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Trans sodium crocetinate has been shown to in-crease oxygen in the setting of hypoxia related to brain tumors and stroke.10,18,19 Reduction of hypox-

ia in malignant brain tumors could yield improvements in their response to radiosurgery, radiation therapy, or chemotherapy.5 In particular, cells surviving hypoxia can lead to increased migration, increased vascular signaling/recruitment, enhanced anaerobic glycolysis, and resis-tance to apoptosis.17 The Licox monitor (Integra) revealed a TSC-associated partial restoration of a hypoxic tumor to more normoxic conditions.20 However, such Licox probes had a limited sampling volume that may not have reflect-ed changes throughout the entire tumor.

Although TSC is postulated to work through en-hancement of oxygen diffusion across blood vessels, it is not known to what extent the administration of TSC results in a functional improvement in hypoxic tissue. If the parenchymal concentration of oxygen increases to a subfunctional degree, the consequences of TSC adminis-tration on hypoxic tissue may not be clinically meaning-ful. Positron emission tomography is a nuclear medicine technique that produces a 3D image of functional pro-cesses within the body; therefore, PET imaging has be-come a very important tool for functional imaging within the brain.21 In particular, Cu-ATSM–based PET imaging has been used to study functional changes in the oxygen levels of tissues.2

Intracranially implanted C6 gliomas have been shown

Trans sodium crocetinate: functional neuroimaging studies in a hypoxic brain tumor

Laboratory investigationJason P. sheehan, M.D., Ph.D., Britney PoPP, B.s., stePhen Monteith, M.D., sushila toulMin, Jennifer toMlinson, Jessica Martin, christoPher P. cifarelli, M.D., Ph.D., Dae-hee lee, Ph.D., anD Deric M. Park, M.D. Department of Neurological Surgery, University of Virginia Health System, Charlottesville, Virginia

Object. Intratumoral hypoxia is believed to be exhibited in high-grade gliomas. Trans sodium crocetinate (TSC) has been shown to increase oxygen diffusion to hypoxic tissues. In this research, the authors use oxygen-sensitive PET studies to evaluate the extent of hypoxia in vivo in a glioblastoma model and the effect of TSC on the baseline oxygenation of the tumor.

Methods. The C6 glioma cells were stereotactically implanted in the right frontal region of rat brains. Forma-tion of intracranial tumors was confirmed on MR imaging. Animals were injected with Copper(II) diacetyl-di(N4-methylthiosemicarbazone) (Cu-ATSM) and then either TSC or saline (6 rats each). Positron emission tomography imaging was performed, and relative uptake values were computed to determine oxygenation within the tumor and normal brain parenchyma. Additionally, TSC or saline was infused into the animals, and carbonic anhydrase 9 (CA9) and hypoxia-inducing factor–1a (HIF-1a) protein expression were measured 1 day afterward.

Results. On PET imaging, all glioblastoma tumors demonstrated a statistically significant decrease in uptake of Cu-ATSM compared with the contralateral cerebral hemisphere (p = 0.000002). The mean relative uptake value of the tumor was 3900 (range 2203–6836), and that of the contralateral brain tissue was 1017 (range 488–2304).

The mean relative hypoxic tumor volume for the saline group and TSC group (6 rats each) was 1.01 ± 0.063 and 0.69 ± 0.062, respectively (mean ± SEM, p = 0.002). Infusion of TSC resulted in a 31% decrease in hypoxic volume. Immunoblot analysis revealed expression of HIF-1a and CA9 in all tumor specimens.

Conclusions. Some glioblastomas exhibit hypoxia that is demonstrable on oxygen-specific PET imaging. It ap-pears that TSC lessens intratumoral hypoxia on functional imaging. Further studies should explore relative hypoxia in glioblastoma and the potential therapeutic gains that can be achieved by lessening hypoxia during delivery of adjuvant treatment. (DOI: 10.3171/2011.5.JNS101954)

key WorDs      •      trans sodium crocetinate      •      glioma      •      hypoxia-inducing factor      • Copper(II) diacetyl-di(N4-methylthiosemicarbazone)      •      rat      •      oncology

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Abbreviations used in this paper: CA9 = carbonic anhydrase 9; Cu-ATSM = Copper(II) diacetyl-di(N4-methylthiosemicarbazone); HIF-1a = hypoxia-inducing factor–1a; PBS = phosphate-buffered saline; SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TSC = trans sodium crocetinate.

This article contains some figures that are displayed in color on line but in black and white in the print edition.

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to have a region of hypoxia on direct measurements with parenchyma Licox oxygen probes and also by other tech-niques.4,20 However, it is not clear whether TSC’s en-hancement of oxygen diffusion represents a functional change in the physiology (that is, cellular metabolism) of cells throughout the entire tumor. In this study, we use Cu-ATSM PET imaging to study the effect of TSC on a hypoxic brain tumor. We also study the expression of HIF-1a and CA9 in this same tumor model.

MethodsThe C6 Glioma Technique

The C6 glioma cells were obtained from the Ameri-can Type Culture Collection. Cells were cultured for 4–5 passages in Ham F-12 medium (GIBCO, Inc.) containing 15% horse serum, 2.5% fetal bovine serum, and 1% anti-biotic-antimycotic solution. The medium was changed at least twice a week, and the cells were grown using 75-cm2 culture flasks. Cells were incubated at 37°C in 5% CO2, passaged weekly by washing with PBS, detached in 10 ml of 0.5 mM EDTA, and subcultured at a ratio of 1:4.

For implantation, cells were washed in PBS, then de-tached in 10 ml of 0.5 mM EDTA. Cells were then pel-leted by centrifugation (1500 G for 4 minutes) and resus-pended in 10 mM PBS-glucose to a final concentration of 106 cells/10 μl. The cell density was determined by a microscope stage micrometer. The time between removal of the cells from incubation to implantation in the rat host was limited to 2 hours or less.

The Stereotactic TechniqueThe anesthetized rat was placed in a stereotactic head

frame (David Kopf Instruments), and a small craniecto-my (2 × 1 mm) was drilled, 3 mm from the midline and 1 mm anterior to the coronal suture. The dura mater was then opened. With a Hamilton syringe, a 2-μl volume of 105 glioma cells was implanted with stereotactic guidance to a depth of 4 mm below the craniectomy, into the right frontotemporal region. The craniectomy was then sealed with bone wax, and the scalp was closed with suture.

The MR Imaging TechniqueTwo weeks after implantation of the C6 glioma cells

in the rats, each animal was assessed using 3-T MR im-aging at the University of Virginia’s Small Animal Mul-timodality Imaging Core. The T1-weighted MR images were obtained before and after addition of contrast mate-rial to determine if tumor was present.

The PET Imaging TechniqueFor the PET experiments, those animals in which a

tumor was confirmed on MR imaging were randomly as-signed to either the TSC or saline infusion group (6 ani-mals each). The PET imaging studies were performed at the University of Virginia’s Small Animal Multimodality Imaging Core. Colleagues in the Department of Radiol-ogy provided a copper nuclide specific for PET imaging to assess oxygenation.

Copper(II) diacetyl-di(N4-methylthiosemicarbazone) has been shown to demonstrate significant selectivity for hypoxic tissues in vivo; this relates to a reduction-oxida-tion trapping mechanism.2 The Cu-ATSM preferentially accumulates in hypoxic cells and delineates hypoxic areas within tumors. The mechanism of Cu-ATSM involves a reduction of Cu(II) to Cu(I), followed by a loss of the ra-diometal from the complex. The Cu-ATSM accumulates in tumor masses with active but hypoxic tumor cells. It has been shown to be effective in both rodent studies and in clinical studies of cancer in humans.1,7,11,13

Each animal was injected with 1 mCi of Cu-ATSM prior to imaging. Each animal then underwent 1.5 hours of PET imaging and 0.5 hours of CT imaging. The PET imaging was continued throughout the intravenous de-livery of 100 μg/kg TSC or an equal volume of saline. All animals were breathing room air (approximately 21% fraction of inspired oxygen).

The degree of uptake of the Cu-ATSM was comput-ed in the hypoxic brain tumor of each animal. The PET 64Cu-ATSM images demonstrated activity concentration (μCi/cm3). Image analysis was performed using AMIDE software (http://amide.sourceforge.net/ [Accessed June 1, 2011]). Regions of interest corresponding to the glioma were contoured, and the volume of hypoxic tissue was computed for each animal by integration of the contoured areas on each slice.9 Hypoxia was defined as a Cu-ATSM uptake at least 2 times greater than normally perfused brain tissue.

The relative hypoxic tumor volume was computed by dividing the volume of hypoxic brain tumor on PET imaging by the total tumor volume. This accounted for slight variations in tumor size between animals. The rela-tive hypoxic tumor volume was computed for each ani-mal in the TSC and saline infusion groups.

Immunoblot AnalysisTumors were implanted and confirmed on MR imag-

ing in 5 animals. Animals were infused with 100 μg/kg TSC (3 rats) or an equivalent volume of saline (2 rats). Tumor tissue was then harvested at 1 day after infusion of TSC or saline. The 3 TSC- and 2 saline-treated animals were killed. Tumor was dissected from the surrounding brain by using microsurgical techniques. From each ani-mal, 100 mg of brain tumor tissue was minced into very small pieces by using a clean razor blade.

Brain tumor tissues were lysed with RIPA (radio-immunoprecipitation assay) buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) containing protease inhibitor cocktail (Sigma Chemical Co.) and phosphatase inhibi-tor cocktail (Sigma). Protein content was measured with BCA Protein Assay Reagent (Pierce). The samples were diluted with 2× Laemmli lysis buffer (2.4 M glycerol; 0.14 M Tris, pH 6.8; 0.21 M SDS; 0.3 mM bromophenol blue) containing 1.28 M b-mercaptoethanol, and equal quanti-ties of protein were loaded on 10% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE and electro-phoretically transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in PBS-Tween 20 (0.1% vol/vol) for 1 hour. The

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membrane was incubated overnight at 4°C with appropri-ate antibodies raised against HIF-1a (anti–HIF-1a 1:500, Novus Biologicals), CA9 (anti-CA9 1:2000, Novus Bio-logicals), and b-actin (1:5000, MP Biomedicals). Horse-radish peroxidase–conjugated antirabbit or antimouse IgG was used as the secondary antibody. Immunoreac-tive protein was visualized by the chemiluminescence protocol (ECL Kit, Amersham). To ensure equal protein loading, each membrane was stripped and reprobed with anti-actin antibody to normalize for differences in protein loading.

The Western blot assay was performed with 5 tumor samples, looking at expression of CA9 (anti-CA9 1:2000, Novus Biologicals); HIF-1a (anti–HIF-1a 1:500, Novus Biologicals); and beta-actin (1:5000, MP Biomedicals).

Statistical AnalysisTo evaluate for differences in PET-defined hypoxic

tissue between treatment groups, a commercially avail-able statistical package (StatView, SAS Institute) was used. A p value ≤ 0.05 was defined as statistically sig-nificant.

ResultsClinical Outcomes

All animals included in the study had demonstrable tumor on postcontrast MR imaging and Cu-ATSM PET studies. However, at the time of MR and PET imaging, no animals demonstrated signs or symptoms of tumor mass effect (for example, weakness, poor grooming, > 20% weight loss). No adverse clinical effects were seen as a result of TSC or saline infusion in these animals.

Tumor Compared with Contralateral Brain Tissue on PET Imaging

The PET imaging studies demonstrated decreased uptake of Cu-ATSM within the C6 glioma compared with the contralateral cerebral hemisphere. All tumors dem-onstrated regions of hypoxia at the time of initial PET imaging. The mean relative uptake value of the tumor was 3900 (range 2203–6836). The mean relative uptake value of the contralateral brain tissue was 1017 (range 488–2304). Thus, the tumor tissue demonstrated a rela-tive hypoxia 3.8 times greater than normal brain tissue (p = 0.000002, t-test).

Differences Between Tumor Tissue in TSC- and Saline-Infused Groups on PET Imaging

After infusion, the median volume of hypoxic tumor in the TSC group was 136 mm3 (Fig. 1 left), and the medi-an volume of hypoxic tumor in the saline group was 192 mm3 (Fig. 1 right). The mean relative hypoxic tumor vol-ume for the saline group (6 rats) was 1.01 ± 0.063 (mean ± SEM). The range for the saline group was 0.86–1.14. For the TSC-infused group (6 rats), the mean relative hypoxic tumor volume was 0.69 ± 0.062 (mean ± SEM). The range for the TSC group was 0.46–0.85. Treatment with TSC reduced the relative volume of hypoxic tumor by 31% (Fig. 2). This difference was statistically significant (p = 0.002, t-test).

Expression of CA9 and HIF-1aImmunoblots revealed that all tumor specimens

showed protein expression for CA9 and HIF-1a (Fig. 3). However, there was no consistent difference in the ex-pression of CA9 and HIF-1a between animals at 24 hours after infusion of either TSC or saline.

DiscussionTrans sodium crocetinate has previously been shown

to reduce hypoxia in gliomas and in stroke.10,20 It has also been shown to have promising therapeutic effects in a recent clinical trial (ClinicalTrials.gov Identifier NCT00725881) for reducing the effects of peripheral vas-cular disease.3 The addition of TSC appears to improve oxygenation in hypoxic tissue by facilitating oxygen dif-fusion in plasma.22

Fig. 1. Left: Coronal Cu-ATSM PET image cut through the region of an intracranially placed C6 glioma. This animal was infused with TSC. The blue regions show minimal uptake, whereas the green and yellow regions show areas of tumor hypoxia. Right: Coronal Cu-ATSM PET image cut through the region of an intracranially placed C6 glioma in an animal infused with saline. The green, yellow, and red regions show areas of increased hypoxia in the tumor.

Fig. 2. Bar graph in which the relative hypoxic tumor volume (mean ± SEM) in saline- and TSC-treated groups (6 rats each) are plotted. The difference between the groups was statistically significant.

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Positron emission tomography imaging most fre-quently uses a glucose analog, FDG, to study a given tis-sue’s metabolic activity based on regional glucose uptake. The use of FDG-PET imaging has been proposed as a means of identifying tissues less responsive to radiation, and of targeting these tissues with an additional boost to cancerous lesions.24 However, other radionuclides have been introduced to study specific aspects of cellular ac-tivity. Copper-64 is one such radioactive nuclide that has proven useful for PET imaging; 64Cu-ATSM is preferen-tially taken up by hypoxic compared with normoxic cells. Copper-64 emits positrons in the cells that take up the tracer. Thus, within physiological systems such as the brain, it can provide real-time images of the oxygenation status of tissues in vivo.

In the current study, all tumors demonstrated a sta-tistically significant uptake of Cu-ATSM compared with brain tissue in the contralateral cerebral hemisphere. Up-take of Cu-ATSM was 3.6 times greater on average in the tumor than in normal brain tissue. This underscores the significant hypoxia within the C6 glioma model. When animals were infused with TSC compared with saline, the relative hypoxia was lessened by approximately 31%. It is also conceivable that Cu-ATSM PET imaging could be used to provide targeting information for boost radia-tion therapy or radiosurgery to high-grade, hypoxic tu-mors such as glioblastoma.16 This type of approach with Cu-ATSM targeting has been shown to work for image-guided radiation therapy.1 It is important to validate this neuroimaging technique and the effect of TSC in other animal glioma models.

Expression of HIF-1a and CA9 stimulates a resis-tance of cells to hypoxic conditions and, in turn, makes

cells more able to withstand adjuvant treatments such as radiosurgery. The expression of HIF-1 has correlated with radiation resistance of C6 xenografts.17 In the cur-rent research, we observed HIF-1a and CA9 expression in the C6 tumors. This is consistent with the previous findings that the C6 glioma is a hypoxic tumor model.20 At 24 hours postinfusion, there were no consistent differ-ences between TSC- and control-treated animals. Evalua-tion of additional time points and varying administration (for example, amounts and frequencies) of TSC should be studied to determine its optimum effects in terms of hypoxic protein expression in glioblastoma. In particular, as opposed to the single TSC dose given in the current experiments, 5 doses of TSC were required to provide a survival advantage in this glioblastoma model.19 An addi-tional amount or duration of TSC administration may be required to alter expression of certain hypoxic proteins.

Hypoxia in gliomas represents a significant therapeu-tic challenge. Tumor hypoxia in high-grade gliomas has been widely described, and it may be responsible for stem cell–induced glioma growth and tumor progression.8,18 Improvements in intratumoral oxygenation have led to therapeutic gains related to radiation therapy and chemo-therapy.12

Novel approaches to reduce tumor hypoxia have been tried. For instance, erythropoietin was shown to improve the response to radiation of GBM (glioblastoma multi-forme) Nan1 and U87 cells xenografted to nude mice.15 Other clinical trials and animal studies in which recombi-nant human erythropoietin was used have had a mixture of responses, including some with significant clinical im-provement.6,23 More recently, by having animals breathe carbogen, partial oxygen concentration in both tumor and contralateral brain tissue increased in an F98 glioma model.5 Interceding with hypofractionated radiation ther-apy (9.3 Gy in 4 fractions) in animals treated with carbo-gen led to a significant decrease in glioma tumor growth.5

In a similar fashion, TSC infusion at or around the time of radiation delivery could be used to lessen intra-tumoral hypoxia and increase the therapeutic gains of radiation therapy or radiosurgery. Also, for those chemo-therapeutic agents in which tumor hypoxia may lessen clinical efficacy, TSC may serve to improve the therapeu-tic response too. The optimal dose and timing of delivery to capitalize on TSC’s apparent lessening of glioma hy-poxia during adjuvant therapy remains to be determined. Further investigation is underway with additional PET imaging studies to evaluate the effects of TSC given con-currently with Temodar. Determining the functional re-sponse of TSC with Temodar will be important, because Temodar is not given concurrently with standard and hy-pofractionated radiation therapy for high-grade gliomas.14

ConclusionsThe C6 gliomas consistently demonstrated signifi-

cant intratumoral hypoxia compared with normal brain tissue. Based on Cu-ATSM PET imaging findings, TSC was noted to lessen intratumoral hypoxia by 31%. The re-duction in tumor hypoxia, if achieved at the time of tumor irradiation, may afford a therapeutic gain. Further pre-

Fig. 3. Western blots showing the effect of TSC in a rat C6 glioma model. The C6 glioma cells (CCL-107 cell line, American Type Culture Collection) were implanted into 1 hemisphere of 5 rats (mean mass 200 g). Animals were treated with TSC (100 μg/kg). Equal amounts of pro-tein (20 μg) from tissue lysates were separated by SDS-PAGE and im-munoblotted with anti–HIF-1a and anti-CA9 antibodies.

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clinical studies will need to be performed to determine the optimal dose and timing of TSC infusion as well as to characterize interactions between TSC and other phar-macological agents (for example, Temodar, Dilantin, and corticosteroids).

Disclosure 

The TSC was provided as a gift from Diffusion Pharmaceuticals (Charlottesville, Virginia). The research was funded by a grant to the University of Virginia from Diffusion Pharmaceuticals. The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Conception and design: Sheehan. Acquisition of data: all authors. Analysis and interpretation of data: Sheehan, Popp, Lee, Park. Drafting the article: Sheehan, Lee, Park. Critically re vising the article: Popp, Monteith, Park.

Acknowledgments

The authors appreciate the assistance of Drs. Dongfeng Pan and Stuart Berr in the University of Virginia Department of Radiology; they assisted with the MR and PET imaging as well as final prepara-tion of the Cu-ATSM.

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Manuscript submitted November 20, 2010.Accepted May 17, 2011.Please include this information when citing this paper: published

online June 17, 2011; DOI: 10.3171/2011.5.JNS101954.Address correspondence to: Jason P. Sheehan, M.D., Ph.D., Box

800212, University of Virginia Health System, Charlottesville, Vir-ginia 22908. email: jps2f@virginia.edu.