Malignant glioma: MR imaging by using 5-aminolevulinic acid in an animal model.

Original Research n Experimental

Studies

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Malignant Glioma: MR Imaging by Using 5-Aminolevulinic Acid in an Animal Model1 Hye Rim Cho, MS Dong Hyun Kim, MD Daehong Kim, PhD Philip Doble, PhD David Bishop, PhD Dominic Hare, PhD Chul-Kee Park, MD, PhD Woo Kyung Moon, MD, PhD Moon Hee Han, MD, PhD Seung Hong Choi, MD, PhD

Purpose:

To evaluate the use of 5-aminolevulinic acid (5-ALA) for the noninvasive detection of malignant gliomas by using in vivo magnetic resonance (MR) imaging in a mouse brain tumor model.

Materials and Methods:

The experiments were animal care committee approved. U-87 glioblastoma cells were exposed to 5-ALA (500 µmol/L) for 6 hours, cells were harvested, and intracellular concentrations of iron, heme, protoporphyrin IX, and ferrochelatase were measured (six in each group). BALB/c nude mice (n = 10) were inoculated with U-87 glioma cells to produce orthotopic brain tumors. T2-weighted imaging was performed 3 weeks after inoculation, and T2* maps were created with a 7-T MR imager before and 24 hours after oral administration of 5-ALA (0.1 mg/g of body weight; n = 6) or normal saline (n = 4). Intratumoral iron concentrations were measured with laser ablation inductively coupled plasma mass spectrometry. For in vitro experiments, differences in the measured data were assessed by using the Mann-Whitney U test with Bonferroni correction. For the in vivo studies, differences in T2* values and iron concentrations of the tumors in the 5-ALA and control groups were assessed by using the Mann-Whitney U test.

Results:

The intracellular concentration of heme and iron was increased at both 24 and 48 hours after 5-ALA exposure (P = .004). 5-ALA promoted expression of ferrochelatase in glioblastoma cells at both 24 and 48 hours after 5-ALA exposure compared with that at 1 hour (P = .004). In vivo MR imaging revealed a lower median T2* value in glioblastomas treated with 5-ALA compared with those in control mice (14.0 msec [interquartile range, 13.0–14.5 msec] vs 21.9 msec [interquartile range, 19.6–23.2 msec]; P = .011), and laser ablation inductively coupled plasma mass spectrometry revealed that iron concentrations were increased in glioblastomas from the 5-ALA group.

Conclusion:

Administration of 5-ALA increased the intracellular iron concentration of glioblastomas by promoting the synthesis of heme, which is the metabolite of 5-ALA. Because intracellular iron can be detected at MR imaging, 5-ALA may aid in the identification of high-grade foci in gliomas.

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From the Departments of Radiology (H.R.C., D.H.K., W.K.M., M.H.H., S.H.C.), Radiation Applied Life Science (H.R.C.), and Neurosurgery (C.K.P.), Seoul National University College of Medicine, 28 Yongon-dong, Chongnogu, Seoul, 110-744, Korea; Research Institute, National Cancer Center, Gyeonggi-do, Korea (D.K.); Department of Chemistry and Forensic Science, University of Technology, Sydney, Australia (P.D., D.B., D.H.); and Center for Nanoparticle Research, Institute for Basic Science (S.H.C.), and School of Chemical and Biological Engineering (S.H.C.), Seoul National University, Seoul, Korea. Received June 25, 2013; revision requested August 27; revision received January 16; accepted January 31, 2014; final version accepted March 14. Supported by the National Research and Development Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (grant 1120300), Korea Healthcare Technology Research and Development Projects, Ministry for Health, Welfare, and Family Affairs (grants A112028 and HI13C0015), and Research Center Program of IBS (Institute for Basic Science) in Korea. Supported by a grant from the Australian Research Council (P.D., D.H.). Address correspondence to S.H.C. (e-mail: [email protected]).

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radiology.rsna.org n Radiology: Volume 272: Number 3—September 2014

EXPERIMENTAL STUDIES: Malignant Glioma: MR Imaging with 5-Aminolevulinic Acid

T

he intratumoral heterogeneity of gliomas poses a risk for the histologic undergrading of these tumors, which may lead to a delay in the administration of adjuvant treatment. Thus, intraoperative identification and sampling of the most malignant area are crucial (1). Although magnetic resonance (MR) imaging is the most popular modality for the preoperative detection of anaplastic foci, signal intensity anomalies limit the reliability of grading gliomas, even with the aid of gadolinium contrast agents (2,3). Advanced MR imaging techniques such as diffusionand perfusion-weighted imaging and MR spectroscopy enable the detection of anaplastic foci in gliomas (4–6), although the quantitative values obtained from these sequences frequently overlap in their characterization of low- and high-grade gliomas. Investigators (7,8) have sought to overcome the limitations of anaplastic foci detection and have included the use of isotopically labeled amino acid

Advances in Knowledge nn Administration of 5-aminolevulinic acid (5-ALA) increases the intracellular iron concentration of glioblastomas by promoting the synthesis of heme, which is the metabolite of 5-ALA. nn In cells treated with 5-ALA, the median concentration and interquartile range (IQR) of intracellular iron increased significantly at both 24 and 48 hours (1.89 mg/mg [IQR, 1.81–1.92 mg/mg] and 1.97 mg/mg [IQR, 1.92–1.98 mg/mg], respectively) compared with that at 1 hour (1.08 mg/mg [IQR, 0.88–1.09 mg/mg]; P =.004). nn Iron accumulation induced with 5-ALA in glioblastomas can be detected at MR imaging, where the median T2* value of U-87 glioblastomas treated with 5-ALA was lower than that of control tumors, which received normal saline (14.0 msec [IQR, 13.0– 14.5 msec] vs 20.8 msec [IQR, 19.6–21.0 msec]; P = .031).

tracers to enhance imaging modalities such as carbon 11 (11C) methionine positron emission tomography (PET) or fluorine 18 fluoroethyl-l-tyrosine PET. In the case of 11C methionine PET, histologic analysis confirmed that voxels with maximum tracer uptake represent the most malignant tumor areas (9– 12). However, PET exposes the patient to radiation (13) and is only available in highly specialized neuro-oncologic centers. Authors of a 2010 study (1) identified 5-aminolevulinic acid (5-ALA) as a new marker for the detection of malignant foci in diffusely infiltrating gliomas. 5-ALA is a nonfluorescent prodrug that leads to the intracellular accumulation of fluorescent protoporphyrin IX (PpIX) in malignant glioma cells (14). The fluorescent signal derived from PpIX provides enhanced contrast between tumors and normal tissue, enabling more complete resection and improving the progression-free survival rate in patients with malignant gliomas (15). However, in low-grade gliomas, the utility of 5-ALA fluorescence has not been studied (16–18). The polymerization of eight 5-ALA monomers results in the synthesis of PpIX, which is critical for heme synthesis (19,20). In the final stages of heme synthesis, iron enters the heme cycle when mitochondrial ferrochelatase (FECH) incorporates ferrous iron into PpIX to generate heme (21). Synthesized heme is degraded by heme oxygenase to produce equimolar quantities of biliverdin, free ferrous iron, and carbon monoxide (22). Results of previous studies (23,24) have shown that high iron concentrations as a result of heme accumulation appear hypointense on T2-weighted images because of the susceptibility effect of iron. The purpose of our study was to use 5-ALA for the noninvasive detection of

Implication for Patient Care nn Because intracellular iron can be detected at MR imaging, 5-aminolevulinic acid–enhanced MR imaging may aid in the identification of high-grade foci in gliomas.

Radiology: Volume 272: Number 3—September 2014 n radiology.rsna.org

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malignant glioma in vivo at MR imaging. We hypothesized that 5-ALA would selectively accumulate in malignant glioma cells and be converted to heme, which could then be detected at MR imaging. To this end, we developed an orthotropic mouse brain tumor model by using the human glioblastoma cell line U-87MG. In addition, we confirmed the accumulation of iron in malignant glioma cells by using laser ablation inductively coupled plasma (ICP) mass spectrometry.

Materials and Methods In Vitro Experiments Figure E1 (online) summarizes the in vitro experiments, which were performed by one author (H.R.C., with 7 years of experience), and the comparisons were performed for the data from six cell culture wells in each group except for the cell viability test (n = 10). First, the U-87 cells (ATCC, Manassas, Va) originating from glioblastoma multiforme, a rapidly growing and highly invasive human brain tumor (25), were cultured at 37°C in a humidified carbon dioxide incubator with Roswell Park Memorial Institute medium and 10% fetal bovine serum. Published online before print 10.1148/radiol.14131459 Content code: Radiology 2014; 272:720–730 Abbreviations: FECH = ferrochelatase ICP = inductively coupled plasma IQR = interquartile range PpIX = protoporphyrin IX RFU = relative fluorescence unit 5-ALA = 5-aminolevulinic acid Author contributions: Guarantor of integrity of entire study, S.H.C.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, H.R.C., D.H.K., S.H.C.; experimental studies, H.R.C., D.K., P.D., D.B., D.H., C.K.P., S.H.C.; statistical analysis, H.R.C., D.H., S.H.C.; and manuscript editing, H.R.C., D.H.K., P.D., D.B., C.K.P., W.K.M., M.H.H., S.H.C. Conflicts of interest are listed at the end of this article.

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The cell viability test was performed with an MTT assay (Sigma-Aldrich, St Louis, Mo) for iron supplementation (Fig E2 [online]). Then, the cells were incubated for 48 hours in media containing ferric ammonium citrate, for which the final concentration was 100 µmol/L. Second, the cells were washed with phosphate-buffered saline for the prevention of further exposure to ferric ammonium citrate and were incubated for 6 hours in Roswell Park Memorial Institute medium with 5-ALA (500 µmol/L) or without 5-ALA. Then, intracellular and extracellular PpIX was measured in the cells without exposure to ferric ammonium citrate. Third, the medium was replaced with Roswell Park Memorial Institute medium without 5-ALA, and the cells were further incubated for 1, 24, or 48 hours. The cells were harvested at 1, 24, or 48 hours after incubation and were used to measure the intracellular concentrations of iron, heme, PpIX, and FECH. Measurement of PpIX in cells after 48-hour exposure to ferric ammonium citrate.—For fluorescence microscopy, U-87 (1 3 105) cells were incubated in six-well plates, which were wrapped in aluminum foil to avoid light exposure, with or without 5-ALA for 6 hours (26– 28), and then, the cells were fixed and mounted. Cellular PpIX was analyzed by using laser scanning microscopy (magnification, ×200; LSM 510 META, Carl Zeiss, Oberkochen, Germany). We optimized a previously described method for quantifying 5-ALA–induced PpIX fluorescence (29). To determine the extracellular fluorescence intensity, the supernatant from incubated samples was removed for analysis. To determine the intracellular fluorescence intensity, cells were lysed and mixed with methanolic perchloric acid (5.6%, 8°C). The fluorescence intensity was measured by using a fluorescence microplate reader (Infinite M200, Magellan software Tecan, Männedorf, Switzerland) at an excitation wavelength of 400 nm 6 30 and an emission wavelength of 645 nm 6 40. The relative fluorescence unit (RFU) values were standardized to 722

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total cellular protein and are shown as RFUs per microgram. Measurement of intracellular iron, heme, and PpIX at 1, 24, and 48 hours after exposure to 5-ALA.—Iron concentration was determined by using a total iron reagent kit (Pointe Scientific, Canton, Mich). The production of heme in the cells was assayed by using a commercially available kit (QuantiChrom Heme assay Kit, DIHM-250; Gentaur, Kampenhout, Belgium). The average concentrations of iron and heme were standardized to total cellular protein levels and are shown in micrograms per milligram. The intracellular accumulation of PpIX was measured by using fluorescence imaging, as described. Measurement of intracellular FECH at 1, 24, and 48 hours after exposure to 5-ALA.—The intracellular concentration of FECH was determined by using a human FECH ELISA kit (E2083h; EIAab Science, Wuhan, China). The intensity was measured in a microplate reader at 450 nm. The average concentrations were standardized to total cellular protein and are shown in nanograms per milligram. In addition, the protein levels of intracellular FECH were evaluated by means of western blot analysis. Cells were lysed in ice-cold lysis buffer (20 mmol/L of Tris hydrochloride [pH 7.5], 150 mmol/L of sodium chloride, 1 mmol/L of disodium edetic acid, 1 mmol/L of ethylene glycol tetraacetic acid, 1% Triton X-100 [Sigma-Aldrich], 2.5 mmol/L of sodium pyrophosphate, 1 mmol/L of b-glycerophosphate, 1 mmol/L of sodium orthovanadate, 1 mg/mL of leupeptin, and 1 mmol/L of protease inhibitor cocktail [Sigma]), and the concentration of lysate protein was evaluated with the bicinchoninic acid method (Pierce Biotechnology, Rockford, Ill). Approximately 30 mg of protein were loaded in each lane of a polyacrylamide denaturing gel for electrophoresis. After electrophoresis, the protein was transferred to nitrocellulose membranes for blotting. We used a rabbit polyclonal antibody to FECH (Santa Cruz Biotechnology, Dallas, Texas), and primary antibodies were detected by using horseradish

peroxidase–conjugated antibodies (Santa Cruz Biotechnology). The relative amount of protein was normalized to b–actin, and the quantification was assessed by using software (Image J; National Institutes of Health, http://rsbweb.nih.gov/nih-image/).

In Vivo Experiments Figure E3 (online) summarizes the in vivo experiments. These experiments were approved by the animal care committee at Seoul National University Hospital. Induction of tumors.—A total of 19 mice with orthotopic brain tumors were used in this study, which was performed by one author (H.R.C.). To produce mice with orthotopic brain tumors, 6-week-old male BALB/c nude mice were anesthetized by means of intraperitoneal injection with a mixture of zolazepam and xylazine and were placed in a stereotaxic device. The mice were inoculated with U-87 glioma cells (1 3 106 cells per 3 mL of serum-free Roswell Park Memorial Institute medium) in the left caudate-putamen region. The cells were injected in the brain by using a Hamilton syringe fitted with a 28-gauge needle, which was positioned with a syringe attachment fitted to the stereotaxic device. The following coordinates, with stereotaxic guidance, were used in the posterior, lateral, and dorsal to the bregma in the left caudate-putamen: 0, 1.4, and 3.0 mm, respectively. Administration of 5-ALA and imaging experiments.—We performed a study to optimize the 5-ALA dose in nine mice, which were given 5-ALA orally (0.02 mg/g of body weight [n = 3]; 0.1 mg/g of body weight [n = 3]; and 0.2 mg/g body weight [n = 3]) 24 hours before MR imaging. For the main study, the mice received 5-ALA orally (0.1 mg/g of body weight) (n = 6) or normal saline (n = 4) 24 hours before MR imaging. We acquired images from anesthetized mice 3 weeks after inoculation by using a 7-T MR imager (BioSpec; Bruker, Ettlingen, Germany). MR imaging experiments were performed by two authors (H.R.C. and D.K., both with 15 years of experience) before and 24 hours after



the oral intake of 5-ALA or normal saline. Coronal imaging was performed at the site of the glioma cells by using T2-weighted spin-echo and T2*weighted gradient-echo sequences. For T2-weighted imaging, the following acquisition parameters were used: repetition time msec/echo time msec, 1500/35; matrix size, 256 3 256; field of view, 20 3 20 mm; section thickness, 0.7 mm; and number of signals acquired, eight. For the estimation of T2*, we used a gradient-echo pulse sequence with the following imaging parameters: 1500/2.76, 6.42, 10.08, 13.75, 17.41, 21.07, 24.73, 28.40; flip angle, 30°; matrix size, 256 3 256; field of view, 20 3 20 mm; section thickness, 0.7 mm; pixel resolution, 0.08 3 0.08 mm2; no intersection gap; and number of signals acquired, one. In total, eight sections were imaged per mouse. For the analysis of T2* in the brain tumors, we used a previously described method (30). After the confirmation of boundaries of each tumor on T2-weighted spin-echo images, regions of interest were defined manually in individual sections imaged with the shortest echo time, and these data were used to generate T2* maps of the regions of interest by using pixel-bypixel analyses with software (MATLAB; MathWorks, Natick, Mass) throughout the eight-point MR imaging examinations, assuming single exponential decay (ie, SI = SI0 · e2TE/T2*, where SI is the signal intensity, SI0 is the proton density, and TE is echo time). A T2* histogram was obtained for each animal by including all pixels in the regions of interest (eg, brain tumor) throughout all sections. In the histogram, the number of pixels on the y-axis was expressed and the pixels were distributed according to T2* value on the x-axis. In terms of T2*-weighted images obtained 24 hours after the oral intake of 5-ALA or normal saline, the number of distributing pixels with T2* values lower than the median value in the histogram of T2* obtained from tumors before administration of 5-ALA was defined as pixels with low T2*. Histologic analysis.—Mice were sacrificed for histologic examination

immediately after the final MR imaging examinations, which were performed by one author (H.R.C.). Before histologic analysis, the brains were fixed in 10% buffered formalin. Paraffin-embedded brains were divided into 4-µm or 10- µm slices. Prussian blue staining of the 4-µm sections was used to visualize iron deposition. Measurement of intratumoral iron concentrations by using laser ablation ICP mass spectrometry.—We measured the intratumoral iron levels of the mice in the 5-ALA (n = 6) and control groups (n = 4) by using laser ablation ICP mass spectrometry, which was performed by one author (P.D., with 17 years of experience). The analysis was performed by using a laser ablation instrument (New Wave Research UP-213; Kenelec Technologies, New South Wales, Australia) fitted with a large format cell. Argon was used as the carrier gas. The laser unit was used with an ICP mass spectrometry instrument (7500cx; Agilent Technologies, Palo Alto, Calif) fitted with a cs lens system, a platinum sampler, and skimmer cones. Before analysis, the system was tuned for sensitivity by using a National Institute of Standards and Technology 612 trace element in glass and according to the tissue standards at our institution. Oxide formation was controlled by limiting the amount of 232Th16O+/232Th+ to less than 0.3% for the ablation of National Institute of Standards and Technology 612. Data were acquired by ablating in adjacent lines on each specimen (10 mm thick) with a beam diameter of 100 mm and an ablation speed of 300 mm · sec21. Mass spectrometer integration times were chosen to maintain the true image dimensions (31) when processed, such that a single pixel represented 100 mm2. Compensation for variation in laser power output and instrument drift was made by means of normalization to the carbon 13 signal (32). Quantitative data were produced by means of representative ablation according to tissue standards by using a previously described method (33,34). Briefly, chicken breast tissue was purchased from a local market and stripped


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of all fatty and connective tissue. Dissected tissue was partially homogenized as 5-gram aliquots by using a tissue homogenizer (OmniTech TH; Kelly Scientific, New South Wales, Australia) fitted with a polycarbonate probe. Then, 10–100 mL aliquots of standard metal solutions were prepared from high-purity iron-nitrate salts (Sigma-Aldrich) and added to the standard preparations, which were then further homogenized. Next, six approximately 250-mg aliquots of each standard were digested in a 3:1 ratio of nitric acid to hydrogen peroxide in a microwave digester (Milestone MLS 1200; John Morris Scientific, Chatswood, Australia), and each standard was analyzed by solution nebulization ICP mass spectroscopy to accurately determine the trace metal concentration and homogeneity of the standards. Data were reduced to multispectral images by using the Interactive Data Imaging Spectral Data Analysis Software developed by the University of Technology, Sydney Computational Research Support Unit. This software is a specialized data-reduction package written in the Python programming language. Images were exported in a visualization toolkit (.vtk) format into a visualization interface (Enthought Mayavi2; http:// code.enthought.com/projects/mayavi/) for color rendering. Quantitative data were extracted by freehand outlining of the regions of interest with Interactive Data Imaging Spectral Data Analysis Software. Immunofluorescence staining for visualization of FECH in glioblastoma tumors.—One author (H.R.C.) performed immunofluorescence staining for visualization of FECH in glioblastoma tumors from an in vivo mice model by using the 4-µm sections. For immunofluorescence staining, prepared paraffin sections were dewaxed, hydrated, and treated with 0.01% protease XXIV (Sigma-Aldrich) in phosphate-buffered saline for 20 minutes at 37°C. Sections were then stained with a primary antibody to FECH according to the manufacturer’s instructions. The expression was visualized with Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, Calif). 723


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Figure 1

Figure 1: Box plots show measurement of intracellular iron, heme, and PpIX after incubation with 5-ALA for 1, 24, and 48 hours. A, In cells treated with 5-ALA, concentration of intracellular iron increased significantly at both 24 and 48 hours compared with that at 1 hour (P = .004). B, Levels of intracellular heme increased significantly (P = .004) with time in cells treated with 5-ALA and controls. At 24 hours, heme values were significantly different between cells treated with 5-ALA and those left untreated (P = .004). C, Intracellular PpIX concentration was increased at 1 hour after exposure to 5-ALA but had decreased by 24 and 48 hours (P = .004).

Statistical Analysis Statistical analyses were performed by using commercially available software (MedCalc, version 11.1.1.0; MedCalc Software, Mariakerke, Belgium). Data are presented as median and interquartile range (IQR, range from the 25th to the 75th percentile). For in vitro experiments, the statistical analyses were as follows: differences in the measured data (cell viability, intracellular and extracellular concentration of PpIX, and intracellular concentrations of iron, heme, PpIX, and FECH) were assessed with the Mann-Whitney U test. With Bonferroni correction to adjust for multiple comparisons, a P value of less than .0125 was considered to be indicative of a statistically significant difference for the concentration of intracellular and extracellular PpIX, and a P value of less than .0083 was considered to indicate a significant difference for cell viability and intracellular concentrations of iron, heme, and FECH. For the in vivo studies, differences in T2* values and iron concentration of the tumors between the 5-ALA and control groups were assessed by using the Mann-Whitney U 724

test, with a P value of less than .05 indicating a significant difference, and differences in T2* values between before and after administration of 5-ALA or saline were assessed by using the Wilcoxon signed-rank test, with a P value of .05 indicating a significant difference.

Results In Vitro Experiments Confocal laser scanning microscopy revealed defined red fluorescence in the cytoplasm of cells treated with 5-ALA in comparison to untreated cells (Fig E4 [online]). After treatment of cells with 5-ALA, the intracellular PpIX concentration was significantly increased compared with that in untreated control cells (203.1 RFU/mg [IQR, 202.5–203.3 RFU/mg] vs 17.3 RFU/mg [IQR, 16.9–17.8 RFU/mg]; P = .004). Moreover, the extracellular concentration of PpIX in the medium from cells treated with 5-ALA was also higher than that detected in the medium from untreated control cells (18.3 RFU/mg [IQR, 17.8–18.8 RFU/

mg] vs 11.4 RFU/mg [IQR, 11.4–11.4 RFU/mg]; P = .004) (Fig E5 [online]). In cells treated with 5-ALA, the concentration of intracellular iron increased significantly at both 24 and 48 hours compared with that at 1 hour (1.89 mg/mg [IQR, 1.81–1.92 mg/mg] and 1.97 mg/mg [IQR, 1.92–1.98 mg/ mg] vs 1.08 mg/mg [IQR, 0.88–1.09 mg/mg]; P = .004) (Fig 1, A). The concentration of intracellular heme also increased significantly with time (1, 24, and 48 hours) in cells treated with or without 5-ALA (Fig 1, B). At 24 hours, a significant difference was also observed between the cells treated with and without 5-ALA (1.20 mg/ mg [IQR, 1.16–1.24 mg/mg] vs 0.82 mg/mg [IQR, 0.80–0.89 mg/mg]; P = .004) (Fig 1, B). Furthermore, the intracellular concentration of PpIX increased dramatically within 1 hour after exposure to 5-ALA but decreased by 24 and 48 hours after treatment (P = .004) (Fig 1, C). After treatment with 5-ALA, the higher intracellular concentration of FECH was observed at 24 hours than that at 1 hour and 48 hours (0.315 ng/ mg of protein [0.290–0.340 ng/mg] vs



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Figure 2

Figure 2: A, Box plot shows measurement of intracellular concentration of FECH after incubation with 5-ALA for 1, 24, and 48 hours. After treatment with 5-ALA, higher intracellular concentration of FECH was observed at 24 hours than that at 1 hour and 48 hours (P = .004), and the intracellular concentration of FECH was significantly lower at 48 hours than at 1 hour and 24 hours in untreated cells (P = .004). At both 24 and 48 hours, intracellular concentration of FECH was higher in cells treated with 5-ALA than in those not treated with 5-ALA (P = .004). B, Western blot analysis and, C, box plot show measurement of FECH expression after incubation with 5-ALA for 1, 24, and 48 hours. Up-regulated FECH protein in cells with treatment of 5-ALA was observed at 24 hours, which was compared with that at 1 hour (P = .004).

0.195 ng/mg [0.184–0.200 ng/mg] and 0.256 ng/mg of protein [0.244–0.260 ng/mg], P = .004), and the intracellular concentration of FECH was significantly lower at 48 hours than that at 1 hour and 24 hours in untreated cells (0.132 ng/mg of protein [IQR, 0.131– 0.135 ng/mg] vs 0.167 ng/mg [IQR, 0.162–0.171 ng/mg] and 0.185 ng/mg of protein [IQR, 0.172–0.207 ng/mg]; P = .004) (Fig 2, A). At both 24 and 48 hours, the intracellular concentration of FECH was higher in cells treated with 5-ALA than in those not treated with 5-ALA (P = .004). This phenomenon was also observed at western blot analysis; the up-regulated FECH protein in the cell with treatment of 5-ALA was observed at 24 hours, which was compared with that at 1 hour (0.93 ng/mg [IQR, 0.90–1.15 ng/ mg] vs 0.57 ng/mg [IQR, 0.41–0.60 ng/mg]; P = .004) (Fig 2, B and C). However, at the same time intervals,

FECH protein was not up-regulated without 5-ALA treatment (P . .05) (Fig 2, B and C).

In Vivo Experiments We regarded 0.1 mg/g of body weight as the optimal dose for 5-ALA–enhanced MR imaging (Fig E6). T2* mapping showed that the median T2* value of U-87 glioblastoma tumors treated with 5-ALA was lower than that of control tumors, which received normal saline (14.0 msec [IQR, 13.0–14.5 msec] vs 21.9 msec [IQR, 19.6–23.2 msec]; P = .011) (Fig 3). There was no significant difference between the median T2* values of brain tumors before and after the administration of saline (23.1 msec [IQR, 20.7–24.1 msec] vs 21.9 msec [IQR, 19.6–23.2 msec]; P = .471; Fig 3, A). However, T2* histograms showed that the number of pixels with low T2* was higher in brain tumors 24 hours after subjects


received 5-ALA compared with the number measured before treatment. Finally, the median T2* value of brain tumors significantly decreased after treatment with 5-ALA (14.0 msec [IQR, 13.0–14.5 msec] vs 20.8 [IQR, 19.6–21.0 msec]; P = .031; Fig 3, B). Prussian blue staining revealed multifocal iron deposits at the border between the tumor and the brain in mice treated with 5-ALA. However, no staining of iron deposits was observed in control mice treated with saline (Fig 4, A). A substantial difference in the concentration of iron was observed between the control animals and the 5-ALA–treated animals (38.8 mg/g [IQR, 31–42 mg/g] vs 51.9 mg/g [IQR, 48–60.5 mg/g]; P = .014; Fig 4, B). In addition, we also observed the increased FECH expression in the glioblastomas from the 5-ALA–treated animals (Fig 5). 725


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Figure 3

Figure 3: T2* maps and graphs show detection of heme synthesized as a result of PpIX generated by 5-ALA treatment in U-87 glioblastomas. Number of pixels (blue bars) was used to calculate median T2* value, and these values were used for detection of hemeinduced changes in T2*. Median T2* value of U-87 glioblastomas treated with 5-ALA was lower than that of control tumors, which received normal saline (P = .011). A, T2* maps showed no significant difference between median T2* values of brain tumors before and after receiving saline (P = .471). B, However, median T2* value of brain tumors significantly decreased after treatment with 5-ALA (P = .031). Arrow indicates U-87 glioblastoma in mouse brain, and numbers are median T2* values of tumors.

Discussion In our study, we observed that U-87 glioblastoma cells had the highest concentration of PpIX at 1 hour after 6 hours of exposure to 5-ALA, and the concentration of PpIX subsequently decreased by 24 and 48 hours after exposure, but 726

the intracellular concentrations of both heme and iron, which are metabolic products of 5-ALA, were increased at both 24 and 48 hours after 5-ALA exposure. In addition, we found that 5-ALA promoted the expression of FECH, a catalytic enzyme that converts PpIX into heme in U-87 glioblastoma cells,

and this expression level was higher at 24 and 48 hours after 5-ALA exposure than at 1 hour after exposure. FECH is likely responsible for iron accumulation in U-87 glioblastoma cells. In vivo MR imaging revealed a lower T2* value in U-87 glioblastomas from mice treated with 5-ALA compared with control



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Figure 4

Figure 4: Determination of heme iron in brain tumors at histologic analysis. A, Multifocal iron deposits (arrows) at border (dotted line) between tumor and brain in mice treated with 5-ALA were stained with Prussian blue. No staining was observed in control mice treated with saline. B, Intratumoral iron levels showed substantial difference in mean concentration of iron (arrow) between control animals and 5-ALA–treated animals measured with laser ablation ICP mass spectrometry (38.8 mg/g [IQR, 31–42 mg/g ] vs 51.9 mg/g [48–60.5 mg/g]; P = .014). T = tumor, B = brain.

mice, and laser ablation ICP mass spectrometry also revealed that the iron concentrations were increased in U-87 glioblastomas from mice after exposure to 5-ALA, which is well correlated with the T2* change of the tumors. 5-ALA is a precursor in the hemoglobin synthesis pathway, and exogenous oral administration of this molecule several hours before surgery leads to the preferential accumulation of PpIX in tumor cells (35,36). Preclinical and clinical study results suggest that the accumulation of PpIX in glioblastoma cells may be caused by various factors (37). Because the normal bloodbrain barrier is impermeable to 5-ALA, the compromised blood-brain barrier in glioblastoma tissue is required for 5-ALA to cross and make contact with glioblastoma cells. 5-ALA enters tumor

cells through transporters such as peptide transporter 2, which is the primary transporter responsible for 5-ALA uptake in astrocytes (38,39). Furthermore, Teng et al (35) demonstrated that FECH messenger RNA expression was significantly downregulated in glioblastomas compared with that in normal brain tissue, which is also responsible for the intracellular accumulation of PpIX. In practice, 5-ALA is given to patients orally 2.5 to 3.5 hours before the administration of anesthesia. Under blue-violet light, the fluorophore PpIX emits light in the red region of the visible spectrum, enabling the identification of tumor tissue that might otherwise be difficult to distinguish from normal brain tissue (36). We observed a similar change in PpIX fluorescence in U-87 cells after exposure to 5-ALA.


In addition, these U-87 cells showed an increase in the concentration of both intracellular iron and heme 24 hours after exposure to 5-ALA. We also observed the increased FECH expression in the glioblastomas from the 5-ALAtreated animals. Thus, we believe that 5-ALA exposure can increase FECH expression to stimulate the metabolism of PpIX to heme in glioblastoma cells, a process that takes approximately 6–24 hours. In routine neurosurgical practice, MR imaging contrast material–enhancement is used to visualize the most malignant areas of a tumor. However, in diffusely infiltrating World Health Organization grades II and III gliomas, such contrast material uptake frequently is not observed; the absence of significant contrast enhancement was reported in 727


Cho et al

Figure 5

Figure 5: Immunofluorescence staining images for visualization of FECH in glioblastoma tumors from in vivo mice model. Glioblastoma tumors from mice treated with 5-ALA showed higher expression of FECH than did control tumors.

up to 55% of World Health Organization grade III gliomas and up to 56% of low-grade gliomas by using contrast media with MR imaging (1). Thus, 5-ALA–induced fluorescence may be effective for guiding the surgical resection of high-grade gliomas in these patients, resulting in significant improvement in completeness of resection and 6-month progression-free survival (36). Some high-grade gliomas can be positive for 5-ALA fluorescence without showing contrast enhancement on MR images. Because 5-ALA is a small but polar amino acid, its uptake into the brain may depend on slight perturbations in the integrity of the blood-brain barrier, although not as severe as those necessary for gadolinium to enter the brain (40). In the present study, in vivo MR imaging revealed a significant decrease in the T2* values of glioblastomas 24 hours after the oral administration of 5-ALA compared with both baseline MR imaging and MR imaging values obtained from mice in the control group. In glioblastoma cells, heme is generated by the incorporation of ferrous iron into the metabolite of 5-ALA, PpIX, by FECH (22). Thus, we believe that ferrous iron incorporation into heme is a major contributor to the T2* 728

contrast of in vivo MR images. Our results suggest that 5-ALA can be used for the detection of malignant foci in gliomas by using MR imaging, especially for postoperative evaluation and follow-up MR imaging. In our study, we used laser ablation ICP mass spectrometry with an in vivo model of glioblastoma to examine iron concentrations in brain sections after MR imaging. Laser ablation ICP mass spectrometry can be used for the in situ analysis of trace metals in biologic tissue, and it is designed to measure and analyze trace levels of elements, unlike other forms of organic mass spectroscopy, which are used to identify and quantify molecular compounds. Furthermore, laser ablation is a sample introduction system for ICP mass spectroscopy that enables the determination of the elemental composition of solid materials, including tissue (33). The iron concentration of glioblastomas exposed to 5-ALA was higher than that of glioblastomas in the control group, which correlated with the MR imaging results. The laser ablation ICP mass spectrometric results further supported the hypothesis that the decreased T2* value of glioblastomas exposed to 5-ALA was due to

the increased iron concentration in the tumors. In this study, we observed the difference in the increase of iron concentration in the glioblastoma cells exposed to 5-ALA compared with that in the control group between in vitro and in vivo experiments: the in vitro experiment revealed that the concentration of intracellular iron was 16.8% higher in glioblastoma cells exposed to 5-ALA than in untreated cells at 24 hours, whereas the mean concentration of iron was measured 44.4% higher in the glioblastomas of the 5-ALA–treated animals than in those of control animals in the in vivo experiment. We believe that this difference between in vitro and in vivo experiments can be explained by the high concentration (100 µM) of ferric ammonium citrate in the culture media (eg, transferrin-bound iron in blood plasma ranges from 9 to 30 µM [41]), which was used for the iron supplement, because a soluble ferric iron complex has been reported to be a good source for iron accumulation in cultured cells (42). In addition, ferric ammonium citrate has been known to increase intracellular iron in cultured cells (43). After application of ferric ammonium citrate, cultured cells tend



to accumulate iron in a time- and concentration-dependent manner. Finally, the glioblastoma cells exposed only to ferric ammonium citrate also accumulated iron, which we believe resulted in less difference of the in vitro intracellular iron concentrations (the cells treated with 5-ALA vs untreated cells) than that of the in vivo iron concentrations of tumors (the 5-ALA-treated animals vs control animals). Our study had some limitations. First, we did not compare 5-ALA–enhanced MR imaging with gadoliniumenhanced MR imaging for the detection of high-grade gliomas. We believe that 5-ALA–enhanced MR imaging can be helpful for the detection of nonenhancing high-grade gliomas, and a future study with both enhancing and nonenhancing high-grade glioma models is warranted to prove our hypothesis. Second, we did not evaluate the effect of the infiltrative glioma cells and their interaction with nontumor cells for 5-ALA uptake and metabolism, which will require another animal model study, because our model simulated mass-forming high-grade gliomas. We believe that 5-ALA–enhanced MR imaging is limited for the detection of microinvasion of glioma cells without blood-brain barrier breakdown, because of the dependency of 5-ALA on the integrity of the blood-brain barrier (40). Future study is also warranted regarding this issue. Third, we did not perform an in vivo study for optimization of the imaging time for 5-ALA–enhanced MR imaging, which we decided was 24 hours after 5-ALA administration according to in vitro data. We believe that there is no substantial difference in 5-ALA metabolism between in vitro and in vivo conditions. Fourth, we used a higher dose of 5-ALA to create sufficient contrast on MR images than that used for usual fluorescence-guided surgery (100 mg/kg vs 20 mg/kg), which has potential adverse effects for future clinical applications including hepatic toxicity, neurotoxicity, and light-induced photosensitization (44). Fifth, we did not standardize the T2* value for the measurement of iron concentration in tissues. Instead, we evaluated the change of T2* values from before to after 5-ALA administration.

We expect that a method such as quantitative susceptibility mapping will be more helpful for the future studies regarding MR imaging with 5-ALA. Sixth, the iron distribution in glioblastomas demonstrated at laser ablation ICP mass spectrometry was slightly different from that on the T2* maps of the tumors. We believe that this phenomenon can be explained by the difference in thickness between the MR imaging sections and slices for laser ablation ICP mass spectrometry (0.7 mm [700 mm] vs 10 mm), where MR imaging reveals a volumeaveraging effect. In conclusion, the results of our study showed that 5-ALA administration increases the intracellular iron concentration of glioblastomas by promoting the synthesis of heme, which is the metabolite of 5-ALA. Because intracellular iron can be detected at MR imaging, we believe that 5-ALA–enhanced MR imaging will aid in the identification of high-grade foci in gliomas, and a future study is warranted regarding clinical applicability of 5-ALA–enhanced MR imaging and its safety. Acknowledgment: The authors would like to acknowledge Adjunct Professor Rudolph Grimm at UC Davis Food Science and Technology for facilitating the collaboration between Seoul National University Hospital and University of Technology, Sydney. Disclosures of Conflicts of Interest: H.R.C. No relevant conflicts of interest to disclose. D.H.K. No relevant conflicts of interest to disclose. D.K. No relevant conflicts of interest to disclose. P.D. No relevant conflicts of interest to disclose. D.B. No relevant conflicts of interest to disclose. D.H. No relevant conflicts of interest to disclose. C.K.P. No relevant conflicts of interest to disclose. W.K.M. No relevant conflicts of interest to disclose. M.H.H. No relevant conflicts of interest to disclose. S.H.C. No relevant conflicts of interest to disclose.

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