular occlusion and hemorrhage usually follows the increase in blood flow in the tumors at the above temperatures. Another pronounced physiological change in tumors by heat is a prompt decrease in intratumor pH. The decrease in intratumor pH would accentuate the thermokilling of tumor cells and also possibly inhibit repair of thermodamage and development of thermotolerance in tumors. The temperature in tumors may rise higher than that in normal tissues during heating because of inefficient heat dissipation from the tumor as a result of decrease blood flow or vascular occlusion. Thus, the differential effects of heat on vascular function and pH in tumors and normal tissues may result in a greater damage in tumors than in surrounding normal tissues. Further investigation is urgently needed to find out whether similar physiological changes occur in human tumors and normal tissues by hyperthermia.     

Reduction of tumour blood flow with KB-R8498 potentiates the response of tumours to hyperthermia.

The anti-tumour activity and the effect on tumour and normal tissue perfusion of a newly discovered anticancer agent, KB-R8498 (Kanebo Ltd., Osaka, Japan), were investigated in FSa II tumours of C3H mice. The tumour perfusion, as measured by the 86Rb-uptake method, markedly decreased with relatively little change in the normal tissue perfusion after an i.v. injection of KB-R8498. Furthermore, the drug potentiated the effect of hyperthermia at 42.5 degrees C for 60 min to suppress the tumour growth. The results suggest that the preferential reduction in tumour blood flow relative to normal tissue blood flow by KB-R8498 may be exploited to enhance the anti-tumour effect of hyperthermia.     

Tumour oxygenation is increased by hyperthermia at mild temperatures

The effects of hyperthermia on the oxygenation status in R3230 AC tumours of Fischer rats were measured using a polarographic oxygen electrode system. The median pO₂ in about 10 mm diameter tumours grown s.c. in the leg of rats was 3.7 ± 0.3 mm Hg and it significantly increased upon heating at modest temperatures. For example, the tumour pO₂ measured within 10–15 min after heating for 30 min at 42.5°C was about three-fold greater than that in the control tumours. About 62% of pO₂ values measured in control tumours were <5 mm Hg. After heating at 42.5°C for 30 min, 37% of pO₂ values were <5 mm Hg. Such an increase in tumour oxygenation or reoxygenation of hypoxic cells appeared to result from an increase in tumour blood flow caused by the mild temperature hyperthermia. The presence of hypoxic cells in tumours is believed to be a major factor in limiting the effectiveness of radiotherapy, certain chemotherapy drugs and phototherapy. Hyperthermia at mild temperatures easily achievable with the use of presently available clinical hyperthermia devices may be an effective means to overcome the hypoxic protection in the treatment of human tumours.     

Mild temperature hyperthermia and radiation therapy: Role of tumour vascular thermotolerance and relevant physiological factors

Here we review the significance of changes in vascular thermotolerance on tumour physiology and the effects of multiple clinically relevant mild temperature hyperthermia (MTH) treatments on tumour oxygenation and corresponding radiation response. Thus far vascular thermotolerance referred to the observation of significantly greater blood flow response by the tumour to a second hyperthermia exposure than in response to a single thermal dose, even at temperatures that would normally cause vascular damage. New information suggests that although hyperthermia is a powerful modifier of tumour blood flow and oxygenation, sequencing and frequency are central parameters in the success of MTH enhancement of radiation therapy. We hypothesise that heat treatments every 2 to 3 days combined with traditional or accelerated radiation fractionation may be maximally effective in exploiting the improved perfusion and oxygenation induced by typical thermal doses given in the clinic.     

Pathophysiological and vascular characteristics of tumours and their importance for hyperthermia: Heterogeneity is the key issue

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 O₂ 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.     

Tumour oxygenation is increased by hyperthermia at mild temperatures

The effects of hyperthermia on the oxygenation status in R3230 AC tumours of Fischer rats were measured using a polarographic oxygen electrode system. The median pO₂ in about 10mm diameter tumours grown s.c. in the leg of rats was 3·7 ± 0·3 mm Hg and it significantly increased upon heating at modest temperatures. For example, the tumour pO₂ measured within 10–15 min after heating for 30 min at 42·5°C was about three-fold greater than that in the control tumours. About 62% of pO₂ values measured in control tumours were < 5 mm Hg. After heating at 42·5°C for 30min, 37% of pO₂ values were < 5 mm Hg. Such an increase in tumour oxygenation or reoxygenation of hypoxic cells appeared to result from an increase in tumour blood flow caused by the mild temperature hyperthermia. The presence of hypoxic cells in tumours is believed to be a major factor in limiting the effectiveness of radiotherapy, certain chemotherapy drugs and phototherapy. Hyperthermia at mild temperatures easily achievable with the use of presently available clinical hyperthermia devices may be an effective means to overcome the hypoxic protection in the treatment of human tumours.     

The Influence of Nitric Oxide on Tumour Vascular Tone

Acetylcholine and sodium nitroprusside, which vasodilate via release of NO by endothelium-dependent and endothelium-independent mechanisms respectively, had little effect on tumour vascular resistance when administered to tissue-isolated tumours perfused in their normal state. However, under phenylephrine-induced vasoconstriction, sodium nitroprusside induced vasodilation whilst acetylcholine induced a small vasoconstriction. Phenylephrine itself induced an oscillatory change in tumour perfusion pressure. The nitric oxide synthase (NOS) inhibitor N^Ω-nitro-L-arginine (L-NNA) caused a dose-dependent increase in vascular resistance in ex vivo perfused tumours which was greater than that in normal perfused hindlimbs. Systemic administration of L-NNA caused a 50% decrease in tumour blood flow which was a larger effect than in any of the normal tissues studied except spleen and skeletal muscle. Modification of NOS activity in tumours is a promising means for selective tumour blood flow modification. Investigation of endothelium-dependent versus endothelium-independent methods for modifying tumour blood flow may provide methods for further selectivity.     

Changes in hepatic blood flow during regional hyperthermia

The influence of liver hyperthermia on hepatic arterial and portal venous blood flow to tumour and normal hepatic tissue was examined in a rabbit VX2 tumour model. Hyperthermia was delivered by 2450 MHz microwave generator to exteriorized livers in 18 rabbits. Blood flow was measured in both portal vein and hepatic artery using radioactive tracer microspheres before, during and 5 min after intense (greater than 43 degrees C) hyperthermia. During hyperthermia a decrease in total liver blood flow was composed primarily of a decrease in hepatic arterial blood flow to tumour tissue. Tumours were supplied almost exclusively by the hepatic artery and thus total tumour blood flow was significantly depressed during heating. The decreased tumour blood flow persisted after the cessation of hyperthermia and was indicative of vascular collapse in the tumour tissue. Temperature differentials in tumour compared to normal tissue ranged from 5 degrees C to 8 degrees C during hyperthermia because of the lower tumour blood flow. The portal vein exerted minimal influence on temperatures attained in the tumour tissue during hyperthermia but would have mediated normal liver tissue heat loss.     

Tumour microcirculation as a target for hyperthermia

A great number of investigators have, independently, shown that tumour blood flow is affected by a hyperthermic treatment to a larger extent than normal tissue blood flow. While the majority of the studies on experimental tumours show a decrease and even a lapse in blood flow within the microcirculation during or after hyperthermia, the data on human tumours are less conclusive. Some of the investigators do not find a decrease in circulation, while others do. Obviously, this is an important field of investigation in the clinical application of hyperthermia because a shut down of the circulation would not only facilitate tumour heating (by reducing venous outflow, this reducing the 'heat clearance' from the tumour), but would also facilitate tumour cell destruction. The same holds for alterations that occur subsequently to the circulatory changes, like a heat-induced decrease of tissue pO2 and pH. If the frequently reported circulatory collapse of the tumour circulation could selectively be stimulated by, e.g. acidification or by vasoactive agents, hyperthermic treatment of patients would possibly be greatly facilitated and intensified. In hyperthermic tumour therapy a number of complex processes and interactions takes place, especially when the treatment is performed in combination with radiation therapy. One of them represents the group of processes related to the random probability of cell sterilization of individual tumour cells resulting in exponential survival curves which are typically evaluated with e.g. cell survival assays. This aspect has not been the issue of this paper. The other group of processes deals with the heat-induced changes in the micro-physiology of tumours and normal tissues which, as discussed before, may not only enhance the exponential cell kill, but which may also culminate in vascular collapse with the ensuing necrosis of the tumour tissue in the areas affected. If this takes place, a process of bulk killing of tumour cells results, rather than the random type of cell sterilization. At present it is not clear to what extent the various separate mechanisms contribute to the total effect of tumour control. With all these considerations in mind, one should be aware of the fact that effects, secondary to heat-induced vascular stasis alone will never be efficient enough to eliminate all tumour cells, even though a heat reservoir is created. This is so because some malignant cells will inevitably have already infiltrated normal, surrounding structures and will therefore not be affected by changes in the tumour vascular bed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Local tumour hyperthermia as immunotherapy for metastatic cancer

Local tumour hyperthermia for cancer treatment is currently used either for ablation purposes as an alternative to surgery or less frequently, in combination with chemotherapy and/or radiation therapy to enhance the effects of those traditional therapies. As it has become apparent that activating the immune system is crucial to successfully treat metastatic cancer, the potential of boosting anti-tumour immunity by heating tumours has become a growing area of cancer research. After reviewing the history of hyperthermia therapy for cancer and introducing methods for inducing local hyperthermia, this review describes different mechanisms by which heating tumours can elicit anti-tumour immune responses, including tumour cell damage, tumour surface molecule changes, heat shock proteins, exosomes, direct effects on immune cells, and changes in the tumour vasculature. We then go over in vivo studies that provide promising results showing that local hyperthermia therapy indeed activates various systemic anti-tumour immune responses that slow growth of untreated tumours. Finally, future research questions that will help bring the use of local hyperthermia as systemic immunotherapy closer to clinical application are discussed.     

Changes in hepatic blood flow during regional hyperthermia

The influence of liver hyperthermia on hepatic arterial and portal venous blood flow to tumour and normal hepatic tissue was examined in a rabbit VX2 tumour model. Hyperthermia was delivered by 2450 MHz microwave generator to exteriorized livers in 18 rabbits. Blood flow was measured in both portal vein and hepatic artery using radioactive tracer microspheres before, during and 5 min after intense (>43°C) hyperthermia. During hyperthermia a decrease in total liver blood flow was composed primarily of a decrease in hepatic arterial blood flow to tumour tissue. Tumours were supplied almost exclusively by the hepatic artery and thus total tumour blood flow was significantly depressed during heating. The decreased tumour blood flow persisted after the cessation of hyperthermia and was indicative of vascular collapse in the tumour tissue. Temperature differentials in tumour compared to normal tissue ranged from 5°C to 8°C during hyperthermia because of the lower tumour blood flow. The portal vein exerted minimal influence on temperatures attained in the tumour tissue during hyperthermia but would have mediated normal liver tissue heat loss.     

Drug-induced alterations in tumour perfusion yield increases in tumour cell radiosensitivity.

The perfusion of human tumour xenografts was manipulated by administration of diltiazem and pentoxifylline, and the extent that observed changes in tumour perfusion altered tumour radiosensitivity was determined. 2 tumour systems having intrinsically different types of hypoxia were studied. The responses of SiHa tumours, which have essentially no transient hypoxia, were compared to the responses of WiDr tumours, which contain chronically and transiently hypoxic cells. We found that relatively modest increases in net tumour perfusion increased tumour cell radiosensitivity in WiDr tumours to a greater extent than in SiHa tumours. Moreover, redistribution of blood flow within WiDr tumours was observed on a micro-regional level that was largely independent of changes in net tumour perfusion. Through fluorescence-activated cell sorting coupled with an in vivo-in vitro cloning assay, increases in the radiosensitivity of WiDr tumour cells at intermediate levels of oxygenation were observed, consistent with the expectation that a redistribution of tumour blood flow had increased oxygen delivery to transiently hypoxic tumour cells. Our data therefore suggest that drug-induced changes in tumour micro-perfusion can alter the radiosensitivity of transiently hypoxic tumour cells, and that increasing the radiosensitivity of tumour cells at intermediate levels of oxygenation is therapeutically relevant.     

Nifedipine improves blood flow and oxygen supply,but not steady-state oxygenation of tumours in perfusion pressure-controlled isolated limb perfusion

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.     

Blood perfusion measurements in human tumours: evaluation of laser Doppler methods

Laser Doppler flowmetry is a simple method of determining, directly and continuously, tissue blood flow. However, its applicability to monitoring tumour blood flow interstitially during hyperthermia treatments is still being evaluated. The purposes of this study were to physically characterize the measurement probes, to evaluate potential sources of artifact with the interstitial use of the probes during hyperthermia treatment, and to obtain measurements in human tumours during hyperthermia sessions. The accuracy of the method in quantifying blood flow, velocity and volume during hyperthermia was found to be unaffected by heating the measurement probe to 42-46 degrees C or by exposing it to various intensities of 915 MHz microwave fields (10-40 W), or 1 MHz ultrasound fields. Catheter insertion methods were developed to place the flow probes interstitially in tumours. Tissue damage was confined to a distance of no greater than 0.12 mm away from the catheter tract, and physical evidence of vascular disruption was within a distance of 0.05 mm as measured in a rat tumour model. This degree of damage/disruption is unlikely to affect LDF measurements which represent blood flow averaged over a 1.0-1.5 mm radius from the probe tip. Concurrently, the device was used to monitor tumour blood flow parameters interstitially in human subjects during hyperthermia treatments given in combination with conventional radiotherapy. Blood-flow data from multiple sites of measurement showed marked heterogeneity within individual tumours (up to 55-fold differences) and between different tumours (greater than 100-fold differences). Measurements made by translating the probe along a tumour radius, beginning at the tumour core and advancing to the tumour edge, were consistent with a two-component tumour perfusion model (shell and core). Data are presented from one patient illustrating a persistent change in perfusion distribution during the hyperthermia treatment course, which occurred concomitantly with increases in thermal data. These results suggest that the technique might be of value in monitoring change in flow between treatments. Responses during hyperthermia treatment sessions were also investigated. Four temporal patterns of flow were observed, ranging from a steady increase in flow to a plateau level to a steady drop in flow during heating. These patterns were not well correlated with average temperature recorded at the site of flow measurement. Further study is needed to determine if this LDF technique is to be useful for evaluation of heat transfer by blood perfusion.     

Resistance to flow through tissue-isolated transplanted rat tumours located in two different sites.

The perfusion characteristics of the P22 carcinosarcoma were investigated in tissue-isolated tumour preparations in the ovarian and inguinal fat pads of BD9 rats. Tumours were perfused with a physiological buffer of known viscosity and changes in perfusion pressure were recorded at different perfusion rates in an ex vivo system. At perfusion pressures exceeding 30-40 mmHg tumour flow rate was directly proportional to the perfusion pressure in all tumours, indicating a constant resistance to flow. An apparent positive pressure difference across the tumour vasculature of 20-30 mmHg occurred under conditions of zero flow in either site. At low perfusion pressures, the flow resistance increased sharply due to increases in the geometric resistance of the tumours. These findings are in accord with previously published data. Geometric resistance increased with tumour volume in both sites and was approximately five times greater in the inguinal tumours than it was in the ovarian tumours, on a weight to weight basis. The dependence of tumour geometric resistance on perfusion pressure differs from the situation in normal tissues and may provide a means of manipulating the tumour microcirculation to the exclusion of the systemic blood supply. The dependence of geometric resistance on tumour site may partly explain why tumours located in different sites respond differently to various forms of therapy.