Hypoxic Fraction Research Articles

Impact of SBRT fractionation in hypoxia dose painting - Accounting for heterogeneous and dynamic tumor oxygenation.

Tumor hypoxia, often found in nonsmall cell lung cancer (NSCLC), implies an increased resistance to radiotherapy. Pretreatment assessment of tumor oxygenation is, therefore, warranted in these patients, as functional imaging of hypoxia could be used as a basis for dose painting. This study aimed at investigating the feasibility of using a method for calculating the dose required in hypoxic subvolumes segmented on 18 F-HX4 positron emission tomography (PET) imaging of NSCLC. Positron emission tomography imaging data based on the hypoxia tracer 18 F-HX4 of 19 NSCLC patients were included in the study. Normalized tracer uptake was converted to oxygen partial pressure (pO2 ) and hypoxic target volumes (HTVs) were segmented using a threshold of 10mmHg. Uniform doses required to overcome the hypoxic resistance in the target volumes were calculated based on a previously proposed method taking into account the effect of interfraction reoxygenation, for fractionation schedules ranging from extremely hypofractionated stereotactic body radiotherapy (SBRT) to conventionally fractionated radiotherapy. Gross target volumes ranged between 6.2 and 859.6cm3 , and the hypoxic fraction<10mmHg between 1.2% and 72.4%. The calculated doses for overcoming the resistance of cells in the HTVs were comparable to those currently prescribed in clinical practice as well as those previously tested in feasibility studies on dose escalation in NSCLC. Depending on the size of the HTV and the distribution of pO2 , HTV doses were calculated as 43.6-48.4Gy for a three-fraction schedule, 51.7-57.6Gy for five fractions, and 59.5-66.4Gy for eight fractions. For patients in whom the HTV pO2 distribution was more favorable, a lower dose was required despite a bigger volume. Tumor control probability was lower for single-fraction schedules, while higher levels of tumor control probability were found for schedules employing several fractions. The method to account for heterogeneous and dynamic hypoxia in target volume segmentation and dose prescription based on 18 F-HX4-PET imaging appears feasible in NSCLC patients. The distribution of oxygen partial pressure within HTV could impact the required prescribed dose more than the size of the volume.

In Silico Model of Critical Cerebral Oxygenation after Traumatic Brain Injury: Implications for Rescuing Hypoxic Tissue.

Cerebral oxygen delivery is central to the modern intensive care of patients with severe traumatic brain injury. Low brain tissue oxygen tension (PbtO2) results from microvascular collapse and diffusion limitation and is associated with adverse outcome. A number of therapies to improve oxygen delivery are known to be effective in improving PbtO2. Their relative effectiveness and microscopic regions of hypoxia, however, may exist/persist even in the presence of normal PbtO2. Unfortunately, there are no methods currently for assessing this quantitatively. We used an in silico (computational) simulation approach to understand the effect of common interventions on the microscopic distribution of brain tissue oxygen tension. We constructed a non-linear mathematical model of cerebral oxygen supply, diffusion, and consumption for a simplified geometry. Model parameters were chosen to agree with clinical parameters. We found that it was possible to create a plausible diffusion-limited scenario with a significant hypoxic fraction by increasing the mean diffusion distance. We found that increasing cerebral blood flow/blood oxygen content or suppressing the cerebral metabolic rate were most effective at improving PbtO2 and reduced the hypoxic fraction. Within the limitations of our modeling assumptions, increasing the arterial oxygen partial pressure was less effective and only improved PbtO2 by creating a region of hyperoxic tissue with no improvement in hypoxic fraction. The in silico simulations can be useful in understanding the likely physiological effect of complex treatments for which measurement techniques do not exist.

DCE-MRI and Quantitative Histology Reveal Enhanced Vessel Maturation but Impaired Perfusion and Increased Hypoxia in Bevacizumab-Treated Cervical Carcinoma

This study had a dual purpose: to investigate (1) whether bevacizumab can change the microvasculature and oxygenation of cervical carcinomas and (2) whether any changes can be detected with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Two patient-derived xenograft models of cervical cancer (BK-12 and HL-16) were included in the study. Immunostained histologic preparations from untreated and bevacizumab-treated tumors were analyzed with respect to microvascular density, vessel pericyte coverage, and tumor hypoxia using CD31, α-SMA, and pimonidazole as markers, respectively. DCE-MRI was performed at 7.05T, and parametric images of Ktrans and ve were derived from the data using the Tofts pharmacokinetic model. The tumors of both models showed decreased microvascular density, increased vessel pericyte coverage, and increased vessel maturation after bevacizumab treatment. Bevacizumab-treated tumors were more hypoxic and had lower Ktrans values than untreated tumors in the BK-12 model, whereas bevacizumab-treated and untreated HL-16 tumors had similar hypoxic fractions and similar Ktrans values. Significant correlations were found between median Ktrans and hypoxic fraction, and the data for untreated and bevacizumab-treated tumors were well fitted by the same curve in both tumor models. Bevacizumab-treated tumors show less abnormal microvessels than untreated tumors do, but because of treatment-induced vessel pruning, the overall function of the microvasculature might be impaired after bevacizumab treatment, resulting in increased tumor hypoxia. DCE-MRI has great potential for monitoring bevacizumab-induced changes in tumor hypoxia in cervical carcinoma.

Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers.

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis- and autophagy signalling cascades are listed.

Fast and sensitive dynamic oxygen-enhanced MRI with a cycling gas challenge and independent component analysis.

There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r=0.91, p=0.0016) but did not correlate in HCT-116 tumors (r=0.13, p=0.81). Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.

18F-EF5 PET-based Imageable Hypoxia Predicts Local Recurrence in Tumors Treated with Highly Conformal Radiation Therapy

Tumor hypoxia contributes to radiation resistance. A non-invasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia targeted therapies. 18F-pentafluorinated etanidazole (18F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by EF5-PET with clinical outcomes after radiation therapy remains limited. We prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment EF5-PET imaging. Depending on the level of 18F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dicholoracetate (DCA), followed by repeat EF5-PET. The hypoxic fraction (HF) of tumor was defined as the proportion of tumor voxels exhibiting higher 18F-EF5 uptake than the 95th percentile of 18F-EF5 uptake in the blood pool. Tumors with HF ≥ 10% on baseline 18F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess correlation between imageable hypoxia and clinical outcomes after treatment. At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with SABR, and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while DCA did not result in significant change under our protocol conditions. Tumors with imageable hypoxia had higher incidence of local recurrence at 12 months (30%) compared to those without (0%) (p < 0.01). Noninvasive hypoxia imaging by EF5-PET identified imageable hypoxia in a substantial proportion of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia.

Combined MR Imaging of Oxygen Consumption and Supply Reveals Tumor Hypoxia and Aggressiveness in Prostate Cancer Patients.

The established role of hypoxia-induced signaling in prostate cancer growth, metastasis, and response to treatment suggests that a method to image hypoxia in tumors could aid treatment decisions. Here, we present consumption and supply-based hypoxia (CSH) imaging, an approach that integrates images related to oxygen consumption and supply into a single image. This integration algorithm was developed in patients with prostate cancer receiving hypoxia marker pimonidazole prior to prostatectomy. We exploited the intravoxel incoherent motion (IVIM) signal in diagnostic diffusion-weighted (DW) magnetic resonance (MR) images to generate separate images of the apparent diffusion coefficient (ADC) and fractional blood volume (fBV). ADC and fBV correlated with cell density (CD) and blood vessel density (BVD) in histology and whole-mount sections from 35 patients, thus linking ADC to oxygen consumption and fBV to oxygen supply. Pixel-wise plots of ADC versus fBV were utilized to predict the hypoxia status of each pixel in a tumor and to visualize the predicted value in a single image. The hypoxic fraction (HFDWI) of CSH images correlated strongly (R2 = 0.66; n = 41) with pimonidazole immunoscore (HSPimo); this relationship was validated in a second pimonidazole cohort (R2 = 0.54; n = 54). We observed good agreement between CSH images and pimonidazole staining in whole-mount sections. HFDWI correlated with tumor stage and lymph node status, consistent with findings for HSPimo Moreover, CSH imaging could be applied on histologic CD and BVD images, demonstrating transferability to a histopathology assay. Thus, CSH represents a robust approach for hypoxia imaging in prostate cancer that could easily be translated into clinical practice.Significance: These findings present a novel imaging strategy that indirectly measures tumor hypoxia and has potential application in a wide variety of solid tumors and other imaging modalities.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4774/F1.large.jpg Cancer Res; 78(16); 4774-85. ©2018 AACR.

PO-272 Optimisation of hypoxia regulated timed systems for genetic ablation and tracking of hypoxic breast cancer cells to study therapy resistance

IntroductionHypoxia plays a role in many types of cancer, and is linked to metastasis, genetic instability, resistance to therapy and poor prognosis. Hypoxic modification has been tested in clinical trials, but results have been inconclusive, in part due to a knowledge gap into the characterisation and behaviour of hypoxic tumour cells. In this project, hypoxic cells will be lineage traced to investigate their dissemination within the primary tumour and for their presence in circulating tumour cells and distant metastasis. To investigate the contribution of the hypoxic fraction to the tumour resistance, hypoxic cells will be specifically ablated, and the growth and response to treatment of tumours with and without this hypoxic population investigated.Material and methodsTo accomplish these aims, two complementary systems will be generated in the 4 T1 mouse mammary gland cell line, a model for stage IV metastatic human breast cancer. The first consists of a HIF-1α-CRE-ERTAM ‘hypoxia sensing’ construct coupled with a loxp DsRed loxp DTR-EGFP cassette (Diphtheria Toxin Receptor: DTR). Under normal conditions the HIF-1α-CRE-ERTAM fusion protein is expressed, but immediately degraded, but under hypoxia HIF-1α degradation is inhibited leading to accumulation. 4-OHT Administration translocates HIF-1α-CRE-ERTAM to the nucleus mediating loxp recombination in the coupled cassette. This allows lineage tracing of hypoxic cells and their progeny and selective ablation throught the DTR using Diphthteria toxin. Secondly, a DTR-UnaG-ODDD-HA construct (Oxygen-dependant degradation domain: ODDD), driven by the hypoxia dependent 5XHRE promoter will be used. This system allows ablation of hypoxic cells through the DTR and identification of hypoxic cells through the UnaG fluorescent protein. Only cells that are hypoxic at the time of administration/analysis will be labelled. Both cell lines will be transduced with luciferase to allow identification of distant metastasis.Results and discussionsThe two systems for this project have been generated and transduced into the 4 T1 cell line. Lineage tracing and selective ablation have been shown to work in vitro and clones are being characterised. In vitro experiments have shown the HIF-1α-CRE-ERTAM system to be extremely tightly regulated in an oxygen and tamoxifen dependant manner.ConclusionOnce optimised, both systems will be used in an in vivo orthotopic mouse model to investigate the role of the hypoxic cells in breast cancer, metastasis and response to radiotherapy.

Abstract 2440: Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids

Abstract Introduction: Chemotherapy is often limited by resistant subpopulations of tumor cells. Tumor hypoxia contributes to resistance of tumor cells to clinical drugs via multiple mechanisms. Hypoxia-activated prodrugs selectively target hypoxic cells, among which SN30000 is a second generation of tirapazamine analogue with superior diffusion coefficient and hypoxia selectivity. Here we hypothesize that SN30000/chemotherapy drug combinations will provide improved activity. Methods: HCT116 and SiHa spheroids were used to screen schedules of SN30000 in combination with 4 clinical drugs (cisplatin, doxorubicin, gemcitabine, paclitaxel) under 5% O2 and 20% O2, with endpoints of spheroid growth delay and clonogenic cell survival. The schedules were tested on HCT116 and SiHa monolayers under 5% O2. HCT116 monolayers and spheroids exposed to SN30000 or the clinical drugs were dissociated for clonogenic survival assay. Tumor growth delay using the same schedules of the most promising combination (SN30000 + gemcitabine) were then tested in HCT116 xenografts in NIH-III mice. Body weight loss and core body temperature of mice in response to the treatments were monitored. Results: Exposing to 25 µM SN30000 and 25 µM cisplatin/5 µM doxorubicin/10 µM gemcitabine simultaneously for 2 hr, and SN30000 3 hr before gemcitabine under 5% O2 significantly delayed HCT116 and SiHa spheroid growth, but their combined activity was compromised under 20% O2. Dosing SN30000 and cisplatin simultaneously, and SN30000 3 hr before gemcitabine significantly delayed HCT116 and SiHa monolayer growth. Clonogenic survival assay demonstrated that HCT116 monolayers were more sensitive than spheroids to doxorubicin, but not the other 3 clinical compounds, consistent with the known limited diffusion of doxorubicin into spheroids.Dosing SN30000 3 hr before gemcitabine or at the same time, but not SN30000 3 hr after gemcitabine significantly delayed tumor growth, which was consistent with the spheroid data. Body weight measurements showed that the combinations of SN30000 and gemcitabine added little additional toxicity while SN30000/doxorubicin was more toxic when administered simultaneously. SN30000 caused hypothermia in mice with recovery after 3 hr. Discussion: Screening schedules of SN30000 in combination with 4 clinical drugs in HCT116/SiHa spheroids and monolayers demonstrated schedule and oxygen dependence of the activity of SN30000 and gemcitabine combinations, which was further demonstrated in HCT116 xenografts. The mechanism of the promising interaction between gemcitabine and SN30000 is being further investigated by flow cytometry and immunohistochemical staining to explore the hypoxic fraction and cell proliferation following treatments, and by evaluating the plasma pharmacokinetics of SN30000 and gemcitabine to investigate the implications of SN30000-induced hypothermia. Citation Format: Xinjian Mao, Sarah McManaway, William Robert Wilson, Kevin Owen Hicks. Potentiation of the action of chemotherapeutic drugs by the hypoxia-activated prodrug SN30000 in multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2440.

Optical spectroscopic sensing of tumor hypoxia.

Tumor hypoxia is a critical indicator of poor clinical outcome in patients with cancers of the breast, cervix, and oral cavity. The ability to noninvasively and reliably monitor tumor oxygenation both prior to and during therapy can aid in identifying poor treatment response earlier than is currently possible and lead to effective changes in treatment regimen. Diffuse reflectance spectroscopy (DRS) has been used in several studies to measure tissue scattering, total hemoglobin content (THb), and vascular oxygenation (sO2) in tissue. In this study, we validate in vivo DRS-based measurements of vascular oxygenation using immunohistochemical staining of tumor hypoxia using pimonidazole, an established hypoxia marker. Using tumor xenografts grown from two different head and neck cell lines-UM-SCC-22B and UM-SCC-47-we demonstrate statistically significant negative correlations between tumor hypoxic fraction (HF) and THb (r = - 0.45; p = 0.04) and sO2 (r = - 0.50; p = 0.02). In addition, we also found a statistically significant positive correlation between HF and mean reduced scattering coefficient (r = 0.60; p = 0.005). Our results demonstrate that DRS-based measures of sO2 can provide reliable indirect measurements of tumor hypoxia that can be of significant utility in preclinical and clinical studies.

The Mitochondrial Citrate Carrier (SLC25A1) Sustains Redox Homeostasis and Mitochondrial Metabolism Supporting Radioresistance of Cancer Cells With Tolerance to Cycling Severe Hypoxia.

Pronounced resistance of lung cancer cells to radiotherapy and chemotherapy is a major barrier to successful treatment. Herein, both tumor hypoxia and the upregulation of the cellular antioxidant defense systems observed during malignant progression can contribute to radioresistance. We recently found that exposure to chronic cycling severe hypoxia/reoxygenation stress results in glutamine-dependent upregulation of cellular glutathione (GSH) levels and associated radiation resistance opening novel routes for tumor cell-specific radiosensitization. Here, we explored the role of the mitochondrial citrate carrier (SLC25A1) for the improved antioxidant defense of cancer cells with tolerance to acute and chronic severe hypoxia/reoxygenation stress and the use of pharmacologic SLC25A1 inhibition for tumor cell radiosensitization. Exposure to acute or chronic cycling severe hypoxia/reoxygenation stress triggered upregulated expression of SLC25A1 in lung cancer, prostate cancer, and glioblastoma cells in vitro. Interestingly, exposure to ionizing radiation (IR) further promoted SLC25A1 expression. Inhibition of SLC25A1 by 1,2,3-benzene-tricarboxylic acid (BTA) disturbed cellular and mitochondrial redox homeostasis, lowered mitochondrial metabolism, and reduced metabolic flexibility of cancer cells. Even more important, combining IR with BTA was able to overcome increased radioresistance induced by adaptation to chronic cycling severe hypoxia/reoxygenation stress. This radiosensitizing effect of BTA-treated cells was linked to increased reactive oxygen species and reduced DNA repair capacity. Of note, key findings could be reproduced when using the SLC25A1-inhibitor 4-Chloro-3-[[(3-nitrophenyl)amino]sulfonyl]-benzoic acid (CNASB). Moreover, in silico analysis of publically available databases applying the Kaplan–Meier plotter tool (kmplot.com) revealed that overexpression of SLC25A1 was associated with reduced survival of lung cancer patients suggesting a potential link to aggressive cancers. We show that SLC25A1 can contribute to the increased antioxidant defense of cancer cells allowing them to escape the cytotoxic effects of IR. Since upregulation of SLC25A1 is induced by adverse conditions in the tumor environment, exposure to IR, or both pharmacologic inhibition of SLC25A1 might be an effective strategy for radiosensitization of cancer cells particularly in chronically hypoxic tumor fractions.

Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation.

To examine the relationship between local oxygen partial pressure and energy metabolism in the tumor, electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) with hyperpolarized [1-13C] pyruvate were performed.SCCVII and HT29 solid tumors implanted in the mouse leg were imaged by EPRI using OX063, a paramagnetic probe and 13C-MRI using hyperpolarized [1-13C] pyruvate. Local partial oxygen pressure and pyruvate metabolism in the two tumor implants were examined. The effect of a single dose of 5-Gy irradiation on the pO2 and metabolism was also investigated by sequential imaging of EPRI and 13C-MRI in HT29 tumors.A phantom study using tubes filled with different concentration of [1-13C] pyruvate, [1-13C] lactate, and OX063 at different levels of oxygen confirmed the validity of this sequential imaging of EPRI and hyperpolarized 13C-MRI. In vivo studies revealed SCCVII tumor had a significantly larger hypoxic fraction (pO2 < 8 mmHg) compared to HT29 tumor. The flux of pyruvate-to-lactate conversion was also higher in SCCVII than HT29. The lactate-to-pyruvate ratio in hypoxic regions (pO2 < 8 mmHg) 24 hours after 5-Gy irradiation was significantly higher than those without irradiation (0.76 vs. 0.36) in HT29 tumor. The in vitro study showed an increase in extracellular acidification rate after irradiation.In conclusion, co-imaging of pO2 and pyruvate-to-lactate conversion kinetics successfully showed the local metabolic changes especially in hypoxic area induced by radiation therapy.

Positron Emission Tomography (PET) and Pharmacokinetics: Classical Blood Sampling Versus Image-Derived Analysis of [18F]FAZA and [18F]FDG in a Murine Tumor Bearing Model.

Pharmacokinetic (PK) data are generally derived from blood samples withdrawn serially over a defined period after dosing. In small animals, blood sampling after dosing presents technical difficulties, particularly when short time intervals and frequent sampling are required. Positron emission tomography (PET) is a non-invasive functional imaging technique that can provide semi-quantitative temporal data for defined volume regions of interest (vROI), to support kinetic analyses in blood and other tissues. The application of preclinical small-animal PET to determine and compare PK parameters for [18F]FDG and [18F]FAZA, radiopharmaceuticals used clinically for assessing glucose metabolism and hypoxic fractions, respectively, in the same mammary EMT6 tumor-bearing mouse model, is reported here. Two study groups were used: normal BALB/c mice under isoflurane anesthesia were intravenously injected with either [18F]FDG or [18F]FAZA. For the first group, blood-sampling by tail artery puncture was used to collect blood samples which were then analyzed with Radio-microTLC. Dynamic PET experiments were performed with the second group of mice and analyzed for blood input function and tumor uptake utilizing a modified two compartment kinetic model. Heart and inferior vena cava vROIs were sampled to obtain image-derived data. PK parameters were calculated from blood samples and image-derived data. Time-activity curves (TACs) were also generated over regions of liver, kidney and urinary bladder to depict clearance profiles for each radiotracer. PK values generated by classical blood sampling and PET image-derived analysis were comparable to each other for both radiotracers. Heart vROI data were suitable for analysis of [18F]FAZA kinetics, but metabolic uptake of radioactivity mandated the use of inferior vena cava vROIs for [18F]FDG analysis. While clearance (CL) and blood half-life (t½) were similar for both [18F]FDG and [18F]FAZA for both sampling methods, volume of distribution yielded larger differences, indicative of limitations such as partial volume effects within quantitative image-derived data. [18F]FDG underwent faster blood clearance and had a shorter blood half-life than [18F]FAZA. Kinetic analysis of tumor uptake from PET image data showed higher uptake and longer tumor tissue retention of [18F]FDG, indicative of the tumor's glucose metabolism rate, versus lower tumor uptake and retention of [18F]FAZA. While [18F]FAZA possesses a somewhat greater hepatobiliary clearance , [18F]FDG clears faster through the renal system which results in faster radioactivity accumulation in the urinary bladder. The present study provides a working example of the applicability of functional PET imaging as a suitable tool to determine PK parameters in small animals. The comparative analysis in the current study demonstrates that it is feasible to use [18F]FDG PET and [18F]FAZA PET in the same model to analyze their blood PK parameters, and to estimate kinetic parameters for these tracers in tumor. This non-invasive imaging-based determination of tissue kinetic parameters facilitates translation from pre-clinical to clinical phases of drug development.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Low dose-rate irradiation with [3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model

Background Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model. Methods Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [3H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography. Results Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [3H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue. Conclusion Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.

Measurement of Tumor Hypoxia in Patients With Locally Advanced Cervical Cancer Using Positron Emission Tomography with 18F-Fluoroazomyin Arabinoside

Toassess cervical tumor hypoxia using the hypoxia tracer 18F-fluoroazomycin arabinoside (18F-FAZA) and compare different reference tissues and thresholds for quantifying tumor hypoxia. Twenty-seven patients with cervical cancer were studied prospectively by positron emission tomography (PET) imaging with 18F-FAZA before starting standard chemoradiation. The hypoxic volume was defined as all voxels within a tumor (T) with standardized uptake values (SUVs) greater than 3 standard deviations from the mean gluteus maximus muscle SUV value (M) or SUVs greater than 1 to 1.4 times the mean SUV value of the left ventricle, a blood (B) surrogate. The hypoxic fraction was defined as the ratio of the number of hypoxic voxels to the total number of tumor voxels. A 18F-FAZA-PET hypoxic volume could be identified in the majority of cervical tumors (89% when using T/M or T/B>1.2 as threshold) on the 2-hour static scan. The hypoxic fraction ranged from 0% to 99% (median 31%) when defined using the T/M threshold and from 0% to 78% (median 32%) with the T/B>1.2 threshold. Hypoxic volumes derived from the different thresholds were highly correlated (Spearman's correlation coefficient ρ between T/M and T/B>1-1.4 were 0.82-0.91), as were hypoxic fractions (0.75-0.85). Compartmental analysis of the dynamic scans showed k3, the FAZA accumulation constant, to be strongly correlated with hypoxic fraction defined using the T/M (Spearman's ρ=0.72) and T/B>1.2 thresholds (0.76). Hypoxia was detected in the majority of cervical tumors on 18F-FAZA-PET imaging. The extent of hypoxia varied markedly between tumors but not significantly with different reference tissues/thresholds.

DCE-MRI of patient-derived xenograft models of uterine cervix carcinoma: associations with parameters of the tumor microenvironment

BackgroundAbnormalities in the tumor microenvironment are associated with resistance to treatment, aggressive growth, and poor clinical outcome in patients with advanced cervical cancer. The potential of dynamic contrast-enhanced (DCE) MRI to assess the microvascular density (MVD), interstitial fluid pressure (IFP), and hypoxic fraction of patient-derived cervical cancer xenografts was investigated in the present study.MethodsFour patient-derived xenograft (PDX) models of squamous cell carcinoma of the uterine cervix (BK-12, ED-15, HL-16, and LA-19) were subjected to Gd-DOTA-based DCE-MRI using a 7.05 T preclinical scanner. Parametric images of the volume transfer constant (Ktrans) and the fractional distribution volume (ve) of the contrast agent were produced by pharmacokinetic analyses utilizing the standard Tofts model. Whole tumor median values of the DCE-MRI parameters were compared with MVD and the fraction of hypoxic tumor tissue, as determined histologically, and IFP, as measured with a Millar catheter.ResultsBoth on the PDX model level and the single tumor level, a significant inverse correlation was found between Ktrans and hypoxic fraction. The extent of hypoxia was also associated with the fraction of voxels with unphysiological ve values (ve > 1.0). None of the DCE-MRI parameters were related to MVD or IFP.ConclusionsDCE-MRI may provide valuable information on the hypoxic fraction of squamous cell carcinoma of the uterine cervix, and thereby facilitate individualized patient management.

147 - Quantitative pO2 Mapping Using Electron Paramagnetic Resonance Imaging in Orthotopic Murine Glioma

Glioblastoma is the most frequent primary malignant brain tumor and its malignant nature is closely associated with hypoxia. Thus, it would be useful for treatment of glioblastomas to evaluate intratumoral distribution of hypoxia for determining the therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI), is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl. In this study, we examined the feasibility of EPRI for assessment of oxygen distribution in the orthotopic glioblastoma models of U251 and U87. Heterogeneous distribution of pO2 between 0 and 50 mmHg was observed throughout the tumors except for the normal brain tissue. U251 glioblastoma was more likely to be exposed to hypoxia than U87 (median pO2; 29.7 mmHg and 18.2 mmHg, P = 0.028, in U87 and U251, respectively). The ratio of hypoxic fraction where pO2 was less than 10 mmHg showed a good correlation with pimonidazole-positive region. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared to U87, suggesting the high population of hypoxic cells. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumor.

Combined endogenous MR biomarkers to predict basal tumor oxygenation and response to hyperoxic challenge.

Hypoxia is a common feature of solid tumors, which translates into increased angiogenesis, malignant phenotype cell selection, change in gene expression and greater resistance to radiotherapy and chemotherapy. Therefore, there is a need for markers of hypoxia to stratify patients, in order to personalize treatment to improve therapeutic outcome. However, no modality has yet been validated for the screening of hypoxia in routine clinical practice. Magnetic resonance imaging (MRI) R1 and R2 * relaxation parameters are sensitive to tissue oxygenation: R1 is sensitive to dissolved oxygen and R2 * is sensitive to intravascular deoxyhemoglobin content. Two rat tumor models with distinct levels of hypoxia, 9L-glioma and rhabdomyosarcoma, were imaged for R1 and R2 * under air and carbogen (95% O2 and 5% CO2 ) breathing conditions. It was observed that the basal tumor oxygenation level had an impact on the amplitude of response to carbogen in the vascular compartment (R2 *), but not in the tissue compartment (R1 ). In addition, the change in tissue oxygenation estimated by ΔR1 correlated with the change in vascular oxygenation estimated by ΔR2 *, which is consistent with an increase in oxygen supply generating an elevated tumor pO2 . At the intra-tumoral level, we identified four types of voxel to which a hypoxic feature was attributed (mild hypoxia, severe hypoxia, normoxia and vascular steal), depending on the carbogen-induced change in R1 and R2 * values for each voxel. The results showed that 9L-gliomas present more normoxic fractions, whereas rhabdomyosarcomas present more hypoxic fractions, which is in accordance with a previous study using 18 F-fluoroazomycin arabinoside (18 F-FAZA) and electron paramagnetic resonance (EPR) oximetry. The response of the combined endogenous MRI contrasts to carbogen challenge could be a useful tool to predict different tumor hypoxic fractions.

Biomarker for Radiation Therapy: The Relationship Between Tissue Oxygenation and Redox Status

Tumor hypoxia is prognostic for poor response to cancer therapy. The prognostic value of tumor hypoxia has been demonstrated in radiotherapy studies of human head and neck and uterine cervix tumors, which show that patients with less hypoxic tumors had a better chance of overall or disease-free survival than did patients with more hypoxic tumors. Furthermore, some chemotherapeutic agents such as bleomycin and doxorubicin exhibit a cytotoxicity that is strongly oxygen dependent, suggesting that hypoxia may be a prognostic factor for chemotherapeutic response as well. Therefore, development of methods allowing measurement of hypoxia in human patients may allow physicians to better manage tumors that exhibit considerable hypoxia. The recent development of a bi-modality magnetic resonance imaging/electron paramagnetic resonance imaging (MRI/EPRI) platform has enabled longitudinal monitoring of both tumor oxygenation and redox status in murine cancer models. The current study used this imaging platform to test the hypothesis that a more reducing tumor microenvironment accompanies the development of tumor hypoxia. To test this, the redox status of the tumor was measured using Tempol as a redox-sensitive MRI contrast agent, and tumor hypoxia was measured with Oxo63, which is an oxygen-sensitive EPRI spin probe. Images were acquired every 1-2 days in mice bearing SCCVII tumors. The median pO2 decreased from 14 mmHg at 7 days after tumor implantation to 7 mmHg at 15 days after implantation. Additionally, the hypoxic fraction, defined as the percentage of the tumor that exhibited a pO2<10 mmHg, increased with tumor size (from 10% at 500 mm3 to 60% at 3,500 mm3). The rate of Tempol reduction increased as a function of tumor volume (0.4 min-1 at 500 mm3 to 1.7 min-1 at 3,500 mm3), suggesting that the tumor microenvironment became more reduced as the tumor grew. The results show that rapid Tempol reduction correlates with decreased tumor oxygenation, and that the Tempol decay rate constant may be a surrogate marker for tumor hypoxia. Nitroxides such as Tempol may provide a clinically feasible magnetic resonance imaging based assay of hypoxia for radiotherapy.

Neoadjuvant olaparib targets hypoxia to improve radioresponse in a homologous recombination-proficient breast cancer model.

Clinical trials are studying the benefits of combining the PARP-1 inhibitor olaparib with chemotherapy and radiotherapy treatment in a variety of cancer increasing the therapeutic ratio for olaparib may come from its ability to modify the tumour microenvironment by targeting homologous recombination-deficient, hypoxic tumour clonogens, and/or increasing tumour-associated vasodilation to improve oxygenation. Herein, we investigated the effect of prolonged neoadjuvant exposure to olaparib on the tumor microenvironment using a genetically-engineered mouse p53−/− syngeneic breast cancer model, which is proficient in homology-directed DNA repair. We observed increased in vivo growth delay and decreased ex vivo clonogenic survival following pre-treatment with olaparib 50 mg/kg bid Olaparib for 7 days ending 48 hours prior to a radiation dose of 12Gy. This increased in vivo radioresponse was associated with a decreased hypoxic fraction. This study suggests that the radiation response in patients can be improved with limited toxicity if olaparib is given in a purely neoadjuvant setting to modify the tumor microenviroment prior to the start of the radiotherapy treatment. Consequently a significant gain can be achieved in therapeutic window and clinical studies are needed to confirm this preclinical data.