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

Stringent glucose demands render the brain susceptible to disturbances in the supply of this main source of energy, and chronic stress may constitute such a disruption. However, whether stress-associated cognitive impairments may arise from disturbed glucose regulation remains unclear. Here we show that chronic social defeat (CSD) stress in adult male mice induces hyperglycemia and directly affects spatial memory performance. Stressed mice developed hyperglycemia and impaired glucose metabolism peripherally as well as in the brain (demonstrated by PET and induced metabolic bioluminescence imaging), which was accompanied by hippocampus-related spatial memory impairments. Importantly, the cognitive and metabolic phenotype pertained to a subset of stressed mice and could be linked to early hyperglycemia 2 days post-CSD. Based on this criterion, ∼40% of the stressed mice had a high-glucose (glucose >150 mg/dL), stress-susceptible phenotype. The relevance of this biomarker emerges from the effects of the glucose-lowering sodium glucose cotransporter 2 inhibitor empagliflozin, because upon dietary treatment, mice identified as having high glucose demonstrated restored spatial memory and normalized glucose metabolism. Conversely, reducing glucose levels by empagliflozin in mice that did not display stress-induced hyperglycemia (resilient mice) impaired their default-intact spatial memory performance. We conclude that hyperglycemia developing early after chronic stress threatens long-term glucose homeostasis and causes spatial memory dysfunction. Our findings may explain the comorbidity between stress-related and metabolic disorders, such as depression and diabetes, and suggest that cognitive impairments in both types of disorders could originate from excessive cerebral glucose accumulation.

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 not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Comparative metabolic analysis in head and neck cancer and the normal gingiva.

OBJECTIVES: Chronic accumulation of lactate in malignant tumor tissue is associated with increased malignancy and radioresistance. For this study, biopsies of primary head and neck squamous cell carcinoma (HNSCC) and of the normal gingiva of the same patient were compared via metabolic profiling to the healthy gingiva from cancer-free patients. MATERIALS AND METHODS: Cryobiopsies of 140 HNSCC patients were used to determine ATP, lactate, and glucose concentrations of the tumor and normal gingiva via induced metabolic bioluminescence imaging (imBI). Additionally, these metabolites were quantified in a collective of 79 healthy (non-tumor-bearing) patients. Furthermore, tumor samples were analyzed via immunofluorescence imaging and quantitative real-time PCR for the expression of lactate and glucose transporters. RESULTS: There were significant differences in ATP concentrations detectable between the tumor, normal gingiva of tumor patients, and gingiva from healthy patients. Lactate concentrations were significantly increased in tumor tissue compared to the normal gingiva of tumor patients as well as the gingiva from healthy patients. Concerning glucose, there was a significant decrease in glucose concentrations detectable in the tumor biopsies compared to the normal gingiva of tumor patients. On the other hand, tumor samples from patients revealed significantly elevated relative expression levels of monocarboxylate transporters (MCT-1 and MCT-4), as well as glucose transporters (GLUT-1 and GLUT-3) compared to the corresponding normal gingiva of each patient. CONCLUSIONS: We could demonstrate that the lactate concentration in HNSCC correlates with primary tumor (T) stage. CLINICAL RELEVANCE: The aim of this study was to identify metabolic parameters to improve early cancer diagnosis, allow predictions on the degree of malignancy, and contribute to a personalized tumor therapy.

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 was in agreement with the result from multiple biopsies in 83 % of all cases. We further demonstrate that the metabolic status of tumor tissue can be preserved at least over 10 years by storage in liquid nitrogen, but not by storage at -80 °C. This means that tissue banking with long-term preservation of the metabolic status is possible at -180 °C, which may be relevant for studies on long-term survival of cancer patients. As with other tumor entities, tissue lactate concentration was shown to be correlated with tumor development and progression in colorectal cancer. At first-time diagnosis, lactate values were low in rectal normal tissue and adenomas, were significantly elevated to intermediate levels in non-metastatic adenocarcinomas, and were very high in carcinomas with distant metastasis. There was an inverse behavior of tissue glucose concentration under corresponding conditions. The expression level of monocarboxylate transporter-4 (MCT4) was positively correlated with the tumor lactate concentration and may thus contribute to high lactate tumors being associated with a high degree of malignancy.

Lactate as a predictive marker for tumor recurrence in patients with head and neck squamous cell carcinoma (HNSCC) post radiation: a prospective study over 15 years.

OBJECTIVES: Lactate as a key regulator of the glycolytic phenotype has been recently described in fueling tumor growth and metastatic spread in head and neck squamous cell carcinoma (HNSCC). However, in context of tumor recurrence following adjuvant radiation, the underlying mechanisms remain uncertain. We therefore investigate the role of lactate towards radioresistance in HNSCC in this prospective study for the first time in vivo. MATERIALS AND METHODS: Herein, we analyzed biopsies of primary squamous cell carcinoma after surgery and adjuvant irradiation in 17 patients. Tumor tissue levels of ATP, glucose, and lactate were detected using induced metabolic bioluminescence imaging (imBI) and correlated with clinical data within an observation period of up to 15 years. RESULTS: High amounts of lactate levels in tumors of HNSCC are significantly negatively correlated with overall patient survival. Moreover, high expression of lactate in a primary tumor site is significantly correlated with tumor recurrence post radiation, whereas ATP and/or glucose showed no such correlation. CONCLUSION: Lactate can be seen not only as a waste product of altered glycolytic metabolism but also as a key master of malignancy as well as resistance mechanism towards irradiation. CLINICAL RELEVANCE: High expression of lactate levels in tumor tissue, obtained by metabolic bioluminescence imaging, may therefore serve as a predictor for overall and recurrence-free survival and could represent a future biomarker in the validation of adjuvant irradiation.

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 hypoxia inducible factor 1α linked vascular remodeling. This cooperation between MYCN and survivin may be important in the genesis of several cancers.

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy.

BACKGROUND: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. METHODS: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. RESULTS: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. CONCLUSION: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

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

The metabolic energy state of sponge tissue in vivo is largely unknown. Quantitative bioluminescence-based imaging was used to analyze the ATP distribution of Suberites domuncula (Olivi 1792) tissue, in relation to differences between the cortex and the medulla. This method provides a quantitative picture of the ATP distribution closely reflecting the in vivo situation. The obtained data suggest that the highest ATP content occurs around channels in the sponge medulla. HPLC reverse-phase C-18, used for measurement of ATP content, established a value of 1.62 µmol ATP g⁻¹ dry mass in sponge medulla, as opposed to 0.04 µmol ATP g⁻¹ dry mass in the cortex, thus indicating a specific and defined energy distribution. These results correlate with the mitochondria localization, determined using primary antibodies against cytochrome oxidase c subunit 1 (COX1) (immunostaining), as well as with the distribution of arginine kinase (AK), essential for cellular energy metabolism (in situ hybridization with AK from S. domuncula; SDAK), in sponge sections. The highest energy consumption seemed to occur in choanocytes, the cells that drive the water through the channel system of the sponge body. Taken together, these results showed that the majority of energetic metabolism in S. domuncula occurs in the medulla, in the proximity of aqueous channels.

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

Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.

Efficacy of catumaxomab in tumor spheroid killing is mediated by its trifunctional mode of action.

Catumaxomab is an intact trifunctional bispecific antibody targeting human EpCAM (epithelial cell adhesion molecule) and CD3 with further binding to Fcgamma receptor type I, IIa and III. We choose multicellular tumor spheroids (MCTS) of human EpCAM-positive FaDu tumor cells in co-culture with human peripheral blood mononuclear cells as an adequate three-dimensional in vitro model for pharmacological testing of catumaxomab. We found a strong dose-dependent antitumor response mediated by catumaxomab, with volume-decreased or completely destroyed tumor spheroids together with a massive immune cell infiltration and decreased signals for cancer cell viability and clonogenicity. In control experiments with F(ab')2 fragments of catumaxomab and the parental antibodies alone or in combination the effects in spheroid volume reduction were less than that of catumaxomab. All binding partners of the postulated tricell complex have to be present to exert catumaxomab's full mode of action. These distinct effects of catumaxomab are based on the unique composition of the trifunctional bispecific antibody. Since, in general, many cancers are treated by chemotherapy in combination with immunological tumor therapy, we additionally analyzed the effects of cisplatin alone and in combination with catumaxomab. For cisplatin alone we detected a dose-dependent response relating to decrease of spheroid volume. The combined approach resulted in a synergistic spheroid volume decrease and the colony formation was reduced to non-detectable levels.

Primary Mucosal Epithelial Cell Cultivation: A Reliable and Accelerated Isolation.

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

Migration of renal carcinoma cells is dependent on protein kinase Cdelta via beta1 integrin and focal adhesion kinase.

Migration and adhesion of tumor cells are essential prerequisites for the formation of metastases in malignant diseases. Protein kinase C (PKC) has been shown to regulate cell migration, adhesion and proliferation. In order to identify a connection between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell migration, adhesion and proliferation and possible influences of the activity of integrins and focal adhesion kinase (FAK) were analyzed in RCC cells. The experiments were performed in the RCC cell line CCF-RC1 after pre-incubation of the cells with the PKC inhibitors GF109203X, GO6976, RO31-8220 and rottlerin. Cell migration and adhesion were assessed through chemotaxis analysis and adhesion to an endothelial monolayer, respectively. Cell proliferation was analysed by a BrdU incorporation assay. The expression and activity of beta1 integrins and FAK were analysed by Western blot analysis. GF109203X reduced cell migration to 69%, the activity of beta1 integrins to 63% and FAK expression to 82% compared to untreated cells. Rottlerin reduced cell migration in a concentration-dependent manner to 36%, cell proliferation to 81%, expression and activity of beta1 integrins to 72 and 79%, and expression and activity of FAK to 56 and 76% of untreated cells, respectively. RO31-8220 also reduced the expression and activity of beta1 integrins as well as the expression of FAK to 84, 66 and 66% of untreated cells, respectively. GO6976 reduced the expression of FAK to 60% of untreated cells. Cell migration was only slightly reduced by GO6976 to 84% of untreated cells, and cell adhesion remained uninfluenced. These findings show a critical role of PKCdelta in the regulation of tumor cell migration, which seems to be caused by affecting the expression and activity of beta1 integrins and FAK. These results can provide a basis for new strategies in preventing metastases of renal cell carcinoma.

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

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 (TCD50) ranged from 47.4 to 129.8 Gy. Concentrations of glucose, lactate, and ATP in viable tumor regions as potential indicators of glycolytic activity were assessed with structure-associated quantitative bioluminescence imaging. RESULTS: Mean lactate concentrations of the different tumor cell lines were in the range of 7.3-25.9 micromol/g. TCD50 values were positively correlated with tumor lactate levels (R = 0.9824, p = 0.0028). CONCLUSIONS: The data obtained support the hypothesis that tissue lactate content correlates with radioresistance in solid human tumors. Furthermore, the results suggest that tumor lactate content determined non-invasively by proton magnetic resonance spectroscopy imaging may be used to predict for radioresistance of malignancies in the clinic; the data also imply that transient inhibition of glycolysis during treatment might possibly sensitize tumors to irradiation.

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.

OBJECTIVE: To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches. MATERIAL AND METHODS: 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 was evaluated in a total of 399 irradiated tumours at 120 days after 30 fractions given within 6 weeks with total doses between 30 and 115 Gy. RESULTS: Tumour lines showed pronounced heterogeneity in both pHF and TCD50. Mean pHF values varied between 5% and 37%, TCD50 values between 47 and 130 Gy. A Cox Proportional Hazards model of time to recurrence with two covariates, dose and pHF, yielded significant contributions of both parameters on local control (p < 0.005) but violated the proportional hazards assumption, suggesting that other factors also influence tumour control. Introduction of histological grade as an example of a confounding factor into the model improved the fit significantly. Local control rates decreased with increasing pHF and this effect was more pronounced at higher doses. CONCLUSIONS: This study confirms that tumour hypoxia measured using Pimonidazole in untreated tumours is a significant determinant of local control after fractionated irradiation. The data support the use of multivariate approaches for the evaluation of a single prognostic biomarker such as Pimonidazole, and more generally, suggest that they are required to establish accurate prognostic factors for tumour response.

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 effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in ß1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during (12)C(+6) irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral mucosa, the superior effectiveness of heavy ions was confirmed by a twofold higher number of foci per nucleus. However, proinflammatory signs were similar for both treatment modalities, e.g., the activation of NFκB and the release of IL6 and IL8. The presence of peripheral blood mononuclear cell increased the radiation-induced release of the proinflammatory cytokines by factors of 2-3. Carbon ions are part of the cosmic radiation. Long-term exposure to such particles during extended space flights, as planned by international space agencies, may thus impose a medical and safety risk on the astronauts by a potential induction of mucositis. In summary, particle irradiation is superior to gamma-rays due to a higher radiobiological effectiveness, a reduced hypoxia-induced radioresistance, a multicellular radiosensitization, and the absence of a radiation-induced cell motility. However, the potential of inducing mucositis is similar for both radiation types.

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.

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

Patients with malignant gliomas have a poor prognosis with average survival of less than 1 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 that was discovered first in this tumor entity. D2HG is generated in large amounts due to various "gain-of-function" mutations in the isocitrate dehydrogenases IDH1 and IDH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (<0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the "oncometabolite" D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0-10 µmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

Feasibility of induced metabolic bioluminescence imaging in advanced ovarian cancer patients: first results of a pilot study.

PURPOSE: The precise determination of energy metabolites is challenged by the heterogeneity of their distribution, their rapid changes after surgical resection and the architectural complexity of malignancies. Induced metabolic bioluminescence imaging (imBI) allows to determine energy metabolites in tissue sections and to co-localize these with histological structures based on consecutive sections stained with HE. In this prospective pilot study patients with suspected advanced ovarian cancer (OC) were enrolled to prove the feasibility of imBI. METHODS: During surgery, suspicious peritoneal metastases were resected and transferred in liquid nitrogen within 30 s. ATP, glucose and lactate concentrations were measured. Furthermore, the expression of monocarboxylate transporters MCT1 and MCT4 was determined by immunofluorescence staining. RESULTS: 16 patients were screened, 12 entered the study. Final histological assessment revealed ten malignant and two benign peritoneal lesions. In all 12 cases high concentrations of ATP suggested that energy metabolism was not altered by the surgical and transport procedures (mean 0.56 µmol/g, range 0.24-1.21 µmol/g). The mean concentration of glucose was 1.95 µmol/g (range 0.58-4.71 µmol/g). The concentration of lactate was drastically higher in the ten OC cases (mean 24.79 µmol/g, range 17.51-37.16 µmol/g) compared to the benign samples (mean 5.98 µmol/g, range 5.43-6.54 µmol/g). Lactate concentrations seem to correlate with MCT1 (spearman rank correlation ρ = 0.624, 0.05 > p > 0.025), but not with MCT4 (spearman rank correlation ρ = 0.018, p > 0.1). CONCLUSIONS: ImBI is feasible in peritoneal metastases of OC and encourages further effort to elucidate the role of glucose, lactate, MCT1 and MCT4 in OC.

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 NK cells, resulting in diminished IFN-γ production. Database analyses revealed negative correlations between LDHA expression and T cell activation markers in human melanoma patients. Our results demonstrate that lactic acid is a potent inhibitor of function and survival of T and NK cells leading to tumor immune escape.

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

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 metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.