Tumor lactate content predicts for response to fractionated irradiation of human squamous cell carcinomas in nude mice

Abstract Background and purpose The present study was performed to test the hypothesis that lactate accumulation correlates with the radioresistance of malignant tumors due to the radical scavenging capacity of lactate or metabolic intermediates of glycolysis, such as pyruvate. Materials and methods Five human head and neck squamous cell carcinoma cell lines (HNSCCs) xenografted in nude mice were treated with a clinically relevant irradiation protocol with 30 fractions within 6 weeks. The radiation dose necessary to locally control 50% of the tumors (TCD 50 ) ranged from 47.4 to 129.8Gy. Concentrations of glucose, lactate, and ATP in viable tumor regions as potential indicators of glycolyti…

Relation between autoradiographically measured blood flow and ATP concentrations obtained from imaging bioluminescence in tumors following hyperthermia.

The effects of moderate local hyperthermia (43.3 degrees C/30 min) on regional blood flow and regional ATP distribution in the amelanotic hamster melanoma A-Mel-3 were investigated by high-resolution techniques. Blood flow and ATP concentrations were measured simultaneously in treated and untreated tumors and in adjacent tissues by means of (14C)-Iodoantipyrine autoradiography and quantitative imaging bioluminescence in consecutive tissue sections at 3, 12 and 24 hr following treatment. Digital image processing and the use of a special algorithm allowed the regional interrelationship of the 2 parameters to be quantified. Measurements revealed a great heterogeneity of blood flow and ATP betw…

Mapping and quantification of biomolecules in tumor biopsies using bioluminescence

Quantitative bioluminescence and single-photon imaging have been applied for mapping concentration distributions of metabolites, such as adenosine triphosphate (ATP), glucose and lactate, in biopsies of cervical cancers in patients. Biopsies were taken before a conventional radiation treatment, and a number of clinically relevant data, such as local tumor control, patient survival, metastatic spread and so forth, were documented. There was no correlation between staging or grading and any of the metabolic parameters measured. Local correlations between ATP, glucose and lactate on a pixel-to-pixel basis were generally positive, with respective Spearman's correlation coefficients less in pati…

Feasibility of an induced metabolic bioluminescence imaging technique in ovarian cancer: Results of a pilot study.

e17093Background: The precise determination of energy metabolites is challenged by the heterogeneity of their distribution, their rapid changes after surgical resection and the architectural comple...

Abstract 3512: MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts

Abstract The oncogenes MYCN and survivin (BIRC5) maintain aggressiveness of diverse cancers including sarcomas. To investigate whether these oncogenes cooperate in initial malignant transformation, we transduced them into Rat-1 fibroblasts. Indeed, survivin enhanced MYCN-driven contact-uninhibited and anchorage-independent growth in vitro. Importantly, upon subcutaneous transplantation into mice, cells overexpressing both instead of either one of the oncogenes generated tumors with shortened latency, marked anaplasia and an increased proliferation-to-apoptosis ratio resulting in accelerated growth. Mechanistically, the increased tumorigenicity was associated with an enhanced Warburg effect …

High‐resolution histographical mapping of glucose concentrations in developing cotyledons of Vicia faba in relation to mitotic activity and storage processes: glucose as a possible developmental trigger

Summary Previous studies provided evidence that the carbohydrate status triggers developmental processes in the growing cotyledons ofVicia faba. We describe here the high-resolution mapping of glucose concentrations in tissue sections of developing faba bean cotyledons by quantitative bioluminescence and single-photon imaging. Patterns of local glucose distributions are compared with tissue cell type, mitotic index and the distribution pattern of starch. During cotyledon differentiation, gradients in the glucose concentration emerge which are related to the particular cell type. Higher concentrations are found in non-differentiated premature regions of the cotyledon whereas mature starch-ac…

ATP distribution and localization of mitochondria in Suberites domuncula (Olivi 1792) tissue

SUMMARY The metabolic energy state of sponge tissue in vivo is largely unknown. Quantitative bioluminescence-based imaging was used to analyze the ATP distribution of Suberites domuncula (Olivi 1792) tissue, in relation to differences between the cortex and the medulla. This method provides a quantitative picture of the ATP distribution closely reflecting the in vivo situation. The obtained data suggest that the highest ATP content occurs around channels in the sponge medulla. HPLC reverse-phase C-18, used for measurement of ATP content, established a value of 1.62 μmol ATP g–1 dry mass in sponge medulla, as opposed to 0.04 μmol ATP g–1 dry mass in the cortex, thus indicating a specific and…

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. …

Energy status and its control on embryogenesis of legumes: ATP distribution within Vicia faba embryos is developmentally regulated and correlated with photosynthetic capacity

To analyse the energy status of Vicia faba embryos in relation to differentiation processes, we measured ATP concentrations directly in cryosections using a quantitative bioluminescence-based imaging technique. This method provides a quantitative picture of the ATP distribution close to the in vivo situation. ATP concentrations were always highest within the axis. In pre-storage cotyledons, the level was low, but it increased strongly in the course of further development, starting from the abaxial region of cotyledons and moving towards the interior. Greening pattern, chlorophyll distribution and photosynthetic O2 production within embryos temporally and spatially corresponded to the ATP di…

Metabolic classification of human rectal adenocarcinomas: a novel guideline for clinical oncologists?

Based on previous findings in SCC of the human uterine cervix and of the head and neck, metastasis- and survival-related metabolic classification was performed in human rectal adenocarcinomas. Such a characterization is suggested as basis of a novel clinical guideline for an appropriate therapy of epithelium-derived malignancies.Tissue concentrations of ATP, glucose, and lactate in viable tumor regions of 33 pretherapeutically taken cryobiopsies from 24 patients were measured at a microscopic level using the technique of imaging bioluminescence.In metastatic carcinomas, lactate levels were significantly higher (mean+/-SD: 13.4+/-3.8 vs 6.9+/-2.5 micro mol/g, P = 0.0046) and glucose levels s…

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

Abstract 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 de…

Correlation between Regional Atp and Blood Flow in Tumors and Surrounding Normal Tissue

Various experimental and human tumors are characterized by a marked heterogeneity in the pathophysiologic micromilieu (1, 2). An inadequate and heterogeneous nutritional blood supply has been suggested to explain the non-uniform distribution of oxygen, pH, and high energy phosphates as it has been observed by many investigators (3, 4). Although there is a distinct understanding of the general interrelationships between these parameters (5), little is known about their actual regional correlation, which cannot be assessed by global measurements. With the autoradiographic method for measurement of blood flow (6, 7) and the metabolic imaging with ATP-induced bioluminescence (8, 9) it has becom…

Lactate: mirror and motor of tumor malignancy

A number of studies have shown 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 can 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 concentrations of lactate were correlated with a high incidence of distant metastasis already in an early stage of the disease. Low lactate tum…

Proliferation-associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture.

The oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in…

Semiautomatic growth analysis of multicellular tumor spheroids.

Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to an…

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI.

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K (trans) and v e were produced by pharmacokinetic analysis o…

Size-Dependent Oxygenation and Energy Status in Multicellular Tumor Spheroids

Multicellular tumor spheroids show numerous analogies to tumor microregions in vivo, such as the development of central necrosis at a certain spheroid size (for reviews see: Mueller-Klieser, 1987; Sutherland, 1988). The histological structure of the cell aggregates suggests that diffusion limitation of oxygen or nutrients in spheroids may cause cell death in the innermost parts of the spheroids. However, measurements with oxygen-sensitive microelectrodes are indicative of necrosis arising in the presence of relatively high oxygen tension (PO2) values, as they were found in normal tissue (Carlsson and Acker, 1985; Mueller-Klieser et al., 1986). Although still controversial in literature, rec…

Tumour-growth inhibition by induced hyperglycaemia/hyperlactacidaemia and localized hyperthermia.

The present study was undertaken to exploit pathophysiological properties of solid tumours for a tumour-specific therapy. Experiments were carried out on DS-sarcomas implanted s.c. in the hind foot dorsum of Sprague Dawley rats. Treatment strategies included tumour acidification, lactate accumulation and disturbance of the microcirculation by induced systemic hyperglycaemia/hyperlact-acidaemia (15-25/10 mmol/L; for 60 min) as well as localized hyperthermia (water-bath; 43 degrees C, 30 min.). A special infusion solution was developed for the systemic treatment containing glucose, lactic acid and organic buffer without inorganic ions. Growth kinetics of tumour volume and animal survival were…

Chronic social stress-induced hyperglycemia in mice couples individual stress susceptibility to impaired spatial memory

Significance Stress-associated mental disorders and diabetes pose an enormous socio-economic burden. Glucose dysregulation occurs with both psychosocial and metabolic stress. While cognitive impairments are common in metabolic disorders such as diabetes and are accompanied by hyperglycemia, a causal role for glucose has not been established. We show that chronic social defeat (CSD) stress induces lasting peripheral and central hyperglycemia and impaired glucose metabolism in a subgroup of mice. Animals exhibiting hyperglycemia early post-CSD display spatial memory impairments that can be rescued by the antidiabetic empagliflozin. We demonstrate that individual stress vulnerability to glucos…

Co-localisation of hypoxia and perfusion markers with parameters of glucose metabolism in human squamous cell carcinoma (hSCC) xenografts

Purpose: To examine relationships between tumour hypoxia, perfusion and metabolic microenvironment at themicroregional level in three different human squamous cell carcinomas (hSCC). Materials and methods: Nude mice bearing FaDu, UT-SCC-15, and UT-SCC-5 hSCC were injected with pimonidazole hypoxia and Hoechst perfusion markers. Bioluminescence imaging was used to determine spatial distribution of glucose and lactate content in serial tumour sections. Metabolite levels were grouped in 10 concentration ranges. Images were co-registered and at each concentration range the proportion of area stained for pimonidazole and Hoechst was determinedin 11–13 tumours per tumour line. Results: The spatia…

Pixel-to-pixel correlation between images of absolute ATP concentrations and blood flow in tumours

Iodo(14C-)antipyrine autoradiography and imaging bioluminescence have been combined to obtain pixel-to-pixel correlations between absolute values for local blood flow and ATP concentrations at a microscopical level within designated areas in hamster melanomas. Positive pixel-to-pixel correlations were obtained in 4 of 6 tumours. Both flow and ATP values were less in mostly necrotic than in mostly viable tumour regions. The data provide evidence for the energetic state of cancer cells being strongly influenced by the efficiency of tumour microcirculation in several but not in all malignancies investigated. Images Figure 1

VEGF-targeted therapy stably modulates the glycolytic phenotype of tumor cells

Abstract Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this met…

Interrelationship Among Morphology, Metabolism, and Proliferation of Tumor Cells in Monolayer and Spheroid Culture

Previous investigations have indicated a positive correlation between the proliferative and metabolic activities of tumor cells in monolayer culture (Freyer et al., 1984; Freyer and Sutherland, 1985; Walenta and Mueller-Klieser, 1987). On the other hand, no difference in the local oxygen consumption has been found between highly proliferating outer cell areas and non-proliferating inner cellular regions in multicellular tumor spheroids (Mueller-Klieser, 1984, 1987). Therefore, the interrelationship among metabolism, proliferation, and cellular morphology was investigated systematically in tumor cells both in monolayer and spheroid culture.

Localizing and Quantifying Metabolites In Situ with Luminometry: Induced Metabolic Bioluminescence Imaging (imBI)

Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique

Abstract Patients with malignant gliomas have a poor prognosis with average survival of less than one year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite, which was discovered first in this tumor entity. D2HG is generated in large amounts due to various “gain-of–function” mutations in the isocitrate dehydrogenases IDH-1 and IDH-2. Meanwhile, D2HG has been detected in se…

Glucose processing may be rate limiting step in R3230Ac mammary carcinoma metabolism

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy.

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but …

MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts

The oncogenes MYCN and survivin (BIRC5) maintain aggressiveness of diverse cancers including sarcomas. To investigate whether these oncogenes cooperate in initial malignant transformation, we transduced them into Rat-1 fibroblasts. Indeed, survivin enhanced MYCN-driven contact-uninhibited and anchorage-independent growth in vitro. Importantly, upon subcutaneous transplantation into mice, cells overexpressing both instead of either one of the oncogenes generated tumors with shortened latency, marked anaplasia and an increased proliferation-to-apoptosis ratio resulting in accelerated growth. Mechanistically, the increased tumorigenicity was associated with an enhanced Warburg effect and a hyp…

Quantitative bioluminescence imaging--a method for the detection of metabolite distributions in frozen tissues

A novel technique allows for measurement of metabolite distributions in tissue cryosections at a microscopic level using bioluminescence, single photon imaging, and computerized image analysis. Metabolites, such as ATP, glucose and lactate are registered in absolute concentration units, and the respective images can be correlated with each other and with histological structures by specific algorithms. One striking difference between malignant tumors and normal tissue is the pronounced heterogeneity of metabolite distributions in malignancies contrasted by rather homogeneous patterns obtained in many normal organs. The heterogeneous distribution of metabolites in solid tumors reflects the ch…

Pathophysiological approaches to identifying tumor hypoxia in patients

The present report summarizes observations of the authors on tumor oxygenation and on techniques for characterizing tumor hypoxia in patients. Cryospectrophotometric measurements of HbO2 saturations in tumor microvessels allow for estimates of the proportion of well oxygenated tissue regions. Labeling of tissue areas at oxygen (O2) tensions (pO2) less than 10 mm Hg with misonidazole may be used for a general characterization of the oxygenation status in patient tumors rather than for the determination of the radiobiologically hypoxic cell fraction. Quantitative bioluminescence and single photon imaging make it possible to determine ATP concentrations in absolute terms with a spatial resolut…

ERGO: A pilot study of ketogenic diet in recurrent glioblastoma

Limiting dietary carbohydrates inhibits glioma growth in preclinical models. Therefore, the ERGO trial (NCT00575146) examined feasibility of a ketogenic diet in 20 patients with recurrent glioblastoma. Patients were put on a low-carbohydrate, ketogenic diet containing plant oils. Feasibility was the primary endpoint, secondary endpoints included the percentage of patients reaching urinary ketosis, progression-free survival (PFS) and overall survival. The effects of a ketogenic diet alone or in combination with bevacizumab was also explored in an orthotopic U87MG glioblastoma model in nude mice. Three patients (15%) discontinued the diet for poor tolerability. No serious adverse events attri…

A bioluminescence technique for quantitative and structure-associated imaging of pyruvate

A novel bioluminescence assay has been developed for measuring pyruvate within sections of snap-frozen tissue in a quantitative manner as well as with a spatial resolution on a microscopical level. The assay was verified via HPLC and two independent photometric tests. The novel assay makes it possible to determine pyruvate concentrations in cryosections in the range of 0-5.0 micromol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviationor =15%. The minimal detectable amount was 0.02 pmol based on a 20 microm thick tissue section with an area of 1 cm(2). Combination of the already established imaging biolumine…

Spatial analysis of plant metabolism: Sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns

During legume embryogenesis the differentiation of the cotyledons proceeds gradually in a wave-like manner. The process is metabolically and genetically controlled and regulated by sugars. In order to perform a spatial and temporal analysis of the sugar distribution pattern a new method was developed to specifically measure sucrose directly in tissues via bioluminescence and single photon counting. This enabled a quantitative sucrose imaging with a resolution close to the single cell level. The procedure was applied on sections of Vicia faba cotyledons covering the main stages of histodifferentiation. Young embryos before the storage phase contained moderate levels of sucrose, which were ev…

Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models

This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, (12)C(+6) in the plateau region, and (12)C(+6) in the Bragg peak, respectively. Similarly, a relative biological e…

LDHA-Associated Lactic Acid Production Blunts Tumor Immunosurveillance by T and NK Cells

Elevated lactate dehydrogenase A (LDHA) expression is associated with poor outcome in tumor patients. Here we show that LDHA-associated lactic acid accumulation in melanomas inhibits tumor surveillance by T and NK cells. In immunocompetent C57BL/6 mice, tumors with reduced lactic acid production (Ldhalow) developed significantly slower than control tumors and showed increased infiltration with IFN-γ-producing T and NK cells. However, in Rag2-/-γc-/- mice, lacking lymphocytes and NK cells, and in Ifng-/- mice, Ldhalow and control cells formed tumors at similar rates. Pathophysiological concentrations of lactic acid prevented upregulation of nuclear factor of activated T cells (NFAT) in T and…

Uncovering Metabolic Effects of Anti-angiogenic Therapy in Tumors by Induced Metabolic Bioluminescence Imaging

Induced metabolic bioluminescence imaging (imBI) is an imaging technique which enables detection of various metabolites associated with glycolysis in tumor sections. Signals captured by imBI can be used to chart the topographic distribution of lactate, glucose, pyruvate, and ATP and quantify their absolute amount. ImBi can enable us to perform metabolic classification of tumors as well as to detect metabolic changes in the glycolytic pathway associated with certain therapies, such as anti-angiogenic drugs.

Growth rates or radiobiological hypoxia are not correlated with local metabolite content in human melanoma xenografts with similar vascular network.

Investigations were carried out on two lines of human melanomas (MF; n = 12 and EE; n = 13) xenografted in nude mice. The tumours were characterised by a similar vascular supply but showed a pronounced difference in the rate of volume growth and in the radiobiologically hypoxic fraction. The distribution of ATP, glucose and lactate in the tumours was investigated using quantitative bioluminescence and single photon imaging. Concentrations of the metabolites were obtained as global values for the entire tumour mass, in regions with densely packed, structurally intact tumour cells ('viable zones'), in areas with necrosis, stromal cells and fibrous material ('necrotic zones') and in adjacent n…

Migration of renal carcinoma cells is dependent on protein kinase Cδ via β1 integrin and focal adhesion kinase

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesi…

In regard to tarnawski et al., IJROBP 2002;52:1271–1276

Full‐thickness tissue engineered oral mucosa for genitourinary reconstruction: A comparison of different collagen‐based biodegradable membranes

Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the ti…

Primary Mucosal Epithelial Cell Cultivation: A Reliable and Accelerated Isolation

We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevert…

Oxygen Consumption Rate of Tumour Cells as a Function of their Proliferative Status

The oxygen consumption rate \(\left( \dot{Q}{{O}_{2}} \right)\) of EMT6/Ro-cells cultured as monolayers was investigated as a function of their proliferative status. The transition of these cells from the exponential to the plateau growth phase was associated with a continuous decrease in \(\dot{Q}{{O}_{2}}\) per single cell. This decrease can partially be attributed to a corresponding decline in cellular volume. In addition, the \(\dot{Q}{{O}_{2}}\) per cell volume was also reduced during the passage of the cells through the growth phase described. The results lead to the conclusion that a reduction in cellular volume and factors which are still unknown may contribute to the metabolic chan…

Glycolytic metabolism and tumour response to fractionated irradiation.

Abstract Background and purpose To study whether pre-therapeutic lactate or pyruvate predict for tumour response to fractionated irradiation and to identify possible coherencies between intermediates of glycolysis and expression levels of selected proteins. Materials and methods Concentrations of lactate, pyruvate, glucose and ATP were quantified via bioluminescence imaging in tumour xenografts derived from 10 human head and neck squamous cell carcinoma (HNSCC) lines. Tumours were irradiated with 30 fractions within 6weeks. Expression levels of the selected proteins in tumours were measured at the mRNA and protein level. Tumour-infiltrating leucocytes were quantified after staining for CD45…

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 an…

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release.

In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown …

Metabolic mapping with bioluminescence: basic and clinical relevance

This review is focused on metabolic mapping in biological tissue with quantitative bioluminescence and single photon imaging. Metabolites, such as ATP, glucose and lactate, can be imaged quantitatively and within microscopic dimensions in cryosections from shock frozen biological specimens using enzyme reactions and light emission by luciferases. The technique has been applied in numerous targets and models of experimental biomedical research, such as multicellular spheroids, various organs of laboratory animals in a physiological or pathophysiological state, and even in plant seeds. Among numerous other aspects, data obtained with this method have contributed to the elucidation of mechanis…

Pimonidazole labelling and response to fractionated irradiation of five human squamous cell carcinoma (hSCC) lines in nude mice: The need for a multivariate approach in biomarker studies

To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches.Five human squamous cell carcinoma lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, and UT-SCC-5) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. Histological material was collected from 60 unirradiated tumours after injection of Pimonidazole. The relative hypoxic area within the viable tumour area (Pimonidazole hypoxic fraction, pHF) was determined in seven serial 10 microm cross-sections per tumour by fluorescence microscopy and computerized image analysis. Local tumour control wa…

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 twodimensional spatial distribution of the bioluminescence int…

Differential changes in purine nucleotides after Doxorubicin treatment of human cancer cells in vitro

The present investigation was performed to elucidate the role of purine nucleotides as potential indicators of chemosensitivity of malignant tumors. Drug-sensitive (s) and -resistant (r) tumor cell lines grown as monolayers (s: T47D, MCF-7 wild-type; r: NCI/ADR-RES, MCF-7/MDR) or as multicellular spheroids (T47D; NCI/ADR-RES) were exposed to 0.1, 1.0, and 10.0 microM Doxorubicin for up to 24 h. Purine nucleotides were assayed using HPLC and with some selected spheroids using imaging bioluminescence. The data show that in the time frame of the experiments reproducible and statistically significant changes in the nucleotides only occur at the highest drug concentration investigated. Under the…

Inhibition of tumor lactate oxidation: consequences for the tumor microenvironment.

Abstract Background and purpose Tumor cells are recognized as being highly glycolytic. However, recently it was suggested that lactate produced in hypoxic tumor areas may be taken up by the monocarboxylate transporter MCT1 and oxidized in well-oxygenated tumor parts. Furthermore, it was shown that inhibition of lactate oxidation using the MCT1 inhibitor α-cyano-hydroxycinnamate (CHC) can radio-sensitize tumors possibly by forcing a switch from lactate oxidization to glycolysis in oxygenated cells, which in turn improves tumor oxygenation and indirectly kills radio-resistant hypoxic tumor cells from glucose starvation. Material and methods To provide direct evidence for the existence of a ta…

Lactate—An Integrative Mirror of Cancer Metabolism

The technique of induced metabolic bioluminescence imaging (imBI) has been developed to obtain a “snapshot” of the momentary metabolic status of biological tissues. Using cryosections of snap-frozen tissue specimens, imBI combines highly specific and sensitive in situ detection of metabolites with a spatial resolution on a microscopic level and with metabolic imaging in relation to tissue histology. Here, we present the application of imBI in human colorectal cancer. Comparing the metabolic information of one biopsy with that of 2 or 3 biopsies per individual cancer, the classification into high versus low lactate tumors, reflecting different glycolytic activities, based on a single biopsy …