Cyclic Hypoxia Research Articles

Targeting cyclic hypoxia to prevent malignant progression and therapeutic resistance of cancers.

Emerging evidence shows that cyclic hypoxia exists in most solid cancers. It is believed that under cyclic hypoxic conditions cancer cells exhibit more malignant biological behaviors than under chronic hypoxic conditions. In this review, we provide a collection of evidence showing the molecular mechanisms by which cyclic hypoxia induces aggressiveness, malignant progression, and therapeutic resistance in cancers. Moreover, we propose that cyclic hypoxia is responsible for the regulation of cancer stem cells, which possess typical biological characteristics of therapeutic resistance. Based on the present findings, some key factors regulated by cyclic hypoxia may serve as potential targets for the prevention of malignant progression and the treatment of solid cancers. Much research is necessary to gain further insights into the biological aspects of cyclic hypoxia in the development and progression of cancers.

SU-E-QI-01: A High-Throughput Quantitative Imaging Method for Immunohistochemistry in Blood Vessels at the Blood-Brain Barrier

Purpose: To develop a simple and high throughput method for the molecular imaging of proteins at the blood-brain barrier using immunohistochemistry quantification. Methods: C57B6 male mice, age 4 months, were exposed to one of four different conditions for 12 hours/day × 2 weeks (a) Sham--exposure to constant air--e.g. 21% oxygen (b) cyclical intermittent hypoxia (CIH) from 21% to 12% (c) CIH from 21% to 10% and (d) CIH from 21% to 5%. Immunohistochemistry was then optimized for proteins GLUT1, p-glycoprotein and CD31 on blood vessels at the bloodbrain barrier. The image data sets of four different conditions were subjected to our image analysis algorithm. Background noise is eliminated using thresholding techniques and blood vessel candidates are delineated using methods based on connected component analysis. Subsequently, relevant features are extracted from the candidate set and are passed through our classification algorithm for capillary identification. We then run a reinforcement algorithm on a subset of images to improve identity classification. This is accomplished by calculating the false positive/negative rate of the subset and feeding these results back to the classification algorithm. We introduce the idea that the gold standard should be computer + human vision. Results: For GLUT1 we analyzed over 5000 images and identified over 400k blood vessels with a false positive rate of 4.1%. For p-glycoprotein we analyzed over 5000 images and identified over 400k blood vessels with a false positive rate of 5%. Conclusion: Our method provides quantitative measurements of proteins on blood vessels at the blood-brain barrier in an objective, reproducible and comparable way using standard immunohistochemistry techniques and bright-field microscopy.

Dichotomous Effects of Chronic Intermittent Hypoxia on Focal Cerebral Ischemic Injury

Obstructive sleep apnea, a condition associated with chronic intermittent hypoxia (CIH), carries an increased risk of stroke. However, CIH has been reported to either increase or decrease brain injury in models of focal cerebral ischemia. The factors determining the differential effects of CIH on ischemic injury and their mechanisms remain unclear. Here, we tested the hypothesis that the intensity of the hypoxic challenge determines the protective or destructive nature of CIH by modulating mitochondrial resistance to injury. Male C57Bl/6J mice were exposed to CIH with 10% or 6% O2 for ≤35 days and subjected to transient middle cerebral artery occlusion. Motor deficits and infarct volume were assessed 3 days later. Intraischemic cerebral blood flow was measured by laser-Doppler flowmetry and resting cerebral blood flow by arterial spin labeling MRI. Ca2+-induced mitochondrial depolarization and reactive oxygen species production were evaluated in isolated brain mitochondria. We found that 10% CIH is neuroprotective, whereas 6% CIH exacerbates tissue damage. No differences in resting or intraischemic cerebral blood flow were observed between 6% and 10% CIH. However, 10% CIH reduced, whereas 6% CIH increased, mitochondrial reactive oxygen species production and susceptibility to Ca2+-induced depolarizations. The influence of CIH on the ischemic brain is dichotomous and can be attributed, in part, to changes in the mitochondrial susceptibility to injury. The findings highlight a previously unappreciated complexity in the effect of CIH on the brain, which needs to be considered in evaluating the neurological effect of conditions associated with cyclic hypoxia.

Imaging Tumor Hypoxia to Advance Radiation Oncology

Most solid tumors contain regions of low oxygenation or hypoxia. Tumor hypoxia has been associated with a poor clinical outcome and plays a critical role in tumor radioresistance. Two main types of hypoxia exist in the tumor microenvironment: chronic and cycling hypoxia. Chronic hypoxia results from the limited diffusion distance of oxygen, and cycling hypoxia primarily results from the variation in microvessel red blood cell flux and temporary disturbances in perfusion. Chronic hypoxia may cause either tumor progression or regressive effects depending on the tumor model. However, there is a general trend toward the development of a more aggressive phenotype after cycling hypoxia. With advanced hypoxia imaging techniques, spatiotemporal characteristics of tumor hypoxia and the changes to the tumor microenvironment can be analyzed. In this review, we focus on the biological and clinical consequences of chronic and cycling hypoxia on radiation treatment. We also discuss the advanced non-invasive imaging techniques that have been developed to detect and monitor tumor hypoxia in preclinical and clinical studies. A better understanding of the mechanisms of tumor hypoxia with non-invasive imaging will provide a basis for improved radiation therapeutic practices.

The roles of TGF-β3 and peroxynitrite in removal of hyper-radiosensitivity by priming irradiation

Purpose: To investigate the mechanisms inducing and maintaining the permanent elimination of low dose hyper-radiosensitivity (HRS) in cells given a dose of 0.3 Gy at low dose-rate (LDR) (0.3 Gy/h).Materials and methods: Two human HRS-positive cell lines (T-47D, T98G) were used. The effects of pretreatments with transforming growth factor beta (TGF-β) neutralizers, TGF-β3 or peroxynitrite scavenger on HRS were investigated using the colony assay. Cytoplasmic levels of TGF-β3 were measured using post-embedding immunogold electron microscopic analysis.Results: TGF-β3 neutralizer inhibited the removal of HRS by LDR irradiation. Adding 0.001 ng/ml TGF-β3 to cells removed HRS in T98G cells while 0.01 ng/ml additionally induced resistance to higher doses. Cytoplasmic levels of TGF-β3 were higher in LDR-primed cells than in unirradiated cells. The presence of the peroxynitrite scavenger uric acid inhibited the effect of LDR irradiation. Furthermore, the permanent elimination of HRS in LDR-primed cells was reversed by treatment with uric acid. The removal of HRS by medium from hypoxic cells was inhibited by adding TGF-β3 neutralizer to the medium before transfer or by adding hypoxia inducible factor 1 (HIF-1) inhibitor chetomin to the cell medium during hypoxia.Conclusions: TGF-β3 is involved in the regulation of cellular responses to small doses of acute irradiation. TGF-β3 activation seems to be induced by low dose-rate irradiation by a mechanism involving inducible nitric oxide (iNOS) and peroxynitrite, or during cycling hypoxia by a mechanism most likely involving HIF-1. The study suggests methods to turn resistance to doses in the HRS-range on (by TGF-β3) or off (by TGF-β3 neutralizer or by peroxynitrite inhibition).

Field study of cyclic hypoxic effects on gene expression in grass shrimp hepatopancreas

Grass shrimp, Palaemonetes pugio, are widely used for ecological and toxicological research. They commonly experience cyclic hypoxia in their natural habitats. The response of grass shrimp to laboratory-controlled cyclic hypoxia has been studied in detail, but little is known about how field acclimatized grass shrimp regulate the gene expression and response to cyclic hypoxia. In this study we examined morphometric parameters, relative fecundity and gene expression of grass shrimp collected from two areas in Weeks Bay (Mobile, Alabama). One is a traditionally normoxic location (WBM), and the other is a traditionally cyclic hypoxic location (WC). In the week preceding grass shrimp collection dissolved oxygen (DO) at the field sites was measured continuously. DO was <2 (mg/L DO) and between 2 and 3 (mg/L DO) for 0 and 255min at WBM, and for 285 and 1035min at WC, respectively. Weight and length of WBM grass shrimp were significantly greater than weight and length of WC shrimp. WBM shrimp had more eggs than WC shrimp, but the difference was not significant. Shrimp from WC had a significant higher number of parasites than those from WBM. A cDNA microarray was utilized to investigate the changes in gene expression in grass shrimp hepatopancreas. Five genes, previously identified as hypoxia/cyclic hypoxia-responsive genes in laboratory exposure studies, were significantly up-regulated in WC shrimp relative to WBM. A total of 5 genes were significantly down-regulated in the field study. Only one of those genes, vitellogenin, has been previously found in chronic and cyclic hypoxic studies. Up and down-regulation of 7 selected genes was confirmed by qPCR. The overall pattern of gene expression in wild shrimp from cyclic DO sites in Weeks Bay showed only weak correlations with gene expression in shrimp from chronic and cyclic hypoxic laboratory studies. It appears therefore that transcriptome profiles of laboratory acclimated animals are of limited utility for understanding responses in field acclimatized animals that are exposed to a broader array of environmental variables.

The Roles of Reactive Oxygen Species and Autophagy in Mediating the Tolerance of Tumor Cells to Cycling Hypoxia

Tumor hypoxia (low oxygenation) causes treatment resistance and poor patient outcome. A substantial fraction of tumor cells experience cycling hypoxia, characterized by transient episodes of hypoxia and reoxygenation. These cells are under a unique burden of stress, mediated by excessive production of reactive oxygen species (ROS). Cellular components damaged by ROS can be cleared by autophagy, rendering cycling hypoxic tumor cells particularly vulnerable to inhibition of autophagy and its upstream regulatory pathways. Activation of the PERK-mediated signaling arm of the unfolded protein response during hypoxia plays a critical role in the defense against ROS, both by stimulating glutathione synthesis pathways and through promoting autophagy.

Hypoxia and metastasis in an orthotopic cervix cancer xenograft model

BackgroundHypoxia can promote tumor metastasis by mechanisms that are believed to result from changes in gene expression. The current study examined the role of putative metastatic genes regulated by cyclic hypoxia in relation to metastasis formation in orthotopic models of cervix cancer. MethodsOrthotopic tumors derived from ME180 human cervix cancer cells or from early generation human cervix cancer xenografts were exposed to cyclic hypoxic conditions during growth in vivo and tumor growth and lymphnode metastases were monitored. Expression of the chemokine receptor CXCR4 and various genes in the Hedgehog (Hh) pathway were inhibited using genetic (inducible shRNA vs CXCR4) small molecule (AMD3100) or antibody (5E1) treatment (CXCR4 and Hh genes, respectively) during tumor growth. ResultsAs reported previously, exposure of tumor bearing mice to cyclic hypoxia caused a reduction of tumor growth but a large increase in metastasis. Inhibition of CXCR4 or Hh gene activity during tumor growth further reduced primary tumor size and reduced lymphatic metastasis to levels below those seen in control mice exposed to normoxic conditions. ConclusionBlocking CXCR4 or Hh gene expression are potential therapeutic pathways for improving cervix cancer treatment.

The role of nitric oxide radicals in removal of hyper-radiosensitivity by priming irradiation

In this study, a mechanism in which low-dose hyper-radiosensitivity (HRS) is permanently removed, induced by low-dose-rate (LDR) (0.2–0.3 Gy/h for 1 h) but not by high-dose-rate priming (0.3 Gy at 40 Gy/h) was investigated. One HRS-negative cell line (NHIK 3025) and two HRS-positive cell lines (T-47D, T98G) were used. The effects of different pretreatments on HRS were investigated using the colony assay. Cell-based ELISA was used to measure nitric oxide synthase (NOS) levels, and microarray analysis to compare gene expression in primed and unprimed cells. The data show how permanent removal of HRS, previously found to be induced by LDR priming irradiation, can also be induced by addition of nitric oxide (NO)-donor DEANO combined with either high-dose-rate priming or exposure to prolonged cycling hypoxia followed by reoxygenation, a treatment not involving radiation. The removal of HRS appears not to involve DNA damage induced during priming irradiation as it was also induced by LDR irradiation of cell-conditioned medium without cells present. The permanent removal of HRS in LDR-primed cells was reversed by treatment with inducible nitric oxide synthase (iNOS) inhibitor 1400W. Furthermore, 1400W could also induce HRS in an HRS-negative cell line. The data suggest that LDR irradiation for 1 h, but not 15 min, activates iNOS, and also that sustained iNOS activation is necessary for the permanent removal of HRS by LDR priming. The data indicate that nitric oxide production is involved in the regulatory processes determining cellular responses to low-dose-rate irradiation.

PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

Principal component analysis enhances SNR for dynamic electron paramagnetic resonance oxygen imaging of cycling hypoxia in vivo

Low oxygen concentration (hypoxia) in tumors strongly affects their malignant state and resistance to therapy. These effects may be more deleterious in regions undergoing cycling hypoxia. Electron paramagnetic resonance imaging (EPRI) has provided a noninvasive, quantitative imaging modality to investigate static pO2 in vivo. However, to image changing hypoxia, EPRI images with better temporal resolution may be required. The tradeoff between temporal resolution and signal-to-noise ratio (SNR) results in lower SNR for EPRI images with imaging time short enough to resolve cycling hypoxia. Principal component analysis allows for accelerated image acquisition with acceptable SNR by filtering noise in projection data, from which pO2 images are reconstructed. Principal component analysis is used as a denoising technique by including only low-order components to approximate the EPRI projection data. Simulated and experimental studies show that principal component analysis filtering increases SNR, particularly for small numbers of sub-volumes with changing pO2 , enabling an order of magnitude increase in temporal resolution with minimal deterioration in spatial resolution or image quality. The SNR necessary for dynamic EPRI studies with temporal resolution required to investigate cycling hypoxia and its physiological implications is enabled by principal component analysis filtering.

Measuring tumor cycling hypoxia and angiogenesis using a side‐firing fiber optic probe

Hypoxia and angiogenesis can significantly influence the efficacy of cancer therapy and the behavior of surviving tumor cells. There is a growing demand for technologies to measure tumor hypoxia and angiogenesis temporally in vivo to enable advances in drug development and optimization. This paper reports the use of frequency-domain photon migration with a side-firing probe to quantify tumor oxygenation and hemoglobin concentrations in nude rats bearing human head/neck tumors administered with carbogen gas, cycling hypoxic gas or just room air. Significant increase (with carbogen gas breathing) or decrease (with hypoxic gas breathing) in tumor oxygenation was observed. The trend in tumor oxygenation during forced cycling hypoxia (CH) followed that of the blood oxygenation measured with a pulse oximeter. Natural CH was also observed in rats under room air. The studies demonstrated the potential of the technology for longitudinal monitoring of tumor CH during tumor growth or in response to therapy.

Production performance of Atlantic salmon (Salmo salarL.) postsmolts in cyclic hypoxia, and following compensatory growth

This study investigated the production performance of the Atlantic salmon postsmolt (Salmo salar L.) subjected to cyclic oxygen reductions (hypoxia) of varying severity. Triplicate groups (N = 955) were kept at constant 80% O2 (control) or subjected to 1 h and 45 min of hypoxia (50, 60 or 70% O2, termed 80:70, 80:60 and 80:50 groups) every 6 h at 16°C for 69 days. Feed was provided in normoxia. One third of the fish were kept further for 30 days in normoxia to study possible compensatory growth. Cyclic hypoxia did not alter the oxygen uptake rates of fish, measured in night-time. Fish subjected to 50% and 60% O2 reduced feeding by 13% and 6% compared with the controls, respectively, with corresponding reductions in specific growth rates. Feed utilization was not reduced. Compensatory growth was observed in fish from the 80:50 group, but full compensation was not achieved. The main conclusions were that feeding in normoxia does not fully alleviate negative effects of cyclic hypoxia on feeding and growth, when oxygen is reduced to 60% or below in hypoxic periods, that feed utilization is maintained, and that compensatory growth may lessen negative effects.

Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply

The purpose of this study was to investigate the effect of acute cyclic hypoxia on tumor vasculature. A-07 human melanoma xenografts growing in dorsal window chambers were used as tumor model. Acute cyclic hypoxia was induced by periodically exposing tumor-bearing mice to a low oxygen atmosphere. The hypoxia treatment consisted of 12cycles of 10min of low O2 (8% O2 in N2) followed by 10min of air for a total of 4hr. The treatment started the first day after tumor initiation, and was given daily for 9days. Vascular morphology was assessed from high-resolution transillumination images, and tumor blood supply was assessed from first-pass imaging movies recorded after a bolus of 155kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Hypoxia-treated tumors showed increased vessel density, decreased interstitial distance, and delayed blood supply compared to control tumors. The increase in vessel density was attributed to an increased number of small vessels. In conclusion, acute cyclic hypoxia induced angiogenesis in A-07 tumors resulting in increased density of small-diameter vessels and delayed tumor blood supply.

Gene expression profile of hepatopancreas from grass shrimp Palaemonetes pugio exposed to cyclic hypoxia

Estuarine organisms often experience periods of cyclic hypoxia characterized by hypoxia in the early morning and normoxia in the afternoon. Here we examine the genomic responses of grass shrimp, Palaemonetes pugio, exposed to cyclic hypoxia in the laboratory. Differentially expressed genes in the hepatopancreas were determined in cyclic hypoxic vs. normoxic control groups after 1, 2, 5 and 10days of exposure to cyclic hypoxia using microarrays printed with 661 annotated transcripts obtained from multiple EST (expressed sequence tag) libraries. Sampling on each day was conducted at two different time series, one in the morning (representing low concentration of dissolved oxygen (DO), designated C-AM) and one in the afternoon (representing high DO concentration, designated C-PM). Distinct differences were observed between the number and identity of specific genes that were significantly down- or up-regulated in shrimp collected at the low DO and high DO points of the cyclic DO cycle. However, cluster analysis showed that the overall response patterns of high (C-PM) and low DO (C-AM) exposures were in the same cluster at 1, 2, and 5days. In contrast, the response patterns on different days were in different clusters. Day 1 was characterized by up-regulation of 17 unknown genes in the morning and a transient down-regulation of several hemocyanin genes, which returned to normoxic control levels in the afternoon. Days 2 and 5 showed significant down-regulation of 10 (C-AM) and 15 (C-PM) unknown genes, respectively. On day 10 the high DO samples showed a dramatic increase in the number of up-regulated genes, including several distinct hemocyanin genes, and this profile did not cluster with any of the other treatment groups. Vitellogenin, cathepsin L, cytochrome c oxidase subunit III, and fatty acid binding protein 10 were the signature down-regulated genes at day 10 (C-AM). According to GO annotation, the most abundant group of genes for both cyclic low (C-AM) and high (C-PM) DO exposure was associated with transport, defense response, and metabolic process. The differentially expressed genes were mapped to KEGG metabolic and regulatory pathways according to the gene distribution in Drosophila pathway database. Cyclic high (C-PM) DO affected a broad range of pathways compared to cyclic low (C-AM) DO.

Chronic and Cyclic Hypoxia in Fractionated Radiation Therapy

Chronic and Cyclic Hypoxia in Fractionated Radiation Therapy

Tumor cycling hypoxia induces chemoresistance in glioblastoma multiforme by upregulating the expression and function of ABCB1

Tumor cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. However, how tumor cycling hypoxia impacts chemotherapy is unclear. In the present study, we explored the impact and the mechanism of cycling hypoxia on tumor microenvironment-mediated chemoresistance. Hoechst 33342 staining and hypoxia-inducible factor-1 (HIF-1) activation labeling together with immunofluorescence imaging and fluorescence-activated cell sorting were used to isolate hypoxic tumor subpopulations from human glioblastoma xenografts. ABCB1 expression, P-glycoprotein function, and chemosensitivity in tumor cells derived from human glioblastoma xenografts or in vitro cycling hypoxic stress-treated glioblastoma cells were determined using Western blot analysis, drug accumulation and efflux assays, and MTT assay, respectively. ABCB1 expression and P-glycoprotein function were upregulated under cycling hypoxia in glioblastoma cells concomitant with decreased responses to doxorubicin and BCNU. However, ABCB1 knockdown inhibited these effects. Moreover, immunofluorescence imaging and flow cytometric analysis for ABCB1, HIF-1 activation, and Hoechst 3342 in glioblastoma revealed highly localized ABCB1 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion in the solid tumor microenvironment. The cycling hypoxic tumor cells derived from glioblastoma xenografts exhibited higher ABCB1 expression, P-glycoprotein function, and chemoresistance, compared with chronic hypoxic and normoxic cells. Tumor-bearing mice that received YC-1, an HIF-1α inhibitor, exhibited suppressed tumor microenvironment-induced ABCB1 induction and enhanced survival rate in BCNU chemotherapy. Cycling hypoxia plays a vital role in tumor microenvironment-mediated chemoresistance through the HIF-1-dependent induction of ABCB1. HIF-1 blockade before and concurrent with chemotherapy could suppress cycling hypoxia-induced chemoresistance.

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.

NADPH oxidase subunit 4 mediates cycling hypoxia-promoted radiation resistance in glioblastoma multiforme

Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve reactive oxygen species (ROS) production. However, details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of an in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence-activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels, and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma; hence, targeting Nox4 may be an attractive therapeutic strategy for blocking cycling hypoxia-mediated radioresistance.

Reply to Nugue and Wion: Targeting cancer stem cells’ hypoxic core

It has become increasingly clear that cancer stem cells (CSCs) flourish in response to low oxygen levels. We recently presented evidence that antiangiogenic therapy stimulated the CSC population in breast cancer by generating intratumoral hypoxia, and we suggested that the addition of CSC-targeting agents was warranted to increase the efficacy of antiangiogenics (1). Therefore, it seems that no matter which therapeutic strategy is used to treat a malignancy (whether it be chemo-, radio-, or antiangiogenic therapy), there is need for the use of CSC-targeting agents in combination. In their work “Angiogenesis and the tumor space–time continuum,” Nugue and Wion (2) make a valid central assertion that, even in the absence of therapeutic agents, the innate defects in tumor vasculature generate areas of cyclic hypoxia. This observation means that, as angiogenesis allows tumors to grow in size, their faulty blood vessels concurrently fuel the CSC population. Naturally, we are compelled to ask how we can use this observation to our advantage. CSCs are known to possess an impressive arsenal of defenses, allowing them to survive under stress and evade traditional therapies, including increased expression of drug transporters and intracellular detoxification enzymes and enhanced DNA damage checkpoint responses (3). It is, therefore, enticing to take advantage of our knowledge of CSCs biology, including their relationship with low oxygen, to target CSCs and combat their defensive mechanisms. The first line of attack may be to directly target the cellular oxygen sensing system, which is predominantly controlled by hypoxia inducible factor-1 (HIF-1). In our studies, we showed that one specific isoform, HIF-1a, was fully necessary for hypoxia to stimulate the CSC population in breast cancer cells (1). In accordance, HIF proteins have also been shown to regulate the tumorigenic potential of glioma cells under hypoxia (4). Blocking HIF signaling, therefore, represents a strong candidate strategy for weakening the hypoxic affect on CSCs. In fact, the work by Wang et al. (5) validated the use of the small-molecule inhibitor of HIFs, echinomycin, in the elimination of acute myeloid leukemia CSCs, in which HIF-1a is selectively activated under all oxygen levels. A second line of attack may be to raise intracellular levels of oxygen in the form of reactive oxygen species (ROS). CSCs are believed to have enhanced free radical scavenging systems, resulting in low levels of ROS (6). Moreover, in addition to low oxygen tension, low levels of ROS are believed to be important components of the CSC niche. Therefore, therapies that can increase ROS within this population may, indeed, be advantageous. Overall, CSCs pose as worthy opponents in the war against cancer, but their dependence on hypoxia may prove to be a lethal flaw in their defenses.