A patterns of care analysis of hyperthermia in combination with radio(chemo)therapy or chemotherapy in European clinical centers.

PURPOSE: The combination of hyperthermia (HT) with radio(chemo)therapy or chemotherapy (CT) is an established treatment strategy for specific indications. Its application in routine clinical practice in Europe depends on regulatory and local conditions. We conducted a survey among European clinical centers to determine current practice of HT. METHODS: A questionnaire with 22 questions was sent to 24 European HT centers. The questions were divided into two main categories. The first category assessed how many patients are treated with HT in combination with radio(chemo)therapy or CT for specific indications per year. The second category addressed which hyperthermia parameters are recorded. Analysis was performed using descriptive methods. RESULTS: The response rate was 71% (17/24) and 16 centers were included in this evaluation. Annually, these 16 centers treat approximately 637 patients using HT in combination with radio(chemo)therapy or CT. On average, 34% (range: 3-100%) of patients are treated in clinical study protocols. Temperature readings and the time interval between HT and radio(chemo)therapy or CT are recorded in 13 (81%) and 9 (56%) centers, respectively. The thermal dose quality parameter "cumulative equivalent minutes at 43 °C" (CEM43°C) is only evaluated in five (31%) centers for each HT session. With regard to treatment sequence, 8 (50%) centers administer HT before radio(chemo)therapy and the other 8 in the reverse order. CONCLUSION: There is a significant heterogeneity among European HT centers as to the indications treated and the recording of thermometric parameters. More evidence from clinical studies is necessary to achieve standardization of HT practice.

Using immunotherapy to enhance the response of a C3H mammary carcinoma to proton radiation.

BACKGROUND: The benefit of combining immunotherapy with photon irradiation has been shown pre-clinically and clinically. This current pre-clinical study was designed to investigate the anti-tumour action of combining immunotherapy with protons. MATERIALS AND METHODS: Male CDF1 mice, with a C3H mammary carcinoma inoculated on the right rear foot, were locally irradiated with single radiation doses when tumours reached 200mm3. Radiation was delivered with an 83-107MeV pencil scanning proton beam in the centre of a 3 cm spread out Bragg peak. Following irradiation (day 0), mice were injected intraperitoneal with anti-CTLA-4, anti-PD-1, or anti-PD-L1 (10 mg/kg) twice weekly for two weeks. Endpoints were tumour growth time (TGT3; time to reach 3 times treatment volume) or local tumour control (percent of mice showing tumour control at 90 days). A Student's T-test (tumour growth) or Chi-squared test (tumour control) were used for statistical analysis; significance levels of p < 0.05. RESULTS: Untreated tumours had a mean (± 1 S.E.) TGT3 of 4.6 days (± 0.4). None of the checkpoint inhibitors changed this TGT3. A linear increase in TGT3 was seen with increasing radiation doses (5-20 Gy), reaching 17.2 days (± 0.7) with 20 Gy. Anti-CTLA-4 had no effect on radiation doses up to 15 Gy, but significantly enhanced 20 Gy; the TGT3 being 23.0 days (± 1.3). Higher radiation doses (35-60 Gy) were investigated using a tumour control assay. Logit analysis of the dose response curve, resulted in a TCD50 value (radiation dose causing 50% tumour control; with 95% confidence intervals) of 48 Gy (44-53) for radiation only. This significantly decreased to 43 Gy (38-49) when mice were treated with anti-CTLA-4. Neither anti-PD-1 nor anti-PD-L1 significantly affected tumour control. CONCLUSION: Checkpoint inhibitors enhanced the response of this C3H mammary carcinoma to proton irradiation. However, this enhancement depended on the checkpoint inhibitor and radiation dose.

Does the combination of hyperthermia with low LET (linear energy transfer) radiation induce anti-tumor effects equivalent to those seen with high LET radiation alone?

INTRODUCTION: The combination of hyperthermia with low LET (linear energy transfer) radiation may have similar anti-tumor effects as high LET radiation alone. This pre-clinical study determined the optimal heating temperature and time interval between radiation and heat to achieve this equivalent effect. METHODS: C3H mammary carcinomas (200 mm3 in size) growing in the right rear foot of CDF1 mice was used in all experiments. Tumors were locally irradiated with graded doses of either 240 kV ortho- or 6 MV mega-voltage X-rays to produce full dose-response curves. Heating (41.0-43.5 °C; 60 min) was achieved by immersing the tumor bearing foot in a water-bath applied at the same time, or up to 4-hours after, irradiating. The endpoint was the percentage of mice showing local tumor control at 90 days, with enhancements calculated from the ratios of the radiation doses causing 50% tumor control (± 95% confidence intervals). RESULTS: Previous published results in this tumor model reported that carbon ions were 1.3-1.7 times more effective than low LET radiation at inducing tumor control. Similar enhancements occurred with a temperature of only 41.0 °C with a simultaneous heat and radiation treatment. However, higher temperatures were needed with the introduction of any interval; at 42.5 °C, the enhancement was 2.5 with a simultaneous treatment, decreasing to a value within the carbon ion range with a 4-hour interval. CONCLUSIONS: Combining hyperthermia with low LET radiation can be as effective as high LET at inducing tumor control, but the temperature needed depended on the time interval between the two modalities.

Tumors Resistant to Checkpoint Inhibitors Can Become Sensitive after Treatment with Vascular Disrupting Agents.

Immune therapy improves cancer outcomes, yet many patients do not respond. This pre-clinical study investigated whether vascular disrupting agents (VDAs) could convert an immune unresponsive tumor into a responder. CDF1 mice, with 200 mm3 C3H mammary carcinomas in the right rear foot, were intraperitoneally injected with combretastatin A-4 phosphate (CA4P), its A-1 analogue OXi4503, and/or checkpoint inhibitors (anti-PD-1, PD-L1, or CTLA-4 antibodies), administered twice weekly for two weeks. Using the endpoint of tumor growth time (TGT5; time to reach five times the starting volume), we found that none of the checkpoint inhibitors (10 mg/kg) had any effect on TGT5 compared to untreated controls. However, CA4P (100 mg/kg) or OXi4503 (5-50 mg/kg) did significantly increase TGT5. This further significantly increased by combining the VDAs with checkpoint inhibitors, but was dependent on the VDA, drug dose, and inhibitor. For CA4P, a significant increase was found when CA4P (100 mg/kg) was combined with anti-PD-L1, but not with the other two checkpoint inhibitors. With OXi4503 (50 mg/kg), a significant enhancement occurred when combined with anti-PD-L1 or anti-CTLA-4, but not anti-PD-1. We observed no significant improvement with lower OXi4503 doses (5-25 mg/kg) and anti-CTLA-4, although 30% of tumors were controlled at the 25 mg/kg dose. Histological assessment of CD4/CD8 expression actually showed decreased levels up to 10 days after treatment with OXi4503 (50 mg/kg). Thus, the non-immunogenic C3H mammary carcinoma was unresponsive to checkpoint inhibitors, but became responsive in mice treated with VDAs, although the mechanism remains unclear.

Dual-tracer PET of viable tumor volume and hypoxia for identification of necrosis-containing radio-resistant Sub-volumes.

Introduction: Positron emission tomography (PET) using hypoxia-selective tracers like FAZA may guide radiation dose-escalation approaches. However, poor resolution combined with slow tracer retention in relatively inaccessible target cells and slow clearance of unbound tracer results in low-contrast images, and areas where viable hypoxic tracer retaining cells and necrosis (no tracer) are intermixed may pass unnoticed during image thresholding. Here we hypothesized that a clinical feasible one-day dual tracer approach that combines a short-lived (e.g., 11C labeled) metabolic tracer that provides voxel-wise information on viable tissue volume (preferably independently of tumor microenvironment) and a hypoxia marker, may limit threshold-based errors. Material and methods: 11C-acetate and 11C-methionine uptake was quantified in tumor cell lines under tumor microenvironment-mimicking conditions of high/low O2 (21%/0%) and pH (7.4/6.7). Next, tumor-bearing mice were administered FAZA and sacrificed 1 h (mimics a clinical low-contrast image scenario) or 4 h (high contrast) later. In addition, all mice were administered pimonidazole (hypoxia) and 14C-methionine 1 h prior to sacrifice. Tumor tissue sections were analyzed using dual-tracer autoradiography. Finally, FAZA, or FAZA normalized to 14C-methionine retention (to adjust for differences in viable tissue volume) was compared to hypoxic fraction (deduced from immune-histological analysis of pimonidazole; ground truth) in PET-mimicking macroscopic pixels with variable extent of necrosis/hypoxia. Results/conclusions: Low pH stimulated 11C-acetate retention in many cell lines, and uptake was further modified by anoxia, compromising its usefulness as a universal marker of viable tumor volume. In contrast, 11C-methionine was largely unaffected by the in vitro microenvironment and was further tested in mice. Necrosis increased the risk of missing hypoxia-containing pixels during thresholding and hypoxic fraction and FAZA signal correlated poorly in the low contrast-scenario. Voxel-based normalization to 14C-methionine increased the likelihood of detecting voxels harboring hypoxic cells profoundly, but did not consistently improve the correlation between the density of hypoxic cells and tracer signal.

Enhancing the radiation response of tumors but not early or late responding normal tissues using a vascular disrupting agent.

INTRODUCTION: Vascular disrupting agents (VDAs) damage tumor vasculature and enhance tumor radiation response. In this pre-clinical study, we combined radiation with the leading VDA in clinical development, combretastatin A-4 phosphate (CA4P), and compared the effects seen in tumors and relevant normal tissues. MATERIAL AND METHODS: Radiation was applied locally to tissues in CDF1 mice to produce full radiation dose-response curves. CA4P (250 mg/kg) was intraperitoneally (i.p.) injected within 30 minutes after irradiating. Response of 200 mm3 foot implanted C3H mammary carcinomas was assessed using percent tumor control at 90 days. Normal tissue effects were evaluated using early responding skin (development of moist desquamation in the foot at 11-30 days), and late responding bladder (50% reduction in reservoir function estimated by cystometry up to 9 months after treatment), and lung (20% increase in ventilation rate measured by plethysmography within 9 months). A Chi-squared test was used for statistical comparisons (significance level of p < .05). RESULTS: The radiation dose controlling 50% of irradiated tumors was 52 Gy. This significantly decreased to 45 Gy with CA4P. The radiation doses inducing a change in skin, bladder and lung response in 50% of mice were 31 Gy, 14 Gy and 12 Gy, respectively. CA4P had no significant effect on the radiation response of any of these normal tissues. CONCLUSIONS: VDAs significantly enhance tumor radiation response, but had absolutely no effect on the radiation response of early or late responding normal tissues.

Hypoxia positron emission tomography imaging: combining information on perfusion and tracer retention to improve hypoxia specificity.

BACKGROUND: Static positron emission tomography (PET) allows mapping of tumor hypoxia, but low resolution and slow tracer retention/clearance results in poor image contrast and the risk of missing areas where hypoxic cells and necrosis are intermixed. Fully dynamic PET may improve accuracy but scan protocols suitable for routine clinical use are warranted. A modeling study proposed that hypoxia specificity can be improved by a clinically feasible blood-flow normalization procedure that only requires a 10- to 15-min dynamic scan (perfusion), followed by a short late static scan, but experimental validation is desired. METHODS: Tumor-bearing mice were administered pimonidazole (hypoxia marker) and the PET hypoxia-tracer 18F-azomycin arabinoside (FAZA) and scanned for 3h. Subsequently, the distributions of FAZA (autoradiography) and hypoxic cells (pimonidazole) were compared on tissue sections. PET images collected in 10-min time intervals between 60 and 90 min post-injection (PETearly), which mimics the image contrast seen in patients, were compared voxel-by-voxel to 3-h PET (PETlate). For comparison, PETearly was normalized to the perfusion peak area, deduced from the first 10 min of the scan (PETperf), and the resulting parameter PETearly/PETperf was compared with PETlate. RESULTS: Tissue analysis revealed a near-perfect spatial match between FAZA signal and hypoxic cell density (pimonidazole) 3 h post-injection, regardless of the tumor type. Only a weak inverse or no correlation between PETperf and PETlate was seen, and the correlation between PETearly/PETperf and PETlate proved inferior to the correlation between PETearly and PETlate. CONCLUSIONS: Late PET scans in rodents, unlike patients, provide an accurate map of hypoxia against which earlier time-point scans can be compared. PETearly and PETlate correlated to a variable extent but the correlation was lowered by normalization to perfusion (PETearly/PETperf). Our study challenges the validity/robustness of a perfusion normalization approach. This may reflect that the chaotic tumor vasculature uncouples microregional blood flow and oxygen extraction.

FDG-PET reproducibility in tumor-bearing mice: comparing a traditional SUV approach with a tumor-to-brain tissue ratio approach.

BACKGROUND: Current [F-18]-fluorodeoxyglucose positron emission tomography (FDG-PET) procedures in tumor-bearing mice typically includes fasting, anesthesia, and standardized uptake value (SUV)-based quantification. Such procedures may be inappropriate for prolonged multiscan experiments. We hypothesize that normalization of tumor FDG retention relative to a suitable reference tissue may improve accuracy as this method may be less susceptible to uncontrollable day-to-day changes in blood glucose levels, physical activity, or unnoticed imperfect tail vein injections. MATERIAL AND METHODS: Fed non-anesthetized tumor-bearing mice were administered FDG intravenously (i.v.) or intraperitoneally (i.p.) and PET scanned on consecutive days using a Mediso nanoScan PET/magnetic resonance imaging (MRI). Reproducibility of various PET-deduced measures of tumor FDG retention, including normalization to FDG signal in reference organs and a conventional SUV approach, was evaluated. RESULTS: Day-to-day variability in i.v. injected mice was lower when tumor FDG retention was normalized to brain signal (T/B), compared to normalization to other tissues or when using SUV-based normalization. Assessment of tissue radioactivity in dissected tissues confirmed the validity of PET-derived T/B ratios. Mean T/B and SUV values were similar in i.v. and i.p. administered animals, but SUV normalization was more robust in the i.p. group than in the i.v. group. CONCLUSIONS: Multimodality scanners allow tissue delineation and normalization of tumor FDG uptake relative to reference tissues. Normalization to brain, but not liver or kidney, improved scan reproducibility considerably and was superior to traditional SUV quantification in i.v. tracer-injected animals. Day-to-day variability in SUV's was lower in i.p. than in i.v. injected animals, and i.p. injections may therefore be a valuable alternative in prolonged rodent studies, where repeated vein injections are undesirable.

The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents.

BACKGROUND: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment. METHODS: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1-13C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology. RESULTS: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1-13C]pyruvate-to-[1-13C]lactate conversion remained unaltered, whereas [1-13C]lactate-to-[13C]bicarbonate (originating from respiratory CO2) ratios increased significantly following treatment. CONCLUSIONS: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1-13C]lactate-to-[13C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

Relative biological effectiveness (RBE) and distal edge effects of proton radiation on early damage in vivo.

INTRODUCTION: The aim of the present study was to examine the RBE for early damage in an in vivo mouse model, and the effect of the increased linear energy transfer (LET) towards the distal edge of the spread-out Bragg peak (SOBP). METHOD: The lower part of the right hind limb of CDF1 mice was irradiated with single fractions of either 6 MV photons, 240 kV photons or scanning beam protons and graded doses were applied. For the proton irradiation, the leg was either placed in the middle of a 30-mm SOBP, or to assess the effect in different positions, irradiated in 4 mm intervals from the middle of the SOBP to behind the distal dose fall-off. Irradiations were performed with the same dose plan at all positions, corresponding to a dose of 31.25 Gy in the middle of the SOBP. Endpoint of the study was early skin damage of the foot, assessed by a mouse foot skin scoring system. RESULTS: The MDD50 values with 95% confidence intervals were 36.1 (34.2-38.1) Gy for protons in the middle of the SOBP for score 3.5. For 6 MV photons, it was 35.9 (34.5-37.5) Gy and 32.6 (30.7-34.7) Gy for 240 kV photons for score 3.5. The corresponding RBE was 1.00 (0.94-1.05), relative to 6 MV photons and 0.9 (0.85-0.97) relative to 240 kV photons. In the mice group positioned at the SOBP distal dose fall-off, 25% of the mice developed early skin damage compared with 0-8% in other groups. LETd,z = 1 was 8.4 keV/µm at the distal dose fall-off and the physical dose delivered was 7% lower than in the central SOBP position, where LETd,z =1 was 3.3 keV/µm. CONCLUSIONS: Although there is a need to expand the current study to be able to calculate an exact enhancement ratio, an enhanced biological effect in vivo for early skin damage in the distal edge was demonstrated.

Hypoxia as a Biomarker and for Personalized Radiation Oncology.

Tumor hypoxia is a clinically relevant cause of radiation resistance. Direct measurements of tumor oxygenation have been performed predominantly with the Eppendorf histograph and these have defined the reduced prognosis after radiotherapy in poorly oxygenated tumors, especially head-and-neck cancer, cervix cancer and sarcoma. Exogenous markers have been used for immunohistochemical detection of hypoxic tumor areas (pimonidazole) or for positron-emission tomography (PET) imaging (misonidazole). Overexpression of hypoxia-related proteins such as hypoxia-inducible factor-1α (HIF-1α) has also been linked to poor prognosis after radiotherapy and such proteins are considered as potential endogenous hypoxia markers.

Realistic biological approaches for improving thermoradiotherapy.

There is now definitive clinical evidence that hyperthermia can successfully improve the response of certain human tumour types to radiation therapy, but, there is still the need for improvement. From a biological standpoint this can be achieved by either targeting the cellular or vascular components of tumours. Strategies include targeting the radiation DNA repair processes, improving drug delivery using nanoparticles, exploiting immunotherapy mechanisms, reducing tumour pH, or modifying the tumour vascular supply. All of these approaches have been combined with either hyperthermia or radiation in preclinical models and clear benefits in tumour response observed. But few of these methods have actually been combined with thermoradiotherapy. Furthermore, very few combinations have been tested in relevant normal tissue studies, despite the fact that it is the normal tissue response that controls the maximal heat or radiation treatment that can be applied. Here we review the most clinically relevant biological approaches that have been shown to enhance thermoradiotherapy, or have the potential to be applied in this context, and suggest how these should be moved forward into the clinic.

Improving efficacy of hyperthermia in oncology by exploiting biological mechanisms.

It has long been established that hyperthermia increases the therapeutic benefit of radiation and chemotherapy in cancer treatment. During the last few years there have been substantial technical improvements in the sources used to apply and measure heat, which greatly increases enthusiasm for the clinical use of hyperthermia. These advances are converging with a better understanding of the physiological and molecular effects of hyperthermia. Therefore, we are now at a juncture where the parameters that will influence the efficacy of hyperthermia in cancer treatment can be optimised in a more systematic and rational manner. In addition, the novel insights in hyperthermia's many biological effects on tumour cells will ultimately result in new treatment regimes. For example, the molecular effects of hyperthermia on the essential cellular process of DNA repair suggest novel combination therapies, with DNA damage response targeting drugs that should now be clinically explored. Here, we provide an overview of recent studies on the various macroscopic and microscopic biological effects of hyperthermia. We indicate the significance of these effects on current treatments and suggest how they will help design novel future treatments.

Targeting tumour hypoxia to improve outcome of stereotactic radiotherapy.

BACKGROUND: Hypoxia is a characteristic feature of solid tumours that significantly reduces the efficacy of conventional radiation therapy. In this study we investigated the role of hypoxia in a stereotactic radiation schedule by using a variety of hypoxic modifiers in a preclinical tumour model. MATERIAL AND METHODS: C3H mammary carcinomas were irradiated with 3 × 15 Gy during a one-week period, followed three days later by a clamped top-up dose to produce a dose response curve; the endpoint was tumour control. The hypoxic modifiers were nimorazole (200 mg/kg), nicotinamide (120 mg/kg) and carbogen (95% O2 + 5% CO2) breathing, OXi4503 (10 mg/kg), and hyperthermia (41.5°C; 1 h). RESULTS: The radiation dose controlling 50% of clamped tumours (TCD50) following 3 × 15 Gy was 30 Gy. Giving nimorazole or nicotinamide+ carbogen prior to the final 15 Gy fraction non-significantly (χ(2)-test; p < 0.05) reduced this TCD50 to 20-23 Gy; when administered with each 3 × 15 Gy fraction these values were significantly reduced to ≤ 2.5 Gy. Injecting OXi4503 or heating after irradiating significantly reduced the TCD50 to 9-12 Gy regardless of whether administered with one or all three 15 Gy fractions. Combining OXi4503 and heat with the final 15 Gy had a significantly larger effect (TCD50 = 2 Gy). CONCLUSIONS: Clinically relevant modifiers of hypoxia effectively enhanced an equivalent stereotactic radiation treatment confirming the importance of hypoxia in such schedules.

Relative biological effectiveness of carbon ions for tumor control, acute skin damage and late radiation-induced fibrosis in a mouse model.

BACKGROUND: The aim of the present study was to compare the biological effectiveness of carbon ions relative to x-rays between tumor control, acute skin reaction and late RIF of CDF1 mice. MATERIAL AND METHODS: CDF1 mice with a C3H mouse mammary carcinoma implanted subcutaneously on the foot of the right hind limb were irradiated with single fractions of either photons, or (12)C ions using a 30-mm spread-out Bragg peak. The endpoint of the study was local control (no tumor recurrence within 90 days). For the acute skin reaction, non-tumor bearing CDF1 mice were irradiated with a comparable radiation scheme, and monitored for acute skin damage between Day 7 and 40. Late RIF was assessed in the irradiated mice. RESULTS: The TCD50 (dose producing tumor control in 50% of mice) values with 95% confidence interval were 29.7 (25.4-34.8) Gy for C ions and 43.9 (39.2-49.2) Gy for photons, with a corresponding Relative biological effectiveness (RBE) value of 1.48 (1.28-1.72). For acute skin damage the MDD50 (dose to produce moist desquamation in 50% of mice) values with 95% confidence interval were 26.3 (23.0-30.1) Gy for C ions and 35.8 (32.9-39.0) Gy for photons, resulting in a RBE of 1.36 (1.20-1.54). For late radiation-induced fibrosis the FD50 (dose to produce severe fibrosis in 50% of mice) values with 95% confidence interval were 26.5 (23.1-30.3) Gy for carbon ions and 39.8 (37.8-41.8) Gy for photons, with a RBE of 1.50 (1.33-1.69). CONCLUSION: The observed RBE values were very similar for tumor response, acute skin damage and late RIF when irradiated with large doses of high- linear energy transfer (LET) carbon ions. This study adds information to the variation in biological effectiveness in different tumor and normal tissue models.

Hyperpolarized magnetic resonance spectroscopy for assessing tumor hypoxia.

INTRODUCTION: Hypoxic tumor cells are radioresistant, therefore, identification of hypoxia is crucial. Hyperpolarized magnetic resonance spectroscopy (HPMRS) allows real time measurements of the conversion of pyruvate to lactate, the final step of anaerobic energy production, and may thus allow non-invasive identification of hypoxia or treatment-induced changes in oxygenation. The aim of the study was to investigate the usefulness of HPMRS as a means to assess tumor hypoxia and its dynamics during intervention. MATERIAL AND METHODS: C3H mammary carcinomas grown in CDF1 mice were used. To manipulate with tumor oxygenation, non-anaesthetized mice were gassed with air, 10% or 100% oxygen prior to administration of hyperpolarized [1-¹³C]pyruvate and HPMRS analysis. A direct assessment of tumor oxygen partial pressure (pO2) distributions were made using the Eppendorf oxygen electrode in a separate, but similarly treated, group of mice. RESULTS: Even though breathing 100% oxygen improved tumor oxygenation as evidenced by pO2 measurements, the mean (with 1 S.E.) [1-¹³C]lactate/[1-¹³C]pyruvate ratio was unaffected by this intervention, being 34 (30-37) in mice breathing air and 37 (33-42) in mice breathing 100% oxygen. In contrast, and in accordance with pO2 measurements, a significant increase in the [1-¹³C]lactate/[1-¹³C]pyruvate ratio was seen in 10% oxygen-breathing mice with a ratio of 46 (42-50). CONCLUSIONS: Although, no metabolic change was observed during 100% O2 breathing using HPMRS, the significant increase in the [1-¹³C]lactate/[1-¹³C]pyruvate ratio during 10% oxygen breathing suggests, that HPMRS can detect hypoxia-driven changes in the metabolic fate of pyruvate. To what extent and for what purposes HPMRS may best supplement or complement established techniques like hypoxia PET needs to be unraveled in future research.

Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 µM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 µM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.

Photoelectron Spectra and Electronic Structures of the Radiosensitizer Nimorazole and Related Compounds.

Soft X-ray photoelectron spectroscopy has been used to investigate the radiosensitizer nimorazole and related model compounds. We report the valence and C, N, and O 1s photoemission spectra and K-edge NEXAFS spectra of gas-phase nimorazole, 1-methyl-5-nitroimidazole, and 4(5)-nitroimidazole in combination with theoretical calculations. The valence band and core level spectra are in agreement with theory. We determine the equilibrium populations of the two tautomers in 4(5)-nitroimidazole and find a ratio of 1:0.7 at 390 K. The NEXAFS spectra of the studied nitroimidazoles show excellent agreement with spectra of compounds available in the literature that exhibit a similar chemical environment. By comparing 1-methyl-5-nitroimidazole (single tautomer) with 4(5)-nitroimidazole, we are able to disentangle the photoemission and photoabsorption spectra and identify features due to each single tautomer.

Therapeutic potential of using the vascular disrupting agent OXi4503 to enhance mild temperature thermoradiation.

PURPOSE: The response of tissues to radiation with mild temperature hyperthermia is dependent on the interval between the two modalities. This study investigated the effect that the vascular disrupting agent OXi4503 had on this time-interval interaction. METHODS: The normal right rear foot of female CDF1 mice or foot-implanted C3H mammary carcinomas were locally irradiated (230 kV X-rays) and heated (41.5 °C for 60 min) by foot immersion in a water bath. OXi4503 (50 mg/kg) was injected intraperitoneally 1.5 h before irradiating. Irradiation was performed either in the middle of the heating period (simultaneous treatment) or at 1 or 4 h prior to starting the heating (sequential treatments). Response was the percentage of mice showing local tumour control at 90 days or skin moist desquamation between days 11-23. From the radiation dose response curves the dose producing tumour control (TCD(50)) or moist desquamation (MDD50) in 50% of mice was calculated. RESULTS: The TCD(50) and MDD50 values for radiation alone were 54 Gy and 29 Gy, respectively. Simultaneously heating the tissues enhanced radiation response, the respective TCD(50) and MDD50 values being significantly (chi-square test, p < 0.05) reduced to 33 Gy and 14 Gy. A smaller enhancement was obtained with a sequential treatment in both tissues. OXi4503 enhanced the radiation response of tumour and skin. Combined with radiation and heat, the only effect was in tumours where OXi4503 prevented the decrease in sensitisation seen with the sequential treatment. CONCLUSION: Combining OXi4503 with a sequential radiation and heat treatment resulted in a 1.4-fold therapeutic gain.

Accumulation of nano-sized particles in a murine model of angiogenesis.

PURPOSE: To evaluate the ability of nm-scaled iron oxide particles conjugated with Azure A, a classic histological dye, to accumulate in areas of angiogenesis in a recently developed murine angiogenesis model. MATERIALS AND METHODS: We characterised the Azure A particles with regard to their hydrodynamic size, zeta potential, and blood circulation half-life. The particles were then investigated by Magnetic Resonance Imaging (MRI) in a recently developed murine angiogenesis model along with reference particles (Ferumoxtran-10) and saline injections. RESULTS: The Azure A particles had a mean hydrodynamic diameter of 51.8 ± 43.2 nm, a zeta potential of -17.2 ± 2.8 mV, and a blood circulation half-life of 127.8 ± 74.7 min. Comparison of MR images taken pre- and 24-h post-injection revealed a significant increase in R2(*) relaxation rates for both Azure A and Ferumoxtran-10 particles. No significant difference was found for the saline injections. The relative increase was calculated for the three groups, and showed a significant difference between the saline group and the Azure A group, and between the saline group and the Ferumoxtran-10 group. However, no significant difference was found between the two particle groups. CONCLUSION: Ultrahigh-field MRI revealed localisation of both types of iron oxide particles to areas of neovasculature. However, the Azure A particles did not show any enhanced accumulation relative to Ferumoxtran-10, suggesting the accumulation in both cases to be passive.