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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.

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 (>0.2°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.

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.

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

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.

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.

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.

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 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)

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.

Advanced melanoma (48 lesions in 40 patients) was treated with external microwave hyperthermia combined with radiation therapy between 1980-1988. Thirty-three lesions in 28 patients were evaluable for tumor response (mean age 64 years, 19 male, 9 female). Evaluable lesions received 13 to 66 Gy (mean 37 +/- 2 Gy) over 5 to 16 fractions (mean of 10) in 14 to 56 elapsed days (mean of 25). Tumor volume (pi/6*length*width*depth) was 62 +/- 16 cm3 (1-377 cm3). Hyperthermia was administered in 6.6 +/- 0.4 sessions (range 1-14), there were 3.2 +/- 0.4 thermal sensors per tumor (range 1-11) and 27 fields were treated twice-weekly (82%). Of the 33 evaluable lesions, 12 exhibited a complete response (36%), and 17 had a partial response (52%). Among the 12 complete responders four recurrences (33%) were observed at 8.6 +/- 1.4 months (median of 8.2 months). In superficial tumors with depthor = 3 cm and with lateral dimensions within 2 cm of the boundaries of the microwave applicator, the complete response rate was 50% (11/22); whereas for patients with deeper tumors with depth3 cm, the complete response rate was 9% (1/11), p = 0.02. The minimal tumor thermal dose during the first hyperthermia treatment session correlated with response (t43min1 = 20 +/- 7 vs. 6 +/- 3 minEq43 degrees C for complete responders and noncomplete responders, respectively, p = 0.06); and 7 of 10 lesions that had t43min 1or = 8 minEq43 degrees C achieved a complete response whereas only 5 of 22 lesions (23%) that had t43min18 minEq43 degrees C did so (p = 0.01). However, neither the minimum tumor temperature during the first treatment, the median minimum tumor temperature over all treatment sessions nor the sum of minimum thermal dose over all treatment sessions correlated with tumor response. Twenty-three patients with 28 lesions died during follow-up (82%). The survival for complete responding patients with superficial lesions was 21.3 +/- 1.5 months compared to 4.5 +/- 0.5 months for patients with superficial lesions that did not experience a complete response (p = 0.0001). For patients with noncomplete responding lesions deeper than 3 cm survival was 4.4 +/- 0.6 months. Twenty lesions were treated without any skin reaction (42%, 20/48). Of the rest, 23 had erythema (48%, 23/48), seven had blistering (14%, 7/48) and one had ulceration of the skin.(ABSTRACT TRUNCATED AT 400 WORDS)

In a prospective study CT scanning was used to evaluate the precision of thermometry catheter placement in tumours in the head and neck or on the chest wall in 30 consecutive patients prior to hyperthermia treatment. Patients had variable-sized tumours from several primary sites. Thermometry catheter placement was guided by palpation with or without a prior CT scan. Catheter placement was confirmed by CT. All lesions were less than 8 x 8 x 6 cm (L x W x D) in size. A mean of 4.2 +/- 0.2 (+/- 1 SEM, range 2-7) closed-end polyurethane catheters were inserted orthogonally by the same experienced radiation oncologist. Horizontal thermometry catheters were intended to traverse the centre and base of the tumour mass, and a vertical catheter was often inserted to intersect a horizontal catheter. After catheter placement, wire cables with 1 cm spacings were inserted into the catheters and positions determined using orthogonal films and CT scans. The success of catheter placement was judged on the following criteria: (1) catheter distribution factor (CDF = proportion of tumour CT slices transected by at least one catheter); (2) catheter hit ratio (CHR = average number of catheters in tumour per CT slice); (3) catheter miss factor (CMF = average number of catheters out of tumour per CT slice); (4) catheter placement index, CPI = [(CHR)(CDF)]-CMF; and (5) distance of nearest catheter from the visually estimated centre of tumour in the most central tumour CT scan. In the first seven lesions with 3-6 cm depth catheter insertion was guided by palpation only. In the next 23 lesions catheter insertion was guided by a prior CT scan. In the latter group, 15 lesions had depth 3-6 cm while eight lesions had depthor = 3 cm. Catheter placement by palpation only, without the benefit of CT scan, was much less accurate in terms of the nearest catheter to the centre of the tumour (p = .001), the proportion of CT slices with catheter in tumour (CDF, p = 0.04) and the probability of a catheter being outside the tumour (CMF, p = 0.01). The catheter placement index (CPI) was a good measure of the accuracy and adequacy of catheter placement in large tumours (p = 0.04). Displacement of normal tissue structures by tumour precluded accurate catheter placement and led to a low CPI. It was difficult to accurately instrument lesionsor = 3 cm depth even with the benefit of a prior CT scan.(ABSTRACT TRUNCATED AT 400 WORDS)

Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T 1 ρ -MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutter-speed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, K trans , that measures vascular perfusion/permeability. K trans was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T 1 ρ values that were significantly higher than those of C8161 melanoma. Measurements of T 1 and T 2 relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp + NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential.

Accurate prediction of tumor metastatic potential would be helpful in treatment planning and in the design of agents that modify the tumor phenotype. We report that three methods that are potentially transferable to the clinic – dynamic contrast enhanced MRI (DCE MRI), T(1ρ)-weighted imaging and low temperature fluorescence imaging (that could be performed on biopsy specimens) – distinguished between relatively indolent (A375P) and aggressive (C8161) metastatic human melanoma xenografts in nude mice, whereas T(1) and T(2) relaxation time measurements did not. DCE MRI data analyzed by the BOLus Enhanced Relaxation Overview (BOLERO) method in conjunction with concurrent measurements of the arterial input function yielded a blood transfer rate constant (K(trans)) which measures perfusion/permeability, that was significantly higher in the core of the indolent tumor than in the core of the aggressive tumor. Histological staining indicated that aggressive tumors had more blood vascular structure but fewer functional vascular structure than indolent tumors. Indolent tumors exhibited T(1ρ) values that were significantly higher than those of aggressive tumors at spin-locking frequencies >500Hz. The mitochondrial redox ratio, Fp/(Fp+NADH), where Fp and NADH are the fluorescence of oxidized flavoproteins and reduced pyridine nucleotides, respectively, of aggressive tumors was much higher (more oxidized) than that of indolent tumors and often showed a bimodal distribution with an oxidized core and a reduced rim. These differences observed between these two types of tumors, one indolent and one aggressive, if generalizable, would be very valuable in predicting human melanoma metastatic potential.

Background: Perfusate acidification with dilute hydrochloric acid augments tumor response rates in a rodent model of isolated limb perfusion (ILP). This study investigates the combination of metaiodobenzylguanidine (MIBG), a mitochondrial inhibitor, and systemic hyperglycemia as a strategy to selectively acidify tumors and thereby sensitize them to ILP. Methods: Human melanoma xenografts were implanted into the hind limbs of athymic rats. When tumors reached 12 to 15 mm in diameter, animals were randomized to ILP with or without melphalan, with or without systemic MIBG, and hyperglycemia of 485 ± 35 mg/dL. Intratumoral pH was measured during MIBG and glucose treatment by using magnetic resonance spectroscopy. Results: MIBG at 30 mg/kg plus hyperglycemia decreased intracellular pH by .6 units and extracellular pH by .8 units. MIBG at 22.5 mg/kg plus hyperglycemia decreased intracellular and extracellular pH by .4 and .5 units, respectively. Tumor growth was unaffected by systemic MIBG and hyperglycemia alone. When MIBG at 30 mg/kg and hyperglycemia were combined with ILP, tumor growth was delayed for 33 days after control ILP and for 44 days after melphalan ILP. However, this dose of MIBG was complicated by a 40% mortality rate after ILP. MIBG at 22.5 mg/kg, in combination with MIBG in the perfusate, did not cause mortality and delayed tumor growth by 51 days after melphalan ILP. Conclusions: MIBG and hyperglycemia improve tumor response rates after ILP in a rodent model of human melanoma. Selective tumor acidification with MIBG and hyperglycemia may offer added benefit to current regional perfusion strategies.

The effects of glucose or meta-iodo-benzylguanidine (MIBG) on oxygen utilization (QO2) of several tumor cell lines were studied using a Clark-type electrode chamber. For in vivo studies, rats bearing R3230 Ac rat mammary adenocarcinomas were utilized. To evaluate changes in tumor oxygenation induced by glucose or MIBG, intratumoral pO2 and skeletal muscle pO2 were measured using Eppendorf Histography. To find the effect of mild hyperglycemia (i.p., 1 g/kg) +/- MIBG (i.p., 20 mg/kg) on the radiation response, a growth delay assay was used. Glucose alone produced a approximately 20% inhibition of QO2 in several tumor cells we tested except Q7 tumor cells. MIBG inhibited QO2 in R3230 Ac tumors. The median tumor pO2 for glucose + MIBG was increased from 5.3 mm Hg to 13.8 mm Hg. We hypothesized that combined treatment with glucose + MIBG significantly enhanced radiation-induced tumoricidal effects on R3230 Ac tumors, mainly due to reduction in QO2 and increase in tumor pO2.

Tumor oxygen tension and extracellular pH (pH(e)) are physiological parameters that can be manipulated to improve current cancer therapies. Many human tumors consist of cells that are chronically exposed to low pH(e). Exposure of tumor cells in culture to glucose decreases oxygen consumption (oxygen sparing or Crabtree effect), and while this effect is absent in low pH-adapted tumor cells, it can be restored by combining the respiratory inhibitor meta-iodo-benzylguanidine (MIBG) with glucose (Burd et al., Cancer Res. 61, 5630-5635, 2001). The effects of hyperglycemia and MIBG on tumor oxygen tension and on pH(e) were investigated in human melanoma xenografts in SCID mice. An oral gavage of 1 M glucose (2 g/kg) increased the average blood glucose concentration from140 mg/dl to approximately 400 mg/dl. Although tumor pH(e) decreased from pH 6.7 to pH 6.5 (P0.01) after about 60 min, no change in tumor oxygen tension was observed. However, when oral glucose and MIBG (15 mg/kg) were administered together, oxygen tension increased from 2.8 mmHg to approximately 17 mmHg, and tumor pH(e) decreased from pH 6.7 to pH 6.3 (P0.01) after about 115 min. In conclusion, administration of glucose together with MIBG increases tumor oxygen tension and also increases the magnitude and duration of acidification. Hyperglycemia plus MIBG has the potential to improve response to radiation therapy as well as to hyperthermia and some chemotherapies.

Melanoma cells in vivo maintain intracellular pH (pHi) in a viable range despite an extracellular tumor pH (pHe) that is typically below 7.0. In general, three families of transporters are capable of removing metabolic protons, but the specific transporters responsible for the maintenance of pHi at low pHe in melanomas have not been identified. Although the transporters exist in most cells, an inhibitor would be predicted to have selectivity for cells located in an acidic tumor bed because cells in that environment would be expected to have transporters chronically activated. In this report, the levels and extent of expression of the Na+/H+ exchanger (NHE-1) and two of the H+-linked monocarboxylate transporters (MCTs) were evaluated in three melanoma cell lines. The effects of inhibitors of each transporter were tested at an extracellular pH (pHe) of 7.3, 6.7, or 6.5 in melanoma cells that were grown at pHe 7.3 or 6.7. The activity of MCT isoform 1 (MCT-1) was up-regulated in three melanoma cell lines at low pHe, but that of NHE-1 was not. Furthermore, NHE-1 activity was lower in the melanomas than in other normal and malignant cell lines that were tested. Reverse transcription-PCR using primers specific for MCT-1, MCT-4, and NHE-1 showed that expression of none of these transporters was reproducibly up-regulated at the level of transcription when cells were grown at pHe 6.7 instead of pHe 7.3. Ex vivo experiments using DB-1 human melanoma xenografts grown in severe combined immunodeficient mice found that MCT-1 and not NHE-1 was a major determinant of DB-1 tumor cell pHi. Taken together, the data indicate that MCTs are major determinants of pH regulation in melanoma. In contrast, keratinocytes and melanocytes under low pHe conditions relied on NHE-1. Inhibitors of MCTs thus have great potential to improve the effectiveness of chemotherapeutic drugs that work best at low pHi, such as alkylating agents and platinum-containing compounds, and they should be selective for cells in an acidic tumor bed. In most tissues, it is proposed that the NHE-1 could compensate for an inhibited MCT to prevent acidification, but in melanoma cells this did not occur. Therefore, MCT inhibitors may be particularly effective against malignant melanoma.

To identify in a multivariate analysis treatment-related factors predisposing patients (pts) with lung cancer to acute esophagitis, expressed as a severity grade or Esophagitis Index (EI).Acute esophagitis is prospectively scored as an RTOG Grade in our institution during and after thoracic radiotherapy. Charts, toxicity forms and digitally reconstructed radiographs (DRRs) of all pts with lung cancer who received thoracic radiotherapy (RT) between 11/95 and 1/99 were reviewed. Esophagitis grades for each time point were verified by review of weekly physician and nursing treatment notes, hospital discharge summaries and referring physician notes and then plotted on graph against time. The area under the curve was calculated for each patient's graph and was defined as an Esophagitis Index. The length of esophagus was measured on each anterior DRR while assuming that esophagus overlies the vertebral bodies on the anterior films and projects over the edge of the vertebral body on the oblique DRRs. This assumption was confirmed in 10 pts by digitizing esophagus on CT simulator-derived slices and visualizing its position on DRRs. To compare RT doses delivered with different fractionation schemes to standard fractionated doses, the equivalent RT doses were calculated using the linear-quadratic formula and alpha/beta ratio of 10. Univariate and multivariate analyses of several factors potentially influencing the maximum esophagitis grade, as well as EI, were performed.A total of 277 pts were identified. Pts were included in the analysis (n = 105) if they fulfilled the following criteria: chart, toxicity form and DRRs were all available; parallel opposed fields (no multiple fields) were used for both the initial and off cord/cone down fields; and an equivalent dose of 45.0 Gy or more was delivered. Seventy-eight pts had Stage III; 32, Stage IV, and the remainder, Stages I, II, or recurrent lung cancer (85 non-small cell and 18, small cell). Seventy-four pts were treated with definitive intent. Chemotherapy was given concurrently with RT in 58 pts (in 7 pts, with twice daily, or b.i.d., RT) and as induction treatment, in 11. Only 2 pts required a treatment break of more than 1 week. Median total and equivalent RT doses, fraction size, and anterior esophageal length were as follows: 59.9 Gy, 59.9 Gy, 2.0 Gy, and 14 cm (range, 4.2-21). The following maximum grades of esophagitis were recorded: 1, in 54 pts; 2, in 17 pts; 3, in 13 pts, and 4, in 1 pt. The mean EI for all pts; pts treated with standard RT alone; induction chemotherapy and standard RT; concurrent chemotherapy and standard (QD) RT; and b.i.d. RT with concurrent chemotherapy, was 41. 5 (range, 0-317); 13.6; 24.5; 52.4; and 132.1, respectively (p0. 001). Three pts developed an esophageal stricture within 3 months beginning RT. In multivariate analysis, the following factors were significantly associated with increasing EI: concurrent chemotherapy with QD RT and concurrent chemotherapy with b.i.d. RT (p0.001, considered jointly). Both factors were also associated with increasing maximum esophagitis grade (p = 0.011). Esophageal length was not associated with increasing EI or esophagitis grade in either univariate or multivariate analyses.Concurrent chemotherapy and twice daily radiotherapy, especially if combined together, were associated with the highest acute maximum esophagitis grade and esophagitis index in pts with lung cancer. The duration of acute esophagitis was also longest in the concurrent chemotherapy/twice daily radiotherapy group. Esophagitis Index appeared to be a more sensitive measure of acute esophagitis than the maximum esophagitis grade. The increasing length of esophagus in the radiation field did not predict for the severity of acute esophagitis.

In this study we performed univariate analyses to analyse the predictive factors for skin reactions, i.e. erythema, thermal blisters and ulceration, that occur during thermoradiotherapy. One hundred and twenty-six fields in 126 patients were treated with thermoradiotherapy using 915 MHz external microwave hyperthermia. Mean age of patients was 62 years. All but 11 lesions received previous therapy. Prior treatment included surgery (75%), chemotherapy (60%) and/or radiation therapy (51%). The mean previous radiation dose was 54 +/- 2 Gy. The concurrent tumour radiation dose was 45 +/- 1 Gy, in 16 fractions, over 35 elapsed days (dose per fraction of 1.6-4.8 Gy). The mean number of heat sessions administered was 5.5 +/- 0.2 (range 1-14). In 83% of cases hyperthermia was administered biweekly. Forty-two patients were treated without any skin reaction (33%), erythema occurred in 59 fields (47%), transient thermal blisters occurred in 25 fields (20%) and ulceration occurred in 23 fields (18%). In 25 cases, two or more skin reactions (20%) were observed concurrently. Concurrent radiation dose correlated with skin reactions (p = 0.02). The incidence of skin reactions was inversely correlated with previous radiation therapy (p = 0.04) and previous radiation therapy dose (p = 0.04) possibly due to fibrosis. None of the tumour or skin thermal parameters correlated with the reaction rate.

Chest wall lesions of advanced breast carcinoma in 23 patients were treated with thermoradiotherapy with clinical intent between January 1987 and March 1992. Treatment consisted of external 915 MHz microwave hyperthermia with commercially available applicators and radiation therapy to doses between 32-58 Gy. Twenty-three large, diffuse lesions were treated with multiple field patchwork hyperthermia. All lesions were diffuse with or without multiple nodulesor = 3 cm depth. All lesions had failed previous therapy. The mean number of hyperthermia fields per patient was 3.2 +/- 0.4 (range of 2-7). The complete response rate was 91% in this group of extensive, diffuse lesions treated by the patchwork technique. Mean total radiation dose administered concurrently with multiple field patchwork hyperthermia was 42 +/- 1 Gy. The recurrence rate was 5%. The mean survival in patients who had a complete response was 9.0 +/- 1.3 months. The reduced survival among patchwork treated patients was due to the extensive tumor burden existing outside of the treated fields in these patients. The skin reactions were minor, causing minimal discomfort. There was no evidence of increased thermal damage to skin, or of tumor recurrence at junctions of hyperthermia field overlap. It is concluded that extensive, diffuse lesions of chest wall recurrence of advanced carcinoma of the breast can be treated effectively with multiple field patchwork thermotherapy.

Advanced tumours in the head and neck 3-6 cm depth are too deep to be completely heated by external 915 MHz microwaves. A preliminary study was performed using interstitial plus external hyperthermia combined with external beam radiation therapy to heat tumours to depthsor = 3 cm. Nine advanced metastatic lesions of squamous cell carcinoma located in the head and neck were treated between 1987 and 1990 with the combined hyperthermia technique and radiation doses of 38-60 Gy (mean of 49 +/- 3 Gy). The mean tumour volume was 58 +/- 9 (SE) cm3 (range 24-94 cm3) with a mean tumour depth of 3.9 +/- 0.3 cm (range 3-5.5 cm). The deeper aspects of the tumour were heated by interstitial 915 MHz microwave antennas and the superficial aspects heated by external 915 MHz applicators. A single plane of polyurethane closed-end catheters, 16 Ga, were inserted under local anaesthesia approximately 1.5-2 cm apart in parallel arrays at the base of a lesion behind the sternomastoid muscle, or an equivalent site in a dissected neck, extending forward and angled deeply no more than 15 degrees. Hyperthermia was administered twice weekly immediately after radiation therapy in a mean of 5.3 +/- 0.7 external heat sessions (range 3-7) and a mean of 3.5 +/- 0.6 interstitial heat sessions (range of 1-6). Interstitial hyperthermia was usually administered in alternating sessions with external hyperthermia, but in some patients all of the sessions of one modality were administered followed by all of the sessions of the other modality. In no case were both interstitial and external heatings performed on the same day. Surface thermometers were used to monitor skin temperature during external hyperthermia sessions. Results showed that by 8 weeks after completion of treatment, six lesions exhibited a complete response (67%) and three a partial response (33%). One of the partial responses continued to regress and became a complete response (78% complete response). The recurrence rate in complete responders was 14% (1/7) with time to recurrence of 7.7 months. Six lesions were recurrence-free at last follow-up of 21.3 +/- 8.8 months. Skin reactions were absent in four fields (44%), erythema was noted in five (56%) and thermal blistering in one (11%). Ulceration occurred only in association with tumour breakdown when the skin was infiltrated by tumour (three patients, 33%).(ABSTRACT TRUNCATED AT 400 WORDS)

One test for thermotolerance development in a clinical situation is to evaluate the effects of altering the hyperthermia fractionation interval on tumour response to thermoradiotherapy. Between 1983 and 1990 44 evaluable advanced superficial tumours of miscellaneous origin in 41 patients were randomized to receive either once-weekly or twice-weekly external microwave hyperthermia treatments combined with radiation therapy. The mean age of patients was 62 years, and 85% had failed previous therapy. All lesions were less than 8 x 8 x 4 cm (L x W x D) and were heated by external 915 MHz microwaves. The mean radiation dose was 44 +/- 3 Gy (mean +/- SE) in the once-weekly group and 46 +/- 3 Gy in the twice-weekly group (p = 0.64). The mean volume of the lesions heated once weekly was 17 +/- 6 versus 23 +/- 5 cm3 for those heated twice weekly (p = 0.45). Hyperthermia was administered once weekly for 4.6 +/- 0.2 sessions (range 3-7) or twice weekly for 8.1 +/- 0.3 sessions (range 4-10). Thermometry was performed using 3.4 +/- 0.2 catheters and 5.1 +/- 0.6 thermal sensors per tumour in the once-weekly group, and 2.7 +/- 0.2 catheters and 5.8 +/- 0.3 thermal sensors per tumour in the twice-weekly group. Of the 44 evaluable randomized lesions a complete response (CR) at 2 months post-treatment was observed in 59% (13/22) heated once weekly and 55% (12/22) in those heated twice weekly. The prognostic factors predictive of tumour complete response were found by logistic regression analysis to be radiation dose and tumour volume, while the prognostic factors predictive of duration of response (Cox proportional hazards analysis) were median minimum tumour temperature (Tmin), minimum tumour temperature during the first heat treatment (Tmin1) and tumour volume. The duration of local control in lesions with Tminor = 39.5 degrees C was 11.7 +/- 1.9 months while for lesions with Tmin39.5 degrees C it was 23.0 +/- 4.2 months (p = 0.01). The ED50 was calculated by logistic regression to be 40 Gy (95% CI = 22-54 Gy) for once- and twice-weekly heated lesions. There was not a significant difference in tumour response or duration of response between populations randomized to receive once- versus twice-weekly hyperthermia treatments. There was also no difference in skin reaction rates between once- and twice-weekly hyperthermia treatments, nor could a correlation be found between any thermal parameter and skin reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

M. W. DEWHIRST, D.V.M., PH.D.,* T. L. PHILLIPS, M.D.,+ T. V. SAMULSKI, PH.D.,+ P. STAUFFER, MSEE,? P. SHRIVASTAVA, PH.D.,+ B. PALIWAL, PH.D.,+ T. PAJAK, PH.D.,+ M. GILLIM, PH.D.,+ M. SAPOZINK, M.D., PH.D.,+ R. MYERSON, M.D., PH.D.,+ F. M. WATERMAN, PH.D.,+ S. A. SAPARETO, PH.D.,+ P. CORRY, PH.D.,+ T. C. CETAS, PH.D.,+ D. B. LEEPER, PH.D.,+ P. FESSENDEN, PH.D.,+ D. KAPP, M.D., PH.D.,+ J. R. OLESON, M.D., PH.D.+ AND B. EMAMI, M.D.*

The effect of heat on blood flow in human tumors was studied as a function of time during 1 hour of local hyperthermia induced by 915 MHz microwaves. Blood flow was determined from the rate of thermal clearance by use of the bio-heat transfer equation. The rate of thermal clearance was measured at intervals of approximately 10 minutes throughout the treatment session by turning off the microwave power for 50 seconds. Tumor blood flow increased by amounts varying from 15 to 250% during the first 20–50 minutes of heating at 41–45°C, after which it remained relatively constant during the remainder of the treatment session. The sharp reduction in blood flow or vascular stasis reported in most transplantable rodent tumors after comparable heating was not observed in human tumors. The maximum blood flow observed in heated human tumors ranged from 10–40 ml/min/100 gm. The systematic error due to thermal conduction was estimated to be equivalent to a blood flow of less than 3 m/min/100 gm.

The effectiveness of hyperthermia in tumor therapy may depend on a lower extracellular pH of tumor compared to that of normal tissue. A technique for measuring extracellular pH in human tumors has been devised to test the usefulness of this parameter as prognostic indicator of tumor hyperthermia response. In a preliminary study 50 of 53 pH readings from 14 human tumors (both heated and unheated) were below normal physiological pH. Tumor pH values ranged between 5.55-7.69 (average for unheated tumors 6.81 +/- 0.09, SEM, only one determination was above 7.40). Although there was considerable heterogeneity of pH within tumors, the accuracy and drift of the 21 gauge needle electrode were not a problem. Fifteen minutes were required for pH stabilization after insertion of an 18 gauge open-ended catheter, and less than 5 min for equilibration after electrode insertion into the catheter. A saline wheal was used for anesthesia to preclude modification of pH by anesthetics. Central portions of tumors were no more acidic than peripheral regions, but large tumors tended to be more acidic than small tumors. The pH of several tumors of various sizes and histologies was also determined immediately before subsequent treatment sessions. These measurements were made by reinsertion of catheters in approximately the same locations at each session. The trend appeared to be that pH increased with the number of treatment sessions. Measurements of pH were made in four patients immediately prior to and at the termination of a heating session (same locations since catheter remained in place during heating sessions). Three of the four tumors showed increased pH readings of 0.25-0.54 units during heating. However, none of the four tumors achieved temperatures exceeding 42 degrees C. The pH measurement technique developed provides a safe and relatively easy method for determining extracellular pH in human tumors. There appears to be a correlation of pH values with tumor size, treatment session, and possibly blood flow.