Low density lipoprotein receptor-related protein 1 mediated endocytosis of β1-integrin influences cell adhesion and cell migration.

The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with β1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and β1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of β1-integrin and decreased β1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model…

Glycolytic phenotype and AMP kinase modify the pathologic response of tumor xenografts to VEGF neutralization.

Abstract VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regress…

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…

Tumor-derived lactic acid modulates dendritic cell activation and antigen expression.

The tumor milieu can influence dendritic cell (DC) differentiation. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTSs) generated from different tumor cell lines. Monocytes invaded the MCTSs and differentiated into tumor-associated dendritic cells (TADCs). The antigen expression was altered on TADCs independent of the culture conditions (immature/mature DCs, Langerhans cells) and IL-12 secretion was reduced. Supernatants of MCTSs could partially transfer the suppressive effect. Conditioned media from urotheli…

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[Formula: see text] distributions with microelectrodes, and determination of cellular oxygen uptake rates (Q˙[Formula: see text]) have been applied. In the ras-transfected, tumorigenic spheroid types, the size d…

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…

Metabolic Imaging in Microregions of Tumors and Normal Tissues With Bioluminescence and Photon Counting

A method has been developed for metabolic imaging on a microscopic level in tumors, tumor spheroids, and normal tissues. The technique makes it possible to determine the spatial distribution of glucose, lactate, and ATP in absolute terms at similar locations within tissues or cell aggregates. The substrate distributions are registered in serial cryostat sections from tissue cryobiopsies or from frozen spheroids with the use of bioluminescence reactions. The light emission is measured directly by a special imaging photon counting system enabling on-line image analysis. The technique has been applied to human breast cancer xenografts, to spheroids originating from a human colon adenocarcinoma…

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. …

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were …

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…

Lactate and Acidity in the Cancer Microenvironment

Fermentative glycolysis, an ancient evolved metabolic pathway, is exploited by rapidly growing tissues and tumors but also occurs in response to the nutritional and energetic demands of differentiated tissues. The lactic acid it produces is transported across cell membranes through reversible H+/lactate−symporters (MCT1 and MCT4) and is recycled in organs as a major metabolic precursor of gluconeogenesis and an energy source. Concentrations of lactate in the tumor environment, investigated utilizing an induced metabolic bioluminescence imaging (imBI) technique, appear to be dominant biomarkers of tumor response to irradiation and resistance to treatment. Suppression of lactic acid formation…

Potential of induced metabolic bioluminescence imaging to uncover metabolic effects of antiangiogenic therapy in tumors

Tumor heterogeneity at the genetic level has been illustrated by a multitude of studies on the genomics of cancer, but whether tumors can be heterogeneous at the metabolic level is an issue which has been less systematically investigated so far. A burning related question is whether the metabolic features of tumors can change either following natural tumor progression (i.e. in primary tumors versus metastasis) or therapeutic interventions. In this regard, recent findings by independent teams indicate that anti-angiogenic drugs cause metabolic perturbations in tumors as well as metabolic adaptations associated with increased malignancy. Induced metabolic bioluminescence imaging (imBI) is an …

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…

The anti-oxidant capacity of tumour glycolysis.

In this mini-review data are summarised which provide evidence for the biological and clinical significance of tumour glycolysis and of its relationship to the redox state of cancer cells.Malignant transformation is associated with an overexpression of numerous glycolysis-related genes in the vast majority of human cancers. At the same time, glycolytic activity and glycolysis-linked metabolic milieu are often variable between individual tumours which induces large variations in treatment response and aggressiveness. Currently, there is no genetic or proteomic marker for the prediction of the therapeutic response for individual tumours, but the prognostic value of tumour lactate accumulation…

Multicellular tumor spheroids: an underestimated tool is catching up again.

The present article highlights the rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms. Unlike classical monolayer-based models, spheroids strikingly mirror the 3D cellular context and therapeutically relevant pathophysiological gradients of in vivo tumors. Some concepts for standardization and automation of spheroid culturing, monitoring and analysis are discussed, and the challenges to define the most convenient analytical endpoints for therapy testing are outlined. The potential of spheroids to contribute to either the elimination of poor drug candidates at the pre-animal and pre-clinical …

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…

Biological response of multicellular emt6 spheroids to exogenous lactate

The influence of elevated lactate concentrations, as found in tumor microregions, on cellular growth, viability, and metabolic state was studied employing the multicellular spheroid model. Spheroids of EMT6/Ro cells were cultured at 37 degrees C in 5% or 20% (v/v) oxygen, using stirred media with various concentrations of exogenous lactate ranging from 0.0 mM (standard conditions) to 20.0 mM. Elevated concentrations of exogenous lactate led to a considerable decrease of the maximum spheroid diameter at growth saturation, e.g., for 20% O2 from around 1700 microns to 700 microns in 0.0 and 20.0 mM lactate respectively. Histological investigations showed that the thickness of the viable cell r…

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…

Glucose Diffusion Coefficients Determined from Concentration Profiles in Emt6 Tumor Spheroids Incubated in Radioactively Labeled L-Glucose

In order to theoretically assess tissue energetic status, conditions for substance exchange need to be known. One group of parameters important in this context are diffusion coefficients of nutrients and metabolic waste products which may be assessed by incubating spheroids in a medium containing tracer amounts of the radioactively labeled substance in question, for a defined period of time. In previous studies, the overall amount of 14C-labeled substance taken up by the spheroids was measured by scintillation counters (e.g.[1]), or the concentration of 3H-labeled substance in the spheroid center was determined by autoradiography and grain counting (e.g. [4]). From a number of such measurem…

Microelectrode Measurements of Oxygen Tension Distributions in Multicellular Spheroids Cultured in Spinner Flasks

Since multicellular spheroids were introduced into cancer research by Sutherland et al. (1970, 1971), they have been widely used for studying the susceptibility of cancer cells to various treatment modalities. Many of these investigations provided evidence for a major role of oxygen in controlling the metabolic and cell cycle state of the tumor cells, thus modifying the responsiveness of the cells to therapy. Several investigators have measured O2 tension (PO2) distributions in spheroids with O2-sensitive microelectrodes (Carlsson et al. 1979; Kaufman et al. 1981; Mueller-Klieser and Sutherland 1982a, b, 1983; Mueller-Klieser et al. 1983). The measurements produced PO2 values that varied co…

Lactate: A Metabolic Key Player in Cancer

Abstract Increased glucose uptake and accumulation of lactate, even under normoxic conditions (i.e., aerobic glycolysis or the Warburg Effect), is a common feature of cancer cells. This phenomenon clearly indicates that lactate is not a surrogate of tumor hypoxia. Tumor lactate can predict for metastases and overall survival of patients, as shown by several studies of different entities. Metastasis of tumors is promoted by lactate-induced secretion of hyaluronan by tumor-associated fibroblasts that create a milieu favorable for migration. Lactate itself has been found to induce the migration of cells and cell clusters. Furthermore, radioresistance has been positively correlated with lactate…

Comparative imaging of structure and metabolites in tumours.

A novel technique for metabolic imaging using quantitative bioluminescence and single photon imaging was used to measure the distribution of ATP and lactate in two types of human melanoma xenografts with different radiobiologically hypoxic cell fractions (MF: 45 +/- 17% and EE: 6 +/- 3%; mean +/- SD). The tumours were s.c. grown in nude mice and were used for measurement at volumes of 153-3072 mm3. For metabolic imaging the rapidly frozen tumours were serially sectioned, and each cryosection was brought into contact with a frozen bioluminescent enzyme cocktail using a specially designed glass sandwich system. After thawing section and cocktail the luminescence was started, and light was emi…

Impact of carbon ion irradiation on epidermal growth factor receptor signaling and glioma cell migration in comparison to conventional photon irradiation

PURPOSE: Radiotherapy of malignant gliomas may be limited by an interference of radiation with the migratory potential of tumor cells. Therefore, the influence of conventional photon and modern carbon ion ((12)C) irradiation on glioblastoma cell migration and on epidermal growth factor receptor-related (EGFR) signaling was investigated in vitro. MATERIALS AND METHods: EGFR overexpressing glioblastoma cell lines U87 EGFR++ and LN229 EGFR++ were irradiated with 0, 2 or 6 Gy photons or (12)C heavy ions. Migration was analyzed 24 h after treatment in a standardized Boyden Chamber assay. At different time points EGFR, protein kinase B (PKB/AKT) and extracellular signal-related kinases (ERK1/2) w…

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…

Impact of exogenous lactate on survival and radioresponse of carcinoma cells in vitro

Tumour lactate levels have been shown to correlate with high radioresistance in tumour models in vivo. The study aimed to evaluate the impact of pathophysiological extracellular lactate concentrations and acidosis on the in vitro survival and radioresponse of various cancer cell lines.HCT-116, HT29 (colorectal) and FaDu (HNSCC) carcinoma cells were studied. Lactate release rates were determined, and expression of the monocarboxylate transporter MCT1 and its cofactor CD147 were monitored by immunofluorescence and flow cytometry. Colony formation was compared for cells exposed to 20 mM exogenous lactate, acidosis (pH 6.4) and lactate plus acidosis relative to control and dose response curves …

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…

Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-beta1.

When lactate accumulation in a tumor microenvironment reaches an average concentration of 10-20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast-endothelial cell co-culture model as in vitro angiogenesis system normally…

Cell Line and Growth Site as Relevant Parameters Governing Tumor Tissue Oxygenation

Experimental rodent tumors commonly used in radiobiology exhibit a large inter-individual variability in the oxygenation status [1,2] and in the hypoxic cell fraction [3,4]. Paramount factors contributing to this variability may be tumor growth stage or tumor size [5], the cell line used, the growth site, the use of anaesthesia and certain tumor-host interactions (e.g. tumor-induced anemia). Concerning the basic pathogenetic mechanisms through which the above mentioned parameters can modulate tumor tissue oxygenation, variations of nutritive blood flow, inherent characteristics of the cell line (e.g. respiration rate or tumor growth rate), and finally changes in the O2 transport capacity of…

Oncogene-Associated Growth Behavior and Oxygenation of Multicellular Spheroids from Rat Embryo Fibroblasts

It is now well documented that naturally occurring and experimentally induced tumors develop by a multistep process involving different stages such as unlimited growth, metastasis, and invasiveness. There is much evidence that malignant transformation involves activation of oncogenes and/or loss of suppressor genes (= anti-oncogenes). The former fundamental class of genes, including the ras and myc families, are associated with cell proliferation and differentiation and may mediate tumor initiation, promotion and progression (for reviews see: Spandidos, 1985; Spandidos and Anderson, 1987, Weinberg, 1989).

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 …

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…

Oxygen Consumption and Oxygen Diffusion Properties of Multicellular Spheroids from two Different Cell Lines

Multicellular spheroids are an in vitro tissue model in which the cells are supplied by diffusion of oxygen and substrates from the environmental growth medium (Sutherland et al., 1971). Since these substances are consumed when diffusing to the spheroid center, their concentration should decrease continuously towards the inner parts of the spheroids. Therefore, the location of the cells within the spheroid is an important determinant of the efficiency of the O2 and nutrient supply. The restriction of the O2 availability in the inner part of the spheroids may influence the metabolic and cell cycle state, and may even cause cell death, indicated by central necrosis in larger spheroids. Also, …

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

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

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.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.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 be…

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…

EFFECT OF MICROENCAPSULATION ON OXYGEN DISTRIBUTION IN ISLETS ORGANS

In islet transplantation, limitation of oxygen supply may restrict graft function, particularly when encapsulated tissue is used. Therefore, oxygen tensions (PO2) in isolated islet organs (Brockmann bodies) of Osphronemus gorami were measured. In a thermostatically (37 degrees C) controlled measuring chamber, PO2 values were recorded at subsequent microelectrode positions on a radial track toward the center of the organ. In 2 independent groups, we studied the effect of fluid convection (n = 12) and microencapsulation (n = 12). In both groups, sigmoidal PO2 profiles were found, which permit differentiation in an oxygen-depleted zone surrounding the surface, a steep decline inside the tissue…

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 restr…

Carbon ions and X‑rays induce pro‑inflammatory effects in 3D oral mucosa models with and without PBMCs.

Oral mucositis is a severe complication of radiotherapy. Hence, it may constitute a serious medical safety risk for astronauts during extended space flights, such as missions to Mars, during which they are exposed to heavy-ion irradiation. For risk assessment of developing radiation-induced mucositis, a three-dimensional (3D) organotypic oral mucosa model was irradiated with 12C heavy ions or X‑rays. The present study focused mainly on early radiation‑induced effects, such as the activation of nuclear factor κB (NFκB) and the expression or release of pro-inflammatory marker molecules. The 3D oral mucosa models with or without peripheral blood mononuclear cells (PBMCs) were irradiated with X…

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 th…

Effectiveness of Respiratory Hyperoxia, of Normobaric and of Hyperbaric Oxygen Atmospheres in Improving Tumor Oxygenation

A restriction and an inhomogeneous distribution of diffusive and convective oxygen supply is considered the main cause for pronounced radioresistance in solid tumors (Thomlinson and Gray, 1955; Tannock, 1972). One way to alleviate this crucial problem in tumor therapy is the direct enhancement of oxygen delivery to the cancer cells. This can be performed either by the tumor host breathing pure O2 (respiratory hyperoxia, RHO) or by whole body exposure to pure O2 atmospheres at normobaric or hyperbaric pressures (normobaric oxygenation NMO, and hyperbaric oxygenation, HPO, respectively). Numerous investigations have been undertaken both in animals and in man using RHO, NMO, or HPO as an adjuv…

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)

Tetrachlorodecaoxide Improves the Oxygenation Status of Multicellular Tumor Spheroids

Insufficient O2 supply to solid tumors has been recognized since many years to be a crucial problem in cancer therapy. Hypoxia and anoxia may increase the resistance of tumor cells to certain antiproliferative drugs or to ionizing radiation. Numerous approaches to overcoming this problem have been reported in the literature. Efforts have been made in mainly two basic directions: (1) the development of therapeutic means which are particularly efficient in hypoxic and nutrient-deprived tissue areas or which are at least independent of the tissue oxygenation, and (2) the development of methods for improving the oxygenation status of solid tumors. Among the former approaches, the cytotoxicity o…

Lactate-induced inhibition of tumor cell proliferation.

Abstract Culture medium that was recovered from tumor cell or fibroblast cultures during the plateau phase, and that was replenished by addition of glucose, glutamine, and serum and readjustment of pH had a distinct growth-inhibiting effect on monolayer cell cultures. The effect, which was not specific for a given cell strain, may be partially responsible for the "density inhibition" commonly observed in malignant cells grown in monolayer cultures. By modifying fresh growth media, it was shown that the growth inhibition observed can be partly attributed to the accumulation of lactate in the culture medium of plateau phase cells. This substance reduced the plating efficiency and the number o…

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…

Growth-related changes of oxygen consumption rates of tumor cells grown in vitro and in vivo

Growth-related changes of oxygen consumption rates of tumor cells, grown in vitro or in vivo, were investigated. For in vitro investigations, L929 and DS-carcinosarcoma cells were cultured in artificial media. For in vivo studies, DS-carcinosarcoma cells were implanted into the abdominal cavity of Sprague-Dawley rats (ascites tumor, containing malignant cells, leukocytes, lymphocytes, and macrophages). Oxygen uptake was measured photometrically. Parameters of the extracellular medium judged to possibly influence the respiratory activity of tumor cells were monitored at different growth stages (glucose, lactate, and amino acid levels, oxygen and carbon dioxide partial pressures, and pH value…

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…

Verapamil Inhibits the Respiration Rate of Cancer Cells

Calcium antagonists have successfully been used in the treatment of hypertension, cardiac arrhythmias and coronary heart disease. Recent evidence has suggested that such agents may also play a role in the treatment of malignant tumors. Verapamil, a calcium entry blocker, has been reported to enhance the cytotoxicity of several anticancer drugs under in vitro- and in vivo-conditions [1–10]. The effects observed could be explained by an enhanced drug accumulation due to a Verapamil-induced inhibition of the drug efflux from the cancer cells.

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…

Influence of growth, measuring conditions, and cell types on oxygen tensions in multicell tumor spheroids determined by microelectrodes

Semiquantitative bioluminescent assay of glutathione

A novel technique has been developed for semiquantitative detection of glutathione (GSH) in small volumes of liquid samples. GSH is detected via enzymatic linkage to the NADP/NADPH + H+ redox system through glutathione reductase. Accumulated NADPH is measured via the bioluminescent FMN oxidoreductase bacterial luciferase reaction. A linear correlation is obtained between bioluminescence intensity of the luciferase reaction and the GSH content of the liquid sample. Possible applications of this procedure are discussed. © 1998 John Wiley & Sons, Ltd.

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 measurem…

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…

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

Three-dimensional cell cultures: from molecular mechanisms to clinical applications.

This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigat…

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…

Effect of Localized Hyperthermia on Tumor Blood Flow and Oxygenation

Hyperthermia has been shown to have a lethal effect in tumor cell cultures, particularly at temperatures above 42°C.1 Moreover, it has been demonstrated that hyperthermia retards the growth rate of certain types of malignant tumors and can be effective in completely eradicating some tumors without any major damage to normal tissue.2,3,4 The differential response of solid tumors in vivo to heat treatment in comparison to normal tissue probably results from several factors. Among these the in vivo micro-environment seems to play a decisive role. In general, the interstitial milieu of solid tumors is characterized by tissue hypoxia and anoxia5,6 and by severe tissue acidosis.7 These unfavorabl…

Test system for trifunctional antibodies in 3D MCTS culture.

The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the …

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…

Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids

To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following…

Oxygenation Status of Rhabdomyosarcoma Spheroids with Different Stages of Differentiation

Multicellular tumor spheroids with regional heterogeneities of proliferation and substrate concentrations have been investigated recently to study the biological properties of small tumor nodules prior to onset of vascularization (for reviews see: Mueller-Klieser, 1987; Sutherland, 1988).

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 …

Glutathione content of V79 cells in two- or three-dimensional culture

The cellular glutathione (GSH) content of two- and three-dimensional cell cultures of V79 hamster lung cells has been studied. As previously described, cells in monolayer cultures show a decrease in GSH when they reach the confluent state. Three-dimensional cell cultures (multicell spheroids) allow a smoother transition from the initial proliferating to the nonproliferating status, and they show a central area of necrosis when a certain diameter is reached. Cellular GSH content in spheroids is variable throughout the culturing period: 1) GSH content (expressed per mg protein) is lower in spheroids with central necrotic areas than in smaller spheroids without necrosis, and 2) results expres…

Changes in O2 Consumption of Multicellular Spheroids During Development of Necrosis

Multicellular spheroids are spherical aggregates of cells that are supplied by diffusion of oxygen and substrates from the surrounding growth medium (Sutherland and Durand, 1976). Metabolic waste products are removed from the cells in these aggregates by diffusion into the growth medium. Cells within multicellular spheroids may be exposed to environmental conditions similar to those in tissue located between nutritive microvessels. Thus, tumor spheroids make it possible to study the impact of the tumor-specific micromilieu on cellular metabolism, cell cycle state, cellular viability or response to treatment. Factors in the microenvironment of tumor cells which may be relevant in this regard…

Hypoxia inducible factor-1alpha inactivation unveils a link between tumor cell metabolism and hypoxia-induced cell death.

Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1alpha (HIF-1alpha) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1alpha inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1alpha in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was a…

Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation

To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo. To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3-dimensional growth under well-defined conditions. The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-tran…

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…

Oxygen Consuming Regions in EMT60/Ro Multicellular Tumour Spheroids Determined by Nonlinear Regression Analysis of Experimental PO2 Profiles

Malignant cells can be studied in vitro, in a tumour-like microenvironment, by growing multicellular tumour spheroids in culture (Sutherland, McCredie and Inch, 1971). Franko and Sutherland (1979) utilized diffusion theory to explain the viable rim thicknesses of spheroids measured histologically. Without PO2 profiles, however, an unequivocal interpretation of their results was not possible. Systematic studies of the PO2 profiles in spheroids have since been made with oxygen microelectrodes by several groups (Carlsson et al., 1979; Kaufman et al., 1981; Mueller-Klieser and Sutherland, 1982a,b). Based on these measurements, new analyses utilizing diffusion theory are being developed to chara…

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 …

Three-dimensional invasion of human glioblastoma cells remains unchanged by X-ray and carbon ion irradiation in vitro.

Purpose Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), a…