Dynamic whole-body [18F]FES PET/CT increases lesion visibility in patients with metastatic breast cancer.

BACKGROUND: Correct classification of estrogen receptor (ER) status is essential for prognosis and treatment planning in patients with breast cancer (BC). Therefore, it is recommended to sample tumor tissue from an accessible metastasis. However, ER expression can show intra- and intertumoral heterogeneity. 16α-[18F]fluoroestradiol ([18F]FES) Positron Emission Tomography/Computed Tomography (PET/CT) allows noninvasive whole-body (WB) identification of ER distribution and is usually performed as a single static image 60 min after radiotracer injection. Using dynamic whole-body (D-WB) PET imaging, we examine [18F]FES kinetics and explore whether Patlak parametric images ( K i ) are quantitative and improve lesion visibility. RESULTS: This prospective study included eight patients with metastatic ER-positive BC scanned using a D-WB PET acquisition protocol. The kinetics of [18F]FES were best characterized by the irreversible two-tissue compartment model in tumor lesions and in the majority of organ tissues. K i values from Patlak parametric images correlated with K i values from the full kinetic analysis, r2 = 0.77, and with the semiquantitative mean standardized uptake value (SUVmean), r2 = 0.91. Furthermore, parametric K i images had the highest target-to-background ratio (TBR) in 162/164 metastatic lesions and the highest contrast-to-noise ratio (CNR) in 99/164 lesions compared to conventional SUV images. TBR was 2.45 (95% confidence interval (CI): 2.25-2.68) and CNR 1.17 (95% CI: 1.08-1.26) times higher in K i images compared to SUV images. These quantitative differences were seen as reduced background activity in the K i images. CONCLUSION: [18F]FES uptake is best described by an irreversible two-tissue compartment model. D-WB [18F]FES PET/CT scans can be used for direct reconstruction of parametric K i images, with superior lesion visibility and K i values comparable to K i values found from full kinetic analyses. This may aid correct ER classification and treatment decisions. Trial registration ClinicalTrials.gov: NCT04150731, https://clinicaltrials.gov/study/NCT04150731.

In Vitro Characterization of the Bacteria-derived Hypoxia-selective Cytotoxin BE-43547.

BACKGROUND/AIM: Hypoxia-activated pro-drugs, such as TH-302, may kill hypoxic treatment-resistant tumor cells, but have failed in clinical trials. This may be related to variable levels of drug-activating reductases. Compounds such as bacteria-derived BE-43547, which target hypoxic cells independently of reductases, may be beneficial. This study characterized the in vitro potency and hypoxia selectivity of BE-43547 and TH-302. MATERIALS AND METHODS: Tumor cells were exposed to different oxygenation levels in the presence/absence of drug, and survival was quantified using total cell number (BE-43547) or clonogenic survival (BE-43547 and TH-302) assays. Half-maximal inhibitory concentration (IC50) values and the hypoxia-cytotoxicity-ratio (HCR: normoxic IC50/hypoxic IC50) were determined from dose-response curves. Finally, both drugs were tested in spheroids exposed to 20% or 0% O2 for 24 h followed by assessment of clonogenic survival. RESULTS: BE-43547 was highly potent and displayed little inter-cell line variability. Strongly enhanced cytotoxicity was observed under oxygen-restricted conditions with HCR's of ~100 and ~20 after 24 h of treatment with 0 or 0.5% O2, respectively. Reducing treatment time somewhat reduced hypoxia selectivity. Hypoxia selectivity was observed regardless of whether the drug was added before or during the hypoxic challenge. TH-302 IC50 values varied 10-fold under oxic conditions, whereas those of the anoxic-to-normoxic HCR varied from 15 to 88. Both BE-43547 and TH-302 were unable to completely sterilize anoxic incubated spheroids. CONCLUSION: BE-43547 is highly hypoxia-selective, and unlike TH-302, displayed minimal variability between cell lines, suggesting that BE-43547 targets a fundamental feature/target that is only present, or of survival importance, during hypoxia. Spheroid experiments suggested inadequate tissue penetrability, which may be overcome by designing novel drug analogs.

Pre-Clinical Models to Study Human Prostate Cancer.

Prostate cancer is a common cancer among men and typically progresses slowly for several decades before becoming aggressive and spreading to other organs, leaving few treatment options. While large animals have been studied, the dog's prostate is anatomically similar to humans and has been used to study spontaneous prostate cancer. However, most research currently focuses on the mouse as a model organism due to the ability to genetically modify their prostatic tissues for molecular analysis. One milestone in this research was the identification of the prostate-specific promoter Probasin, which allowed for the prostate-specific expression of transgenes. This has led to the generation of mice with aggressive prostatic tumors through overexpression of the SV40 oncogene. The Probasin promoter is also used to drive Cre expression and has allowed researchers to generate prostate-specific loss-of-function studies. Another landmark moment in the process of modeling prostate cancer in mice was the orthoptic delivery of viral particles. This technology allows the selective overexpression of oncogenes from lentivirus or the use of CRISPR to generate complex loss-of-function studies. These genetically modified models are complemented by classical xenografts of human prostate tumor cells in immune-deficient mice. Overall, pre-clinical models have provided a portfolio of model systems to study and address complex mechanisms in prostate cancer for improved treatment options. This review will focus on the advances in each technique.

The effect of single bout and prolonged aerobic exercise on tumor hypoxia in mice.

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.

Refinement of an Established Procedure and Its Application for Identification of Hypoxia in Prostate Cancer Xenografts.

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.

Imaging of Tumor Hypoxia for Radiotherapy: Current Status and Future Directions.

Tumor regions that are transiently or chronically undersupplied with oxygen (hypoxia) and nutrients, and enriched with acidic waste products, are common due to an abnormal and inefficient tumor vasculature, and a deviant highly glycolytic energy metabolism. There is compelling evidence that tumor hypoxia is strongly linked to poor prognosis since oxygen-deprived cells are highly resistant to therapy including radio- and chemotherapy, and survival of such cells is a primary cause of disease relapse. Despite a general improvement in cancer survival rates, hypoxia remains a formidable challenge. Recent progress in radiation delivery systems with improved spatial accuracy that allows dose escalation to hypoxic tumors or even tumor subvolumes, and the development of hypoxia-selective drugs, including bioreductive prodrugs, holds great promise for overcoming this obstacle. However, apart from one notable exception, translation of promising preclinical therapies to the clinic have largely been disappointing. A major obstacle in clinical trials on hypoxia-targeting strategies has been the lack of reliable information on tumor hypoxia, which is crucial for patient stratification into groups of those that are likely to benefit from intervention and those who are not. Further, in many newer trials on hypoxia-selective drugs the choice of cancer disease and combination therapy has not always been ideal, especially not for clinical proof of principle trials. Clearly, there is a pending need for clinical applicable methodologies that may allow us to quantify, map and monitor hypoxia. Molecular imaging may provide the information required for narrowing the gap between potential and actual patient benefit of hypoxia-targeting strategies. The grand majority of preclinical and clinical work has focused on the usefulness of PET-based assessment of hypoxia-selective tracers. Since hypoxia PET has profound inherent weaknesses, the use of other methodologies, including more indirect methods that quantifies blood flow or oxygenation-dependent flux changes through ATP-generating pathways (eg, anaerobic glycolysis) is being extensively studied. In this review, we briefly discuss established and emerging hypoxia-targeting strategies, followed by a more thorough evaluation of strengths and weaknesses of clinical applicable imaging methodologies that may guide timely treatment intensification to overcome hypoxia-driven resistance. Historically, most evidence for the linkage between hypoxia and poor outcome is based on work in the field of radiotherapy. Therefore, main emphasis in this review is on targeting and imaging of hypoxia for improved radiotherapy.

In vitro hypoxia responsiveness of [18F] FDG and [18F] FAZA retention: influence of shaking versus stagnant conditions, glass versus polystyrene substrata and cell number down-scaling.

BACKGROUND: In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose ([18F]FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, [18F]FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. METHODS: Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2 h to 21%, 0.5% or 0% O2 and [18F] FDG or [18F] FAZA was added, followed by cell harvest and analysis of radioactivity 1 h ([18F]FDG) or 3 h ([18F]FAZA) after. Experiments were conducted with/without orbital shaking. RESULTS: The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven [18F] FAZA, and to some extent [18F] FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. CONCLUSIONS: Tracer retention was similar under stagnant and forced convection conditions suggesting that the former approach may be appropriate even when accurate control of oxygen and tracer availability is required. In contrast, conventional plasticware should be used with caution when studying tracers and drugs that are metabolized and retained or activated at low O2 levels. Downscaling of cell number, by reducing the effective growth area, was feasible, without compromising accuracy.

Characterization and radiosensitivity of HPV-related oropharyngeal squamous cell carcinoma patient-derived xenografts.

Background: Oropharyngeal squamous cell carcinomas (OPSCC) are rising rapidly in incidence due to Human Papillomavirus (HPV) and/or tobacco smoking. Prognosis is better for patients with HPV-positive disease, but may also be influenced by tobacco smoking and other factors. There is a need to individualize treatment to minimize morbidity and improve prognosis. Patient-derived xenografts (PDX) is an emerging pre-clinical research model that may more accurately reflect the human disease, and is an attractive platform to study disease biology and develop treatments and biomarkers. In this study we describe the establishment of PDX models, compare PDX tumors to the human original, and assess the suitability of this model for radiotherapy research and biomarker development. Material and methods: Tumor biopsies from 34 patients with previously untreated OPSCC were implanted in immunodeficient mice, giving rise to 12 squamous cell carcinoma PDX models (7 HPV+, 5 HPV-). Primary and PDX tumors were characterized extensively, examining histology, immunohistochemistry, cancer gene sequencing and gene expression analysis. Radiosensitivity was assessed in vivo in a growth delay assay. Results: Established PDX models maintained histological and immunohistochemical characteristics as well as HPV-status of the primary tumor. Important cancer driver gene mutations, e.g., in TP53, PIK3CA and others, were preserved. Gene expression related to cancer stem cell markers and gene expression subtype were preserved, while gene expression related to hypoxia and immune response differed. Radiosensitivity studies showed high concordance with clinical observations. Conclusion: PDX from OPSCC preserves important molecular characteristics of the human primary tumor. Radiosensitivity were in accordance with clinically observed treatment response. The PDX model is a clinically relevant surrogate model of head and neck cancer. Perspectives include increased understanding of disease biology, which could lead to development of novel treatments and biomarkers.

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.

APD-Containing Cyclolipodepsipeptides Target Mitochondrial Function in Hypoxic Cancer Cells.

The natural product family of macrocyclic lipodepsipeptides containing the 4-amido-2,4-pentadienoate functionality possesses intriguing cytotoxic selectivity toward hypoxic cancer cells. These subpopulations of cancer cells display increased metastatic potential and resistance to chemo- and radiotherapy. In this paper, we present studies on the mechanism of action of these natural products in hypoxic cancer cells and show that this involves rapid and hypoxia-selective collapse of mitochondrial integrity and function. These events drive a regulated cell death process that potentially could function as a powerful tool in the fight against chemo- and radiotherapy-resistant cancer cells. Toward that end, we demonstrate activity in two different mouse tumor models.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

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.

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.

Results from 11C-metformin-PET scans, tissue analysis and cellular drug-sensitivity assays questions the view that biguanides affects tumor respiration directly.

The anti-diabetic biguanide drugs metformin (METF) and phenformin (PHEN) may have anti-cancer effects. Biguanides suppress plasma growth factors, but nonetheless, the view that these mitochondrial inhibitors accumulate in tumor tissue to an extent that leads to severe energetic stress or alleviation of hypoxia-induced radioresistance is gaining ground. Our cell studies confirm that biguanides inhibits cell proliferation by targeting respiration, but only at highly suprapharmacological concentrations due to low drug retention. Biodistribution/PET studies of 11C-labeled metformin (11C-METF) revealed that plasma bioavailability remained well below concentrations with metabolic/anti-proliferative in vitro effects, following a high oral dose. Intraperitoneal administration resulted in higher drug concentrations, which affected metabolism in normal organs with high METF uptake (e.g., kidneys), but tumor drug retention peaked at low levels comparable to plasma levels and hypoxia was unaffected. Prolonged intraperitoneal treatment reduced tumor growth in two tumor models, however, the response did not reflect in vitro drug sensitivity, and tumor metabolism and hypoxia was unaffected. Our results do not support that direct inhibition of tumor cell respiration is responsible for reduced tumor growth, but future studies using 11C-METF-PET are warranted, preferably in neoplasia's originating from tissue with high drug transport capacity, to investigate the controversial idea of direct targeting.

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.

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.

Simultaneous Hypoxia and Low Extracellular pH Suppress Overall Metabolic Rate and Protein Synthesis In Vitro.

BACKGROUND: The tumor microenvironment is characterized by regions of hypoxia and acidosis which are linked to poor prognosis. This occurs due to an aberrant vasculature as well as high rates of glycolysis and lactate production in tumor cells even in the presence of oxygen (the Warburg effect), which weakens the spatial linkage between hypoxia and acidosis. METHODS: Five different human squamous cell carcinoma cell lines (SiHa, FaDuDD, UTSCC5, UTSCC14 and UTSCC15) were treated with hypoxia, acidosis (pH 6.3), or a combination, and gene expression analyzed using microarray. SiHa and FaDuDD were chosen for further characterization of cell energetics and protein synthesis. Total cellular ATP turnover and relative glycolytic dependency was determined by simultaneous measurements of oxygen consumption and lactate synthesis rates and total protein synthesis was determined by autoradiographic quantification of the incorporation of 35S-labelled methionine and cysteine into protein. RESULTS: Microarray analysis allowed differentiation between genes induced at low oxygen only at normal extracellular pH (pHe), genes induced at low oxygen at both normal and low pHe, and genes induced at low pHe independent of oxygen concentration. Several genes were found to be upregulated by acidosis independent of oxygenation. Acidosis resulted in a more wide-scale change in gene expression profiles than hypoxia including upregulation of genes involved in the translation process, for example Eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), and Ribosomal protein L37 (RPL37). Acidosis suppressed overall ATP turnover and protein synthesis by 50%. Protein synthesis, but not total ATP production, was also suppressed under hypoxic conditions. A dramatic decrease in ATP turnover (SiHa) and protein synthesis (both cell lines) was observed when hypoxia and low pHe were combined. CONCLUSIONS: We demonstrate here that the influence of hypoxia and acidosis causes different responses, both in gene expression and in de novo protein synthesis, depending on whether the two factors induced alone or overlapping, and as such it is important for in vivo studies to take this into account.

The usability of a 15-gene hypoxia classifier as a universal hypoxia profile in various cancer cell types.

BACKGROUND AND PURPOSE: A 15-gene hypoxia profile has previously demonstrated to have both prognostic and predictive impact for hypoxic modification in squamous cell carcinoma of the head and neck. This gene expression profile may also have a prognostic value in other histological cancer types, and could potentially have a function as a universal hypoxia profile. The hypoxia induced upregulation of the included genes, and the validity of the previously used reference genes was established in this study, in a range of different cell lines representing carcinomas of the prostate, colon, and esophagus. MATERIALS AND METHODS: Eleven adenocarcinoma and one squamous cell lines: Six colon carcinomas (HTC8, HT29, LS174T, SW116, SW948 and T48), 3 prostate carcinomas (LNCaP, DU-145 and PC-3) and 3 esophagus carcinoma cell lines (OE19, OE21 and OE33) were cultured under normoxic or hypoxic conditions (0% O2) for 24hours. Total RNA was extracted and gene expression levels measured by qPCR. For each tissue type, individual reference genes were selected and applied in the normalization of the relative expression levels. RESULTS: In all three tissue types, individual, optimal, reference genes were selected. In the analysis of the hypoxia induced genes, both the original reference genes and the new selected reference genes were used. There was no significant difference in the obtained data. The gene expression analysis demonstrated cell line specific differences in the hypoxia response of the 15 genes, with BNIP3 not being upregulated at hypoxic conditions in 3 out of 6 colon cancer cell lines, and ALDOA in OE21 and FAM162A and SLC2A1 in SW116 only showing limited hypoxia induction. Furthermore, in the esophagus cell lines, the normoxic and hypoxic expression levels of LOX and BNIP3 were below the detection limit in OE19 and OE33, respectively. However, a combined analysis of the 15 genes in both adenocarcinoma cell lines and squamous carcinoma cell lines demonstrated a very consistent expression pattern in hypoxic induced gene expression across all cell lines. CONCLUSION: This study addressed the tissue type dependency of hypoxia induced genes included in a 15-gene hypoxic profile in carcinoma cell lines from prostate, colon, and esophagus cancer, and demonstrated that in vitro, with minor fluctuations, the genes in the hypoxic profile are hypoxia inducible, and the hypoxia profile may be applicable in other sites than HNSCC.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Effect of radiation on cell proliferation and tumor hypoxia in HPV-positive head and neck cancer in vivo models.

BACKGROUND/AIM: Human papilloma virus-associated head and neck squamous cell carcinomas (HNSCC) represent a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. There is a range of unresolved questions regarding the different biology and clinical outcome of HPV-positive HNSCC. The purpose of the present project was to obtain insight into the biology of treatment responsiveness of HPV-related HNSCC. MATERIALS AND METHODS: Tumor xenografts were established from HPV-negative (FaDuDD,) and HPV-positive (UD2 and UMSCC47) HNSCC cell lines. Tumors were treated with 10 Gy or 20 Gy and the effect on the tumor microenvironment was studied at different time points after treatment. Cryosections were imaged for cell proliferation, hypoxia, vessel density and vessel perfusion. RESULTS: In the HPV-positive tumor models the levels of cell proliferation decreased significantly following irradiation. This was not seen in the HPV-negative model (FaDuDD). Furthermore, it was found that the tumor hypoxic fraction decreased over time after treatment in irradiated HPV-positive tumors and not in the HPV-negative tumors. CONCLUSION: The radiosensitivity previously observed in vitro could be applied in vivo in respect to a radiation-induced decrease in proliferating cells. A decreasing hypoxic fraction following irradiation in the HPV-positive tumors could explain the lack of benefit from hypoxic modifiers observed in patients.