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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. 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. 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. Checkpoint inhibitors enhanced the response of this C3H mammary carcinoma to proton irradiation. However, this enhancement depended on the checkpoint inhibitor and radiation dose. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate 1) the effect of acute aerobic exercise on tumor hypoxia and blood perfusion, 2) the impact of exercise intensity, 3) the duration of the effect, and 4) the effect of prolonged training on tumor hypoxia and tumor growth. Female CDF1 mice were inoculated with the C3H mammary carcinoma either in the mammary fat pad or subcutaneously in the back. For experiments on the effect of different intensities in a single exercise bout, mice were randomized to 30-min treadmill running at low-, moderate-, or high-intensity speeds or no exercise. To investigate the prolonged effect on hypoxia and tumor growth, tumor-bearing mice were randomized to no exercise (CON) or daily 30-min high-intensity exercise averaging 2 wk (EX). Tumor hypoxic fraction was quantified using the hypoxia marker Pimonidazole. Initially, high-intensity exercise reduced tumor hypoxic fraction by 37% compared with CON [P = 0.046; 95% confidence interval (CI): 0.1; 10.3] in fat pad tumors. Low- and moderate-intensity exercises did not. Following experiments investigating the duration of the effect--as well as experiments in mice with back tumors--failed to show any exercise-induced changes in hypoxia. Interestingly, prolonged daily training significantly reduced hypoxic fraction by 60% (P = 0.002; 95% CI: 2.5; 10.1) compared with CON. Despite diverging findings on the acute effect of exercise on hypoxia, our data indicate that if exercise has a diminishing effect, high-intensity exercise is needed. Prolonged training reduced tumor hypoxic fraction--cautiously suggesting a potential clinical potential. NEW & NOTEWORTHY This study provides novel information on the effects of acute and chronic exercise on tumor hypoxia in mice. In contrast to the few related existing studies, diverging findings on tumor hypoxia after acute exercise were observed, suggesting that tumor model and location should be considered in future studies. Highly significant reductions in tumor hypoxia following chronic high-intensity exercise propose a future clinical potential but this should be investigated in patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 mm3foot 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 ofp < .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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction:The aim of the present study was to examine the RBE for early damage in anin vivomouse 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 MDD50values 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 = 1was 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 =1was 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 effectin vivofor early skin damage in the distal edge was demonstrated. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background. Radiation-induced fibrosis (RIF) is a dose-limiting complication of cancer radiotherapy and causes serious problems, i.e. restricted tissue flexibility, pain, ulceration or necrosis. Recently, we have successfully treated RIF in a mouse model by intraperitoneal administration of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. Methods. Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. Results. We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. Conclusion. We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan/siRNA nanoparticle may constitute a general treatment for inflammatory diseases using the natural homing potential of macrophages to inflammatory sites. [ABSTRACT FROM AUTHOR]

Purpose. This pre-clinical study was designed to investigate the effect of various vascular disrupting agents (VDAs) that have undergone or are in clinical evaluation, had on the oxygenation status of tumours and what effects that could have on the combination with radiation. Material and methods. The tumour model was a C3H mammary carcinoma grown in the right rear foot of female CDF1 mice and treated when at 200 mm3 in size. The VDAs were the flavenoid compounds flavone acetic acid (FAA) and its more recent derivative 5,6-dimethylxanthenone-4-acetic acid (DMXAA), and the leading tubulin binding agent combretastatin A-4 phosphate (CA4P) and the A-1 analogue OXi4503. Oxygenation status was estimated using the Eppendorf oxygen electrode three hours after drug injection. Radiation response was determined following single or fractionated (10 fractions in 12 days) irradiations with a 240 kV x-ray machine using either a tumour re-growth or local tumour control assay. Results. All VDAs significantly reduced the oxygenation status of the tumours. They also influenced radiation response, but the affect was time and sequence dependent using single radiation schedules; an enhanced effect when the VDAs were injected at the same time or after irradiating, but no or even a reduced effect when given prior to irradiation. Only OXi4503 showed an increased response when given before the radiation. CA4P and OXi4503 also enhanced a fractionated radiation treatment if the drugs were administered after fractions 5 and 10. Conclusions. VDAs clearly induced tumour hypoxia. This had the potential to decrease the efficacy of radiation. However, if the appropriate timing and scheduling were used an enhanced effect was observed using both single and fractionated radiation treatments. [ABSTRACT FROM AUTHOR]

Background. Tumor hypoxia contributes to loco-regional failure, and for optimal treatment planning, knowledge about tumor hypoxia in individual patients is required. Nitroimidazole-based tracers, which are retained in hypoxic cells, allow PET-based assessment of tumor hypoxia, but current tracers are characterized by slow tracer retention and clearance, resulting in low inter-tissue contrast. Pimonidazole is an immune detectable hypoxia marker widely used for detection of hypoxia in tumor samples. Pimonidazole has excellent chemical properties for hypoxia imaging, but labeling for non- invasive assay has not been attempted. Here we labeled pimonidazole with 18F ([18F]FPIMO). Material and methods. [18F]FPIMO was produced by fluorination of 1-[2- O-tosyl-3-(2-nitroimidazole-1-yl)-propyl]-piperidine, which resulted in two isomeric interchangeable forms (named '5' and '6') with a radiochemical purity of 91-100%. [18F]FPIMO was tested by incubation of two different tumor cell lines at high and low oxygen levels. [18F]FPIMO was also administered to tumor-bearing mice and tracer retention in tumors, non-hypoxic reference tissues and tissues involved in drug metabolism/clearance was evaluated by various techniques. Results and conclusions. Retention of [18F]FPIMO was strongly hypoxia-driven in vitro, but isomeric form '5' was particularly promising and reached impressive anoxic-to-oxic retention ratios of 36 and 102, in FaDuDD and SiHa cells, respectively, following three hours of tracer incubation. This was equal to or higher than ratios measured using the established hypoxia tracer [18F]FAZA. [18F]FPIMO also accumulated in tumors grown in mice, and reached tumor levels that were two to six-fold higher than in muscle three hours post-administration. Furthermore, the intra-tumoral distribution of [18F]FPIMO (autoradiography) and unlabeled pimonidazole (immunohistochemistry) was largely identical. Nonetheless, [18F]FPIMO proved inferior to [18F]FAZA, since absolute tumor signal and intra-tumoral contrast was low, thus compromising PET imaging. Low tumor signal was coupled to extensive tracer accumulation in liver and kidneys, and analysis of blood metabolites revealed that [18F]FPIMO was metabolized rapidly, with little parent compound remaining 15 minutes post-administration. Ongoing work focuses on the possibility of labeling pimonidazole in different positions with 18F to improve tracer stability in vivo. [ABSTRACT FROM AUTHOR]

Background. Currently, radiation treatments are being optimised based on in vivo imaging of radioresistant, hypoxic tumour areas. This study aimed at detecting nicotinamide's reduction of acute hypoxia in a mouse tumour model by two clinically relevant magnetic resonance imaging (MRI) methods at ultra-high magnetic field strength. Material and methods. The C3H mammary carcinoma was grown to 200 mm3 in the right rear foot of CDF1 mice. The mice were anaesthetised with ketamine and xylazine prior to imaging. A treatment group received nicotinamide intraperitoneally (i.p.) at the dose 1000 mg/kg, and a control group received saline. MRI was performed at 16.4 T with a spatial resolution of 0.156 × 0.156 × 0.5 mm3. The imaging protocol included BOLD imaging and two DCE-MRI scans. Initial area under the curve (IAUC) and the parameters from the extended Toft's model were estimated from the DCE-MRI data. Tumour median values of 1) T2* mean, 2) T2* standard deviation, 3) DCE-MRI parameters, and 4) DCE-MRI parameter differences between scans were compared between the treatment groups using Student's t-test (significance level p < 0.05). Results. Parametric maps showed intra- and inter-tumour heterogeneity. Blood volume was significantly larger in the nicotinamide-treated group, and also the blood volume difference between the two DCE-MRI scans was significantly larger in the treatment group. Conclusion. Higher blood volume and blood volume variation was observed by DCE-MRI in the treatment group. Other DCE-MRI parameters showed no significant differences, and the higher blood volume was not detected by BOLD MRI. The higher blood volume variation seen with DCE-MRI may be influenced by the drug effect reducing over time, and furthermore the anaesthesia may play an important role. [ABSTRACT FROM AUTHOR]

Introduction. The efficacy of the vascular disrupting agent combretastatin A-4 phosphate (CA4P) depends on several factors including tumor size, nitric oxide level, interstitial fluid pressure, and vascular permeability. These factors vary among tumor types. The aim of this study was to investigate all these factors in two tumor models that respond differently to CA4P. Material and methods. Mice bearing C3H mammary carcinomas or KHT sarcomas (200 to 800 mm3) were intraperitoneally injected with CA4P (100 mg/kg). Tumor size and the effect of a nitric oxide inhibitor nitro-L-arginine (NLA) administered intravenously were evaluated by necrotic fraction histologically assessed at 24 hours. Interstitial fluid pressure (IFP) was measured using the wick-in-needle technique, and vascular characteristics were assessed with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Results. Initial necrotic fraction was about 10% in both tumor models at 200 mm3, but only increased significantly with tumor size in the C3H mammary carcinoma. In this tumor, CA4P significantly induced further necrosis by about 15% at all sizes, but in the KHT tumor, the induced necrotic fraction depended on tumor size. For both tumor types, NLA with CA4P significantly increased necrotic fraction above that for each drug alone. CA4P significantly decreased IFP in all tumors except in the 800 mm3 C3H tumor, which had an initially non-significant lower value. Interstitial volume estimated by DCE-MRI increased in all groups, except the 800 mm3 C3H tumors. DCE-MRI vascular parameters showed different initial characteristics and general significant reductions following CA4P treatment. Conclusions. Both tumor models showed differences in all factors before treatment, and in their response to CA4P. Perfusion and permeability as estimated by DCE-MRI play a central role in the CA4P response, and interstitial volume and IFP seemed related. These factors may be of clinical value in the planning of CA4P treatments. [ABSTRACT FROM AUTHOR]

Background. Genes upregulated by low oxygen have been suggested as endogenous markers for tumor hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to concerns about their ability to be true hypoxia makers. Previous studies have demonstrated that expression of hypoxia induced genes can be affected by extracellular pH (pHe). Methods. Five different human cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were exposed to different oxygen concentrations and pH (7.5 or 6.3), and gene expression analyzed with microarray (Affymetrix - Human Genome U133 Plus 2.0 Array). Results. An analysis of two of the cell lines using SAM identified 461 probesets that were able to separate the four groups “Normal oxygen, normal pH”, “Low oxygen, normal pH”, “Normal oxygen, low pH” and “Low oxygen, low pH”. From here it was possible to identify a fraction of probesets induced at low oxygen independent of pH in these two cell lines, this fraction included HIG2, NDRG1, PAI1 and RORA. Further verification by qPCR highlighted the necessity of using more cell lines to obtain a robust gene expression profiles. To specifically select pH independent hypoxia regulated genes across more cell lines, data for FaDuDD, UTSCC5, UTSCC14 and UTSCC15 were analyzed to identify genes that were induced by hypoxia in each cell line, where the induction was not affected by low pH, and where the gene was not significantly induced by low pH alone. Each cell line had 65–122 probesets meeting these criteria. For genes to be considered as target genes (hypoxia inducible pH independent), genes had to be present in three of four cell lines. Conclusion. The result is a robust hypoxia profile unaffected by pH across cell lines consisting of 27 genes. This study demonstrates a way to identify hypoxia markers by microarray, where other factors in the tumor microenvironment are taken into account. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the possible relationship between radiobiological hypoxia in a C3H mouse mammary carcinoma and osteopontin (OPN) levels measured in mouse serum. Material and methods: Experiments were performed in CDF1 mice that were either non-tumour bearing or with different sized tumours implanted in the right rear foot. Osteopontin levels in extracted mouse blood serum and tissue from the transplanted tumours were measured using an ELISA assay. The tumour oxygenation status was estimated using the Eppendorf Histograph and the fraction of oxygen partial pressure (pO2) values =5 mm Hg (HF5) was calculated. Necrosis was measured in haematoxylin and eosin-stained sections. Tumour hypoxia was increased by placing animals in a low-oxygen (10%) environment. Single radiation doses (240 kV x-rays) were given locally to tumours under ambient or clamped conditions and response assessed using a tumour control assay. Results: Serum OPN levels increased linearly with increasing tumour volume and this increase correlated with tumour OPN. HF5 and necrosis also increased with increasing tumour volume, but this increase was non-linear. Converting the HF5 results into equivalent tumour volume gave results that were directly correlated to OPN serum levels. Placing mice in a 10% oxygen environment for 12 hours significantly increased HF5. However, serum OPN only increased if reoxygenation occurred before measurement. Radiobiological hypoxic fraction in this tumour model did not change with increasing tumour size, but the total number of hypoxic cells did increase. Conclusions: These findings suggest that serum OPN measurement may predict the proportion of hypoxic cells in this tumour model, although increased serum OPN levels simply resulting from an increased tumour burden can not be ruled out. [ABSTRACT FROM AUTHOR]

: PurposeThe aim of this study was to investigate the pathophysiologic effects induced by the novel vascular targeting agent ZD6126 in a C3H mouse mammary carcinoma and to evaluate the agent''s ability to inhibit tumor growth either when given alone or in combination with radiation.: Methods and materialsA C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm3 in size. ZD6126 was dissolved in saline and injected intraperitoneally. Blood perfusion was measured using the RbCl extraction procedure, tumor oxygen (pO2) status was assessed with the Eppendorf electrode, and tumor necrosis was estimated from histologic sections. Radiation (240-kV X-rays) was locally administered to tumors of restrained nonanesthetized mice, and response was assessed using a tumor growth assay.: ResultsZD6126 induced a significant dose- and time-dependent decrease in tumor perfusion, reaching a maximal 70% reduction around 3 h after injecting 150–300 mg/kg. However, full recovery was seen within 6 h. A 200 mg/kg dose significantly decreased tumor oxygenation status at 3 h (median pO2 decreased from 7 to 3 mm Hg; % pO2 values ≤2.5 mm Hg increased from 30% to 55%) and by 24 h had significantly increased necrotic fraction from 14.5% to 25.2%. This ZD6126 dose resulted in a small, yet significant, 1.4 days inhibition of tumor growth when given alone. It also enhanced the tumor response to radiation, giving rise to a significant 1.3-fold increase in the slope of the radiation dose–response curve. Of the normal tissues, only muscle (at 3 h) and spleen (at 6 h) showed any significant reduction in perfusion after injecting 200 mg/kg, but these transient decreases were only 32% and 49%, respectively.: ConclusionsOur preclinical studies clearly demonstrate a tumor-specific reduction in blood perfusion by ZD6126. Although these changes were transient, they were sufficient to increase tumor necrosis, inhibit tumor growth, and enhance radiation response. [Copyright &y& Elsevier]

Simple Summary: Regions of low oxygen status (hypoxia) are a characteristic feature of solid tumors. This hypoxia is a major cause of resistance to treatment, especially radiation therapy, and it can increase the likelihood of metastatic spread. Being able to accurately identify tumor hypoxia will allow us to predict a patient's response to therapy and find alternative approaches to improve outcome. We have refined a dissection method that involves autoradiography and laser-guided microdissection of hypoxic areas in tumors. Using this approach, we were able to test the feasibility of applying a 15-gene signature that was previously developed for head and neck cancer patients to identify hypoxia in pre-clinical models of prostate cancer. Our results demonstrated the potential of this method to identify hypoxia in this tumor type and suggest its applicability for use in patients with prostate cancer. Background: This pre-clinical study was designed to refine a dissection method for validating the use of a 15-gene hypoxia classifier, which was previously established for head and neck squamous cell carcinoma (HNSCC) patients, to identify hypoxia in prostate cancer. Methods: PC3 and DU-145 adenocarcinoma cells, in vitro, were gassed with various oxygen concentrations (0–21%) for 24 h, followed by real-time PCR. Xenografts were established in vivo, and the mice were injected with the hypoxic markers [18F]-FAZA and pimonidazole. Subsequently, tumors were excised, frozen, cryo-sectioned, and analyzed using autoradiography ([18F]-FAZA) and immunohistochemistry (pimonidazole); the autoradiograms used as templates for laser capture microdissection of hypoxic and non-hypoxic areas, which were lysed, and real-time PCR was performed. Results: In vitro, all 15 genes were increasingly up-regulated as oxygen concentrations decreased. With the xenografts, all 15 genes were up-regulated in the hypoxic compared to non-hypoxic areas for both cell lines, although this effect was greater in the DU-145. Conclusions: We have developed a combined autoradiographic/laser-guided microdissection method with broad applicability. Using this approach on fresh frozen tumor material, thereby minimizing the degree of RNA degradation, we showed that the 15-gene hypoxia gene classifier developed in HNSCC may be applicable for adenocarcinomas such as prostate cancer. [ABSTRACT FROM AUTHOR]

The study was designed to investigate the binding of [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) in a C3H mouse mammary carcinoma. Non-anaesthetized tumour-bearing animals breathing either normal air or carbogen (to reduce tumour hypoxia) were examined by PET after tracer injection. Tumours were identified by radioactive labelling and methods of defining regions of interest (ROI) in the tumours were investigated. Reference tissue was selected elsewhere in the mice and the ratio between mean radioactivity in tumour and reference tissue was compared. The results showed a correlation between the methods of identifying ROIs and a significantly lower tumour to reference tissue ratio for carbogen-treated mice compared with controls when using [18F]FMISO. Only one of the methods showed a significant difference in the tumour labelling between treatment groups using [18F]FDG. The study supports the contention that [18F]FMISO may be able to identify hypoxia in tumours, whereas a similar role for [18F]FDG is more doubtful. [ABSTRACT FROM AUTHOR]

Purpose: To examine the feasibility of using the MRI blood pool agent NC100150 for evaluation of tumor blood volume (TBV) estimates by both dynamic contrast-enhanced MRI (DCE-MRI) and susceptibility contrast MRI assays in an experimental tumor. Contrast agent clearance (K(trans); depends on perfusion and permeability) from the DCE-MRI time curves was estimated, and changes in TBV and K(trans) were measured after administration of two drugs that reduce perfusion by different mechanisms.Methods and Materials: The DCE-MRI experiments were simulated with expected physiologic values for the C3H mouse mammary carcinoma. The C3H tumor was examined by DCE-MRI and susceptibility contrast MRI with NC100150 (NC100150 Injection, Clariscan; Amersham Health, Oslo, Norway) after treatment with either hydralazine or combretastatin (Oxigene, Boston, MA).Results: Simulations showed that reliable estimates of changes in TBV and K(trans) could be performed with DCE-MRI. Hydralazine was shown to reduce TBV as measured by both assays and to reduce K(trans). Dynamic contrast-enhanced MRI also suggested that TBV and K(trans) were reduced in combretastatin-treated tumors, and the TBV reduction was confirmed by susceptibility contrast MRI. Data suggested the drug to affect mainly the total TBV, whereas microvessels as such seemed less altered.Conclusion: The study supports the use of the combined DCE-MRI and susceptibility contrast MRI assay with a blood pool agent in characterizing tumors and their response to treatment. [ABSTRACT FROM AUTHOR]