Glycolysis-related gene induction and ATP reduction during fractionated irradiation. Markers for radiation responsiveness of human tumor xenografts.

BACKGROUND AND PURPOSE: Lactate was previously shown to be a prognostic but not a predictive pre-therapeutic marker for radiation response of tumor xenografts. We hypothesize that metabolic changes during fractionated irradiation may restrict the predictiveness of lactate regarding tumor radiosensitivity. MATERIALS AND METHODS: Tumor xenografts were generated in nude mice by implanting 4 head and neck squamous cell carcinoma lines with different sensitivities to fractionated irradiation. Tumors were irradiated with up to 15 fractions of 2 Gy over a period of 3 weeks, and ATP and lactate levels were measured in vital tumor areas with induced metabolic bioluminescence imaging. Corresponding changes in mRNA expression of glycolysis-related genes were determined by quantitative RT-PCR. RESULTS: Lactate content decreased significantly in 3 out of 4 cell lines in the course of irradiation showing no correlation with cell line-specific radiosensitivity. Radiation-induced changes in ATP levels and glycolysis-related mRNA expression, however, only occurred in radiosensitive or intermediately radioresistant xenografts, whereas these parameters remained unchanged in radioresistant tumors. CONCLUSION: Sensitivity-related differences in the transcriptional response of tumors to radiotherapy may be exploited in the clinic for better individualization of tumor treatment.

Effects of three modifiers of glycolysis on ATP, lactate, hypoxia, and growth in human tumor cell lines in vivo.

BACKGROUND: High pretreatment tumor lactate content is associated with poor outcome after fractionated irradiation in human squamous cell carcinoma (hSCC) xenografts. Therefore, decreasing lactate content might be a promising approach for increasing tumor radiosensitivity. As the basis for such experiments, the effects of the biochemical inhibitors pyruvate dehydrogenase kinase dichloroacetate (DCA), lactate dehydrogenase oxamate, and monocarboxylic acid transporter-1 α-cyano-4-hydroxycinnamate (CHC) on tumor micromilieu and growth were investigated. MATERIALS AND METHODS: Oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured in FaDu and UT-SCC-5 hSCC in response to DCA in vitro. Mice bearing FaDu, UT-SCC-5, and WiDr colorectal adenocarcinoma received either DCA in drinking water or DCA injected twice a day, or CHC injected daily. WiDr was also treated daily with oxamate. FaDu and UT-SCC-5 were either excised 8 days after treatment for histology or tumor growth was monitored. WiDr tumors were excised at 8 mm. Effect of inhibitors on ATP, lactate, hypoxia, and Ki67 labeling index (LI) was evaluated. RESULTS: DCA increased OCR and decreased ECAR in vitro. None of the treatments with inhibitors significantly changed lactate content, hypoxia levels, and Ki67 LI in the three tumor lines in vivo. ATP concentration significantly decreased after only daily twice injections of DCA in FaDu accompanied by a significant increase in necrotic fraction. Tumor growth was not affected by any of the treatments. CONCLUSION: Overall, tumor micromilieu and tumor growth could not be changed by glycolysis modifiers in the three tumor cell lines in vivo. Further studies are necessary to explore the impact of metabolic targets on radiation response.

[Metabolic micromilieu in tumours]. / Das Tumormikromilieu.

UNLABELLED: Solid malignant tumours are characterized by a heterogeneous metabolic micromilieu with the intra-individual variability within single tumours being substantially smaller than the inter-individual differences between tumours. Despite this variability, there are some hallmarks which are characteristic for the majority of malignancies. They include hypoxia, tissue acidosis, and abnormal microcirculation. Peculiarities of the carbohydrate metabolism and specifically of glycolysis in tumours receive increasing attention in experimental and clinical research. As shown by our research with induced bioluminescence, different tumours from various entities exhibit a large spectrum of lactate accumulation. Interestingly, primary lesions with metastasis contain significantly higher amounts of lactate as compared to non-metastatic tumours. Classification into high and low lactate tumours according to the median lactate concentration in combination with a Kaplan-Meier analysis reveals that survival of patients with high lactate tumours is significantly worse than that with low lactate carcinomas. Furthermore, there is a positive correlation between tumour lactate content and radio-resistance. CONCLUSION: High lactate tumours are characterized by a higher degree of malignancy and therapeutic resistance.

Manipulation of glycolysis in malignant tumors: fantasy or therapy?

After Warburg stated his hypothesis on tumor cell metabolism about 80 years ago, the field of carbohydrate metabolism of cancer cells and solid tumors is experiencing a boom for the past few years. Numerous studies have been focused on the characteristics of cancer metabolism and its accessibility to novel therapeutic interventions. Malignant transformation is associated with an increase in glycolytic flux, mainly caused by an upregulation of numerous glycolysis-related genes in the majority of human cancers. As a consequence of these alterations, tumor cells are producing lactate at higher levels compared to non-malignant tissue, even in the presence of oxygen, a phenomenon termed "aerobic glycolysis" or "Warburg effect". A correlation between alterations in glycolytic pathways and therapy resistance in tumors is partially due to radical scavenger properties of specific metabolites which may decrease therapy-induced radical formation. Glycolytic activity and glycolysis-linked metabolic milieu are often variable between individual tumors resulting in variations in treatment response and aggressiveness. The peculiarities of tumor cell metabolism can be utilized for cancer diagnostics, such as metabolic imaging techniques and metabolic tumor markers. An emerging field of research is the manipulation of tumor cell metabolism for therapeutic purposes; respective studies include approaches of glycolysis inhibition or forcing mitochondrial respiration, respectively, based on biochemical and molecular principles. Up to now, such approach could eliminate tumors in patients without side effects when applied as single drug. Nevertheless, several agents that manipulate tumor metabolism may become a supportive therapy in combination with established cancer treatments.

The impact of conventional and heavy ion irradiation on tumor cell migration in vitro.

PURPOSE: The influence of X-ray and (12)C heavy ion irradiation on tumor cell migration and of beta(3) and beta(1) integrin expression was investigated. MATERIAL AND METHODS: Two different tumor cell lines (U87 glioma and HCT116 colon carcinoma cells) were irradiated with 1, 3, or 10 Gy X-rays or (12)C heavy ions. 24 h after irradiation a standardized Boyden Chamber assay for migration analysis was performed and cells were lysed for Western blotting. RESULTS: Radiation-induced influences were cell line- and radiation type-dependent. X-rays decreased HCT116 migration at higher doses and appear to increase U87 migration after 3 Gy. Heavy ions decreased migration of both cell lines dose-dependently. A trend of increased beta(3) and beta(1) integrin expression in U87 cells after both radiation types was observed. beta(1) integrin expression in HCT116 cells was increased after X-rays but decreased after heavy ion irradiation. CONCLUSIONS: Results suggest that irradiation of tumor cells can modulate their migratory behavior. An increased migration, as shown with U87, leaves a higher probability of metastatic induction after irradiation of solid tumors in vivo, whereas an invariably reduced tumor cell migration, as shown after heavy ion treatment, could diminish the hazard of radiation-induced metastasis. As integrin expression and migration were only partially correlated, other migration-related surface molecules may be more relevant for radiation effects on tumor cell motility.

Radiobiological characterization of human tumor cell multilayers after conventional and particle irradiation.

The goal of this study was to establish planar multilayers from human tumor cells (WiDr and SiHa) as a model for irradiation of solid tumors. In addition to using conventional X rays (250 kV) as a reference standard, multilayers were tested for their suitability in cell survival studies with heavy-ion irradiation ((12)C(6+)) in the plateau and the extended Bragg peak with a scanned ion beam. Multilayers of both cell lines showed decreased survival compared to the corresponding monolayers after both X and heavy-ion irradiation. This multicellular sensitization effect is in contrast to the multicellular resistance or contact effect commonly described in the literature. Flow cytometry measurements showed an arrest of irradiated SiHa cells in G(2)/M phase. In contrast to the transient arrest of the monolayers, the multilayers stayed in a prolonged arrest. After Bragg-peak irradiation of monolayers, the arrest time was increased by 12-24 h, and more cells were arrested than with X rays. For multilayers, there were no differences between G(2) arrest after X rays and heavy ions for the entire observation period.

[Lactate and redox status in malignant tumors]. / Laktat und Redoxstatus in malignen Tumoren.

This article summarizes data from experimental and clinical oncology which are indicative of a pivotal role of tumor carbohydrate metabolism in malignant behavior and outcome of treatment. In primary tumors, such as cervix carcinomas, head and neck squamous cell carcinomas or rectum adenocarcinomas, elevated lactate levels as a mirror of a high glycolytic activity, are correlated even at the initial diagnosis with a high level of malignancy as indicated by increased formation of metastases or an elevated radiotherapy resistance. The relationship between therapeutic resistance and glycolysis may at least partially be due to the radical scavenging potential of glycolytic intermediates, mainly pyruvate and lactate and to the link between these metabolites and the cellular redox status. On the basis of these data and other considerations, a novel technique has been developed for imaging the lactate/pyruvate ratio in tumor biopsies using quantitative bioluminescence. More research effort should, therefore, be focussed on the redox status of tumors in oncological studies in the future.

Lactate in solid malignant tumors: potential basis of a metabolic classification in clinical oncology.

A number of studies have demonstrated that malignant transformation is associated with an increase in glycolytic flux and in anaerobic and aerobic cellular lactate excretion. Using quantitative bioluminescence imaging in various primary carcinomas in patients (uterine cervix, head and neck, colorectal region) at first diagnosis of the disease, we showed that lactate concentrations in tumors in vivo could be relatively low or extremely high (up to 40 micromol/g) in different individual tumors or within the same lesion. In all tumor entities investigated, high molar concentrations of lactate were correlated with a high incidence of distant metastasis already in an early stage of the disease. Low lactate tumors (< median of approx. 8 micromol/g) were associated with both a longer overall and disease free survival compared to high lactate lesions (lactate > approx. 8 micromol/g). Lactate dehydrogenase was found to be upregulated in most of these tumors compared to surrounding normal tissue. Numerous recent reports support these data by demonstrating various biological activities of lactate that can enhance the malignant behavior of cancer cells. These mechanisms include the activation of hyaluronan synthesis by tumor-associated fibroblasts, upregulation of VEGF and of HIF-1 alpha, and direct enhancement of cellular motility which generates favorable conditions for metastatic spread. Thus, lactate accumulation not only mirrors but also actively enhances the degree of tumor malignancy. We propose that determination of lactate in primary tumors may serve as a basis for a novel metabolic classification which can lead to an improvement of prognosis and therapy in clinical oncology.

Response of Chinese hamster v79 multicellular spheroids exposed to high-energy carbon ions.

Chinese hamster V79-379A spheroids 200 +/- 30 microm (+/- SD) in diameter were irradiated in agitated medium in different oxygen atmospheres with (1) 227 MeV/nucleon (12)C(+6) ions (plateau region) to model tissue in the entrance channel during therapy, (2) carbon ions in the extended Bragg peak modeling tissue in the target volume, or (3) X rays as a reference modality. Cell survival curves were similar for modes (1) and (3), indicating the absence of a contact effect and the presence of a pronounced oxygen effect with oxygen enhancement ratios (OERs) of 2.8 and 2.9, respectively. In contrast, the oxygen effect was substantially smaller in mode (2) with an OER of 1.4. Under normal or restricted oxygen supply conditions (external pO(2) = 145 or 0 mmHg), the relative biological effectiveness (RBE) was 2.1 or 4.3, respectively, for Bragg-peak irradiation. This modality induced apoptosis and a dose-dependent accumulation of cells in G(2)/M phase even at pO(2) = 0 mmHg. The volume ratios of treated to untreated spheroids exhibited cyclic variations after heavy-particle treatment that were not directly attributable to cell cycle synchronization. In summary, the results suggest that carbon ions in the extended Bragg peak are more effective than conventional X rays, particularly under hypoxic conditions.

Bioactivity of well-defined green tea extracts in multicellular tumor spheroids.

The effect of green tea extracts (GTE) of a reproducible, well-defined composition on cellular viability, proliferation, and antioxidant defense was investigated in multicellular spheroids derived from WiDr human colon adenocarcinoma cells. The maximum GTE concentration investigated, i.e. 100 micro g GTE/ml, was equivalent to the plasma concentration commonly measured in humans drinking 6-10 cups of green tea per day. This GTE concentration lead to a substantial retardation of spheroid volume growth with diameters reaching only half the size of untreated aggregates. Flow cytometric analysis and immunocytochemistry showed an enhanced accumulation of cells in G2/M and in the non-proliferating compartment, respectively. The emergence of central necrosis occurred at larger spheroid diameters compared to control conditions leading to a significant increase (p<0.05) in the thickness of the viable cell rim (mean +/- SD) from 240+/-49.9 micro m to 294+/-69.5 micro m. This was associated with an elevation of the intracellular GSH concentration and, thus, of cellular antioxidant defense, as shown by HPLC analysis. A considerable toxicity, however, was found at these GTE levels in single cells. Cells did not adhere to culture dishes nor did they aggregate to form spheroids when plated as a suspension with GTE already in the culture medium. The findings show that green tea constituents interfere with early phases of tumorigenesis at a cellular level, e.g., by reducing cell-substratum and cell-cell interaction, enhancing G2/M arrest, and retarding spheroid volume growth. The differences in GTE effects between single cells and cell spheroids underline the importance of inclusion of spheroids in pharmaco-/toxicological testing.

Tissue gradients of energy metabolites mirror oxygen tension gradients in a rat mammary carcinoma model.

PURPOSE: It has been shown that oxygen gradients exist in R3230AC tumors grown in window chambers. The fascial surface is better oxygenated than the tumor surface. The purpose of the present study was to determine whether gradients exist for energy metabolites and other end points related to oxygen transport. METHODS AND MATERIALS: Imaging bioluminescence was used to measure ATP, glucose, and lactate in cryosections of R3230AC tumors. Mean vessel density and hypoxic tissue fraction were assessed using immunohistochemistry. Tumor redox ratio was assessed by redox ratio scanning. RESULTS: Lactate content and hypoxic fraction increased, whereas ATP, glucose, redox ratio, and vessel density decreased from the fascial to the tumor surface. CONCLUSIONS: The data support a switch from aerobic to anaerobic metabolism concomitant with the PO2 gradient. The vascular hypoxia that exists in perfused vessels at the tumor surface leads to macroscopic tissue regions with restricted oxygen availability and altered metabolic status. Methods to reduce tumor hypoxia may have to take this into account if such gradients exist in human tumors. The results also have implications for hypoxia imaging, because macroscopic changes in PO2 (or related parameters) will be easier to see than PO2 gradients limited to the diffusion distance of oxygen.

Elevated tumor lactate concentrations predict for an increased risk of metastases in head-and-neck cancer.

PURPOSE: Hypoxia shifts the balance of cellular energy production toward glycolysis with lactate generation as a by-product. Quantitative bioluminescence imaging allows for the quantitation of lactate concentrations in individual tumors. We assessed the relationship between pretreatment tumor lactate concentrations and subsequent development of metastatic disease in patients with newly diagnosed head-and-neck cancer. METHODS AND MATERIALS: At the time of biopsy of the primary site, a separate specimen was taken and flash-frozen for subsequent quantitation of lactate concentration using a luciferase bioluminescence technique. The two-dimensional spatial distribution of the bioluminescence intensity within the tissue section was registered directly using a microscope and an imaging photon counting system. Photon intensity was converted to distributions of volume-related tissue concentrations (micromol per gram wet weight). Treatment consisted of either surgery and postoperative radiotherapy or primary radiotherapy, based on presenting disease stage and institutional treatment policies. The subsequent development of metastatic disease constituted the primary clinical endpoint. RESULTS: Biopsies obtained from 40 patients were evaluable in 34. The larynx was the most frequent primary site (n = 25). Other sites included oropharynx (n = 5), hypopharynx (n = 3), and oral cavity (n = 1). Most patients (74%) presented with an advanced stage T3 or T4 primary tumor. Nodal involvement was present in 19 (54%) patients. The median tumor lactate concentration was 7.1 micromol/g. Tumors were classified as having either low or high lactate concentrations according to whether these values were below or above the median. The median follow-up time for surviving patients is 27 months. Two-year actuarial survival was 90% for patients with low-lactate-concentration tumor vs. 35% for patients with high-lactate-concentration primaries (<0.0001). Two-year metastasis-free survival was adversely influenced by high tumor lactate concentrations (90% vs. 25%, p < 0.0001). The median lactate concentration for tumors that subsequently metastasized was 12.9 micromol/g vs. 4.8 micromol/g for patients who remained continuously free of disease (p < 0.005). Lactate concentration was not correlated with presenting T stage or N stage. DISCUSSION: Elevated tumor lactate concentrations are associated with the subsequent development of nodal or distant metastases in head-and-neck cancer patients. This more aggressive malignant phenotype is probably associated with hypoxia-mediated radioresistance and the upregulation of metastasis-associated genes.

Endothelial capillaries chemotactically attract tumour cells.

Directional migration of capillaries towards tumour implants is generally assumed to be regulated by chemotaxis. Preliminary evidence has also been presented for the existence of a reverse chemotactic signalling pathway, with capillaries attracting tumour cells via paracrine factors. By using a variety of endothelial cell types and tumour cell lines, this study has systematically investigated chemotaxis between endothelial cells and tumour cells in two- and three-dimensional systems. Checkerboard analysis revealed faint attraction of human umbilical vein endothelial cells (HUVECs), but not porcine aortic endothelial cells (PAECs), by tumour cells. In reverse, both PAECs and HUVECs potently induced chemotactic migration of tumour cells. Using a microcarrier-based fibrin gel assay, directional migration of endothelial cells towards tumour cells was not observed. In reverse, tumour cells were strongly attracted by endothelial cells. Identification of endothelium-derived chemotactic molecules may provide a valuable approach for the treatment of tumour metastasis.

Tumor biology and experimental therapeutics.

Recent research using multicellular tumor spheroids has resulted in new insights in the regulation of invasion and metastasis, angiogenesis and cell cycle kinetics. The onset and expansion of central necrosis in tumor spheroids has been characterized to be a complex interaction of several mechanisms; in a number of cases, necrosis is not a consequence of hypoxia or anoxia, but emerges as secondary necrosis following an accumulation of apoptosis in spheroids. Recent therapeutically oriented studies have been directed towards novel hypoxic markers, targeted therapy, multicellular-mediated drug resistance, and heavy ion irradiation of spheroids. Research efforts should be enhanced mainly in the fields of tumor tissue modeling by heterotypic three-dimensional (3D) cultures and of apoptotic versus necrotic cell death. Based on the fundamental differences between monolayer and 3D cultures, spheroids should become mandatory test systems in therapeutic screening programs.

Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts.

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.

High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers.

Pathophysiological parameters such as vascular density and tissue oxygen pressure can influence tumor malignancy and patient survival. Observations from our group showed that metastatic spread of carcinomas of the uterine cervix and of head and neck cancers was closely correlated with the lactate concentration in the primary lesion. Because these results were obtained in a low number of patients, the present investigation was performed to verify such a correlation in a larger population. Cryobiopsies were taken at first diagnosis of cervical cancer from 34 patients. Tissue concentrations of ATP, glucose, and lactate in viable tumor regions of these biopsies were measured microscopically using the technique of imaging bioluminescence. There was no correlation between stage or grade and any of the metabolic parameters measured. ATP and glucose concentrations were not significantly different in metastatic and nonmetastatic primary tumors (P>0.05). However, lactate concentrations were significantly higher (P = 0.001) in tumors with metastatic spread (mean +/- SD, 10.0+/-2.9 micromol/g; n = 20) compared with malignancies in patients without metastases (6.3+/-2.8 micromol/g; n = 14). The majority of patients who suffered a recurrence of the disease (17 of a total of 22 patients) or died (15 of 20) within the observation period of up to 8 years belonged to the metastatic, i.e., high lactate group. A Kaplan-Meier analysis of the data showed that the overall and disease-free survival probabilities of patients having low tumor lactate values were significantly higher compared with patients with high tumor lactate concentrations (P = 0.015 and 0.014, respectively). We conclude that tumor lactate content may be used as a prognostic parameter in the clinic. Furthermore, these findings are in accordance with data from the literature showing that the presence of hypoxia in cervical tumors is associated with a poorer patient survival.

Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture.

Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of PO(2) distributions with microelectrodes, and determination of cellular oxygen uptake rates (Qc(O(2))) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of approximately 830 microm for Rat1-T1 and approximately 970 microm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as "metabolic switch"). Qc(O(2)) decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement of Qc(O(2)) with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Qc(O(2)), and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not ras transfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy-consuming process.

Hyperlactacidaemia in isolated hyperthermic perfusion of tumour bearing rat limbs: a study of feasibility using a novel infusion solution.

PURPOSE: In a methodological study the applicability of hyperlactacidaemia in isolated hyperthermic perfusion of tumour-bearing rat limbs was investigated. METHODS: In 50 Sprague Dawley rats, DS-sarcoma growth was initiated on the right food dorsum by subcutaneous injection of 0.5 ml ascites cells. In the anaesthetized animals isolated limb perfusion was performed under steady state conditions for 60min using a miniature equipment. Thereafter tumour volume was measured daily. (a) Investigation of feasability: 40 rats were allocated to four groups. Group I: Normothermic perfusion at 38 degrees C, n = 10; Group II: Hyperthermic perfusion at 40-41 degrees C, n = 10; Group III: Normothermic perfusion at 38 degrees C and hyperlactacidaemia of 10 mmol/l, n = 10; Group IV: Hyperthermic perfusion at 40-41 degrees C and hyperlactacidaemia of 10 mmol/l, n = 10. (b) Investigation of survival and histological changes: In group V hyperthermic perfusion at 40-41 degrees C and hyperlactacidaemia of 10 mmol/l, n = 10 was performed. After the animals had died, hip disarticulation of the tumour-bearing limb was performed for histological examination. RESULTS: Normothermic and hyperthermic perfusion of tumour-bearing rat limbs using miniature equipment was feasible and tolerated by the animals. Regional hyperlactacidaemia of 10 mmol/l could be maintained throughout the perfusions. After combined treatment with hyperthermia and hyperlactacidaemia, tumour volume decreased and extensive tumour necrosis occurred, while in other animals aggressive tumour growth with bone infiltration could be observed. CONCLUSIONS: The present study demonstrates the applicability of hyperlactacidaemia in hyperthermic isolated limb perfusion in the rat and proved a tumour growth delay due to an induction of tumour necrosis thereafter. Further investigations in other tumour entities and experimental models are required to confirm this impressive therapeutic effect of hyperthermia in combination with hyperlactacidaemia.