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The increasing development of novel anticancer agents demands parallel advances in the methods used to rapidly assess their therapeutic efficacy (TE) in the preclinical phase. We evaluated the ability of small-animal PET, using the 18F-fluorodeoxy-D-glucose (FDG) radiotracer, to predict the TE of a number of anticancer agents in the rat C6 glioma model following 3 days of treatment. Semi-quantitative measurements of changes in FDG uptake during the course of treatment (standardized uptake value response [SUVr]) were found to be significantly lower in tumors treated with the hypoxia-inducible factor-1α inhibitor YC-1 (15 mg/kg) than in tumors in the control group. No significant SUVr change was observed following a similar 3-day regimen with the proapoptotic agent NS1619 (20 μg/kg), the combination of YC-1 and NS1619, or the alkylating agent temozolomide (7.5 mg/kg). Quantitative immunohistochemical studies demonstrated significantly lower levels of glucose transporter-1 (GLUT-1) expression in the YC-1 – treated tumors, thereby correlating with the low SUVr observed in this group. The ability of SUVr to predict gold-standard outcomes of TE was further validated as YC-1 – treated tumors had decreased volumes compared to control tumors. As such, we successfully demonstrated the ability of FDG-PET to rapidly determine the TE of novel agents for the treatment of glioma in the preclinical phase of evaluation.
The development of effective treatments for cancer has risen dramatically in recent years. In 2005, approximately 400 anticancer medicines developed by 178 biopharmaceutical companies and the U.S. National Cancer Institute were in clinical trials or under review by the U.S. Food and Drug Administration.1 The rising costs of bringing effective agents to market have prompted the drug development industry to seek new markers of therapeutic efficacy (TE). In the preclinical phase of the drug development pipeline, these markers should, ideally, provide for rapid screening of efficacy and reduce research and development costs through earlier termination of ineffective agents and accelerated development of promising compounds.
The use of noninvasive imaging modalities, such as MRI and PET, to provide objective, longitudinal, primary, or surrogate outcome measures of TE in clinical trials has become a standard practice.2 The recent advent of dedicated small-animal imaging systems, including microPET; high-field, small-bore MRI; and optical imaging scanners, has led to the evaluation of these technologies for rapid and cost-effective evaluation of agents in preclinical studies.3 PET is particularly well suited for preclinical TE studies because high-resolution, small-animal PET provides excellent three-dimensional spatial localization, and a wide variety of tracers exist for metabolic and molecular imaging.4
Aliaga et al.5 recently demonstrated the use of 18F-fluorodeoxy-D-glucose (FDG) to monitor the time course of the metabolic response of breast carcinomas to chemotherapy and hormone therapy in murine models. The ability to detect a response within 24 h of treatment initiation and, subsequently, to follow the differential efficacies of the various therapeutic regimens over a long period of time, points to the promise of this technique for preclinical oncology TE studies. While these elegant studies demonstrated the feasibility of this technique for monitoring tumor metabolism and response to standard chemotherapeutic intervention, the acceptance of small-animal PET as a valid marker of TE in preclinical studies of targeted therapies requires extensive correlation with gold-standard measures of therapeutic response, including histology, immunohistochemistry (IHC), and tumor volumetry.
In this study, we evaluated the use of small-animal FDG-PET to determine the TE of a number of anti-cancer agents, with different mechanisms of action, in a rat model of glioma. Specifically, we assessed the compounds NS1619, YC-1, and temozolomide (TMZ) in the intracranial rat C6 glioma model. NS1619 is an activator of calcium-activated potassium channels, which are overexpressed in both glioma cells and the endothelial cells of the blood – tumor barrier (BTB).6 The activation of these channels by NS1619 has been shown to inhibit migration of human glioma cells7 and induce apoptosis in rodent models of glioma.6 YC-1, an inhibitor of hypoxia-inducible factor-1α (HIF-1α ), has been shown to significantly reduce tumor growth in a number of human xenograft models.8 Further, the close association between hypoxia and the progression of glioma9 and the antiangiogenic effects of HIF-1α inhibitors10 suggest the potential utility of YC-1 in the treatment of glioma. Given the different mechanisms of action of NS1619 and YC-1 and the ability of both agents to increase the BTB permeability in animal models of glioma,11 we also investigated the possibility of a synergistic, combined effect of these two agents. Finally, we utilized TMZ, an alkylating agent that is currently used in the clinical setting for the treatment of glioma, as a model of a chemotherapeutic agent with a conventional, well- characterized mechanism of action.12
The semiquantitative measures of tumor metabolism determined from the PET studies described in this article were rigorously correlated with tumor morphology on histological sections, as well as IHC markers of proliferation, microvascular density, glucose transporter expression, and macrophage/microglial infiltration, to provide insight into the underlying pathophysiological mechanisms of the glucose metabolic changes identified on the FDG-PET images. Changes in tumor glucose metabolism were also compared to postmortem tumor volume measurements to validate the ability of FDG-PET to accurately predict TE.
Rat C6 glioma cells13 were purchased from American Type Culture Collection (Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 125 U/ml penicillin G, 125 μg/ml streptomycin sulfate, and 2.2 μg/ml amphotericin B (Fungizone). All culture reagents were obtained from Gibco BRL (Invitrogen, Burlington, ON, Canada). Cultures were grown in monolayers and maintained at 37ºC in a humidified atmosphere of 5% CO2. Upon reaching confluency, spheroids were prepared using the hanging drop method previously described by Del Duca et al.14 Briefly, 20-μl drops of DMEM containing 15,000 cells each were suspended from the lids of culture dishes and the resulting aggregates were transferred to culture dishes base-coated with agar after 72 h. The resulting spheroids were adequate for in vivo implantation after 48 h of incubation on agar.
The spheroid implantation technique was selected for these studies as this method has been shown to produce fully vascularized rapid-growing tumors with many of the characteristics of glioblastoma multiforme, including pseudopalisading necrosis.15 Furthermore, as the tumors grow in a spheroidal fashion, the tumor – brain interface can be readily defined,15 making this model well suited for imaging studies. All animal experiments were conducted in accordance with the guidelines of the Canadian Council on Animal Care and the Montreal Neurological Institute and McGill University Animal Ethics Committees. Male, Sprague-Dawley rats (40 – 50 g; Charles River Canada, St. Constant, QC, Canada) were anesthetized with 50 mg/kg ketamine and 10 mg/kg xylazine. The right cortical surface in the parietal-occipital region was exposed by craniectomy using a high-powered drill (Dremel, Racine, WI, USA), and the underlying dura and its vessels were carefully removed under a surgical microscope. A piece of the cortex was removed to expose the underlying white matter, and a single spheroid was placed into the surgical defect. The craniectomy was covered with bone wax (Ethicon, Peterborough, ON, Canada), and the overlying skin was sutured. Following recovery from anesthesia, the animals were fed and had access to water ad libitum.
NS1619 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), YC-1 (Cayman Chemical, Ann Arbor, MI, USA), and TMZ (ChemPacific Corp., Baltimore, MD, USA) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Oakville, ON, USA). Fourteen days post-tumor implantation, the rats were randomly assigned to five different therapeutic groups: vehicle (DMSO 1 g/kg i.v.), NS1619 (20 μg/kg i.v.), YC-1 (15 mg/kg i.v.), YC-1 (15 mg/kg i.v.) + NS1619 (20 μg/kg i.v.), or TMZ (7.5 mg/kg i.p.),16 as summarized in Table 1. All agents were administered daily for 3 consecutive days via tail-vein or intraperitoneal injections, as appropriate. During this period, the animals were weighed and monitored daily for signs of pain and neurological symptoms.
PET images were acquired immediately prior to the initiation of treatment and upon completion of the treatment regimen (i.e., day 17 post-tumor implantation). The animals were fasted for 5 h prior to PET scans, and blood glucose levels were normalized to approximately 7 mM,17 as measured using a standard glucometer (ACCU-CHEK Aviva System, Roche Diagnostics Canada, Laval, QC, Canada) in order to minimize any anesthesia-related variations in FDG uptake.18 The rats were anesthetized with 50 mg/kg ketamine and 10 mg/kg xylazine and injected intravenously with 200 – 400 μl of normal saline containing 100 – 300 μCi of FDG. Images were acquired using a CTI Concorde R4 microPET scanner (Siemens/CTI Concorde, Knoxville, TN, USA). Imaging studies were performed while the animal was anesthetized and placed in the supine position on the bed of the scanner at the center of the field of view. At each time point, a transmission scan was acquired for 10 min, followed by an emission scan initiated exactly 45 min after the administration of FDG and lasting 20 min. All images were reconstructed using the maximum a posteriori algorithm. The Acquisition Sinogram and Image Processing Software (Siemens/CTI Concorde, Knoxville, TN, USA) was used to draw three-dimensional regions of interest (ROI) around the regions of the tumors with high FDG uptake. In order to normalize for differences in FDG uptake between the rats, three-dimensional ROIs were also drawn around the normal brain in the contralateral hemisphere. The standardized uptake values (SUVs) for both tumor (SUVt) and normal brain (SUVb) were then measured based on the average activity obtained in the ROIs as follows:
The results were then expressed as “SUV response” (SUVr), defined as the percentage change of the normalized SUV of the tumor, before and after treatment:
Following the completion of the PET studies, rats were euthanized by anesthetic overdose and cervical dislocation and decapitated, and the brains were removed for histopathological/IHC studies. The brains were fixed in 10% neutral-buffered formalin, dehydrated through graded alcohols and xylene, and embedded in paraffin wax. Sections 5 μm thick were cut through the tumor, and each section was stained with hematoxylin and eosin (H&E). For IHC studies, sections were treated with 0.3% H2O2 in methanol, followed by antigen-retrieval in boiling 10 mM citrate buffer (pH 6.1) at elevated pressure. All IHC studies were performed on a Lab Vision Autostainer 360 (Lab Vision Corporation, Fremont, CA, USA) with the following antibodies, dilutions, and incubation times: monoclonal Ki-67 (1:200, 60 min; Neomarkers, Fremont, CA, USA), polyclonal GLUT-1 (1:200, 60 min; Neomarkers), polyclonal Iba1 (1:200, 60 min; Wako Chemicals USA, Inc., Richmond, VA, USA), and polyclonal collagen IV (1:50, 120 min; Chemicon International Inc., Temecula, CA, USA); the IHC studies were followed by visualization with the Ultravision LP detection system and AEC chromogen (Lab Vision Corporation), and counterstaining with hematoxylin. All sections were digitized at × 400 magnification at the invasive edge, central, and perinecrotic regions of the tumors using a Nikon Eclipse 55i microscope (Nikon Canada Inc., Mississauga, ON, Canada) equipped with a QICAM 12-bit Fast 1394 digital camera (QImaging, Surrey, BC, Canada). The percentage of positively stained cells or pixels in each region was calculated using a fully automated image segmentation and quantification algorithm developed in-house.
As a significant SUVr was only found in the YC-1 – treated group (see “Results”), the treatment regimen was extended to 7 days in a separate group of rats in order to validate the true TE of YC-1 using gold-standard measures. Tumor volumetry is a gold-standard method commonly used to detect the TE of novel therapeutic agents developed for the treatment of glioma.16,19–21 In this experiment, rats were treated with YC-1 (15 mg/kg, n = 6) or vehicle (DMSO, 1 g/kg, n = 6) for a 7-day period, starting 14 days post-tumor implantation; 72 h prior to the start of treatment, a permanent catheter was implanted in the right jugular vein of each rat. Briefly, the rats were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg), and the right jugular vein was isolated under a surgical microscope. A rat thoracic jugular vein catheter (Braintree Scientific Inc., Braintree, MA, USA) containing heparinized saline was inserted into the vein and secured using a 4.0 silk suture. The injection port was sutured in the subcutaneous space at the dorsal midline. The catheter was flushed daily with 20 μl heparinized saline, and the port was filled with 10 μl lock solution (Braintree Scientific Inc.). Following the completion of therapy (i.e., day 21 post-tumor implantation), the rats were euthanized and the entire head was fixed in 10% neutral-buffered formalin for 24 h. The head was then decalcified in Cal-EX solution (Fisher Scientific, Ottawa, ON, Canada) for 4 days at 25ºC, after which serial 1-mm-thick coronal sections were cut using an adjustable acrylic brain matrix (Braintree Scientific Inc.). An image of each serial section was captured using a digital camera (Canon EOS 20D; Canon Canada Inc., Mississauga, ON, Canada). Tumors were segmented using Adobe Photoshop CS2 (Adobe Systems Inc., San Jose, CA, USA), and tumor volumes were measured using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Quantitative results are expressed as mean ± SEM. Comparisons were made by the Student’s two-tailed t-tests and by analysis of variance (ANOVA), as appropriate. Significant associations in ANOVA were further analyzed by Dunnett’s multiple comparison post hoc tests. Differences were considered significant at p < 0.05.
In order to assess the ability of FDG-PET to rapidly detect the TE of the various therapeutic agents, the SUVr of the tumor was calculated for each of the five treated groups. As shown in Fig. 1A, treatment with YC-1 resulted in a significantly lower SUVr (1.06 ± 6.55) than that observed in the control group (26.11 ± 10.08) (Table 1). Representative FDG-PET images of the DMSO-treated and YC-1 – treated groups, before and after treatment, are shown in Fig. 1B. The combined YC-1 + NS1619 group also demonstrated a lower SUVr (6.43 ± 10.22) than did the control group. However, this difference was not found to be statistically significant. The SUVr values upon administration of NS1619 (24.25 ± 7.56) and TMZ (18.92 ± 9.94) were not statistically different from those observed in the control group.
Qualitative analysis of the H&E-stained tissue sections revealed varying levels of necrosis and edema within the tumors of all therapeutic groups, with no observable difference between the control and treated rats. As small necrotic areas are generally undetectable on PET images, we compared tumors with similar extents of necrosis from the control, YC-1, and YC-1 + NS1619 therapeutic groups to determine whether the low SUVs observed were due to the averaging of small necrotic areas in our ROIs. YC-1 and YC-1 + NS1619 consistently showed lower SUVs than control when corrected for necrosis, suggesting that the differences in SUVs between the different therapeutic groups were not the result of small necrotic areas, but rather were most likely associated with molecular processes that cannot be determined on the H&E-stained tissue sections.
In order to gain further insight into the cellular and physiological processes responsible for the differences in SUVr observed between the therapeutic groups, IHC was performed for various markers (Fig. 2A). Since the metabolic activity of macrophages and microglia infiltrating the gliomas was expected to be high, IHC was performed against the macrophage/microglial marker Iba1 to detect whether therapy affected the extent of macrophage and microglia infiltration in the tumors and thereby contributed to the differences in SUVr observed. Quantitative analysis of the levels of Iba1 staining revealed no significant differences between the control (22.69 ± 7.46) and the groups treated with NS1619 (35.06 ± 8.66), YC-1 (47.33 ± 4.83), YC-1 + NS1619 (36.31 ± 9.57), and TMZ (29.43 ± 14.84) (Fig. 2B).
Tumor proliferation indices (PIs) and levels of GLUT-1 expression have been reported to correlate with FDG uptake in glioma at a single time point.22 Therefore, we performed IHC staining for the Ki-67 proliferation marker and the GLUT-1 transporter to determine whether differences in the number of proliferating cells, tumor cellularity, or GLUT-1 expression significantly contributed to the SUVr over the time course of therapy. As shown in Fig. 2C, the PIs were consistent across the control (54.10 ± 5.39), NS1619 (52.93 ± 3.23), YC-1 (57.28 ± 6.84), YC-1 + NS1619 (58.17 ± 7.77), and TMZ (64.57 ± 4.00) therapeutic groups. The extent to which tumor cellularity affected the metabolic demand of the tumor was also measured from the Ki-67 images by averaging the total number of nuclei in three × 400 fields and was not found to be significantly different between the various groups (Fig. 2D). IHC against the GLUT-1 transporter revealed two different patterns of staining in the tumors. As exemplified in the control group (Fig. 2A), GLUT-1 staining was primarily associated with the vasculature at the invasive edge of the tumor. Further, the stabilization of HIF-1α in the hypoxic regions, resulting in the up-regulation of GLUT-1 in the macrophages/microglia and in the tumor cells,23 yielded intense staining in the perinecrotic regions.
Quantification of GLUT-1 staining over the entire tumor (Fig. 2E) showed levels of GLUT-1 expression in the NS1619-treated group (16.03 ± 3.6) comparable to those observed in the control tumors (18.35 ± 7.24). On the other hand, significantly lower levels of GLUT-1 expression were observed in the YC-1 – treated group (1.82 ± 0.43, p = 0.02). The YC-1 + NS1619 (5.26 ± 2.18) and TMZ (9.11 ± 0.02) treatments also resulted in lower levels of GLUT-1 expression, but this change was not found to be statistically significant. In order to determine whether the SUVr observed in the YC-1 group represented an antiangiogenic effect, we performed IHC for the vascular basement membrane marker collagen IV.
Qualitative assessment of collagen IV staining revealed a decreased number and smaller caliber of vessels in the YC-1 – treated group when compared to the control and other therapeutic groups (Fig. 3A). This observation was further confirmed by the quantitative analysis of the IHC data, which revealed a significantly lower percentage of stained pixels in the YC-1 – treated group (3.52 ± 1.62, p = 0.02) compared to the control group (8.92 ± 0.32) (Fig. 3B). No significant difference in collagen IV staining was observed between the NS1619 (8.12 ± 0.44), YC-1 + NS1619 (8.00 ± 1.24), TMZ (10.87 ± 1.24), and control groups (8.92 ± 0.32) (Fig. 3A, B).
In order to validate the ability of the SUVr to predict TE, longitudinal studies were performed to observe differences in tumor growth between YC-1 – treated and control rats over 7 days of therapy. No toxicity related to the daily intravenous administration of YC-1 was observed. As shown in Fig. 3C, a significant arrest in tumor growth was observed with YC-1 treatment (138.08 ± 47.83 mm3, p = 0.03) compared to the control group (300.95 ± 54.97 mm3).
The recent upsurge in the discovery of anticancer agents has been facilitated by new, target-based approaches in therapeutic design and the increasingly high-throughput nature of in vitro assays. The growing number of agents entering preclinical studies necessitates the development and validation of rapid, cost-effective methods for determining TE to facilitate advancement of promising agents to clinical trials. In this study, we successfully demonstrated, for the first time, the ability of noninvasive FDG-PET imaging to predict the TE of novel anticancer agents in vivo in the intracranial rat C6 glioma model. Traditional methods of preclinical evaluation of TE in intracranial glioma models, including postmortem tumor volumetry and/or survival studies, are lengthy and require large numbers of animals due to the intrinsic biological variability of these tumors. In contrast, our serial, in vivo FDG-PET scans allowed for robust prediction of TE after just 3 days of treatment in small cohorts of animals.
These studies revealed the ability of YC-1 to slow the growth of the C6 glioma and the capacity of FDG-PET to provide insights into early molecular events regulating tumor growth. YC-1 is a novel HIF-1α inhibitor that decreases the growth of tumors through multiple mechanisms, including antiangiogenic effects.24 HIF-1α is a protein that is stabilized in hypoxic microenvironments, such as those typically found in rapidly growing neoplasia, including high-grade gliomas. The dimerization of HIF-1α with the constitutively expressed HIF-1β results in the formation of the transcription factor HIF-1,25 which up-regulates approximately 40 different genes, including glucose transporters, glycolytic enzymes, vascular endothelial growth factor (VEGF), and other key elements of angiogenesis.23 The positive therapeutic effects of YC-1 have been well characterized in models of hepatoma and neuroblastoma, as well as gastric, cervical, and renal carcinomas.26 Our study is the first demonstration of the efficacy of YC-1 in a glioma model.
The SUVr observed with YC-1 treatment was found to be primarily due to a decrease in GLUT-1 expression, resulting in a low FDG uptake at the completion of treatment. These findings were consistent with previous studies demonstrating a positive correlation between FDG uptake and the extent of GLUT-1 expression in glioma.22 Furthermore, the low levels of GLUT-1 expression in the YC-1 – treated group confirmed the ability of this compound to prevent the stabilization of HIF-1α and the subsequent up-regulation of proteins involved in the glycolytic pathway.8,23,27,28
While FDG uptake has previously been shown to correlate with cellular proliferation in glioma,22 we did not find any differences between the PIs of the YC-1 – treated and control groups. One possible explanation for this discrepancy may be related to the short therapeutic time frame utilized in our study. While our PET studies were able to detect the early HIF-1α inhibitory effect of YC-1, due to its direct effect on the GLUT-1 transporter expression and FDG uptake, this small temporal window may have been insufficient to detect later, downstream events, such as cell cycle arrest and cell death. Alternatively, the fact that the Ki-67 proliferation marker is expressed in cells at all active phases of the cell cycle (i.e., cells not in G0 phase)29 may explain the poor correlation between SUVr and PI in the YC-1 – treated group. As YC-1 has been shown to arrest cell cycle at the S-phase in models of hepatoma and renal carcinoma,30 Ki-67 may not have been the most appropriate marker of proliferation for this particular drug, as it is not capable of detecting S-phase arrested cells.
The lack of significant SUVr in the TMZ and NS1619 groups may be explained by the relatively early time point chosen to conduct the FDG-PET studies. The 3-day interval used to detect an SUVr using these agents was selected in accordance with previous studies that had detected an SUVr to doxorubicin in breast cancer models as early as 1 day following the initiation of therapy.31 As TMZ is commonly used in the treatment of glioma12 and the TE of NS1619 has been previously reported in glioma models,6 it can be concluded that this period was insufficient to detect a significant change in SUVr for these two agents. Furthermore, the unmethylated status of the O6-methylguanine-DNA-methyltransferase enzyme in the rat C6 glioma cell line,32 which has been negatively correlated with tumor response to TMZ,33 may have hindered detection of TE of TMZ in this model.
Contrary to the predicted synergistic effect of NS1619 and YC-1, this combination did not seem to offer a therapeutic advantage, as the efficacy of YC-1 was diminished by the coadministration of NS1619. A possible explanation for this finding may be related to an increase in intracellular calcium7 and induction of nitric oxide34 by NS1619 leading to an increase in HIF-1α transcription/activity35 and the stabilization of HIF-1α ,36 thereby counteracting the effect of YC-1.
The ability of our FDG-PET studies and SUVr measurements to accurately predict TE was extensively validated against gold-standard outcome studies. Longitudinal assessments of tumor growth over an extended, 7-day treatment period resulted in significantly smaller tumors in the YC-1 – treated group, confirming the TE of YC-1 in glioma models. Consequently, the SUVr observed after 3 days of YC-1 treatment was indeed representative of the TE of YC-1 in glioma, pointing to the ability of FDG-PET studies to detect the TE of novel therapeutic agents much earlier than traditional methods.
While these studies successfully demonstrated the feasibility of FDG-PET for determination of TE of YC-1 in the intracranial rat C6 glioma model after just 3 days of treatment, we plan to conduct further studies that will employ multiple time points and longer treatment periods to determine the optimal imaging window for a broad range of therapeutic agents with various mechanisms of action. Future studies will also compare metabolic PET to in vivo tumor volumetric MRI studies in order to determine whether PET provides temporal superiority for TE assessments, as well as the potential advantage of combining the respective information provided by these two modalities. Finally, other PET tracers, such as 18F-fluorothymidine or 5-[124I]iododeoxyuridine, should be explored for their ability to provide complementary information to FDG-PET studies. In conclusion, these studies provide compelling evidence for the use of PET in evaluating TE of antiglioma agents in rodent models and warrant further exploration for the integration of multimodal, noninvasive imaging techniques into pre-clinical oncology studies.
Preliminary data from this study were presented at the 11th Annual Scientific Meeting of the Society for Neuro-Oncology. We express our sincere appreciation to Dr. Pedro Rosa-Neto for reviewing the manuscript; Dr. Simone Zehtner, Rozica Bolovan, and Kurt Hemmings for their extensive technical support in the immunohistochemical studies; and Carmen Sabau for her technical support in establishing and maintaining the cell cultures. This work was partially supported by funds from the Montreal Neurological Institute, the Goals for Lily, the Alex Pavanel Family, the Franco Di Giovanni and Raymond and Tony Boeckh Brain Tumor Research Funds, the Montreal English School Board, Brainstorm, and Maggie De Fontes Foundation.