Figures
Abstract
Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using 18F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as 18F-Fluorocholine (18F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer 18F-FCH/18F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnosis based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to 18F-FCH and 18F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The 18F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in 18F-FCH uptake between tumor and normal brain tissue (2.65 ± 0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by 18F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of 18F-FCH/ 18F-FDG-PET imaging in this study, while the false-positive caused by 18F-FCH can be eliminated. Dual tracer 18F-FCH/18F-FDG PET imaging has the potential to improve the visualization of low grade glioma. 18F-FCH delineates tumor areas and the tumor can be identified by subtracting the 18F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.
Citation: Fu Y, Ong L-C, Ranganath SH, Zheng L, Kee I, Zhan W, et al. (2016) A Dual Tracer 18F-FCH/18F-FDG PET Imaging of an Orthotopic Brain Tumor Xenograft Model. PLoS ONE 11(2): e0148123. https://doi.org/10.1371/journal.pone.0148123
Editor: Russell R. Lonser, Ohio State University, UNITED STATES
Received: April 17, 2015; Accepted: January 13, 2016; Published: February 4, 2016
Copyright: © 2016 Fu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information file.
Funding: The authors acknowledge the funding support from Biomedical Research Council (BMRC), A*STAR under the grant numbers BMRC/07/1/21/19/508. CHW and WZ acknowledge the support from the National Research Foundation (NRF), Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme. Grant Number R-706-001-101-281, National University of Singapore.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Gliomas (glioblastoma multiforme, GBM) are the most common primary tumors of the cerebral hemisphere. They are highly malignant and possess a poor prognosis. Similar to all other cancers, the key to survival for glioma patients is early diagnosis. Unfortunately, the early symptoms of gliomas are highly non-specific and patients are usually diagnosed at the late stage of this disease.
Currently, the diagnosis of glioma mainly relies on CT and MRI. Although CT and MR can provide high resolution images, the tumor diagnosis can be delayed since these two imaging techniques rely on the detection of anatomical changes resulted by tumor growth. Contrastingly, PET imaging detects biochemical abnormalities that precedes anatomical changes, and hence may provide an earlier diagnosis of glioma, particularly low grade gliomas. However, the appropriate radiotracer for PET imaging of glioma has not been determined yet.
As the most commonly used PET radiopharmaceutical, 18F-fluorodeoxyglucose (18F-FDG) is a glucose analogue and is taken up significantly by normal brain cells relative to glioma, making the use of PET in the diagnosis of glioma inefficient and inconclusive except in cases of advanced stage glioma [1–5].
18F-Fluorocholine (18F-FCH), a choline analogue, is a radiotracer developed for the imaging of prostate cancer. Choline is a precursor for phospholipids and is incorporated into phosphatidylcholine, a primary component for cell membrane construction through the activity of choline kinase. Tumor cells often present an elevated level of choline kinase, resulting in an increased uptake of 18F-FCH [6–8]. Nuclear Magnetic Spectroscopy data revealed a high choline content in gliomas [9]. Animal studies showed that 18F-FCH is also taken up by glioma with a high tumor to background ratio [10]. Clinical experiments represented that 18F-FCH accumulates significantly in high grade glioma [11], but little is known about its distribution into low grade gliomas. Recent clinical studies demonstrated a high sensitivity of 18F-FCH PET/CT detection for low-grade glioma based on experiments involving 18 patients [12], with no false positive being observed. However, it still has been reported that 18F-FCH can accumulate in normal brain cells [6, 10] to result in false positive.
In this study, the possibility of using dual tracer of 18F-FCH/18F-FDG for PET imaging in low grade glioma diagnosis is investigated. The main assumption is: 18F-FCH may effectively demarcate the glioma from normal cerebral tissues and the presence of tumor may be confirmed on the merged 18F-FCH/18F-FDG images. The basic principle of the dual tracer technique in this study is owing to the potential false-positive introduced by possible accumulation of 18F-FDG and 18F-FCH in normal brain cell in the low grade glioma diagnosis with PET imaging applied, the overlapping on the merged 18F-FDG/18F-FCH PET images is able to effectively identify the tumor and hence improve the diagnosis accuracy.
As indicated in this article, a CT scan is obtained as a reference to determine the brain regions in the 18F-FDG and 18F-FCH PET images. Since 18F-FDG scan may falsely label the region of the normal tissue as the tumor, and 18F-FCH scan is able to provide an improved tumor delineation, a tumor Region of Interest (ROI) is firstly drawn manually on the 18F-FCH image. The 18F-FDG and the 18F-FCH images are then merged. The tumor ROI from the 18F-FCH can be transferred onto the merged images and the 18F-FCH counts are eventually subtracted from the merged images to obtain the finial tumor 18F-FDG counts.
Materials and Methods
Animal preparation
All animal experiments were approved by the SingHealth Institutional Animal Care and Use Committee (Approved: IACUC #2007/SHS/305 and IACUC #2007/SHS/305A) and carried out in an AAALAC-accredited facility. Five weeks old BALB/c nude mice were used for the study, as 14 mice in the normal control group and 10 mice in the tumor bearing group, respectively. The animals were provided with sterilized food and water ad libitum, and housed in negative pressure isolators.
Generation of intracranial glioblastoma in mice
U87 MG-luc2, a human glioblastoma cell line was purchased from Caliper Life Sciences, Hopkinton, MA. The cells were grown in Minimum Essential Medium (Invitrogen) with 10% fetal bovine serum (Gibco, Invitrogen) and 1% penicillin-streptomycin (Gibco, Invitrogen) in humidified atmosphere containing 5% CO2 at 37°C. Cells growing exponentially in vitro were trypsinized and harvested for tumor inoculation. All animal manipulations were performed under sterile conditions. In order to obtain intracranial glioblastoma bearing mice, BALB/c nude mice were anaesthetized with intraperitoneal injection of a ketamine/diazepam solution (50 mg/kg ketamine and 5 mg/kg diazepam) and immobilized on a stereotactic head frame (Thermo Fisher Scientific). For subcutaneous administration of baytril, a total dose of 5 mg/kg was injection before operative procedure. Briefly, a midline incision was made on the scalp and a burr hole was drilled 2 mm lateral to the sagittal sinus at the midpoint between the bregma and lambda, and 300,000 U87-MG-luc2 cells suspended in 10 μL of MEM were injected into the hole to a depth of ~ 2.5 mm by using a hypodermic needle (Hamilton Co., Reno, NV). The incision was sutured closed with 5/0 prolene. Postoperatively, the mice were given a subcutaneous injection of 5 mg/kg caprofen. PET scan was performed on the mice one week after the tumor inoculation.
Micro-PET imaging by using dual tracers of 18F-FCH/18F-FDG
Each mouse was subjected to 18F-FCH scan followed by 18F-FDG scan on consecutive days. To perform 18F-FCH imaging, the animals were injected with approximately 5.5MBq of 18F-FCH intravenously and conscious uptake of the radiotracers was allowed for 30 min before initiating micro-PET imaging with the R4 microPET scanner (Concordes Microsystems Inc.). The animals were then left to recover and to allow complete the decay of radioactivity. Prior to 18F-FDG micro-PET imaging, the mice were fasted overnight, with water being available. On the day of 18F -FDG imaging, the mice were pre-warmed to a body temperature of 37°C before an approximate dose of 5.5MBq of 18F-FDG (0.6mM) was administered intravenously. The body temperature was maintained at 37°C with heating pad throughout the uptake period. Micro-PET imaging was performed 45 min after 18F-FDG injections. The duration of both 18F-FCH and 18F-FDG-PET imaging was 10 min for each mouse. During the micro-PET imaging, the mice were placed in an imaging chamber and kept under 2% isoflurane anesthesia. Both 18F-FDG and 18F-FCH were obtained from the Department of Nuclear Medicine, Singapore General Hospital.
Micro-PET image reconstruction and analysis
Images were reconstructed by using 3-D reprojection reconstruction (3DRP) without scatter or attenuation correction. A CT scan (described below) was obtained as a reference to determine the brain regions in the 18F-FDG and 18F-FCH PET images. Since the PET image with 18F-FCH can deliver an improved tumor delineation, a tumor ROI was drawn manually on the 18F-FCH image first. The 18F-FDG and 18F-FCH images were then merged, and the tumor ROI from the 18F-FCH was transferred onto the merged images to obtain the finial tumor 18F-FDG counts by subtracting the 18F-FCH counts from the merged images. The tumor to background ratio, which is defined as 18F-FDG counts per pixel within the tumor compared to 18F-FDG counts per pixel in the normal brain area, was used for further analysis. No other scoring system or interpretation criteria of metabolic activity was used in this study.
Micro-CT procedure
A Rigaku micro-CT scanner (JMorita Engineering Co., Ltd, Japan) was adopted in the CT imaging. This in vivo Micro X-ray CT system consists of an X-ray tube (X-ray energy, max 90 kV), a semiconductor detector with 200 × 200 μm pixel size (acceptance surface size, 124.8 × 124.8 mm), an image reconstruction part, an optical system arm, a 3-axis sample stage part. The detector and X-ray source rotated around a fixed bed, allowing the mice to be kept in the same horizontal position in the CT scanner as in the PET scanner. The mice were transported and positioned in the CT scanner right after the PET scan. The X-ray voltage and anode current were set at 90 kVp and 50 μA, respectively. Images of the whole animal were obtained after one revolution that lasted for 17 s. No X-ray contrast medium was used.
Bioluminescence imaging (BLI)
Anesthesia was induced with 4% isoflurane and maintained at 2% isoflurane. Mice were injected intraperitoneally with 150 mg/kg of D-luciferin potassium salt (Caliper Life Sciences), and were then incubated for 10 min to allow the distribution of D-luciferin. An integration time of 30 s was used for luminescent image acquisition by using the Xenogen Spectrum optical imaging system (Xenogen Corp., Alameda, CA). Tumor ROIs were drawn and quantified based on the Living Image software version 2.5. Specific signal was reported as the ratio of the bioluminescence signal in the ROI over the bioluminescence signal in a background region without tumor.
Histology and immunohistochemical staining
The brain tissues were harvested 35–74 days after tumor cell inoculation. Later the tissues were stained as follows. Briefly, tissues were fixed in 4% buffered formaldehyde for 24 h, processed in paraffin, and stained with hematoxylin and eosin for histopathological examination. For immunoreactivity staining, 5 μm deparaffinized polysin-coated sections were treated with 3% hydrogen peroxide for getting rid of the endogenous oxides. A mouse anti-human Ki-67 antibody (Dako, Glostrup, Denmark) was employed for immunohistochemical staining using the peroxidase-antiperoxidase technique.
Results
18F-FCH PET provides better contrast between tumor and normal tissues but with high false positive
Glioblastoma bearing mice and normal control mice were subjected to PET scan with single tracer administrated at one time, the images of two perpendicular cross-sections are compared in Fig 1. As expected, no tumor region can be read on the images of normal control mice with either of the tracers injected (Fig 1A and 1C). However, the image with tracer 18F-FDG fails to show the tumor region (Fig 1B). Only 18F-FCH provides a clear contrast between tumor and normal cerebral tissues (Fig 1D). It is observed that normal cerebral tissues typically represent low 18F-FCH uptake [12] as shown in Fig 2A. However, in some of the control animals, 18F-FCH uptake is unexpectedly observed in normal brain tissue (Fig 2B), resulting in potential false-positive findings. In total, 4 false positives out of 14 normal mice experiments are observed in this study, indicating a false positive rate of 28.6%. Therefore, although 18F-FCH can delineate the glioma clearly, the high false positive rate renders it unsuitable as a glioma imaging radiotracer.
A and C: normal control mice, B and D glioblastoma bearing mice; A and B: with 18F-FDG, C and D: with 18F-FCH. The low grade glioma is illustrated by arrows.
The brain region with high 18F-FCH concentration is pointed by arrows. (A) No or trivial uptake of 18F-FCH in normal brain, (B) high concentration of 18F-FCH in normal brain.
Combined 18F-FCH/18F-FDG-PET for imaging of glioma xenograft
In order to detect the low grade glioma, the 18F-FCH and 18F-FDG-PET images are merged first and ROIs guided by the region with high 18F-FCH concentration are then manually drawn on the 18F-FDG images. This process is specified in Fig 3(A). As shown in Fig 3(A) A-D, no tumor can be observed in the images of normal control mice with either of the imaging techniques applied. Although 18F-FDG image fails to represent the tumor, the brain region can be clearly sketched in Fig 3(A) E. On the contrary, the tumor region is shown in Fig 3(A) F where 18F-FCH is applied as the tracer. However, the brain region is blur, resulting in the difficulty to identify the tumor boundary. The low grade glioma can only be clear observed by merging E and F, as shown in Fig 3(A) G. The result of tumor detection is also proved by the BLI image of Fig 3(A) H.
(a) PET and BLI images of a representative normal mice (A-D) and glioblastoma-bearing mice (E-H). ‘T’ denotes tumor ROIs which is further indicated by arrows. A close look of the tumor on CT image is given in E-1. (b) Morphological features of normal brain (A and C) and glioblastoma (B and D). Shown are representative histological sections stained with haematoxylin and eosin (100×, A and B) and Ki-67 expression (400×, C and D). (c) Representative microCT and/or PET images of a normal (A-D) and glioblastoma-bearing mouse (E-H). A and E: CT images without contrast agent; B and F: 18F-FCH PET images; C and G: 18F-FDG PET images; D and H: Merged images of 18F-FDG (in white) and 18F-FCH. Arrows indicate presence of tumors.
The target to background ratio (Mean ± SD) from the 18F-FCH and 18F-FDG images for each of the 4 non-tumor bearing control mice (4 false positive) and 10 tumor-bearing mice are summarized in Table 1. It is difficult to differentiate the U87 xenografts from normal brain tissues by using 18F-FDG only [13]. However, the tumor area delineated by 18F-FCH shows an increased target to background ratio, ranging from the minimum of 1.12 ± 0.02 to the maximum of 2.35 ± 0.06, with a group mean of 1.47 ± 0.37. Results show that there are 4 false positives predicted by the 18F-FCH images. As an improvement, all the tumor can be successfully detected by the merged 18F-FCH/18F-FDG images. The target to background ratio of the false positive area ranges from 0.98 ± 0.01 to 1.14 ± 0.07, with a group mean of 1.04 ± 0.07. This is significantly lower than the tumor to background ratio described in the non-false positive cases (P < 0.01).
The detection of low grade glioma with small size is shown in Fig 3(C). As shown in Fig 3(E), the tumor is difficult to be observed based on the CT image owing to its small dimension. However, the tumor can be clearly identified in the tumor bearing mice after subtracting the 18F-FCH counts from the merged 18F-FCH/18F-FDG image, as shown in Fig 3(C) F-H. The same experiments were carried out on normal control mice, and results are represented in Fig 3(C) A-D, with no tumor can be observed.
Bioluminescence imaging, histology and micro-CT
Both control and tumor-bearing mice were subjected to bioluminescence imaging (Fig 3A), but only tumor-bearing mice showed positive readouts. The growth of the xenograft is confirmed by necropsy and with standard histology techniques (Fig 3B). The tumor–bearing group shows glioblastoma xenograft on H&E–stained slices and marks by proliferation marker (Ki-67 positivity). The xenograft can be defined in CT images (the diameter of the tumor ranged from 2.8 to 4.2 mm) and corresponds well to the 18F-FCH PET image (Fig 3C).
This technique can also be applied to monitor progression of tumor. Fig 4 illustrates the growth of glioblastoma xenograft at 20, 33, 60 and 74 days after inoculation of U87 MG-luc2 cells in a nude mouse. Increased radioactivity can be observed in the tumor from day 33 to 74 after inoculation. This is confirmed by a marked increase of BLI signal intensity from day 20 to 74.
The growth was monitored at 20, 33, 60 and 74 days after the inoculation. Tumor ROIs are indicated by arrows in PET fusion images (B, C and D) and show increase in tracer uptake from day 33 to 74. Implanted luciferase expressing U87 MG-luc2 cells are illustrated in the brain of mouse (E-H) with a red–blue color bar as a reference of BLI signal intensity, red color indicating the highest BLI signal intensity. Increased BLI signal intensity is marked from day 20 to 74, indicating progressive growth of glioblastoma xenograft.
Using this dual tracer technique, we also monitored tumor metabolic activity quantitatively in terms of 18F-FDG uptake ratio between tumor and background. As indicated in Fig 5, the metabolic activity of the tumor increased significantly from day 35 to day 50 after inoculation in nude mice.
The graph indicates a significant increase in metabolic activity of U87 MG-luc2 cells in nude mice from day 35 to day 50 after inoculation measured by dual tracer technique (N = 3 to 6 per time point, error bars represent standard deviation).
Discussion
18F-FDG is by far the most commonly used PET radiopharmaceutical in cancer imaging. Chemically, it is a glucose analogue and the positron emitting radioactive isotope 18F substitutes the hydroxyl group at the 2’ position of the glucose molecule. As a glucose analog, 18F-FDG is taken up by cells that significantly consume glucose such as brain, kidney and cancer cells. Once being up-taken, glucose and 18F-FDG undergo phosphorylation at the 6 position carbon. Unlike glucose, 18F-FDG will not undergo further glycolysis because the 2' hydroxyl group has been replaced by an 18F atom. Nevertheless, 18F-FDG distribution is a good reflection of the distribution of glucose uptake and tumor metabolism. Cancer cells, in order to support its fast metabolism and energy demand, require a much higher intake of energy source such as glucose. 18F-FDG, therefore, localizes in cancer cells. However, other cells with fast metabolism such as brain cells also take up comparative amount of glucose and 18F-FDG. As a result, it is difficult to differentiate low grade gliomas from surrounding normal brain cells based on 18F-FDG uptake only [14–16].
18F-FDG is primarily applied in monitoring effects of various glioma treatment modalities to determine the prognosis in patients with these lesions and to differentiate recurrent tumor due to therapy. However, its application as a first line diagnosis is limited [17–19]. Our results in this study support such findings.
18F-FCH is a developed PET tracer for the imaging of prostate cancer. In vitro and in vivo studies have provided substantial evidences that increased choline metabolism is a prominent feature of many cancers. Choline, a precursor for phospholipids, is transported into mammalian cells by a low affinity sodium-independent transport system and then phosphorylated by choline kinase. It is further metabolized to phosphatidylcholine, which is a primary component of cell membrane. Tumor cells usually demonstrate an elevated choline uptake compared to normal cells, which can be explained by the upregulation of choline kinase due to an increased demand of membrane constituents [20, 21].
Carbon-11 labeled Choline has been successfully used in glioma imaging, but the short half-life (20 min) of 11C limits its applications in clinic [22–24]. The 18F with a longer half-life of 120 min is able to overcome this shortcoming. As an avid substrate for choline kinase, 18F-FCH also undergoes phosphorylation to produce 18F-phosphorylfluorocholine. In cultured PC-3 (human prostate cancer) cells incubated with 18F-FCH, most of the intracellular 18F radioactivity is accounted for by the production of 18F-phosphorylfluorocholine [5]. Clinical data suggested that 18F-FCH is favorable in the imaging of many kinds of cancers, including primary brain tumors and prostate cancer that are ineffectively imaged with 18F-FDG [24–26].
In addition to 18F-FCH, there are other PET tracers which may have potential applications in glioma imaging, particularly 18F-labelled amino acid such as 18F Fluoroethyltyrosine (18F-FET) [27, 28]. A recent animal study has compared several potential PET tracers including 18F-FCH, 18F-FET and 18F-FDG for glioma imaging. Although 18F-FDG and 18F-FET have higher uptake in glioma than 18F-FCH, they also represent higher uptake in collateral normal cerebral tissues. The radioactivity ratio between glioma and normal tissue was 3.77 for 18F-FCH, 2.58 for 18F-FET and 1.98 for 18F-FDG [10]. Hence 18F-FCH was chosen for this study.
18F-FCH was applied to clinical brain tumor imaging by DeGrado et al. for a patient with biopsy-proven recurrent anaplastic astrocytoma [6]. Their findings were consistent with the results obtained in this study, in which a high tumor-to-normal cortex ratio was achieved. Results also showed that physiological uptake of 18F-FCH did occur in the pituitary gland and choroids plexus. This could possibly account for the false-positive results found in our normal control mice, as shown in Fig 2. It is worth noting that no such false positive being observed in Ref. [12] based on patient-specific studies. This difference may be caused by the in vivo environment of each patient, the location, growth stage and dimension of the tumor, and PET scanner, etc. Further investigations on the pharmacokinetics and pharmacodynamics of 18F-FCH are needed to understand the mechanism of 18F-FCH concentrating in normal brain cells.
Dual tracer technique has been successfully applied in the imaging of other cancers [29, 30]. One example is the imaging of parathyroid cancers in which patients are injected with 99mTc-Sestamibi, which localizes in both the thyroid and parathyroid, followed by administration with 99mTcO4 which localizes in thyroid only. Combining the 99mTc-Sestamibi and 99mTcO4 scan followed by subtraction of the thyroid radioactivity would enhance the sensitivity of visualization of the parathyroid cancer. However, such technique has not been applied to PET imaging. An advantage of PET over SPECT is the ability to perform quantitative imaging. As such, dual imaging technique may be even more accurate in PET than in SPECT.
We hence investigated on the potential application of a dual tracer technique in the early diagnosis of low grade glioma. The basic principle in this study is that the 18F-FDG concentrates in both the normal brain cells and glioma cells, making it difficult to differentiate tumor from normal tissue, particularly in low-grade glioma diagnosis. On the other hand, 18F-FCH would delineate the malignant area. The overlapping of the 18F-FDG and 18F-FCH images can provide an improved delineation to identify the tumor regions, and therefore develop the application of PET imaging in glioma diagnosis. A recent simulation study also confirmed dual tracer technique is superior to the single tracer in glioma PET imaging [31].
Our results demonstrated that such a dual tracer PET imaging protocol could be effective to avoid false- positive findings in 18F-FCH PET scan as well as identification of glioma. Guided by 18F-FCH delineating “hotspot” on merged images (Fig 3A) and then followed by subtraction of the 18F-FCH counts, all the xenografts of tumor bearing mice were successfully identified in this study, indicating a high sensitivity in diagnosis of low grade glioma.
Quantitatively, there is also a significant difference in target / background ratio between the 18F-FCH false- positive image and the merged 18F-FDG images (P<0.01). The tumor detected by the dual-tracer PET protocol is confirmed by histology (Fig 3B) and micro-CT (Fig 3C).
There are other tracers showing promise in imaging cancers such as 18F-fluoro-levo-thymidine (18F-FLT). It would be interesting to have further investigations to verify the possible application of dual tracer of 18F-FLT/18F-FDG in glioma diagnosis.
Although this study provide insights into the application of a dual tracer PET imaging technique in the low grade glioma diagnosis, there are still limitations for this technique. The availability of 18F-FCH is still limited to larger medical institutions with expertise in radiochemistry. Our experience with the synthesis of 18F-FCH is that it will take 60 to 90 minutes to synthesize 18F-FCH and the yield is around 8 to 15% only. Also, the additional cost, logistics and radiation adsorption to the patient due to an extra PET scan may be substantial.
Conclusions
The potential application of a dual tracer technique for PET imaging in low grade glioma diagnosis is investigated based on a mouse orthotopic xenograft model. Results show that the dual tracer of 18F-FCH/18F-FDG with PET scan is able to improve the diagnosis of low grade glioma. By using 18F-FCH to clearly locate and delineate brain tumors, it is possible to effectively identify and quantify the metabolic activity of the brain tumors on the merged 18F-FCH/18F-FDG PET images. Such findings might have great impact on clinical studies for early detection of low grade glioma. Further investigations are warranted to evaluate potential of this technique in clinical settings.
Supporting Information
S1 ARRIVE Checklist. ARRIVE Guidelines Checklist Animal Research: Reporting In Vivo Experiments.
- Title: Page 1.
- Abstract: Page 3.
- Background: Pages 4–5. Introduction
- Objectives: Page 5.
- Ethical statement: Page 5. Section 2.
- Study design: Pages 5–8. Section 2.
- Experimental procedures: Pages 5–8. Section 2.
- Experimental animals: Pages 5–6. Sections 2.1 & 2.2.
- Housing and husbandry: Page 5. Section 2.1.
- Sample size: Page 5. Section 2.1.
- Allocating animals to experimental groups: Page 8. Section 2.8.
- Experimental outcomes. Pages 8–11. Section 3.
- Statistical methods: Page 8. Section 2.8.
- Baseline data: Page 8. Section 3.1.
- Numbers analyzed: Pages 8–11. Section 3.
- Outcomes and estimation: Page 8. Section 2.8.
- Adverse event: Not applicable.
- Interpretation/ Scientific implications: Pages 11–14. Section 4.
- Generalization / Translation: N/A.
- Funding: Page 15. Acknowledgements.
https://doi.org/10.1371/journal.pone.0148123.s001
(PDF)
Author Contributions
Conceived and designed the experiments: YF LCO SHR LZ IK SY PKHC CHW. Performed the experiments: YF LCO SHR LZ IK SY PKHC CHW. Analyzed the data: YF LCO SHR LZ IK WZ SY. Contributed reagents/materials/analysis tools: YF LCO SHR LZ IK SY. Wrote the paper: YF LCO SHR LZ IK WZ SY PKHC CHW. Revised the manuscript: YF LCO SHR LZ IK WZ SY PKHC CHW.
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