Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells.

A network model for the determination of tumor metabolic fluxes from (13)C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH

(31) P and (1) H MRS of DB-1 melanoma xenografts: lonidamine selectively decreases tumor intracellular pH and energy status and sensitizes tumors to melphalan.

In vivo (31) P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg intraperitoneally) exhibit a decrease in intracellular pH (pH(i) ) from 6.90 ± 0.05 to 6.33 ± 0.10 (p \u3c 0.001), a slight decrease in extracellular pH (pH(e) ) from 7.00 ± 0.04 to 6.80 ± 0.07 (p \u3e 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p \u3c 0.001) relative to the baseline level. Both bioenergetics and pH(i) decreases were sustained for at least 3 h following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p \u3e 0.05) at 20 min post-LND, with no significant change in pH(e) and a small transient decrease in bioenergetics (32.9 ± 10.6%, p \u3e 0.05) at 40 min post-LND. No changes in pH(i) or adenosine triphosphate/inorganic phosphate were detected in the brain (pH(i) , bioenergetics; p \u3e 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p \u3e 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by (1) H MRS with a selective multiquantum pulse sequence with Hadamard localization increased approximately three-fold (p = 0.009). Treatment with LND increased the systemic melanoma response to melphalan (LPAM; 7.5 mg/kg intravenously), producing a growth delay of 19.9 ± 2.0 days (tumor doubling time, 6.15 ± 0.31 days; log(10) cell kill, 0.975 ± 0.110; cell kill, 89.4 ± 2.2%) compared with LND alone of 1.1 ± 0.1 days and LPAM alone of 4.0 ± 0.0 days. The study demonstrates that the effects of LND on tumor pH(i) and bioenergetics may sensitize melanoma to pH-dependent therapeutics, such as chemotherapy with alkylating agents or hyperthermia

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate-malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to (13)C magnetic resonance spectroscopy ((13)C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable (13)C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by (1)H MRS and perhaps also by FDG-PET and hyperpolarized (13)C MRS methods

Mechanism of antineoplastic activity of lonidamine

Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells. It has been reported to alter the bioenergetics of tumor cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggested that it also inhibited L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Recent studies have demonstrated that LND potently inhibits MPC activity in isolated rat liver mitochondria (K(i) 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K(0.5) and Hill Coefficient values of 36–40 μM and 1.65–1.85, respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC(50) ~7 μM) than other substrates including glutamate (IC(50) ~20 μM). LND inhibits the succinate-ubiquinone reductase activity of respiratory Complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through Complex II and has been reported to promote cell death by suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux, Complex II and glutamine/glutamate oxidation

Feasibility of removable balloon implant for simultaneous magnetic nanoparticle heating and HDR brachytherapy of brain tumor resection cavities.

AIM: Hyperthermia (HT) has been shown to improve clinical response to radiation therapy (RT) for cancer. Synergism is dramatically enhanced if HT and RT are combined simultaneously, but appropriate technology to apply treatments together does not exist. This study investigates the feasibility of delivering HT with RT to a 5-10mm annular rim of at-risk tissue around a tumor resection cavity using a temporary thermobrachytherapy (TBT) balloon implant. METHODS: A balloon catheter was designed to deliver radiation from High Dose Rate (HDR) brachytherapy concurrent with HT delivered by filling the balloon with magnetic nanoparticles (MNP) and immersing it in a radiofrequency magnetic field. Temperature distributions in brain around the TBT balloon were simulated with temperature dependent brain blood perfusion using numerical modeling. A magnetic induction system was constructed and used to produce rapid heating (\u3e0.2°C/s) of MNP-filled balloons in brain tissue-equivalent phantoms by absorbing 0.5 W/ml from a 5.7 kA/m field at 133 kHz. RESULTS: Simulated treatment plans demonstrate the ability to heat at-risk tissue around a brain tumor resection cavity between 40-48°C for 2-5cm diameter balloons. Experimental thermal dosimetry verifies the expected rapid and spherically symmetric heating of brain phantom around the MNP-filled balloon at a magnetic field strength that has proven safe in previous clinical studies. CONCLUSIONS: These preclinical results demonstrate the feasibility of using a TBT balloon to deliver heat simultaneously with HDR brachytherapy to tumor bed around a brain tumor resection cavity, with significantly improved uniformity of heating over previous multi-catheter interstitial approaches. Considered along with results of previous clinical thermobrachytherapy trials, this new capability is expected to improve both survival and quality of life in patients with glioblastoma multiforme

Improved Tumor Control Following Radiosensitization with Ultrasound-Sensitive Oxygen Microbubbles and Tumor Mitochondrial Respiration Inhibitors in a Preclinical Model of Head and Neck Cancer

Tumor hypoxia (oxygen deficiency) is a major contributor to radiotherapy resistance. Ultrasound-sensitive microbubbles containing oxygen have been explored as a mechanism for overcoming tumor hypoxia locally prior to radiotherapy. Previously, our group demonstrated the ability to encapsulate and deliver a pharmacological inhibitor of tumor mitochondrial respiration (lonidamine (LND)), which resulted in ultrasound-sensitive microbubbles loaded with O2 and LND providing prolonged oxygenation relative to oxygenated microbubbles alone. This follow-up study aimed to evaluate the therapeutic response to radiation following the administration of oxygen microbubbles combined with tumor mitochondrial respiration inhibitors in a head and neck squamous cell carcinoma (HNSCC) tumor model. The influences of different radiation dose rates and treatment combinations were also explored. The results demonstrated that the co-delivery of O2 and LND successfully sensitized HNSCC tumors to radiation, and this was also enhanced with oral metformin, significantly slowing tumor growth relative to unsensitized controls (p \u3c 0.01). Microbubble sensitization was also shown to improve overall animal survival. Importantly, effects were found to be radiation dose-rate-dependent, reflecting the transient nature of tumor oxygenation

Acidification-induced sensitization to thermoradiotherapy in breast cancer

Hyperthermia is an extensively studied cytotoxic agent, with strong radio- and chemosensitizing potential. Recent positive clinical trials combining superficial or deep heating techniques with radiation therapy strongly support a role for hyperthermia as an adjuvant to radiation. Many in vitro and in vivo studies have shown that acute extracellular acidification will compromise fundamental protective cellular responses and enhance tumor response to hyperthermia and chemotherapy. Breast cancers, like most other tumors, exhibit elevated levels of lactate production that provides a basis for selective acidification. A phase I/II clinical trial is underway to test the hypothesis that hyperglycemia-induced acute acidification will sensitize carcinoma of the breast to thermoradiotherapy. Six patients consented to fast for at least 4 h and ingest oral glucose (2 g/kg, 0.44 g/ml) 1.5 h before each hyperthermia treatment (HT) during a course of thermoradiotherapy. Hyperglycemia reduced tumor pHe before the first hyperthermia session by 0.10 ± 0.04 pH unit (-0.29 to +0.08) from 7.12 ± 0.11 (6.65-7.52); and during the third week of treatment hyperglycemia reduced tumor pHe in five patients by 0.01 ± 0.04 pH unit (-0.06 to +0.1). The three patients with a CR (60%) exhibited tumor acidification during both sessions, in contrast to the two patients with a PR (40%) who exhibited tumor acidification only during one session. Tumor acidification may indicate tumor response. Human tumor cells adapted to growth at pHe 6.7 do not show thermosensitization until pHe is below 6.3 (pHi \u3c 6.45). Combining an inhibitor of respiration such as MIBG with hyperglycemia blocks mitochondrial respiration and increases lactate production. Thus, tumor oxygenation occurs coincidentally with acute acidification. Rats bearing the R3230 Ac rat mammary adenocarcinoma were administered 1 g/kg glucose ip, and/or 20 mg/kg MIBG ip. The median pO2 for glucose plus MIBG was increased from 5.3 to 13.8 mmHg. A single ip injection of glucose or MIBG in rats fasted for 24 h before irradiation did not show an increase in tumor growth delay compared with 5 Gy radiation alone. However, combined treatment with glucose plus MIBG significantly inhibited tumor growth delay. Radiation therapy and glucose plus MIBG was more than additive. These results support our hypothesis that hyperglycemia plus an inhibitor of respiration will sensitize tumors to radiation by oxygenation, in addition to enhanced hyperthermia sensitization by acute acidification