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Hyperpolarized magnetic resonance spectroscopy (MRS) is a technology for characterizing tumors in vivo based on their metabolic activities. The conversion rates (kpl) of hyperpolarized [1-13C]pyruvate to [1-13C]lactate depend on monocarboxylate transporters (MCT) and lactate dehydrogenase (LDH); these are also indicators of tumor malignancy. An unresolved issue is how glucose and glutamine availability in the tumor microenvironment affects metabolic characteristics of the cancer and how this relates to kpl-values. Two breast cancer cells of different malignancy (MCF-7, MDA-MB-231) were cultured in media containing defined combinations of low glucose (1 mM; 2.5 mM) and glutamine (0.1 mM; 1 mM) and analyzed for pyruvate uptake, intracellular metabolite levels, LDH and pyruvate kinase activities, and 13C6-glucose-derived metabolomics. The results show variability of kpl with the different glucose/glutamine conditions, congruent with glycolytic activity, but not with LDH activity or the Warburg effect; this suggests metabolic compartmentation. Remarkably, kpl-values were almost two-fold higher in MCF-7 than in the more malignant MDA-MB-231 cells, the latter showing a higher flux of 13C-glucose-derived pyruvate to the TCA-cycle metabolites 13C2-citrate and 13C3-malate, i.e., pyruvate decarboxylation and carboxylation, respectively. Thus, MRS with hyperpolarized [1-13C-pyruvate] is sensitive to both the metabolic program and the nutritional state of cancer cells.

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The metabolism of cancer cells is an issue of dealing with fluctuating and limiting levels of nutrients in a precarious microenvironment to ensure their vitality and propagation. Glucose and glutamine are central metabolites for catabolic and anabolic metabolism, which is in the limelight of numerous diagnostic methods and therapeutic targeting. Understanding tumor metabolism in conditions of nutrient depletion is important for such applications and for interpreting the readouts. To exemplify the metabolic network of tumor cells in a model system, the fate 13C6-glucose was tracked in a breast cancer cell line growing in variable low glucose/low glutamine conditions. 13C-glucose-derived metabolites allowed to deduce the engagement of metabolic pathways, namely glycolysis, the TCA-cycle including glutamine and pyruvate anaplerosis, amino acid synthesis (serine, glycine, aspartate, glutamate), gluconeogenesis, and pyruvate replenishment. While the metabolic program did not change, limiting glucose and glutamine supply reduced cellular metabolite levels and enhanced pyruvate recycling as well as pyruvate carboxylation for entry into the TCA-cycle. Otherwise, the same metabolic pathways, including gluconeogenesis, were similarly engaged with physiologically saturating as with limiting glucose and glutamine. Therefore, the metabolic plasticity in precarious nutritional microenvironment does not require metabolic reprogramming, but is based on dynamic changes in metabolite quantity, reaction rates, and directions of the existing metabolic network.

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In cancers, tumor cells are exposed to fluctuating nutrient microenvironments with limiting supplies of glucose and glutamine. While the metabolic program has been related to the expression of oncogenes, only fractional information is available on how variable precarious nutrient concentrations modulate the cellular levels of metabolites and their metabolic pathways. We thus sought to obtain an overview of the metabolic routes taken by 13C-glucose-derived metabolites in breast cancer MCF-7 cells growing in combinations of limiting glucose and glutamine concentrations. Isotopologue profiles of key metabolites were obtained by gas chromatography/mass spectrometry (GC/MS). They revealed that in limiting and standard saturating medium conditions, the same metabolic routes were engaged, including glycolysis, gluconeogenesis, as well as the TCA cycle with glutamine and pyruvate anaplerosis. However, the cellular levels of 13C-metabolites, for example, serine, alanine, glutamate, malate, and aspartate, were highly sensitive to the available concentrations and the ratios of glucose and glutamine. Notably, intracellular lactate concentrations did not reflect the Warburg effect. Also, isotopologue profiles of 13C-serine as well as 13C-alanine show that the same glucose-derived metabolites are involved in gluconeogenesis and pyruvate replenishment. Thus, anaplerosis and the bidirectional flow of central metabolic pathways ensure metabolic plasticity for adjusting to precarious nutrient conditions.

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Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 (gal-3) is expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate gal-3 targeting as a specific method to detect non-radioiodine-avid thyroid cancer in thyroid orthotopic tumor models. Methods: Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cell lines were characterized via Western blot and polymerase chain reaction for gal-3 and sodium-iodide symporter (NIS) expression. An 89Zr-labeled F(ab')2 antigal-3 was generated and characterized for binding versus 125I on 2- and 3-dimensional cell cultures. The thyroid carcinoma cells were inoculated into the left thyroid lobe of athymic nude mice, and the orthotopic tumor growth was monitored via ultrasound and fluorescence molecular tomography. Head-to-head PET/CT comparison of 124I versus 89Zr-deferoxamine (DFO)-F(ab')2 antigal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. Results: The thyroid carcinoma cells investigated were invariably gal-3-positive while presenting low or lost NIS expression. 89Zr-DFO-F(ab')2 antigal-3 tracer showed high affinity to gal-3 (dissociation constant, ∼3.9 nM) and retained immunoreactivity (>75%) on 2-dimensional cell cultures and on tumor spheroids. 125I internalization in FRO82-1, BcPAP, and CAL62 was directly dependent on NIS expression, both in 2-dimensional and tumor spheroids. PET/CT imaging showed 89Zr-DFO-F(ab')2 antigal-3 signal associated with the orthotopically implanted tumors only; no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using 124I showed background accumulation in tumor-infiltrated lobe, a condition simulating the presence of non-radioiodine-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. Conclusion: A specific and selective visualization of thyroid tumor by targeting gal-3 was demonstrated in the absence of radioiodine uptake. Translation of this method into the clinical setting promises to improve the management of patients by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.

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Tumors are metabolic entities wherein cancer cells adapt their metabolism to their oncogenic agenda and microenvironmental influences. Metabolically different cancer cell subpopulations collaborate to optimize nutrient delivery with respect to immediate bioenergetic and biosynthetic needs. They can also metabolically exploit host cells. These metabolic networks are directly linked with cancer progression, treatment, resistance, and relapse. Conversely, metabolic alterations in cancer are exploited for anticancer therapy, imaging, and stratification for personalized treatments. These topics were addressed at the 4th annual meeting of the International Society of Cancer Metabolism (ISCaM) in Bertinoro, Italy, on 19-21 October 2017.

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Cancers are metabolic entities wherein cancer cells adapt their metabolism to their oncogenic agenda and microenvironmental influences. Metabolically different cancer cell subpopulations collaborate to optimize nutrient delivery with respect to immediate bioenergetic and biosynthetic needs. They can also metabolically exploit host cells. These metabolic networks are directly linked with cancer progression, treatment resistance and relapse. Conversely, metabolic alterations in cancer are exploited for anticancer therapy, imaging and personalized medicine. These topics were addressed at the 3rd annual meeting of the International Society of Cancer Metabolism (ISCaM) in Brussels, Belgium, on 26-29 October 2016.

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Virtually everyone working in cancer research is familiar with the "Warburg effect", i.e., anaerobic glycolysis in the presence of oxygen in tumor cells. However, few people nowadays are aware of what lead Otto Warburg to the discovery of this observation and how his other scientific contributions are seminal to our present knowledge of metabolic and energetic processes in cells. Since science is a human endeavor, and a scientist is imbedded in a network of social and academic contacts, it is worth taking a glimpse into the biography of Otto Warburg to illustrate some of these influences and the historical landmarks in his life. His creative and innovative thinking and his experimental virtuosity set the framework for his scientific achievements, which were pioneering not only for cancer research. Here, I shall allude to the prestigious family background in imperial Germany; his relationships to Einstein, Meyerhof, Krebs, and other Nobel and notable scientists; his innovative technical developments and their applications in the advancement of biomedical sciences, including the manometer, tissue slicing, and cell cultivation. The latter were experimental prerequisites for the first metabolic measurements with tumor cells in the 1920s. In the 1930s-1940s, he improved spectrophotometry for chemical analysis and developed the optical tests for measuring activities of glycolytic enzymes. Warburg's reputation brought him invitations to the USA and contacts with the Rockefeller Foundation; he received the Nobel Prize in 1931. World politics and world wars heavily affected Warburg's scientific survival in Berlin. But, after his second postwar recovery, Warburg's drive for unraveling the energetic processes of life, both in plants and in tumor cells, continued until his death in 1970. The legacy of Otto Warburg is not only the Warburg effect, but also the identification of the "respiratory ferment" and hydrogen-transferring cofactors and the isolation of glycolytic enzymes. His hypothesis of respiratory damage being the cause of cancer remains to be a provocative scientific issue, along with its implications for cancer treatment and prevention. Warburg is therefore still stimulating our thinking, as documented in a soaring increase in publications citing his name in the context of tumor metabolism.

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OBJECTIVE: (13)C metabolic MRI using hyperpolarized (13)C-bicarbonate enables preclinical detection of pH. To improve signal-to-noise ratio, experimental procedures were refined, and the influence of pH, buffer capacity, temperature, and field strength were investigated. MATERIALS AND METHODS: Bicarbonate preparation was investigated. Bicarbonate was prepared and applied in spectroscopy at 1, 3, 14 T using pure dissolution, culture medium, and MCF-7 cell spheroids. Healthy rats were imaged by spectral-spatial spiral acquisition for spatial and temporal bicarbonate distribution, pH mapping, and signal decay analysis. RESULTS: An optimized preparation technique for maximum solubility of 6 mol/L and polarization levels of 19-21% is presented; T1 and SNR dependency on field strength, buffer capacity, and pH was investigated. pH mapping in vivo is demonstrated. CONCLUSION: An optimized bicarbonate preparation and experimental procedure provided improved T1 and SNR values, allowing in vitro and in vivo applications.

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Characteristic of the tumor microenvironment are fluctuating gradients of reduced nutrient levels and released lactate. A fundamental issue is how tumor cells modulate their metabolic activity when both glucose and glutamine levels become limiting in the presence of high exogenous lactate. For functional analyses, the activities of pyruvate kinase, lactate dehydrogenase (LDH) and plasma membrane NADH oxidase (NOX) as well as cell growth were measured in breast cancer MCF-7 cells cultured in medium containing various concentrations of these metabolites. After 3 days at glucose concentrations below 2.5 mM, cell number was higher with 0.1 mM than with 1.0 mM glutamine, indicating that the glucose/glutamine balance is important for growth. On the other hand, NOX activity increased with increasing glucose >2.5 mM, but only with low glutamine (0.1 mM). Pyruvate kinase activity also increased, with LDH activity remaining 2-3-fold lower. Here NOX could have a complementary role in reoxidizing NADH for glycolysis. Exogenous lactate supported cell survival at limiting concentrations of glucose and glutamine while increasing NOX and pyruvate kinase activities as well as NADH levels. It is proposed that lactate supports cell survival by fuelling gluconeogenesis and/or the TCA cycle in mitochondria, from where NADH could be shuttled to the cytosol and reoxidized by NOX. Cell survival and the metabolic phenotype are thus interrelated to the dynamics of NADH and plasma membrane NOX activity, which are regulated by the balance of glucose/glutamine levels, in conjunction with lactate in a precarious tumor microenvironment.

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UNLABELLED: Abnormalities of tumor metabolism can be exploited for molecular imaging. PET imaging of (18)F-FDG is a well-established method using the avid glucose uptake of tumor cells. (13)C MR spectroscopic imaging (MRSI) of hyperpolarized [1-(13)C]pyruvate and its metabolites, meanwhile, represents a new method to study energy metabolism by visualizing, for example, the augmented lactate dehydrogenase activity in tumor cells. Because of rapid signal loss, this method underlies strict temporal limitations, and the acquisition of data-encoding spatial, temporal, and spectral information within this time frame-is challenging. The object of our study was to compare spectroscopic images with (18)F-FDG PET images for visualizing tumor metabolism in a rat model. METHODS: (13)C MRSI with IDEAL (Iterative Decomposition of water and fat with Echo Asymmetry and Least-squares estimation) chemical shift imaging in combination with single-shot spiral acquisition was used to obtain dynamic data from 23 rats bearing a subcutaneous hepatocellular carcinoma and from reference regions of the same animals. Static and dynamic analysis of (18)F-FDG PET images of the same animals was performed. The data were analyzed qualitatively (visual assessment) and quantitatively (magnitude and dynamics of (18)F-FDG uptake, (13)C MRSI dynamics, and physiologic parameters). RESULTS: In most animals increased [1-(13)C]lactate signals in the tumor could be detected by simple display of integrated [1-(13)C]lactate images with corresponding enhanced (18)F-FDG uptake. Low [1-(13)C]pyruvate or [1-(13)C]lactate signals did not correlate with histologic or physiologic parameters. Significantly less pyruvate reached the tumors than the gastrointestinal tract, but in tumors a significantly higher amount of pyruvate was converted to lactate and alanine within seconds after intravenous administration. CONCLUSION: This study reveals that PET and (13)C MRSI can be used to visualize increased glycolytic flux in malignant tissue. The combination of signals will allow the quantitative dissection of substrate metabolism, with respect to uptake and downstream metabolic pathways. Although hyperpolarized [1-(13)C]pyruvate increases the sensitivity of MR imaging, signal-to-noise ratio constraints still apply for spatially and temporally resolved (13)C MRSI, emphasizing the need for further MR methodologic development. These first imaging data suggest the feasibility of (13)C MRSI for future clinical use.

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The detection of tumors noninvasively, the characterization of their progression by defined markers and the monitoring of response to treatment are goals of medical imaging techniques. In this article, a method which measures the apparent diffusion coefficients (ADCs) of metabolites using hyperpolarized (13) C diffusion-weighted spectroscopy is presented. A pulse sequence based on the pulsed gradient spin echo (PGSE) was developed that encodes both kinetics and diffusion information. In experiments with MCF-7 human breast cancer cells, we detected an ADC of intracellularly produced lactate of 1.06 ± 0.15 µm(2) /ms, which is about one-half of the value measured with pyruvate in extracellular culture medium. When monitoring tumor cell spheroids during progressive membrane permeabilization with Triton X-100, the ratio of lactate ADC to pyruvate ADC increases as the fraction of dead cells increases. Therefore, (13) C ADC detection can yield sensitive information on changes in membrane permeability and subsequent cell death. Our results suggest that both metabolic label exchange and (13) C ADCs can be acquired simultaneously, and may potentially serve as noninvasive biomarkers for pathological changes in tumor cells.

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The effective isolation and purification of proteins from biological fluids is the most crucial step for a successful protein analysis when only minute amounts are available. While conventional purification methods such as dialysis, ultrafiltration or protein precipitation often lead to a marked loss of protein, SPE with small-sized particles is a powerful alternative. The implementation of particles with superparamagnetic cores facilitates the handling of those particles and allows the application of particles in the nanometer to low micrometer range. Due to the small diameters, magnetic particles are advantageous for increasing sensitivity when using subsequent MS analysis or gel electrophoresis. In the last years, different types of magnetic particles were developed for specific protein purification purposes followed by analysis or screening procedures using MS or SDS gel electrophoresis. In this review, the use of magnetic particles for different applications, such as, the extraction and analysis of DNA/RNA, peptides and proteins, is described.

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Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB 231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy.

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The recent development of an electronic test system based on silicon sensor-chips allows the continuous parallel recording of relative changes in extracellular acidification, oxygen consumption and electric impedance in living cells. The objective of this proof-of-principle study therefore, was to clarify whether this system can also be applied to live tissue slices thus providing a device for an ultimately envisioned chemosensitivity testing apparatus for individualized treatment schemes in cancer therapy. A prototype of the testing apparatus equipped with six individual measuring devices has been used to simultaneously analyze changes in extracellular acidification, oxygen consumption and electronic impedance in live liver tissue and compared to data obtained from a tumor cell line. In contrast to tumor cells, tissue slices showed low rates of extracellular acidification but high rates of oxygen consumption. Monitoring of electrical impedance values, reflecting cellular morphology, revealed that the compact cell structure of the tissue slices was able to function as electric insulator and actively change the impedance values of the system. Exposure of tumor cells to 1 microM cytochalasin B, a fungal metabolite known to interact with the cytoskeleton and influence glucose metabolism, resulted in the rapid decline of extracellular acidification, increased oxygen consumption rates and increased values in capacitance. In tissue slices upon addition of 1 microM cytochalasin B, a decline of both extracellular acidification and electrical impedance was observed within 1 h. Determination of ATP content in the tissue slices revealed that decreasing ATP content paralleled diminishing oxygen consumption. This new technique offers the possibility of generating metabolic profiles for cells and tissues by studying oxygen consumption, extracellular acidification and electrical impedance.

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Chemotherapeutic drugs affect the metabolism of tumor cells regardless of the specific target of action. Basic parameters of cell metabolism are extrusion of acids into the microenvironment and oxygen consumption. To analyze these changes on living cells in real-time, a test system based on multiparametric chips with an array of sensors for monitoring pH and O(2) as well as electric impedance has been developed. Cells are cultivated on these chips and supplied with medium by a fluid perfusion set-up which mimics microphysiological conditions and allows for drug addition and removal. Human colon carcinoma cells LS174T were used as a model to test the effect of drugs. Cells growing on chips were monitored for 24 h and longer. Untreated cells showed a continuous increase in the rate of acidification, while the rate of respiration remained fairly constant. Addition of chloroacetaldehyde (50 microM) rapidly attenuated O(2) consumption with a gradual decrease in acidification following. In contrast, with cisplatin (16.7 microM) a delayed and gradual decrease in both the rates of acidification and respiration effect occurred over 2-3 days. These results provide insights to the mechanisms of action of these drugs, which are coherent with those already known. Thus, multiparametric sensor chips provide elementary information on drug action.

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Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

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An alternative assay for replacing animal experiments should serve the specific microphysiological needs of the cells and be endowed with multiparametric signal monitoring. These requirements are provided by a test system in which the key elements are biocompatible electronic sensor-chips. It is also connected to a medium perfusion set-up, which allows to control the supply of nutrients and test compounds, and the removal of culture medium. The chips are equipped with sensors that continuously monitor basic metabolic parameters and membrane-associated changes of living cells: pH-ISFETs for extracellular acidification, amperometric O2-sensors for oxygen consumption, and IDES for electrical impedance of the cell layer. Experiments with LS174T colon carcinoma cells in culture show the metabolic and electrical changes upon incubation with Zytochalasin B and chloroacetaldehyde. The signal patterns vary and indicate different mechanisms of action for these test compounds. With this test system it is possible to detect effects of unknown substances and mixtures, and to analyse the cellular probe for prolonged times.

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Tumor interstitium exhibits a microenvironment that differs from corresponding normal tissues. Tumor phenotype shows, for example, an elevated intracellular pH (pHi), a lowered extracellular pH (pHe), a low oxygen concentration and low glucose levels. These differences are caused by cell biological (so called intrinsic) factors, e.g. a higher acidification rate, as well as by more systemic (extrinsic) factors, e.g. poor tumor vascularization. They represent important factors for invasiveness, immune suppression and proliferation, and they imply possibilities for diagnosis, prognosis and therapy. We have developed an experimental data-based computer model, which has simulated the potential role of metabolic effects on tumor progression. We show an experiment on cellular metabolism demonstrating the immunosuppressive impact of low pHe on peripheral blood mononuclear cells. Finally, we review important findings on the tumor microenvironment leading to possibilities for therapy which are currently evolving and which promise higher effectiveness for cancer therapy.

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A constraint in the reliability of predictive chemosensitivity assays is linked to the fact that they analyze only a single cellular or biochemical parameter. A multiparametric test system using microsensor chips has been developed which can detect online microphysiological changes in living cells. Tumor cells were grown directly on glass- or silicon-based electronic sensor chips. Changes in extracellular pH and pO(2), reflecting metabolic activities, and changes in impedance, reflecting morphological properties, were monitored. In this study, colon and breast cancer cells as well as doxorubicin-sensitive and doxorubicin-resistant sarcoma cell lines were exposed to cytochalasin B, chloroacetaldehyde, or doxorubicin. Results show (1) reduction in medium acidification, (2) marked and rapid changes in O(2) consumption, and (3) modulations in impedance correlating with morphological changes observed in the microscope. Drug-resistant cells do not show these changes. Therefore, this microphysiological monitoring is a versatile tool for chemosensitivity testing of tumor cells.

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