Tumor Oxygenation Status Research Articles

Master Role of Hypoxia in Cancer Progression: Major Insights During ISOTT's Half-Century.

On the occasion of ISOTT's half-century, in this chapter, tumour hypoxia (i.e. critically reduced oxygen levels on macro- and microscopic scales), recently classified as an additional hallmark of cancer, its various aetiology-related classifications (diffusion- or perfusion-limitations, hypoxaemic), time-frames of exposure (acute, chronic, cyclic), and different levels (moderate, mild, severe) within and across tumours, and its Janus-face-like role ("dichotomy") in tumour regression (e.g. apoptosis, necrosis) versus "adaptive" tumour progression have been updated and summarised. This latter knowledge is, to a great extent, based on (a) direct, reliable assessments and mapping of the heterogeneous tumour oxygenation status using minimally invasive polarographic pO2 microsensors in clinical settings since the late 1980s, and (b)the discovery of the hypoxia-inducible factors (HIFs) in the early 1990s. These data have clarified the role of hypoxia in stimulating a variety of biologic responses that mediate cancer progression through changes (a) in the genome (associated with clonal selection and expansion), (b) in the transcriptome, and (c) in the proteome, as well as its role as a barrier to the effectiveness of anti-cancer therapies.

Feasibility and sensitivity study of radiomic features in photoacoustic imaging of patient-derived xenografts

Photoacoustic imaging is an increasingly popular method of exploring the tumour microenvironment, which can provide insight into tumour oxygenation status and potentially treatment response assessment. Currently, the measurements most commonly performed on such images are the mean and median of the pixel values of the tumour volumes of interest. We investigated expanding the set of measurements that can be extracted from these images by adding radiomic features. In particular, we found that Skewness was sensitive to differences between basal and luminal patient derived xenograft cancer models with an eta ^2 of 0.86, and that it was robust to variations in confounding factors such as reconstruction type and wavelength. We also built discriminant models with radiomic features that were correlated with the underlying tumour model and were independent from each other. We then ranked features by their importance in the model. Skewness was again found to be an important feature, as were 10th Percentile, Root Mean Squared, and several other texture-based features. In summary, this paper proposes a methodology to select radiomic features extracted from photoacoustic images that are robust to changes in acquisition and reconstruction parameters, and discusses features found to have discriminating power between the underlying tumour models in a pre-clinical dataset.

Evaluation of Betulinic Acid Derivatives as PET Tracers for Hypoxia-Induced Carbonic Anhydrase IX (CA IX) Expression.

Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.

Improved Oxygenation of Human Skin, Subcutis and Superficial Cancers Upon Mild Hyperthermia Delivered by WIRA-Irradiation.

Clinical trials have shown that mild hyperthermia (HT) serves as an adjunct to cancer treatments such as chemo- and radiotherapy. Recently, a high efficacy of mild HT immediately followed by hypofractionated radiotherapy (RT) in treatment of recurrent breast cancer has been documented if temperatures of 39-43°C are achieved for 40-60 min. In the present study, temperature and oxygenation profiles were measured in superficial tissues of healthy volunteers exposed to water-filtered infrared-A- (wIRA)- irradiation, to verify that adequate thermal doses together with the improved tumor oxygenation necessary for radiosensitisation are obtained. Experiments were performed using a wIRA-system equipped with two wIRA-radiators, each with a thermography camera for real-time monitoring of the skin surface temperature. Temperatures within the abdominal wall were measured with fibre optic sensors at defined tissue depths (subepidermal, and 1-20 mm inside the tissue). The corresponding tissue pO2 values were assessed with fluorometric microsensors. In selected situations, hyperspectral tissue imaging was used to visualise the oxygenation status of normal skin and superficial tumours in patients. Pre-treatment skin surface temperature was 34.6°C. Upon wIRA exposure, average skin surface temperatures reached 41.6°C within 5-12 min. Maximum tissue temperatures of 41.8°C were found at a tissue depth of 1mm, with a steady decline in deeper tissue layers (41.6°C @ 5mm, 40.8°C @ 10mm, 40.6°C @ 15mm, and 40.1°C @ 20mm). Effective HT levels ≥39°C were established in tissue depths up to 25 mm. Tissue heating was accompanied by a significant increase in tissue pO2 values [e.g., at a tissue depth of 13 mm mean pO2 rose from 46mmHg to 81mmHg (@ T=40.5°C). In the post-heating phase (+ 5min), pO2 was 79mmHg (@ T=38°C) and 15 min post-heat pO2 was 72mmHg (@ T=36.8°C)]. pO2 values remained elevated for 30-60 min post-heat. Non-invasive monitoring of normal skin and of recurrent breast cancers confirmed the improved O2 status by wIRA-HT. In conclusion, wIRA-irradiation enables effective tissue heating (T=39-43°C) associated with distinct increases in blood flow and pO2. These adjustments unequivocally meet the requirement for effective radiosensitisation.

Assessment of hypoxia by pimonidazole staining following radiotherapy.

The rapid proliferation of cancer cells and the aberrant vasculature present in most solid tumors frequently result in the lack of oxygen generating a hypoxic tumor microenvironment. Low levels of oxygen not only affect the tumor cell biology and tumorigenesis, but also the other components of the tumor microenvironment such as the tumor stroma and the immune infiltrate, promoting a more suppressive environment. In addition, tumor hypoxia has been associated with reduced sensitivity to chemotherapy (CH) and radiotherapy (RT), leading to poor outcomes in cancer patients. Therefore, the evaluation of tumor oxygen status has become clinically relevant. Tumor hypoxia can be assessed by different methods that include the analysis of the oxygen concentration or the expression of endogenous markers directly related to hypoxia. In this paper, we focus on the use of the hypoxia-specific marker pimonidazole as a straightforward way to measure tumor hypoxia following radiotherapy in a preclinical melanoma model.

Correction to: Oxygenation Status of Malignant Tumors vs. Normal Tissues: Critical Evaluation and Updated Data Source Based on Direct Measurements with pO2 Microsensors

Correction to: Oxygenation Status of Malignant Tumors vs. Normal Tissues: Critical Evaluation and Updated Data Source Based on Direct Measurements with pO2 Microsensors

Assessment of Hypoxic Tissue Fraction and Prediction of Survival in Cervical Carcinoma by Dynamic Contrast-Enhanced MRI.

Tumor hypoxia is a major cause of treatment resistance and poor survival in locally-advanced cervical carcinoma (LACC). It has been suggested that K trans and v e maps derived by dynamic contrast-enhanced magnetic resonance imaging can provide information on the oxygen supply and oxygen consumption of tumors, but it is not clear whether and how these maps can be combined to identify tumor hypoxia. The aim of the current study was to find the optimal strategy for calculating hypoxic fraction and predicting survival from K trans and v e maps in cervical carcinoma. K trans and v e maps of 98 tumors of four patient-derived xenograft models of cervical carcinoma as well as 80 patients with LACC were investigated. Hypoxic fraction calculated by using K trans maps correlated strongly (P < 0.0001) to hypoxic fraction assessed with immunohistochemistry using pimonidazole as a hypoxia marker and was associated with disease-free and overall survival in LACC patients. Maps of v e did not provide information on hypoxic fraction and patient outcome, and combinations of K trans and v e were not superior to K trans alone for calculating hypoxic fraction. These observations imply that K trans maps reflect oxygen supply and may be used to identify hypoxia and predict outcome in cervical carcinoma, whereas v e is a poor parameter of oxygen consumption and does not provide information on tumor oxygenation status.

Polymerized human hemoglobin facilitated modulation of tumor oxygenation is dependent on tumor oxygenation status and oxygen affinity of the hemoglobin-based oxygen carrier

Administration of hemoglobin-based oxygen carriers (HBOCs) into the systemic circulation is a potential strategy to relieve solid tumor hypoxia in order to increase the effectiveness of chemotherapeutics. Previous computational analysis indicated that the oxygen (O2) status of the tumor and HBOC O2 affinity may play a role in increased O2 delivery to the tumor. However, no study has experimentally investigated how low- and high-affinity HBOCs would perform in normoxic and hypoxic tumors. In this study, we examined how the HBOC, polymerized human hemoglobin (PolyhHb), in the relaxed (R) or tense (T) quaternary state modulates O2 delivery to hypoxic (FME) and normoxic (LOX) human melanoma xenografts in a murine window chamber model. We examined microcirculatory fluid flow via video shearing optical microscopy, and O2 distributions via phosphorescence quenching microscopy. Additionally, we examined how weekly infusion of a 20% top-load dose of PolyhHb influences growth rate, vascularization, and regional blood flow in the FME and LOX tumor xenografts. Infusion of low-affinity T-state PolyhHb led to increased tissue oxygenation, decreased blood flow, decreased tumor growth, and decreased vascularization in hypoxic tumors. However, infusion of both T-state and R-state PolyhHbs led to worse outcomes in normoxic tumors. Of particular concern was the high-affinity R-state PolyhHb, which led to no improvement in hypoxic tumors and significantly worsened outcomes in normoxic tumors. Taken together, the results of this study indicate that the tumor O2 status is a primary determinant of the potency and outcomes of infused PolyhHb.

Molecular Imaging of the Tumor Microenvironment Reveals the Relationship between Tumor Oxygenation, Glucose Uptake, and Glycolysis in Pancreatic Ductal Adenocarcinoma.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. SIGNIFICANCE: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

Hyperoxygenation as a Therapeutic Supplement for Treatment of Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) refers to a group of biologically aggressive breast cancers that do not express estrogen, progesterone or epidermal growth factor receptor 2 hormone receptors. Each subset of TNBC has a unique molecular profile and may require specific treatments. A combination of surgery and chemotherapy followed by radiation therapy is the standard treatment mode for TNBC patients. Tumor oxygen status (hypoxia) is a key factor that may compromise the effectiveness of radiation treatment, as it is known that hypoxia can confer radiation resistance. In this study, we characterized MDA-MB-231 orthotropic xenograft tumors with respect to tumor oxygen level and their response to supplemental oxygen therapy in combination with paclitaxel and radiation therapy. We observed that the TNBC tumors became severely hypoxic (pO2 < 4 mmHg) within 1 week of tumor growth and responded poorly to administration of respiratory hyperoxygenation (100% O2) to mitigate hypoxia. However, periodic administration of supplemental oxygen (100% O2; 60 min/day for 21 days) showed a significant inhibitory effect on tumor volume when compared to control (1,023 ± 32 mm3 vs. 1,378 ± 114 mm3; p < 0.05). Combination of supplemental oxygen with paclitaxel and radiation therapy led to a significant reduction in tumor growth when compared to radiation alone (239 ± 40 mm3 vs. 390 ± 32 mm3; p < 0.05). The therapeutic enhancement by supplemental oxygen is possibly attributed to increase in tumor oxygenation with paclitaxel at the time of radiation treatment. These findings may have important implications in the understanding of the role of oxygen and supplemental oxygen therapy for the treatment of TNBC patients.

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.

Effect of HNSCC Radiochemotherapy on Imaging Biomarker T2* MRI and its Relation to FMISO-PET Derived Hypoxia

Previous studies using hypoxia PET have shown significant reduction of tumor hypoxia (reoxygenation) during primary radiochemotherapy (RCT) of HNSCC and a significant correlation between reoxygenation on hypoxia PET and local control. Assessment of tumor hypoxia using MRI would offer independence from hypoxia PET tracer availability. The apparent MRI transverse relaxation time T2* has been proposed as an imaging biomarker and as a potential surrogate for hypoxia PET. The aim of this study was to assess the effect of primary RCT on T2* at an early (week 2) and late (week 5) time point during RCT and to analyse the relation between T2* and established hypoxia PET tracer 18F-misonidazole PET/CT (FMISO PET) and standard 18F-FDG PET/CT (FDG PET). In 10 T2-4N+ HNSCC patients, FDG PET was obtained at baseline and repeat FMISO PET and 3 Tesla MRI T2* in weeks 0, 2 and 5. MRI gross tumor volumes for tumor and lymph nodes (GTV-T, GTV-LN) were contoured and FMISO PET derived hypoxic tumor/lymph node subvolumes (HSV-T, HSV-LN) and complementary non-hypoxic subvolumes (nonHSV-T, nonHSV-LN) were generated using a threshold level of 1.4 times the mean SUV FMISO within muscle. Volumes were contoured for all time points (week 0, 2 and 5) individually and mean values for T2* and SUVmean FDG PET were obtained. Within GTVs (T, LN), r2 (FMISO to T2*) was calculated for [FMISO SUVmax GTV/SUVmean muscle] to [T2* mean GTV] and r2 (FMISO to FDG) was calculated for [FMISO SUVmax GTV/SUVmean muscle] to [FDG SUVmax GTV/SUVmean muscle]. From week 0 to 5 GTV-T and GTV-LN decreased by -56% and -63%, respectively and HSV-T and HSV-LN nearly completely resolved (n = 10). Mean T2* signal showed no significant change for GTV-T or GTV-LN. Within HSV-T mean T2* values were smaller compared to nonHSV-T: 15.0+/-4.6 vs. 18.3+/-2.9 (p = 0.051), whereas FDG SUVmean was significantly higher within hypoxic as compared to non-hypoxic regions: HSV-T 12.1+/-5.5 vs. nonHSV-T 6.1+/-2.6 and HSV-LN 10.2+/-3.9 vs. nonHSV-LN 4.7+/-1.9 (week 0, p≤0.026 and p≤0.011). Correlation between FMISO PET and FDG PET was higher than between FMSIO PET and T2*: r2 for GTV-T (FMISO/FDG)=0.81, r2 for GTV-T (FMISO/T2*)=0.32. Marked reduction of tumor hypoxia between week 0, 2 and 5 found on FMISO PET was not accompanied by a significant T2*change within GTVs over time. At baseline, smaller T2* values and significantly larger FDG SUVmean were found within HSV-T as compared to nonHSV-T. These results suggest a relation between tumor oxygenation status and T2* at baseline, however with the correlation coefficient r2 FMISO/T2* being lower than r2 FMISO/FDG. Overall, T2* quantitation was feasible and the findings on T2* imaging warrant further investigations but do not indicate a simple surrogate role for T2* as hypoxia imaging marker due to lack of correlation to established hypoxia marker FMISO PET. Our findings on reduction of FMISO uptake during RCT were in line with previous reports.

Effect of radiochemotherapy on T2* MRI in HNSCC and its relation to FMISO PET derived hypoxia and FDG PET

BackgroundTo assess the effect of radiochemotherapy (RCT) on proposed tumour hypoxia marker transverse relaxation time (T2*) and to analyse the relation between T2* and 18F-misonidazole PET/CT (FMISO-PET) and 18F-fluorodeoxyglucose PET/CT (FDG-PET).MethodsTen patients undergoing definitive RCT for squamous cell head-and-neck cancer (HNSCC) received repeat FMISO- and 3 Tesla T2*-weighted MRI at weeks 0, 2 and 5 during treatment and FDG-PET at baseline. Gross tumour volumes (GTV) of tumour (T), lymph nodes (LN) and hypoxic subvolumes (HSV, based on FMISO-PET) and complementary non-hypoxic subvolumes (nonHSV) were generated. Mean values for T2* and SUVmean FDG were determined.ResultsDuring RCT, marked reduction of tumour hypoxia on FMISO-PET was observed (T, LN), while mean T2* did not change significantly. At baseline, mean T2* values within HSV-T (15 ± 5 ms) were smaller compared to nonHSV-T (18 ± 3 ms; p = 0.051), whereas FDG SUVmean (12 ± 6) was significantly higher for HSV-T (12 ± 6) than for nonHSV-T (6 ± 3; p = 0.026) and higher for HSV-LN (10 ± 4) than for nonHSV-LN (5 ± 2; p ≤ 0.011). Correlation between FMISO PET and FDG PET was higher than between FMSIO PET and T2* (R2 for GTV-T (FMISO/FDG) = 0.81, R2 for GTV-T (FMISO/T2*) = 0.32).ConclusionsMarked reduction of tumour hypoxia between week 0, 2 and 5 found on FMISO PET was not accompanied by a significant T2*change within GTVs over time. These results suggest a relation between tumour oxygenation status and T2* at baseline, but no simple correlation over time. Therefore, caution is warranted when using T2* as a substitute for FMISO-PET to monitor tumour hypoxia during RCT in HNSCC patients.Trial registrationDRKS,DRKS00003830. Registered 23.04.2012.

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.

Matching the reaction-diffusion simulation to dynamic [18F]FMISO PET measurements in tumors: extension to a flow-limited oxygen-dependent model

Positron-emission tomography (PET) with hypoxia specific tracers provides a noninvasive method to assess the tumor oxygenation status. Reaction-diffusion models have advantages in revealing the quantitative relation between in vivo imaging and the tumor microenvironment. However, there is no quantitative comparison of the simulation results with the real PET measurements yet. The lack of experimental support hampers further applications of computational simulation models. This study aims to compare the simulation results with a preclinical [18F]FMISO PET study and to optimize the reaction-diffusion model accordingly. Nude mice with xenografted human squamous cell carcinomas (CAL33) were investigated with a 2 h dynamic [18F]FMISO PET followed by immunofluorescence staining using the hypoxia marker pimonidazole and the endothelium marker CD 31. A large data pool of tumor time-activity curves (TAC) was simulated for each mouse by feeding the arterial input function (AIF) extracted from experiments into the model with different configurations of the tumor microenvironment. A measured TAC was considered to match a simulated TAC when the difference metric was below a certain, noise-dependent threshold. As an extension to the well-established Kelly model, a flow-limited oxygen-dependent (FLOD) model was developed to improve the matching between measurements and simulations. The matching rate between the simulated TACs of the Kelly model and the mouse PET data ranged from 0 to 28.1% (on average 9.8%). By modifying the Kelly model to an FLOD model, the matching rate between the simulation and the PET measurements could be improved to 41.2–84.8% (on average 64.4%). Using a simulation data pool and a matching strategy, we were able to compare the simulated temporal course of dynamic PET with in vivo measurements. By modifying the Kelly model to a FLOD model, the computational simulation was able to approach the dynamic [18F]FMISO measurements in the investigated tumors.

Tumor Oxygenation Status: Facts and Fallacies.

In this chapter we allude to a series of facts and fallacies often encountered in the description of tumor hypoxia, a relevant trait of the tumor microenvironment and a paramount driver of tumor aggressiveness and treatment resistance. The critical role of diffusion distances, terminological inconsistencies considering O2 partial pressures vs. O2 concentrations and with it the use of inept units, the impact of O2 depletion on proliferation and cell viability, the switch in the Warburg dogma , the distribution of hypoxic subvolumes within a tumor, the involvement of O2 diffusion shunts in the development of chronic hypoxia, and the role of endogenous biomarkers as surrogates for the assessment of hypoxia are discussed in more detail. Special emphasis is put on the clinical relevance of these misconceptions and misinterpretations and their impact on the assessment of hypoxia as well as hypoxia-targeted treatment planning.

Special Issue: Selected papers from the Topical Problems of Biophotonics conference

The ensuing seven papers represent the sampling of the science presented at the Topical Problems in Biophotonics (TPB) conference that took place aboard a cruise ship on the river Volga and its tributaries in July 2015. This bi-annual gathering of active researchers and clinicians in the field of biomedical optics has, over the last decade, become globally renowned for its high-quality science, ample discussion opportunities, informal atmosphere, active participation of experienced and novice researchers alike, beautiful background scenery of the Russian “heartland”, and establishment of both scientific and personal connections and collaborations. It was the fifth conference organized by scientific and educational institutions of Nizhny Novgorod (Institute of Applied Physics RAS, Nizhny Novgorod State Medical Academy, and Nizhny Novgorod State University). We are grateful to the Journal of Biophotonics for providing the opportunity to showcase this special conference. The breadth and depth of TPB's science was reflected in its three sub-conferences (Optical BioImaging, Nanobiophotonics, and Novel Laser Bio-Applications) and two workshops (Clinical Biophotonics and Tumour & Stem Cell Biology). These seven representative Journal of Biophotonics papers were selected from various tracks of the conference, have undergone the journal's rigorous peer-review process, and span the range from volumetric optical imaging to novel contrast agents to advanced microscopies. Specifically, Liu et al. 1 report on a mouse study using optical coherence tomography (OCT) to enable high diagnostic accuracy for glomerulonephritis based on structural and elastic tissue parameters. Wang et al. 2 employed Doppler OCT for four-dimensional live monitoring of hemodynamics in a mammalian embryonic heart with quantification of blood flow velocity. Yashchenok et al. 3 describe the fabrication methodology of polyelectrolyte microcapsules characterized by very high absorption in NIR, with promising potential as contrast agents for both photoacoustic imaging and for therapy (theranostics). Another class of photonic contrast agents are nanodiamonds that are amenable to bio-functionalization; their nanotoxicity in a zebrafich embryo model is examined by Lin et al. 4. Razali et al. 5 report on imaging photoluminescent nanoruby probes with wide-field time-gated photoluminescence microscopy, demonstrating significant increase in imaging contrast. Kalinina et al. 6 present an approach to mapping cell metabolic parameters and pO2 by combined use of FLIM and PLIM, demonstrating an attractive imaging platform for ex vivo monitoring of tumor oxygen status (a key determinant for proper treatment strategy selection). In vivo monitoring of oxygen saturation in the brain by NIR spectroscopy provides unique opportunities for non-invasive brain disorder diagnostics and monitoring; thus, Clancy et al. 7 present MRI-based numerical simulation of NIR spectroscopy towards improving the accuracy of quantitative mapping of oxygen saturation in brain. As seen from this brief overview of the selected titles, research in biophotonics presented at TPB-15 is interesting and exciting indeed. On behalf of the TPB's organizing committee and its enthusiastic attendees, we wish you pleasant “light” reading of these papers and look forward to your active participation in TPB-17!

ERRα-Regulated Lactate Metabolism Contributes to Resistance to Targeted Therapies in Breast Cancer.

Imaging studies in animals and in humans have indicated that the oxygenation and nutritional status of solid tumors is dynamic. Furthermore, the extremely low level of glucose within tumors, while reflecting itsrapid uptake and metabolism, also suggests that cancer cells must rely on other energy sources in some circumstances. Here, we find that some breast cancer cells can switch to utilizing lactate as a primary source of energy, allowing them to survive glucose deprivation for extended periods, and that this activity confers resistance to PI3K/mTOR inhibitors. The nuclear receptor, estrogen-related receptor alpha (ERRα), was shown to regulate the expressionof genes required for lactate utilization, and isotopomer analysis revealed that genetic or pharmacological inhibition of ERRα activity compromised lactate oxidation. Importantly, ERRα antagonists increased the invitro and invivo efficacy of PI3K/mTOR inhibitors, highlighting the potential clinical utility of this drug combination.

Towards Human Oxygen Images with Electron Paramagnetic Resonance Imaging.

Electron paramagnetic resonance imaging (EPRI) has been used to noninvasively provide 3D images of absolute oxygen concentration (pO2) in small animals. These oxygen images are well resolved both spatially (~1 mm) and in pO2 (1-3 mmHg). EPRI preclinical images of pO2 have demonstrated extremely promising results for various applications investigating oxygen related physiologic and biologic processes as well as the dependence of various disease states on pO2, such as the role of hypoxia in cancer. Recent developments have been made that help to progress EPRI towards the eventual goal of human application. For example, a bimodal crossed-wire surface coil has been developed. Very preliminary tests demonstrated a 20 dB isolation between transmit and receive for this coil, with an anticipated additional 20 dB achievable. This could potentially be used to image local pO2 in human subjects with superficial tumors with EPRI. Local excitation and detection will reduce the specific absorption rate limitations on images and eliminate any possible power deposition concerns. Additionally, a large 9 mT EPRI magnet has been constructed which can fit and provide static main and gradient fields for imaging local anatomy in an entire human. One potential obstacle that must be overcome in order to use EPRI to image humans is the approved use of the requisite EPRI spin probe imaging agent (trityl). While nontoxic, EPRI trityl spin probes have been injected intravenously when imaging small animals, and require relatively high total body injection doses that would not be suitable for human imaging applications. Work has been done demonstrating the alternative use of intratumoral (IT) injections, which can reduce the amount of trityl required for imaging by a factor of 2000- relative to a whole body intravenous injection. The development of a large magnet that can accommodate human subjects, the design of a surface coil for imaging of superficial pO2, and the reduction of required spin probe using IT injections all are crucial steps towards the eventual use of EPRI to image pO2 in human subjects. In the future this can help investigate the oxygenation status of superficial tumors (e.g., breast tumors). The ability to image pO2 in humans has many other potential applications to diseases such as peripheral vascular disease, heart disease, and stroke.

Hypoxia-Driven Adenosine Accumulation: A Crucial Microenvironmental Factor Promoting Tumor Progression.

Systematic studies on the oxygenation status of solid tumors have shown that the development of hypoxic/anoxic tissue subvolumes is a pathophysiological trait in a wide range of human malignancies. As a result of this characteristic property, adenosine (ADO) accumulation (range: 50-100 μM) occurs caused by intra- and extracellular generation of ADO. Extracellular nucleotide catabolism by hypoxia-/HIF-1α-sensitive, membrane-associated ecto-5'-nucleotidases most probably is the major source of ADO in the halo of cancer cells upon specific genetic alterations taking place during tumor growth. Extracellular ADO can act through autocrine and paracrine pathways following receptor-binding and involving different intracellular signalling cascades. Hypoxia-driven receptor activation can lead to a broad spectrum of strong immune-suppressive properties facilitating tumor escape from immune control (e.g., inhibition of CD4+, CD8+, NK and dendritic cells, stimulation of Treg cells). In addition, tumor growth and progression is promoted by ADO-driven direct stimulation of tumor cell proliferation, migration, invasion, metastatic dissemination, and an increase in the production of molecules stimulating tumor angiogenesis. Hypoxia- driven ADO accumulation in the tumor microenvironment thus plays a critical role in tumor growth and progression at multiple pathophysiological levels.