Pimonidazole Binding Research Articles

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma

Background and purposeTumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methodsTumour samples (n=201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). ResultsThe assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001–10ng/μl). Intra- (CoV=6.9%) and inter- (CoV=2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p<0.01) and positive pimonidazole scores (p=0.005). ConclusionsGene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material.

Increased hemoglobin O2affinity protects during acute hypoxia

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.

Abstract LB-440: Low density array profiling of head and neck squamous cell carcinoma utilizing a 25-gene signature can reliably identify hypoxic tumors

Abstract Purpose: Administering hypoxia modifying therapy to patients with head and neck squamous cell carcinoma (HNSCC) improves outcomes in those with identified hypoxic tumors. As no method for measuring tumor hypoxia has demonstrated clinical utility, this study aimed to validate use of a gene expression signature with a TaqMan Low Density Array (TLDA) platform. Experimental design: Tumor samples (n=201) from 80 HNSCC patients were collected prospectively from two centers. Fifty-three patients received pimonidazole prior to surgery. Hypoxia scores (TLDA HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customized TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Relationships with outcome were determined by log-rank analysis. Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/µl). Intra- (CoV=0.5%) and inter- (CoV=0.1%) assay reproducibility were excellent. Intra-tumor heterogeneity was lower for TLDA HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA HS in HNSCC cell lines increased with decreasing pO2 and was reduced but not completely abrogated in HIF1A silenced cells. TLDA HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p&amp;lt;0.01) and pimonidazole scores (p=0.021). High TLDA HS was associated with worse overall (p=0.037) and recurrence free (p=0.036) survival. Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumor heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for application in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-440. doi:1538-7445.AM2012-LB-440

Abstract 4371: RRx-001 modulates intratumor blood flow in SCCVII and U87 tumors

Abstract The tumor-specific provascular effects of the anti-cancer agent RRx-001, a novel nitrogen oxide donor and allosteric hemoglobin modifier in Phase 1 clinical trials were investigated using contrast enhanced ultrasound (CEUS) and whole tissue section quantitative immunohistological staining. Changes in tumor blood flow by CEUS in SCCVII tumor bearing mice following RRx-001 (3, 6, 12 mg/kg) were dose and time dependent, reaching maximum perfusion 6 h after treatment, returning to baseline within 72 h. Microregional effects of RRx-001 (15 mg/kg) on tumor blood flow and oxygenation by immunohistological staining were studied in mouse SCCVII and human U87 tumors. SCCVII tumors exhibited regions of intermittent perfusion exemplified by co-localization of vessels with the hypoxia marker pimonidazole commonly occurring throughout the tissue. Only 21% of vessels showed perfusion after a bolus dose of the perivascular stain DiOC7. This increased to 28% 12 h post RRx-001. Paradoxically, an increase in tissue oxygenation was not seen while modestly increased pimonidazole binding was observed. However, preliminary radiobiological assessment suggested tumors were radiosensitized when irradiated 10 min after 12 mg/kg RRx-001. In the U87 tumor a decrease in vessel perfusion at 90 min post RRx-001 administration was observed. Blood flow over large areas of treated tumors was completely shut down. These areas stained positive for CD31 and tissue did not appear necrotic. Analysis indicated 25% reduction in the fraction of perfused vessels and un-perfused tissue by area at 90 min returning to near normal levels at 12 h. There was no significant increase in the fraction of necrosis at 12 h suggesting that the large un-perfused regions seen at 90 min had recovered rather than become necrotic. No significant change in tumor hypoxia was seen at 90 min or 12 h. For both tumor types, RRx-001 appeared to cause the loss of perfusion in large regions of the tumor however, at the 12 h time point, both tumor types showed increase in vessel perfusion but no significant decrease in hypoxia. These data suggest a redistribution of blood flow in both tumor types while differences between the tumors were related to tumor architecture and distribution of α-SMA. RRx-001 shows promise for short-term blood flow redistribution in tumors with a pericyte-rich vasculature with contractile proteins such as α-SMA. Expression of α-SMA in tumor vasculature could therefore be useful for predicting tumor response to RRx-001. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4371. doi:1538-7445.AM2012-4371

308 DETECTION OF FUNCTIONAL CHANGES IN RENAL CELL CARCINOMA WITH POSITRON EMISSION TOMOGRAPHY

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2012308 DETECTION OF FUNCTIONAL CHANGES IN RENAL CELL CARCINOMA WITH POSITRON EMISSION TOMOGRAPHY David W. Chapman, Melinda Wuest, Leonard I. Wiebe, and Ronald B. Moore David W. ChapmanDavid W. Chapman Edmonton, Canada More articles by this author , Melinda WuestMelinda Wuest Edmonton, Canada More articles by this author , Leonard I. WiebeLeonard I. Wiebe Edmonton, Canada More articles by this author , and Ronald B. MooreRonald B. Moore Edmonton, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.367AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Broad-spectrum tyrosine kinase inhibitors (TKIs) are clinically used for the therapy of metastasizing renal cell carcinoma (RCC). It has been shown in vivo that multitargeted TKIs such as sunitinib exert antiangiogenic effects directly on endothelial cells, but also by blocking compensatory changes in the activity of hypoxia-inducible factors (HIF), mainly HIF-1α. The present study investigated whether sunitinib-induced changes in the microenvironment of a mouse RCC tumor model can be detected utilizing positron-emission tomography (PET). METHODS Caki-1 tumors were grown for 4 weeks in Balb c/nu-nu mice after implantation subcutaneously or into the renal subcapsule. Both models were analyzed first with dynamic small animal PET with [18F]FDG and [18F]FAZA up to 3h post-injection (p.i.). In a second setup, mice bearing 2 subcutaneous Caki-1 tumors were sorted into 2 groups: a) receiving 40mg/kg/d sunitinib i.p. for 5 days prior to analysis with [18F]FAZA PET, b) vehicle control injections. Tumor uptake of [18F]FAZA and immunohistochemical tissue staining with pimonidazole HCl and CD-31 were determined. RESULTS Functional analysis of subcutaneous Caki-1 tumors with PET revealed a mean standardized uptake value (SUV) after 3h p.i. of 0.60±0.02 for [18F]FDG, which is indicative of glucose metabolism within the tumor, and 0.32±0.02 (n=6/3) for [18F]FAZA determining tumor's hypoxia status. Based on the clearance patterns, orthotopically grown tumors were not detectable with PET alone. The SUV3h for [18F]FAZA was significantly lower in sunitinib group compared to controls in the subcutaneous model: 0.23±0.02 vs. 0.42±0.05 (n=4/2). The time-activity curve indicated significant [18F]FAZA washout from treated tumors as compared to the control. This observation was confirmed by the biodistribution of [18F]FAZA, resulting in SUV3h of 0.29±0.03 in treated vs. 0.45±0.06 (n=8/4) in control mice. Pimonidazole binding was reduced by 9% in the treated tumors. CD31 binding (%) was reduced in sunitinib treated groups: 1.8±0.1% vs. 0.6±0.1% (n=80/4). CONCLUSIONS Reduced trapping of [18F]FAZA in this RCC tumor model, in vivo, indicates that TKI inhibition with mutitargeting sunitinib leads to changes in tumor oxygenation, which is functionally detectable with PET. Furthermore, sunitinib led to a significant reduction in vascular density. Monitoring early response to TKI therapy in RCC tumor patients could potentially utilize [18F]FAZA. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e125 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information David W. Chapman Edmonton, Canada More articles by this author Melinda Wuest Edmonton, Canada More articles by this author Leonard I. Wiebe Edmonton, Canada More articles by this author Ronald B. Moore Edmonton, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression of EGFR Under Tumor Hypoxia: Identification of a Subpopulation of Tumor Cells Responsible for Aggressiveness and Treatment Resistance

Overexpression of epidermal growth factor receptor (EGFR) and tumor hypoxia have been shown to correlate with worse outcome in several types of cancer including head-and-neck squamous cell carcinoma. Little is known about the combination and possible interactions between the two phenomena. In this study, 45 cases of histologically confirmed squamous cell carcinomas of the head and neck were analyzed. All patients received intravenous infusions of the exogenous hypoxia marker pimonidazole prior to biopsy. Presence of EGFR, pimonidazole binding, and colocalization between EGFR and tumor hypoxia were examined using immunohistochemistry. Of all biopsies examined, respectively, 91% and 60% demonstrated EGFR- and pimonidazole-positive areas. A weak but significant association was found between the hypoxic fractions of pimonidazole (HFpimo) and EGFR fractions (F-EGFR) and between F-EGFR and relative vascular area. Various degrees of colocalization between hypoxia and EGFR were found, increasing with distance from the vasculature. A high fraction of EGFR was correlated with better disease-free and metastasis-free survival, whereas a high degree of colocalization correlated with poor outcome. Colocalization of hypoxia and EGFR was demonstrated in head-and-neck squamous cell carcinomas, predominantly at longer distances from vessels. A large amount of colocalization was associated with poor outcome, which points to a survival advantage of hypoxic cells that are also able to express EGFR. This subpopulation of tumor cells might be indicative of tumor aggressiveness and be partly responsible for treatment resistance.

Rete mirabile in mouse retina?

AbstractPurpose One possible definition for rete mirabile, the “wonderful net”, is a capillary network interrupting one arteriole, as happens in kidney. In a glomerulus, the afferent and efferent arterioles are communicated by a capillary tuft. It is generally accepted that in mouse retina, capillaries communicate directly arterioles with venules. However, this must be difficult in a tissue where normally there is not concordance between the number of arterioles and veins. In the current study, we explored the presence of retia mirabilia in mouse retina which could explain the mismatch between retinal arterioles and venules.Methods Retinas from C57BL6 and CD1 mice were used. After partially clamping of the common carotid arteries, hypoxia was detected using the Hypoxiprobe‐1 Kit (Chemicon). Mice were intravenously injected with 60 mg pimonidazole/kg body weight in phosphate‐buffered saline. Pimonidazole specifically binds to proteins in hypoxic cells at an oxygen pressure equal to or lower than 10 mmHg. Thirty minutes after injection animals were euthanized and pimonidazole binding proteins were detected in the retina by immunohistochemistry.Results Direct capillary connections between retinal arterioles were found in C57BL6 and CD1 mice, indicating the presence of retia mirabilia. No venules were found between arterioles in the rete mirabile areas. In order to know if these retia mirabilia have some function in retinal oxygenation, partially clamping of common carotid arteries was performed and hypoxia was detected in the retina using the Hyposiprobe‐1 Kit. Interestingly, rete mirabile areas were less hypoxic than conventional capillary areas.Conclusion There are retia mirabilia in mouse retina and these vascular structures could be important in retinal oxygenation.

Abstract 2063: Fate of hypoxic cancer cells in microscopic and fully developed macroscopic tumors

Abstract We previously reported, in mouse cancer models of human colorectal cancer HT29 cells, microscopic tumors (&amp;lt;1 mm) were severe hypoxia and poor perfusion, in contrast, larger ones (1-4 mm) were well-perfused and not significantly hypoxic, as tumors increased in size beyond this level, the typical pattern of macroscopic tumor hypoxia reappeared (Cancer Res, 2007; 67: 7646). The fate of hypoxic cells at each stage of cancer development is largely unknown. This issue is critically important for planning anticancer therapy; hypoxia induces cancer therapy resistance. Methods: We injected HT29 cells into peritoneal cavity, ascites tumors (floating in ascites, in dormant status and &amp;lt;1mm in diameter) were evident 4-5 wk later, ascites tumors were harvested 1-7 d followed exogenous hypoxia marker pimonidazole (PIMO) administration, the spatial localization of PIMO adducts visualized by histochemical staining and their relating to viable cells or cell debris in necrosis was used to estimate the lifespan of hypoxic cancer cells, fate of hypoxic cells was also observed in fully developed HT29 subcutaneous xenografts (∼10 mm in diameter). Changes in hypoxia during early stage of tumor development were investigated in an intradermal tumor model using dual hypoxia markers staining technique: various days after cancer cells inoculation (i.e. various size of tumors), we injected PIMO to hosts 1h before their sacrifice to label “current” hypoxic cells, this compared with hypoxia induced protein carbonic anhydrase 9 (CA9) expression, CA9 is able to mark “historic” hypoxia because it has 3 days half-life even after reoxygenation. Stroma formation and angiogenesis were also observed. Results: PIMO adducts closely associated with viable cancer cells in dormant ascites tumors 7 d followed PIMO injection, there was little necrotic tissue in ascites tumors. However, Major PIMO adducts were in necrotic zones 2-3 d later in fully developed macroscopic xenografts. Changes in hypoxia during early stage of tumor development were investigated: 3 to 5 d after tumor initiation, a sole cluster of pure cancer cells (&amp;lt;1 mm) was found, majority of cancer cells had high PIMO binding and high CA9 expression. Seven days after cell inoculation when tumors reached to 2-3 mm in diameter, cancer cells had little PIMO binding but still strong CA9 expression, abundant stroma along with functional blood vessels were evident in the tumors, Which indicated previous hypoxic cells had been recently oxygenated due to stroma formation/angiogenesis switch. Conclusion: Hypoxic cancer cells in submillimeter dormant microscopic tumors have very long lifespan, which maybe oxygenate after angiogenesis and tumor growth. Hypoxic cell in fully developed tumors generally go to necrosis. Hypoxia is critical important in microscopic tumors, hypoxic cells may resist cancer therapies and microscopic tumors develop to macroscopic tumors in a future time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2063. doi:10.1158/1538-7445.AM2011-2063

Hypoxia, hypoxia-inducible factor-1α and vascular endothelial growth factor in a murine model of Schistosoma mansoni infection

Schistosomiasis mansoni is a chronic parasitic disease where much of the symptomatology is attributed to granuloma formation, an immunopathological reaction against Schistosoma eggs. To more clearly understand the immunopathology of schistosomiasis, the tissue microenvironment generated by S. mansoni infected mice was investigated. Using the hypoxia marker pimonidazole, we provide immunohistochemical evidence that hypoxia occurred in inflammatory cells infiltrated around the eggs and cells surrounding granulomas in the liver, intestine, spleen and lungs of infected mice. Hypoxia-inducible factor-1α (HIF-1α) was mainly expressed in inflammatory cells surrounding the eggs and in hepatocytes surrounding cellular and fibrocellular granulomas in infected mouse liver. HIF-1α expression was also verified in granulomas in the other tissues tested (intestine, spleen and lungs). Vascular endothelial growth factor (VEGF) expression was observed in the extracellular space surrounding inflammatory cells in liver granuloma. The VEGF expression pattern verified in infected mouse liver was very similar to that observed in the other tissues tested. A strong positive correlation occurred between pimonidazole binding and HIF-1α and VEGF expression in the tissues tested, except for lung. This work is the first evidence that infection by a helminth parasite, S. mansoni, produces a hypoxic tissue microenvironment and induces HIF-1α and VEGF expression.

Oxygen Tension Regulates Hematopoietic Stem Cell Migration Into An in-Vitro Niche Beneath Mesenchymal Stromal Cells.

Abstract 1443Poster Board I-466 BackgroundHematopoietic stem cells (HSCs) are located mainly in the bone marrow interacting with a specific microenvironment called “stem cell niche”. The niche has been proven to be critical for stem cell regulation. Coculture with mesenchymal stromal cells (MSCs) has been used as an in vitro model to investigate the interaction between HSCs and MSCs. In our study we investigated the impact of normoxia and hypoxia on the distribution of HSC subsets with regard to their spatial localization in cocultures during ex-vivo expansion. Design and MethodsThree HSC subsets are defined: (i) cells in supernatant (non-adherent cells); (ii) cells adhering on the MSC layer surface (phase-bright cells); (iii) cells beneath the MSC layer (phase-dim cells). Using pimonidazole binding we investigated the spatial distribution of hypoxic cells in various cell subsets. Cell cycle, cell division, immunophenotype and migratory capacity of the three HSC subsets under distinct oxygen tension were studied. In addition the impact of oxygen tension on HSCs via VEGF-A and SDF-1 were analyzed by ELISA and gene knockdown with siRNA. ResultsFirst we could show that phase-bright cells contained the highest proportion of cycling progenitors. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype. Next pimonidazole binding revealed that the most hypoxic area in the coculture is the compartment beneath MSC layer. Then we investigated the impact of hypoxia conditions on HSCs in cocultures. We could demonstrate that under hypoxic conditions phase-bright cells were significantly diminished and phase-dim cells were increased. Interestingly, the migratory capacity of phase-bright cells from cocultures performed under hypoxic conditions was consistently enhanced in comparison to normoxia (32.7 ±2.2.0% vs 17.6 ±2.6%, p<0.01). Surprisingly, the SDF-1 concentration was lower after hypoxic coculture (189 ±33μg/ml vs 352 ±40μg/ml, p<0.05). In contrast, the VEGF-A concentration was significantly increased compared to normoxic conditions (7.7 ±1.2ng/ml vs 4.5 ±1.0ng/ml, p<0.01). In addition we could demonstrate that the lower adhesion and higher migratory capacity of HSCs under hypoxia can be partially inversed by silencing VEGF-A with siRNA in MSCs. ConclusionsOur data indicate that under our experimental conditions, the MSC surface is the dominant location where HSCs proliferate, whereas the compartment beneath the MSC layer seems to be a hypoxic niche dedicated to the maintenance of HSC stemness. The lower levels of SDF-1 in the supernatant may be explained by the increased internalization of SDF-1 by MSCs when cultured together with HSC. This hypothesis will require the concomitant analysis of protein and SDF-1 mRNA in MSC. In addition our data suggest that low oxygen tension facilitates HSC migration into the in-vitro niche provided by MSCs which preserves immaturity of HSCs and modifies the cytokine profile of MSCs. DisclosuresNo relevant conflicts of interest to declare.

Detection of hypoxia in microscopic tumors using 131I-labeled iodo-azomycin galactopyranoside (131I-IAZGP) digital autoradiography

Previous studies have shown that tumors less than 1 mm diameter derived from HT29 colorectal cancer cells are extremely hypoxic when grown intraperitoneally or intradermally in nude mice, whereas those of greater size (approximately 1-4 mm diameter) are not significantly hypoxic. The object of this study was to determine if digital autoradiography using the radiolabeled hypoxia imaging tracer iodo-azomycin galactopyranoside ((131)I-IAZGP) could detect hypoxia in this model. Microscopic HT29 tumors were grown as disseminated peritoneal disease and intradermally in nude mice. Tumors ranged in size from a few hundred microns to several millimeters in diameter. Animals were intravenously administered (131)I-IAZGP and pimonidazole 2 h before sacrifice. Following sacrifice, the intratumoral distribution of (131)I-IAZGP was assessed by digital autoradiography and compared with immunofluorescence microscopic images of pimonidazole binding and carbonic anhydrase IX (CAIX) expression. The distributions of (131)I-IAZGP, pimonidazole, and CAIX expression were similar. Tumors less than 1 mm diameter displayed high (131)I-IAZGP uptake; these tumors also stained strongly for pimonidazole and CAIX. Larger tumors (approximately 1-4 mm diameter) were not significantly hypoxic and had low (131)I-IAZGP accumulation. (131)I-IAZGP can detect hypoxia in microscopic tumors. Microscopic tumors are useful models for the validation of hypoxia radiotracers, and digital autoradiography is an appropriate technique for studying the distribution of hypoxia radiotracers in microscopic tumors.

Hypoxia in larynx carcinomas assessed by pimonidazole binding and the value of CA-IX and vascularity as surrogate markers of hypoxia

Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics. One hundred and three patients with T2–T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2 h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0–19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0–34%) was observed. In 67% of the tumours, hypoxia involved ⩾1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak ( p = 0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) ( p = 0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage. From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.

Comparison between hypoxic markers pimonidazole and glucose transporter 1 (Glut-1) in murine fibrosarcoma tumours after electrochemotherapy

Background. Tumour hypoxia occurs as a result of an inadequate supply of blood-borne oxygen due to the disorganized and chaotic vascular network that develops in tumours. Because tumour hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is a recognized need for a method of measuring tumour hypoxia that is suitable for widespread clinical use. The aim of the current study was to compare the expression of Glut-1 with the binding of the bioreductive hypoxia marker pimonidazole and to elucidate the characteristics and pitfalls when they are used as hypoxic markers. Materials and methods. In the study, SA-1 solid subcutaneous tumours in A/J mice were treated by bleomycin given i.v. (1 mg/kg), or the application of electric pulses (8 pulses, 1400 V, 100 μs, 1Hz), or a combination of the two - electrochemotherapy. Pimonidazole was injected 16 hours before tumour excision. Tumours were excised at different time points (0.5, 1, 2, 8, 14 and 24 hours) after therapy. Immunohistochemistry for Glut-1 and pimonidazole adduct was carried out on two consecutive tumour sections and the percentages of positive staining areas were determined. Results. Glut-1 staining was membranous and typically expressed peri-necrotically, whereas pimonidazole staining, although showing substantial co-localisation with Glut-1, was cytoplasmatic. More than 65% of the stained areas showed a high degree of colocalization when the two markers were compared. Our results show that Glut-1 expression significantly correlates with the level of pimonidazole binding (r = 0.41; p = 0.028). Conclusions. Our study confirms that HIF-1 regulated genes, such as Glut-1, have potential for future use as predictors of a decreased sensitivity of tumours to radio- and chemotherapy mediated by hypoxia.

No Detectable Hypoxia in Malignant Salivary Gland Tumors: Preliminary Results

Hypoxia is detected in most solid tumors and is associated with malignant progression and adverse treatment outcomes. However, the oxygenation status of malignant salivary gland tumors has not been previously studied. The aim of this study was to investigate the potential clinical relevance of hypoxia in this tumor type. Twelve patients scheduled for surgical resection of a salivary gland tumor were preoperatively injected with the hypoxia marker pimonidazole and the proliferation marker iododeoxyuridine. Tissue samples of the dissected tumor were immunohistochemically stained for blood vessels, pimonidazole, carbonic anhydrase-IX, glucose transporters-1 and -3 (Glut-1, Glut-3), hypoxia-inducible factor-1alpha, iododeoxyuridine, and epidermal growth factor receptor. The tissue sections were quantitatively assessed by computerized image analysis. The tissue material from 8 patients was of sufficient quality for quantitative analysis. All tumors were negative for pimonidazole binding, as well as for carbonic anhydrase-IX, Glut-1, Glut-3, and hypoxia-inducible factor-1alpha. The vascular density was high, with a median value of 285 mm(-2) (range, 209-546). The iododeoxyuridine-labeling index varied from <0.1% to 12.2% (median, 2.2%). Epidermal growth factor receptor expression levels were mostly moderate to high. In one-half of the cases, nuclear expression of epidermal growth factor receptor was observed. The absence of detectable pimonidazole binding, as well as the lack of expression of hypoxia-associated proteins in all tumors, indicates that malignant salivary gland tumors are generally well oxygenated. It is unlikely that hypoxia is a relevant factor for their clinical behavior and treatment responsiveness.

Importance of Antibody Concentration in the Assessment of Cellular Hypoxia by Flow Cytometry: EF51and Pimonidazole

The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of (14)C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO(2) from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO(2). The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] - pimonidazole << EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO(2) and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO(2). The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO(2) dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO(2).

Early fetal hypoxia leads to growth restriction and myocardial thinning

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired O2 from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of E11.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18-24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate.

Endothelin receptor (ETR) blockade combined with phosphodiesterase‐5 (PDE5) inhibition prevents right ventricular hypoxia in Pulmonary Hypertension

BackgroundPulmonary hypertension (PH) may cause right ventricular (RV) hypoxia, which may contribute to RV failure.GoalTo study the effects of current PH therapies on the presence of hypoxia in the RV.MethodsPH was induced in Wistar rats by monocrotaline (MCT) (40 mg/kg). From day 14 to 28, rats were treated with Bosentan (ETR antagonist, 300 mg/kg/day), Sildenafil (PDE5 inhibitor, 10 mg/kg/day) or both (all n=3). Healthy rats served as controls (n=3). 1.5 hour prior to sacrifice hypoxyprobe (pimonidazole, 60 mg/kg) was administered i.v. Cryosections (5 μm) of the hearts were incubated with hypoxyprobe antibody and for succinate dehydrogenase (SDH) activity. RV capillary density and cardiomyocyte cross‐sectional area (CSA) were determined.ResultsPH significantly increased RV pimonidazole binding, and cardiomyocyte CSA, and decreased capillary density, and SDH activity (all p&lt;0.05 vs control). Single treatment with Bosentan or Sildenafil had no effect, whereas combination treatment normalized all parameters (p&lt;0.05 vs MCT rats). Pimonidazole binding related linearly to CSA (r=0.6), SDH activity (r=−0.7), and capillary density (r=−0.8) (all p&lt;0.05).ConclusionHypoxia in the RV of PH rats is caused by cardiomyocyte hypertrophy, a lowering in mitochondrial activity and a diminished capillary density, and can be prevented by combining ETR blockade and PDE5 inhibition.

Measurement of hypoxia using invasive oxygen-sensitive electrode, pimonidazole binding and 18F-FDG uptake in anaemic or erythropoietin-treated mice bearing human glioma xenografts

Relationship between haemoglobin levels and tumour oxygenation has been already reported. The purpose of this work was to compare in human malignant glioma-bearing mice the sensitivity of two well established techniques of tumour hypoxia assessment, especially their ability to detect expected weak variations of tumour oxygenation status associated to haemoglobin level modifications. The relationship between tumour hypoxia and glucose metabolism was also investigated. Experiments were performed on a human malignant glioma (GBM Nan1) xenografted into nude mice. Twenty-four hours after tumour implantation, animals were randomized into three groups: 'Anaemia' for mice subjected to repeated blood samplings, 'Control', and 'rHuEPO' for mice receiving recombinant human erythropoietin. Once the tumours reached a volume of 300+/-100 mm(3), tumour hypoxia was assessed both using the pO(2)-Histograph, Eppendorftrade mark and the pimonidazole binding assay. Glucose metabolism was evaluated by (18)F-FDG autoradiography and compared with the pimonidazole binding distribution pattern. Repeated blood samplings significantly reduced mean haemoglobin levels (10.9+/-2.0 g/dl), inducing chronic anaemia in mice, while daily administration of rHuEPO led to increase of haemoglobin levels (15.8+/-2.0 g/dl). Oxygenation status evaluated by a microelectrode was worsened in anaemic mice (mean pO(2) in tumour = 6.9+/-0.8 mmHg) and improved in rHuEPO-treated animals (mean pO(2)in tumour = 11.4+/-1.2 mmHg). No correlation was observed between the oxygen-sensitive probe and pimonidazole labelling results: both techniques give different but complementary information about tumour hypoxia. Areas of high pimonidazole binding and areas of high (18)F-FDG uptake superimposed well. Present results confirm that modification of haemoglobin levels leads to alteration of tumour oxygenation status. These variations were detectable using the oxygen-sensitive electrode but not the pimonidazole binding assay. The strong correlation between pimonidazole labelling and (18)F-FDG uptake suggests a positive relationship between hypoxia and increased glucose metabolism in this tumour model.

Tumour cell proliferation under hypoxic conditions in human head and neck squamous cell carcinomas

Two mechanisms of radiotherapy resistance of major importance in head and neck cancer are tumour cell repopulation and hypoxia. Hypoxic tumour cells that retain their clonogenic potential can survive radiation treatment and lead to local recurrences. The aim of this study was to quantify this cellular population in a cohort of human head and neck carcinomas and to investigate the prognostic significance. The proliferation marker iododeoxyuridine (IdUrd) and the hypoxia marker pimonidazole were administered intravenously prior to biopsy taking in patients with stage II-IV squamous cell carcinoma of the head and neck. Triple immunohistochemical staining of blood vessels, IdUrd and pimonidazole was performed and co-localization of IdUrd and pimonidazole was quantitatively assessed by computerized image analysis. The results were related with treatment outcome. Thirty-nine biopsies were analyzed. Tumours exhibited different patterns of proliferation and hypoxia but generally the IdUrd signal was found in proximity to blood vessels whereas pimonidazole binding was predominantly at a distance from vessels. Overall, no correlations were found between proliferative activity and oxygenation status. The fraction of IdUrd-labelled cells positive for pimonidazole ranged from 0% to 16.7% with a mean of 2.4% indicating that proliferative activity was low in hypoxic areas and occurring mainly in the well-oxygenated tumour compartments. IdUrd positive cells in hypoxic areas made up only 0.09% of the total viable tumour cell mass. There were no associations between the magnitude of this cell population and local tumour control or survival. Co-localization between proliferating cells and hypoxia in head and neck carcinomas was quantified using an immunohistochemical triple staining technique combined with a computerized simultaneous analysis of multiple parameters. The proportion of cells proliferating under hypoxic conditions was small and no correlation with treatment outcome could be found.

Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia

The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.