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Radiation therapy (RT) causes DNA damage through ionization, leading to double-strand breaks. In addition, it generates reactive oxygen species (ROS), which are toxic to tumor cells and the vasculature. However, hypoxic regions in the tumor have been shown to not only decrease treatment response but also increase the likelihood of recurrence and metastasis. Ultrasound-sensitive micro-bubbles are emerging as a useful diagnostic and therapeutic tool within RT. Contrast-enhanced ultrasound (CEUS) has shown great promise in early prediction of tumor response to RT. Ultrasound-triggered micro-bubble cavitation has also been shown to induce bio-effects that can sensitize angiogenic tumor vessels to RT. Additionally, ultrasound can trigger the release of drugs from micro-bubble carriers via localized micro-bubble destruction. This approach has numerous applications in RT, including targeted oxygen delivery before radiotherapy. Furthermore, micro-bubbles can be used to locally create ROS without radiation. Sonodynamic therapy uses focused ultrasound and a sonosensitizer to selectively produce ROS in the tumor region and has been explored as a treatment option for cancer. This review summarizes emerging applications of ultrasound contrast agents in RT and ROS augmentation.

The feasibility of activating phase change contrast agents (PCCA) made from Definity (Lantheus Medical Imaging) using X-rays was investigated. A 10 mL of Definity PCCA (0.1 mL PCCA/mL) were injected into gelatin phantoms and irradiated using doses of 0, 30, 50, and 100 Gy. Size distribution and PCCA activation were measured after irradiation. Definity PCCAs were activated at a threshold of 50 Gy. Changes were visible with microscopy, visual inspection of T-flasks, and ultrasound imaging of gelatin phantoms. Moreover, increasing the radiation dose above 50 Gy appeared to further activate PCCA. These results indicate Definity PCCAs may be useful for ultrasound-based radiation dosimetry.

Prior work has shown that microbubble-assisted delivery of oxygen improves tumor oxygenation and radiosensitivity, albeit over a limited duration. Lonidamine (LND) has been investigated because of its ability to stimulate glycolysis, lactate production, inhibit mitochondrial respiration, and inhibit oxygen consumption rates in tumors but suffers from poor bioavailability. The goal of this work was to characterize LND-loaded oxygen microbubbles and assess their ability to oxygenate a human head and neck squamous cell carcinoma (HNSCC) tumor model, while also assessing LND biodistribution. In tumors treated with surfactant-shelled microbubbles with oxygen core (SE61O(2)) and ultrasound, pO(2) levels increased to a peak 19.5 ± 9.7 mmHg, 50 s after injection and returning to baseline after 120 s. In comparison, in tumors treated with SE61O(2)/LND and ultrasound, pO(2) levels showed a peak increase of 29.0 ± 8.3 mmHg, which was achieved 70 s after injection returning to baseline after 300 s (p < 0.001). The co-delivery of O(2) and LND via SE61 also showed an improvement of LND biodistribution in both plasma and tumor tissues (p < 0.001). In summary, ultrasound-sensitive microbubbles loaded with O(2) and LND provided prolonged oxygenation relative to oxygenated microbubbles alone, as well as provided an ability to locally deliver LND, making them more appropriate for clinical translation.

Lonidamine (LND) has been widely explored due to its ability to target metabolic glycolysis and mitochondrial respiration in tumors [4]. However, clinical translation has stalled due to poor bioavailability after oral administration. In this study, we explored three incubation time points of loading oxygen-filled microbubbles with LND (SE61O2-LND) for radiotherapy sensitization by modifying our published methods for creating surfactant-shelled oxygen microbubbles (SE61 O2). Acoustic enhancement and stability were quantified in vitro in a continuously insonated sample holder using a 5 MHz single element transducer which generated a peak positive pressure of 0.69 MPa and a peak negative pressure of 0.25 MP at a PRF of 100 Hz and in a pulsatile flow phantom insonated at a single point using a C1-6 transducer on a GE Logiq E9 system (GE Healthcare) operating at 4.0 MHz at a mechanical index of 0.12. Flow cytometry was performed to analyze the total bubble count for each incubation time point. Lonidamine encapsulation was determined using liquid chromatography-mass spectrometry (LC-MS), the loading approach consisted of incubating the LND and TPGS at 37°C at 0, 24, and 48 hours. Bubbles incubated for 24 hours had an average encapsulation of 2.64 ± 0.32 µg LND/ mL MB, and those incubated for 48 hours had an average of 2.97 ± 0.36 µg LND/ mL MB, while non-incubated micelles had an average of 1.51 ± 0.05 µg LND/ mL MB, both TPGS micelle methods that were incubated showed a statistically significant improvement p < 0.001 compared to non-incubated. This work demonstrates the feasibility of fabricating lonidamine-loaded microbubbles and an improved approach for increasing drug encapsulation. The microbubbles retained their acoustic properties, and by incubating the lonidamine with the TPGS to form micelles, drug loading was increased.

Much of the volume of solid tumors typically exists in a chronically hypoxic microenvironment that has been shown to result in both chemotherapy and radiation therapy resistance. The purpose of this study was to use localized microbubble delivery to overcome hypoxia prior to therapy.In this study, surfactant-shelled oxygen microbubbles were fabricated and injected intravenously to locally elevate tumor oxygen levels when triggered by noninvasive ultrasound in mice with human breast cancer tumors. Changes in oxygen and sensitivity to radiation therapy were then measured.In this work, we show that oxygen-filled microbubbles successfully and consistently increase breast tumor oxygenation levels in a murine model by 20 mmHg, significantly more than control injections of saline solution or untriggered oxygen microbubbles (P .001). Using photoacoustic imaging, we also show that oxygen delivery is independent of hemoglobin transport, enabling oxygen delivery to avascular regions of the tumor. Finally, we show that overcoming hypoxia by this method immediately prior to radiation therapy nearly triples radiosensitivity. This improvement in radiosensitivity results in roughly 30 days of improved tumor control, providing statistically significant improvements in tumor growth and animal survival (P .03).Our findings demonstrate the potential advantages of ultrasound-triggered oxygen delivery to solid tumors and warrant future efforts into clinical translation of the microbubble platform.

OBJECTIVES—: Hypoxic cancer cells have been shown to be more resistant to radiation therapy than normoxic cells. Hence, this study investigated whether ultrasound (US)-induced rupture of oxygen-carrying microbubbles (MBs) would enhance the response of breast cancer metastases to radiation. METHODS—: Nude mice (n = 15) received stereotactic injections of brain-seeking MDA-MB-231 breast cancer cells into the right hemisphere. Animals were randomly assigned into 1 of 5 treatment groups: no intervention, 10 Gy radiation using a small-animal radiation research platform, nitrogen-carrying MBs combined with US-mediated MB rupture immediately before 10 Gy radiation, oxygen-carrying MBs immediately before 10 Gy radiation, and oxygen-carrying MBs with US-mediated MB rupture immediately before 10 Gy radiation. Tumor progression was monitored with 3-dimensional US, and overall survival was noted. RESULTS—: All groups except those treated with oxygen-carrying MB rupture and radiation had continued rapid tumor growth after treatment. Tumors treated with radiation alone showed a mean increase in volume ± SD of 337% ± 214% during the week after treatment. Tumors treated with oxygen-carrying MBs and radiation without MB rupture showed an increase in volume of 383% ± 226%. Tumors treated with radiation immediately after rupture of oxygen-carrying MBs showed an increase in volume of only 41% ± 1% (P = 0.045), and this group also showed a 1 week increase in survival time. CONCLUSIONS—: Adding US-ruptured oxygen-carrying MBs to radiation therapy appears to delay tumor progression and improve survival in a murine model of metastatic breast cancer.

Lonidamine (LND), a metabolic modulator, sensitizes DB-1 human melanoma to doxorubicin (DOX) chemotherapy by acidifying and de-energizing the tumor. This report compares the effects of LND on two human melanoma lines, DB-1 and WM983B, which exhibit different metabolic properties. Using liquid chromatography mass spectrometry and Seahorse analysis, we show that DB-1 was more glycolytic than WM983B in vitro. 31P magnetic resonance spectroscopy (MRS) indicates that LND (100 mg/kg, i.p.) induces similar selective acidification and de-energization of WM983B xenografts in immunosuppressed mice. Over three hours, intracellular pH (pHi) of WM983B decreased from 6.91 ± 0.03 to 6.59 ± 0.10 (p = 0.03), whereas extracellular pH (pHe) of this tumor changed from 7.03 ± 0.05 to 6.89 ± 0.06 (p = 0.19). A decline in bioenergetics (β-NTP/Pi) of 55 ± 5.0% (p = 0.03) accompanied the decline in pHi of WM983B. Using 1H MRS with a selective multiquantum pulse sequence and Hadamard localization, we show that LND induced a significant increase in tumor lactate levels (p

We seek to exploit the natural tendency of melanomas and other tumors to convert glucose to lactate as a method for the selective intracellular acidification of cancer cells and for the potentiation of the activity of nitrogen-mustard antineoplastic agents. We performed this study to evaluate whether the induction of hyperglycemia (26 mM) could enhance the effects of lonidamine (LND, 100 mg/kg; intraperitoneally) on the induction of intracellular acidification, bioenergetic decline and potentiation of the activity of melphalan (LPAM) against DB-1 melanoma xenografts in mice. Intracellular pH (pHi ), extracellular pH (pHe ) and bioenergetics (β-nucleoside triphosphate to inorganic phosphate ratio, β-NTP/Pi) were reduced by 0.7 units (p 0.05) and 51.4% (p < 0.05), respectively. The therapeutic response to LPAM (7.5 mg/kg; intravenously) + LND (100 mg/kg; intraperitoneally) was reduced by about a factor of three under hyperglycemic conditions relative to normoglycemia, producing a growth delay of 7.76 days (tumor doubling time, 5.31 days; cell kill, 64%) compared with LND alone of 1.70 days and LPAM alone of 0.29 days. Under normoglycemic conditions, LND plus LPAM produced a growth delay of 17.75 days, corresponding to a cell kill of 90% at the same dose for each of these agents. The decrease in tumor cell kill under hyperglycemic conditions correlates with an increase in tumor ATP levels resulting from increased glycolytic activity. However, hyperglycemia substantially increases lactic acid production in tumors by a factor of approximately six (p < 0.05), but hyperglycemia did not increase the effects of LND on acidification of the tumor, most probably because of the strong buffering action of carbon dioxide (the pKa of carbonic acid is 6.4). Therefore, this study demonstrates that the addition of glucose during treatment with LND diminishes the activity of this agent.

We demonstrate that the effects of lonidamine (LND, 100 mg/kg, i.p.) are similar for a number of xenograft models of human cancer including DB-1 melanoma and HCC1806 breast, BT-474 breast, LNCaP prostate and A2870 ovarian carcinomas. Following treatment with LND, each of these tumors exhibits a rapid decrease in intracellular pH, a small decrease in extracellular pH, a concomitant monotonic decrease in nucleoside triphosphate and increase in inorganic phosphate over a 2–3 hr period. We previously demonstrated that selective intracellular tumor acidification potentiates response of this melanoma model to melphalan (7.5 mg/kg, i.v.), producing an estimated 89% cell kill based on tumor growth delay analysis. We now show that in both DB-1 melanoma and HCC1806 breast carcinoma, LND potentiates response to doxorubicin producing 95% cell kill in DB-1 melanoma at 7.5 mg/kg, i.v. doxorubicin and 98% cell kill at 10.0 mg/kg doxorubicin, and in HCC1806 breast carcinoma producing a 95% cell kill at 12.0 mg/kg doxorubicin. Potentiation of doxorubicin can result from cation trapping of the weakly basic anthracycline. Recent experience with the clinical treatment of melanoma and other forms of human cancer suggests that these diseases will probably not be cured by a single therapeutic procedure other than surgery. A multimodality therapeutic approach will be required. As a potent modulator of tumor response to N-mustards and anthracyclines as well as tumor thermo- and radiosensitivity, LND promises to play an important clinical role in the management and possible complete local control of a number of prevalent forms of human cancer.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and the fastest growing malignancy in the United States. With a 5-year survival rate below 12%, effective therapies for HCC are needed. Current treatments for HCC include microwave and radiofrequency ablation, high intensity focused ultrasound, liver transplant, surgical resection, and localized embolizations. However, each of these approaches has some limitation, making it imperative to develop improved methods for sensitizing tumors prior to therapy. We hypothesized that the use of ultrasound-triggered microbubble destruction (UTMD), which sensitizes tumors to radiotherapy by inducing vascular endothelial cell apoptosis, will selectively sensitize malignant tissue to radiotherapy and improve outcomes. To test this, 18 nude rats were inoculated in the right liver lobe with Hu7.5 HCC cells and after tumor formation, received 5 Gy radiotherapy, UTMD, or UTMD prior to radiotherapy. Compared to radiotherapy alone, there was a 170% reduction in tumor growth 7 days post treatment and a 3.2X improvement in median survival time when radiotherapy was combined with UTMD. These results indicate that UTMD is an effective adjunct when combined with radiotherapy to treat HCC.

BACKGROUND/AIM: Since temozolomide (TMZ) is activated under alkaline conditions, we expected lonidamine (LND) to have no effect or perhaps diminish its activity, but initial results suggest it may actually enhance either or both short- and long-term activity of TMZ in melanoma xenografts. MATERIALS AND METHODS: Cohorts of 5 mice with subcutaneous xenografts ~5 mm in diameter were treated with saline (control (CTRL)), LND only, TMZ only or LND followed by TMZ at t=40 min (time required for maximal tumor acidification). RESULTS: Mean tumor volume for LND+TMZ for the period between 6 and 26 days was reduced compared to TMZ alone (repeated measures ANOVA F (1, 8), p=0.006), suggesting a pronounced impact of LND on this phenomenon. TMZ and LND+TMZ produced median growth delays of 82 and 106 days, respectively. CONCLUSION: The use of TMZ alone and in combination with LND deserves further investigation in treatment of melanoma and other malignancies.

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 (Ki 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 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 (IC50 ~ 7 μM) than other substrates including glutamate (IC50 ~ 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.

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.

Purpose To implement clinical stereotactic body radiation therapy (SBRT) using a small animal radiation research platform (SARRP) in a genetically engineered mouse model of lung cancer. Methods and Materials A murine model of multinodular Kras -driven spontaneous lung tumors was used for this study. High-resolution cone beam computed tomography (CBCT) imaging was used to identify and target peripheral tumor nodules, whereas off-target lung nodules in the contralateral lung were used as a nonirradiated control. CBCT imaging helps localize tumors, facilitate high-precision irradiation, and monitor tumor growth. SBRT planning, prescription dose, and dose limits to normal tissue followed the guidelines set by RTOG protocols. Pathologic changes in the irradiated tumors were investigated using immunohistochemistry. Results The image guided radiation delivery using the SARRP system effectively localized and treated lung cancer with precision in a genetically engineered mouse model of lung cancer. Immunohistochemical data confirmed the precise delivery of SBRT to the targeted lung nodules. The 60 Gy delivered in 3 weekly fractions markedly reduced the proliferation index, Ki-67, and increased apoptosis per staining for cleaved caspase-3 in irradiated lung nodules. Conclusions It is feasible to use the SARRP platform to perform dosimetric planning and delivery of SBRT in mice with lung cancer. This allows for preclinical studies that provide a rationale for clinical trials involving SBRT, especially when combined with immunotherapeutics.

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 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p 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 > 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 > 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p > 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.

A network model for the determination of tumor metabolic fluxes from 13C 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 [1,6-13C2]glucose under normoxic conditions at 37 °C and monitored by 13C 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.

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit 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). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 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 (IC50 ∼ 7 μM) than other substrates including glutamate (IC50 ∼ 20 μM). In isolated DB-1 melanoma cells 1–10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. 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 and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND on L-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC).

The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that 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, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Exposure of cells to heat inhibits a number of nuclear activities associated with semi-conservative replication of DNA including the incorporation of radiolabelled precursors into acid-insoluble DNA, the initiation of new replicons, the elongation of the DNA fibre at the replication fork, the synthesis and deposition of new histones into chromatin and the reorganization of nascent DNA into mature chromatin. These effects are likely to underlie the heat sensitivity of S-phase cells and may contribute to the radiosensitization observed in this phase of the cell cycle. While some of these effects may be explained as 'passive' consequences of heat-induced damage on chromatin structures experiments reviewed here point to the activation of a checkpoint as a contributing factor to the observed inhibition of DNA replication. Activation of a heat responsive S-phase checkpoint targets the activity of RPA via interaction with nucleolin. Nucleolin, a major nucleolar protein, is found normally sequestered in the nucleolus. Exposure of cells to heat causes a rapid translocation of nucleolin from the nucleolus into the nucleoplasm that enables RPA/nucleolin interaction. This interaction inhibits functions of RPA associated with the initiation of DNA replication and contributes to the immediate inhibition of DNA synthesis observed after heat shock. The results suggest that the nucleolus serves as a sequestration centre for the temporary inactivation of regulatory molecules, such as nucleolin, capable of regulating essential cellular functions after heat shock. It is speculated that this regulatory process is integrated in the network of responses that determine cell sensitivity to heat and that it may be involved in heat radiosensitization to killing as well.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Although p53 responses after DNA damage have been investigated extensively, p53 responses after heat shock, which exerts cytotoxic action by mechanisms other than direct induction of DNA damage, are less well characterized. We investigated, therefore, the effect of hyperthermic exposures on the levels and DNA-binding activity of p53. Experiments were carried out with U2OS and ML-1 cells, known to express wild-type p53 protein. Although heating at 41 degrees C for up to 6 h had only a small effect on p53 levels or DNA binding activity, exposure to temperatures between 42.5 and 45.5 degrees C caused an immediate decrease in protein levels that was associated with a reduction in DNA binding activity. This observation is compatible with a high lability of p53 to heat shock, or heat sensitivity of the pathway regulating p53 levels in non-stressed cells. When cells were heated to 42.5 degrees C and returned to normal temperatures, a strong p53 response associated with an increase in protein levels and DNA binding activity was observed, suggesting the production of p53-inducing cellular damage. At higher temperatures, however, this response was compromised in an exposure-time-dependent manner. The increase in DNA binding activity was more heat sensitive than the increase in p53 levels and was inhibited at lower temperatures and shorter exposure times. Thus, the pathway of p53 activation is itself heat sensitive and compromised at high levels of exposure. Compared to p53 activation after exposure to ionizing radiation, heat-induced activation is rapid and short lived. When cells were exposed to combined heat and radiation, the response observed approximated that of cells exposed to heat alone. Wortmannin at 10 microM inhibited p53 activation for up to 2 h after heat shock suggesting the involvement of wortmannin-sensitive kinases, such as DNA-PK and ATM. Heat shock causes phosphorylation of p53 at Serine-15, but there is no correlation between phosphorylation at this site and activation of the protein. The results in aggregate indicate p53 activation in the absence of DNA damage by a heat-sensitive mechanism operating with faster kinetics than radiation-induced p53 activation. The former response may induce pathways preventing other stimuli from activating p53, as heat-induced activation of p53 is dominant over activation of p53 by DNA damage in combined-treatment experiments. These observations suggest means for abrogating p53 induction after DNA damage with the purpose of potentiating response and enhancing cell killing.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42 degrees C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2 h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42 degrees C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42 degrees C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42 degrees C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.

Non-invasive thermometry is pivotal to the future advances of regional hyperthermia as a cancer treatment modality. Current magnetic resonance (MR) thermometry methods suffer from poor thermal resolution due to relatively weak dependence of chemical shift of the (1)H water signal on temperature. This study evaluated the feasibility of using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA(-)) for MR thermometry. TmDOTA(-) is non-toxic and the gadolinium complex of DOTA(4-) is widely used as a MR contrast agent. The results demonstrate that the temperature dependence of the TmDOTA(-) proton shifts are about two orders of magnitudes higher than the water proton and, thus, provide excellent accuracy and resolution. In addition, TmDOTA(-) proton shifts are insensitive to the paramagnetic complex concentration, pH, Ca(2+) or presence of plasma macromolecules and ions. Because hyperthermia is known to produce changes in tissue pH and other physiological parameters, these properties of TmDOTA(-) greatly simplify the procedures for using the lanthanide complex for MR thermometry. Application of TmDOTA(-) for measurement of temperature in a subcutaneously implanted human melanoma xenograft is demonstrated. Finally, the feasibility of imaging one of the (1)H resonances of the lanthanide complex is demonstrated in phantom experiments. Overall, TmDOTA(-) appears to be a promising probe for MR thermometry in vivo.

Betulinic acid is a known inducer of apoptosis in human melanoma that is most effective under conditions of low pH. It was hypothesized that betulinic acid, in combination with acute acidification and/or hyperthermia, would induce higher levels of apoptosis and cytotoxicity in low pH-adapted human melanoma cells than in cells grown at pH 7.3. DB-1 human melanoma cells, adapted to a tumour-like growth pH of 6.7, were exposed to hyperthermia (2h at 42 degrees C) and/or betulinic acid (4-10 microg/ml) and compared with cells grown at a physiological pH of 7.3 or after acute acidification from pH 7.3-6.3 or pH 6.7-6.3. Betulinic acid induced higher levels of apoptosis and cytotoxicity in low pH-adapted cells than in cells grown at pH 7.3, as measured by the terminal deoxynucleotidyl transferase (TdT) DNA fragmentation assay (TUNEL), the MTS cell viability assay, and single cell survival. Acute acidification of low pH adapted cells rendered them more susceptible to betulinic acid-induced apoptosis and cytotoxicity. In the presence of hyperthermia at 42 degrees C for 2 h, cells grown at pH 7.3 were not sensitized to heat killing by betulinic acid, whereas cells grown at pH 7.3 and acutely acidified to pH 6.3, cells adapted to growth at pH 6.7 and cells adapted to growth at pH 6.7 and acutely acidified to pH 6.3 were all similarly sensitized to heat killing by betulinic acid, with survival values of 5, 9 and 2%, respectively. It is concluded that betulinic acid may be useful in potentiating the therapeutic efficacy of hyperthermia as a cytotoxic agent in acidotic areas of tumours with minimal effect in normal tissues growing at pH 7.3.

Rationale and Objectives The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level ± standard error of the mean, 26 mmol/L ± 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), α-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4,4′-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). Materials and Methods The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. Results In small tumors (≤8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the β-nucleoside triphosphate (NTP)/inorganic phosphate (P i ) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P i and β-NTP/P i ratios, respectively. The combination of hyperglycemia, MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. Conclusion These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermia or other pH-sensitive therapeutic modalities.

Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44 degrees C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60-90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20-30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPA-nucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.

This study tested the ability of lonidamine (LND), a clinically applicable inhibitor of monocarboxylate transporters (MCT), to thermally sensitise human melanoma cells cultured at a tumour-like extracellular pH (pHe) 6.7.Human melanoma DB-1 cells cultured at pHe 6.7 and pHe 7.3 were exposed to 150 µM LND for 3 h, beginning 1 h prior to heating at 42 °C (2 h). Intracellular pH (pHi) was determined using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and whole spectrum analysis. Levels of heat shock proteins (HSPs) were determined by immunoblot analysis. Cell survival was determined by colony formation.Treatment with LND at pHe 6.7 reduced pHi to 6.30 ± 0.21, reduced thermal induction of HSPs, and sensitised cells growing at pHe 6.7 to 42 °C. When LND was combined with an acute acidification from pHe 6.7 to pHe 6.5, pHi was reduced to 6.09 ± 0.26, and additional sensitisation was observed. LND had negligible effects on cells cultured at pH 7.3.The results show that LND can reduce pHi in human melanoma cells cultured at a tumour-like low pHe so that the 42 °C induction of HSPs are abrogated and the cells are sensitised to thermal therapy. Cells cultured at a normal tissue-like pHe 7.3 were not sensitised to 42 °C by LND. These findings support the strategy that human melanoma cells growing in an acidic environment can be sensitised to thermal therapy in vivo by exposure to an MCT inhibitor such as LND.

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H+ transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4–2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pHe) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pHi) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pHe was observed by monitoring pHi after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pHe was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pHi. This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification. J. Cell. Physiol. 183:373–380, 2000. © 2000 Wiley-Liss, Inc.

Introduction: Meta-iodobenzylguanidine (MIBG) in its 131 I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, i.e. , MIBG reportedly inhibits mitochondrial respiration in vitro . The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo . Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo . Materials and Methods: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo . 31 P-NMR was used to determine alterations in tumor cell pH in vivo . Results: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro . The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro . Changes in intracellular pH measured with 31 P-NMR, in vivo , precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. Conclusions: We conclude that 31 P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

The response of human tumour blood flow to a fractionated course of thermoradiotherapy was documented in four superficial but bulky tumours (three adenocarcinomas, one melanoma). Blood flow was measured 15, 30, 45, and 60 min after the onset of heating. These measurements were made at the same intra-tumour point during each heat fraction by use of a modified thermal clearance technique in which a correction was made for the heat dissipated by thermal conduction. This point was at least 2 cm beneath the surface in the central portion of the tumour. Extracellular pH was measured within 1 cm of this point prior to the first heat fraction and 2-3 weeks later. Hyperthermia was administered for 60 min, twice a week for 4 weeks by use of a 16-channel 915 MHz microwave applicator. Each patient also received a radiation dose of 40 Gy fractionated at 2 Gy/fx, five times a week (adenocarcinomas) or 4 Gy/fx, twice a week (melanoma). Blood flow remained relatively constant during heating after steady state conditions were attained. However, an overall decrease in tumour blood flow was observed in each patient over the course of thermoradiotherapy. In each case, a relatively small decrease in blood flow occurred between most heat fractions which resulted in an overall decrease which ranged from 50-100%. However, there was a tendency for blood flow to increase following the initial heat fraction at points where the steady state temperature was approximately 41 degrees C or less. Extracellular pH increased in two of three patients and decreased in the other.

The purpose of this study was to determine whether intravenous or combined intravenous plus oral glucose administration was more effective inducing acute tumour acidification. Seventeen nondiabetic patients at the Henan Tumour Hospital with superficial tumour deposits of various histologies and size were administered, after fasting, either 50 g glucose intravenously (i.v., in 100 ml over 10 min) or 50 g i.v. glucose (in 100 ml over 10 min) combined with 100 g oral glucose (in 200 ml; i.v. + oral). Extracellular tumour pH (pHe) was determined with one or two indwelling needle combination pH microelectrodes. Blood glucose concentration was determined every 15-20 min by finger stick with Chem-Strips and a Glucometer. Ten patients received i.v. glucose, and seven patients received i.v. + oral glucose. Blood glucose rose to 430 +/- 15 mg/dL in both groups. However, the rate of clearance of blood glucose was greater for the i.v. glucose than for the i.v. + oral glucose group (p0.00002), and thus the blood glucose levels remained elevated longer after i.v. + oral than after i.v. glucose administration. Relative to the initial fasting blood glucose concentration, blood glucose was -2 +/- 7 mg/dL at 110 min after glucose administration by the i.v. route, whereas, blood glucose relative to initial values was 143 + 23 mg/dL by 110 min after glucose administration by the i.v. + oral route, p = 0.000004. The initial pHe values in the two groups of tumours were similar, 7.34 +/- 0.09 (6.78-7.71) and 7.35 +/- 0.08 (6.99-7.61), respectively. After i.v. glucose, tumour acidification occurred in nine of ten patients (-0.16 + 0.02 pH unit, range -0.24 to -0.05), and after i.v. + oral glucose tumour acidification occurred in six of seven patients (-0.19 +/- 0.07 pH unit, range -0.43 to -0.06). When the initial fasting blood glucose concentration was in excess of 82 mg/dL, all patients (12/12) exhibited tumour acidification during hyperglycaemia, whereas, only 3/5 patients exhibited tumour acidification when the initial blood glucose concentration was less than 82 mg/dL (p = 0.07). The time to maximum decrease in tumour pHe was significantly shorter after i.v. + oral glucose than after i.v. glucose (e.g., 67 +/- 11 versus 102 +/- 8 min, p = 0.02) and correlated with the rate of clearance of blood glucose (p = 0.02, r = 0.55). Larger tumours tended to exhibit a greater decrease in pHe (p = 0.08, r = 0.04). The only side effects of hyperglycaemia were transient nausea and increased urinary output. The effect of hyperglycaemia induced by administration of 200 g oral glucose was similar to i.v. administration in that 83% of tumours exhibited acidification of 0.14 +/- 0.02 pH unit by 91 +/- 7 min. We conclude that i.v. and i.v. + oral glucose administration are equally effective inducing tumour acute acidification, but no more effective than 200 g oral glucose, for investigation of hyperglycemic sensitization to thermoradiotherapy.

Cells which have been adapted to growth at low extracellular pH (pHe) typically develop both an upregulation of steady state intracellular pH (pHi) and an ability to develop thermotolerance to 42 degrees C hyperthermia. These properties were acquired at different times, however. Days were required at pHe = 6.70 for two cell lines to adapt to low pHe by the thermotolerance criterion, but both had elevated steady state pHi values after only 4 hours at pHe = 6.70. A better correlation with adaptation to low pHe (as defined by hyperthermia) was found with changes in proton extrusion and the rate of pHi recovery after cytosolic acidification.

Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/ H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl-/HCO(-3)exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification.

As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO−3. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells. © 1996 Wiley-Liss, Inc.

Murine bone marrow granulocyte-macrophage progenitors (CFU-GM) are capable of developing thermotolerance during exposure to temperatures < 42.5 degrees C. Bone marrow from the tibia and femora was heated to 40-42 degrees C (i.e. chronic hyperthermia), and challenged immediately with 15 min at 44 degrees C at regular intervals during treatment (step-up heating). CFU-GM were heated and cultured in McCoy's 5A medium + 15% FBS (fetal bovine serum) and lung-conditioned medium (source of colony stimulating factor) in semisolid agar. The kinetics of thermotolerance development and decay, and the magnitude of the thermotolerance during chronic heating with temperatures of 40-41.5 degrees C were similar. Survival increased rapidly to a maxima by approximately 120 min of hyperthermia (temperatures of 40-41.5 degrees C) and thereafter decreased with a slope similar to the controls. Normalization for cell killing by chronic hyperthermia that occurred during "step-up' heating permitted analysis of thermotolerance in the surviving cells. The surviving fraction after 15 min at 44 degrees C, during incubation at 40, 41 and 41.5 degrees C increased from 0.13 to maxima of 0.56 +/- 0.04, 0.71 +/- 0.03 and 0.82 +/- 0.03 respectively, by 150 min and did not decrease for up to 480 min during chronic hyperthermia. The surviving fraction after 15 min at 44 degrees C during incubation at 42 degrees C increased more slowly than during incubations at 40-41.5 degrees C. The survival of thermotolerant cells after exposure to 15 min at 44 degrees C during 42 degrees C chronic hyperthermia was maximal at 0.87 +/- 0.08 by 120 min and then decreased after approximately 150 min of exposure to 42 degrees C. The thermotolerance ratios (TTR's) were 4.0, 5.4, 6.7 and 6.9 for temperatures of 40, 41, 41.5 and 42 degrees C respectively. The results suggest that chronic hyperthermia temperatures (i.e. 40-42 degrees C) induce rapid thermotolerance development in CFU-GM during the thermal exposure and protect this normal marrow progenitor during whole body hyperthermia or ex vivo purging of leukaemic cells.

Fluorescent measurements of intracellular H(+) and Na(+) are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength "windows" in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na(+)]i with excellent linearity over the range 2-150 mM and with an accuracy of approximately 5 mM.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Previous NMR studies demonstrated that lonidamine (LND) selectively diminishes the intracellular pH (pHi) of DB-1 melanoma and mouse xenografts of a variety of other prevalent human cancers while decreasing their bioenergetic status (tumor βNTP/Pi ratio) and enhancing the activities of melphalan and doxorubicin in these cancer models. Since melphalan and doxorubicin are highly toxic agents, we have examined three other nitrogen (N)-mustards, chlorambucil, cyclophosphamide and bendamustine, to determine if they exhibit similar potentiation by LND. As single agents LND, melphalan and these N-mustards exhibited the following activities in DB-1 melanoma xenografts; LND: 100% tumor surviving fraction (SF); chlorambucil: 100% SF; cyclophosphamide: 100% SF; bendamustine: 79% SF; melphalan: 41% SF. When combined with LND administered 40 min prior to administration of the N-mustard (to maximize intracellular acidification) the following responses were obtained; chlorambucil: 62% SF; cyclophosphamide: 42% SF; bendamustine: 36% SF; melphalan: 10% SF. The effect of LND on the activities of these N-mustards is generally attributed to acid stabilization of the aziridinium active intermediate, acid inhibition of glutathione-S-transferase, which acts as a scavenger of aziridinium, and acid inhibition of DNA repair by O6-alkyltransferase. Depletion of ATP by LND may also decrease multidrug resistance and increase tumor response. At similar maximum tolerated doses, our data indicate that melphalan is the most effective N-mustard in combination with LND when treating DB-1 melanoma in mice, but the choice of N-mustard for coadministration with LND will also depend on the relative toxicities of these agents, and remains to be determined.

Extracellular pH (pHc) was determined by needle microelectrodes in 67 tumour nodules in 58 patients. The objective was to evaluate the relationship between pHe, tumour histology and tumour volume. The mean age of the patients was 62 years, mean depth of the lesions was 2.7 +/- 0.2 cm, and mean tumour volume was 187 +/- 60 cm3. Lesions were located in readily accessible areas such as on the limbs, neck or chest wall. Tumour histologies included: 48% adenocarcinoma; 34% squamous cell carcinoma; 8% soft tissue sarcoma; and 10% malignant melanoma. The mean tumour pHe for the entire group of tumours was 7.06 +/- 0.05 (range 5.66-7.78). Variation in pHe measurements between tumours was greater than the variation in measurements within tumour (F = 7.11, p < 0.01). In adenocarcinomas pHe was 6.93 +/- 0.08 (range 5.66-7.78), in soft tissue sarcomas 7.01 +/- 0.21 (6.25-7.45), in squamous cell carcinomas 7.16 +/- 0.08 (6.2-7.6), and in malignant melanomas 7.36 +/- 0.1 (6.98-7.77). Tumour pHe was significantly different between the four histological groups (p < 0.001). When adenocarcinoma and soft tissue sarcoma lesions were grouped together, pHe was 6.94 +/- 0.08 compared with 7.20 +/- 0.07 in squamous cell carcinomas and malignant melanomas lesions (p < 0.01). Tumour pHe increased as a function of the logarithm of tumour volume at 0.07 +/- 0.02 pH unit/ln cm3 (p = 0.006, r = 0.34). In conclusion, tumour histology and tumour volume were the most important factors determining the range of pHe's.(ABSTRACT TRUNCATED AT 250 WORDS)

A network model has been developed for analysis of tumor glucose metabolism from 13C MRS isotope exchange kinetic data. Data were obtained from DB1 melanoma cells grown on polystyrene microcarrier beads contained in a 20-mm diameter perfusion chamber in a 9.4 T Varian NMR spectrometer; the cells were perfused with 26 mM [1,6-13C2]glucose under normoxic conditions and 37°C and monitored by 13C NMR spectroscopy for 6 h. The model consists of ∼150 differential equations in the cumomer formalism describing glucose and lactate transport, glycolysis, TCA cycle, pyruvate cycling, the pentose shunt, lactate dehydrogenase, the malate-aspartate and glycerophosphate shuttles, and various anaplerotic pathways. The rate of oxygen consumption (CMRO2) was measured polarographically by monitoring differences in pO2. The model was validated by excellent agreement between model predicted and experimentally measured values of CMRO2 and glutamate pool size. Assuming a P/O ratio of 2.5 for NADH and 1.5 for FADH2, ATP production was estimated as 46% glycolytic and 54% mitochondrial based on average values of CMRO2 and glycolytic flux (two experiments).

Tumor extracellular pH measurements in 26 human tumors were evaluated for the purpose of prognostic indication of response to thermoradiotherapy.Twenty-six patients (10 male, 16 female; mean age 62 years, range 18-89) were treated with external microwave hyperthermia (915 MHz) combined with radiation therapy. Tumor histologies included: 46% adenocarcinoma, 38% squamous cell carcinoma, 12% soft tissue sarcoma, and 4% malignant melanoma. The mean tumor depth was 1.6 +/- 0.2 cm (range 0.4-3 cm) and the mean tumor volume was 73 +/- 11 cm3 (range 1-192 cm3). The mean radiation dose administered concurrently with hyperthermia was 39 +/- 1 Gy (range 24-60 Gy, median of 40 Gy), in 15 fractions (range 8-25), over 32 elapsed days (range 15-43). The mean number of hyperthermia sessions administered was 5.4 +/- 0.5 (range 2-10). A battery operated pH meter and combination 21 ga recessed glass, beveled needle microelectrodes were used for tumor pH measurements. Calibration in pH buffers was performed before and after each pH measurement. The needle microelectrodes were 2.5 cm in length.A complete response (CR) was obtained in 20 of 26 patients (77%) and a partial response in six (23%). The mean extracellular tumor pH was 6.88 +/- 0.09 in CR patients while it was 7.24 +/- 0.09 in noncompletely responding (NCR) patients (p = 0.08). Logistic regression analysis indicated that the probability of obtaining a complete response was influenced by the tumor volume (p = 0.02), tumor depth (p = 0.05), and extracellular tumor pH (p = 0.08). Lesions in the pH range of 6.00-6.40 and lesions in the pH range of 6.41-6.80 exhibited a CR rate of 100%, while those lesions in the pH range of 6.81-7.20 exhibited a CR of 90% and those in the pH range of 7.21-7.52 exhibited a CR of 50% (p = 0.002). In lesions with depthor = 1.5 cm, the CR rate was 100% when the tumor pH was7.15 and 75% when the tumor pH wasor = 7.15. In lesions with depth between 1.5 and 3 cm, the CR rate was 66% when the tumor pH was7.15 and 43% when the tumor pH wasor = 7.15 (p = 0.02). In small tumors, that is,or = 20 cm3, tumor pH increased with volume, whereas in larger tumors, that is,20 cm3, tumor pH decreased as a function of tumor volume.Tumor extracellular pH may be useful as a prognostic indicator of tumor response to thermoradiotherapy.

Extensive recurrences on the chest wall of advanced carcinoma of the breast in 20 patients were treated with multiple field patchwork hyperthermia combined with radiation therapy between 1987-1991. The objective of the study was to evaluate the feasibility, tumour response and complications of treating extensive lesions with multiple, overlapping fields of hyperthermia. All lesions were diffuse encompassing up to 2900 cm2 in area with or without multiple nodulesor = 3 cm deep. All lesions had failed previous therapy with all but three failing previous radiotherapy. Hyperthermia consisted of 282 hyperthermia applicator fields and 357 hyperthermia treatments with external 915 MHz microwaves using commercially available applicators. Hyperthermia applicator fields were defined by the surface 50% SAR distribution of a particular applicator, and hyperthermia fields were abutted to cover the entire tumour bearing area. Radiation therapy consisted of 81 fields to a mean dose of 40 +/- 1 Gy (SE), 88% of fields received between 30 and 50 Gy. The equivalent dose was 42 +/- 1 Gy, based on the linear-quadratic model and alpha/beta = 25 (Fowler 1989). Overlapping hyperthermia fields were separated by an interval of at least three days. Up to four heat sessions per week were required to cover the entire tumour in a rotating fashion. The hyperthermia treatment time was 60 min. Hyperthermia treatments were continued for the duration of radiation therapy. Each hyperthermia applicator field was heated at least once. Patients were exposed to a mean of 14 +/- 3 hyperthermia applicator fields (range of 3-46 fields) and a mean of 18 +/- 3 hyperthermia treatments (range of 6-61) delivered over a mean of 7.5 +/- 0.9 weeks (range of 3-17 weeks). Each field was heated an average of 1.3 times. The tumour complete response rate was 95% with a recurrence rate of 5%. Nevertheless, the mean survival of patients with a complete response was only 10.8 +/- 1.7 months (range of 2-28 months) because of the systemic tumour burden existing outside of the treated fields in these patients. Neither complete response, local control nor survival after thermoradiotherapy correlated with the disease free interval between initial mastectomy and recurrence. There was no evidence of increased thermal damage to skin nor evidence of tumour recurrence at junctions of hyperthermia field overlap. It is concluded that recurrent advanced carcinoma of the breast presenting as extensive, diffuse lesions on the chest wall can be treated as effectively with multiple field patchwork thermoradiotherapy as can nodular lesions treated with single hyperthermia fields.

Previously we reported that three imaging methods, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), T(1ρ)-MRI, and ultralow temperature NADH/flavoprotein fluorescence imaging (redox scanning), could differentiate the less metastatic human melanoma cell line A375P from a more metastatic line C8161 growing as mouse xenografts in nude mice (Li LZ et al. Adv. Exp. Med. Biol., 2007, 599:67–78; PNAS, 2009, 106:6608–6613). The more metastatic C8161 tumor was characterized by less blood perfusion/permeability, a more oxidized mitochondrial redox state in the tumor core, and a smaller T(1ρ) relaxation time constant averaged across the entire tumor section. In the current study, we have further probed the bioenergetic status and tissue microenvironment of these tumors by applying whole tumor phosphorous magnetic resonance spectroscopy ((31)P-MRS) to these two xenografts in a vertical bore 9.4-T Varian magnet. The phosphomonoester (PME)/βNTP ratio and intracellular pH value (pH(i)) were determined. The phosphomonoester (PME)/βNTP was higher in the more metastatic C8161 tumors (n=4) than in the less metastatic A375P tumors (n=4) (p < 0.1). No significant difference between the pH(i) of C8161 and A375P was observed.

Previously we reported that three imaging methods, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), T1ρ-MRI, and ultralow temperature NADH/flavoprotein fluorescence imaging (redox scanning), could differentiate the less metastatic human melanoma cell line A375P from a more metastatic line C8161 growing as mouse xenografts in nude mice (Li LZ et al. Adv. Exp. Med. Biol., 2007, 599:67-78; PNAS, 2009, 106:6608-6613). The more metastatic C8161 tumor was characterized by less blood perfusion/permeability, a more oxidized mitochondrial redox state in the tumor core, and a smaller T1ρ relaxation time constant averaged across the entire tumor section. In the current study, we have further probed the bioenergetic status and tissue microenvironment of these tumors by applying whole tumor phosphorous magnetic resonance spectroscopy (31P-MRS) to these two xenografts in a vertical bore 9.4-T Varian magnet. The phosphomonoester (PME)/βNTP ratio and intracellular pH value (pHi) were determined. The phosphomonoester (PME)/βNTP was higher in the more metastatic C8161 tumors (n=4) than in the less metastatic A375P tumors (n=4) (p < 0.1). No significant difference between the pHi of C8161 and A375P was observed.