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In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography

In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using... Hindawi Publishing Corporation Journal of Oncology Volume 2009, Article ID 596560, 8 pages doi:10.1155/2009/596560 Research Article In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography 1 2 3 4 Karen E. Pollok, Michael Lahn, Nathan Enas, Ann McNulty, 4 1 1 1 Jeremy Graff, Shanbao Cai, Jennifer R. Hartwell, Aaron Ernstberger, 2 2 5 Donald Thornton, Les Brail, and Gary Hutchins Section of Pediatric Hematology & Oncology, Herman B Wells Center for Pediatric Research, Indiana University Simon Cancer Center, 1044 West Walnut Street R4 321, Indianapolis, IN 46202, USA Therapeutic Area Oncology, Lilly Research Laboratories, Indianapolis, IN 46285, USA Program Phase Statistics, Lilly Research Laboratories, Indianapolis, IN 46285, USA Oncology Discovery, Lilly Research Laboratories, Indianapolis, IN 46285, USA Department of Radiology, Indiana University Cancer Center, Indianapolis, IN 46202, USA Correspondence should be addressed to Michael Lahn, mlahn@lilly.com Received 21 July 2008; Accepted 13 March 2009 Recommended by Bruce Baguley 18 18 Background. The use of 2-[ F]fluoro-2-deoxy-D-glucose ([ F]FDG) may help to establish the antitumor activity of enzastaurin, a novel protein kinase C-beta II (PKC-βII) inhibitor, in mouse xenografts. Methods. The hematologic cell line RAJI and the solid tumor cell line U87MG were each implanted in NOD/SCID mice. Standard tumor growth measurements and [ F]FDG PET imaging were performed weekly for up to three weeks after tumor implantation and growth. Results. Concomitant with caliper measurements, [ F]FDG PET imaging was performed to monitor glucose metabolism. Heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment. This heterogeneity may limit the use of [ F]FDG PET imaging to measure enzastaurin-associated changes in xenograft tumors. Conclusion.[ F]FDG PET imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin. Future studies are needed to determine the use of [ F]FDG PET imaging in preclinical models. Copyright © 2009 Karen E. Pollok et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction [2], non-Hodgkin lymphoma [3], and glioblastoma [4]. Because of this increased utility in oncology [5–7], [ F]FDG Imaging techniques play an important role in the diagnosis, PET is being evaluated as a tool to assess antitumor effects of staging, and follow-up of cancer patients. While standard standard or novel anticancer drugs in both human subjects imaging techniques, such as computed tomography (CT), and in animal models of cancer [8]. PET imaging may are based on differences in the anatomical structure of the provide evidence of biological responses of novel anticancer compounds, which, in turn, can facilitate the transition of tissues, positron emission tomography (PET) uses radiola- beled molecular probes to assess differences in biological or compounds from preclinical to clinical investigation. One biochemical properties of tissues [1]. The most commonly such novel compound is enzastaurin, which was developed used tracer for PET is the glucose analogue 2-[ F]fluoro- as a Protein kinase C-beta (PKC-β) inhibitor. 2-deoxy-D-glucose ([ F]FDG), which provides an estimate The family of Protein Kinase C (PKC) has been impli- of tissue glucose utilization. Today, [ F]FDG PET is widely cated in processes that control tumor growth, survival, and used in clinical diagnoses of cancer, including lung cancer progression [9]. In particular, PKC-β activation has been 2 Journal of Oncology recognized as an important contributor to malignant growth time period from 45–60 minutes posttracer administration. in diffuse large cell B cell lymphoma [10] and in glioblastoma Indices of tumor FDG uptake were generated by calculating [11]. Recently, enzastaurin has shown antitumor activity in a ratio of the tumor to muscle uptake and by calculating the xenograft models of the colorectal cancer cell line HCT116 standardized uptake value (SUV): and in the glioblastoma cell line U87MG [12]. While enzastaurin was designed as a selective PKC-β inhibitor, C (t)dt SUV = . (1) recent studies suggest that its antitumor activity is modulated FDG ρ × ID /M by activation of GSK-3β and the PI3K/AKT pathway [12]. In this study we evaluated the extent to which [ F]FDG PET In (1) C (t) is the concentration of [F-18] in the tumor imaging can accurately characterize the antitumor activity of from the PET image, M is the mass of the animal, ID is the enzastaurin in two different mouse xenograft tumor models. dose of the tracer injected into the animal, and ρ is tissue density. [ F]FDG was prepared by the Hamacher method 2. Material and Methods using a commercial synthesis unit provided by PETNET/CTI (Knoxville, Tenn, USA) [16]. 2.1. Animal Model. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were raised and cared 2.6. Statistical Analyses. Endpoints included tumor volume for by the Indiana University Simon Cancer Center Trans- via calipers, tumor volume via CT, PET-determined mea- plant and Xenograft Core following the institutional guide- sures for tumor, muscle, tumor/muscle ratio, and a stan- lines of animal care. The study was conducted from October dardized measure of uptake. Changes from baseline for each 2004 to July 2005. endpoint were analyzed for each cell line (U87MG, RAJI) and each time point (Week 2, Week 3) using nonparametric 2.2. Cell Lines. The glioblastoma cell line, U87MG, and statistical methods (Wilcoxon rank-sum test) to compare Burkitt’s lymphoma cell line, RAJI, were obtained from the treated versus control animals. Due to the exploratory nature American Type Culture Collection (ATCC, Manassas, Va, of the analyses, P-values were used to indicate trends and USA) and cultured as recommended by ATCC. As previously potential future research hypotheses, rather than to test and published, U87MG and RAJI cells express high levels of PKC- confirm prespecified hypotheses. β [12, 13]. 3. Results 2.3. Xenograft Tumor Studies. Prior to subcutaneous (s.c.) injection, cells were resuspended in a 1 : 1 ratio of tumor We first investigated whether two tumor cell lines provided cells : matrigel (BD Biosciences, Bedford, Mass, USA) and acceptable tumor growth in NOD/SCID mice to allow Burkitt’s lymphoma cell line, RAJI was resuspended in a 1 : 2 reproducible imaging with [ F]FDG PET (Figure 1). We ratio of tumor cells : matrigel. Each mouse was injected s.c. used U87MG cells as a representative cell line for solid in the right flank with 5 × 10 cells. Mice were monitored tumors, and RAJI as a model for hematologic cancers. Both daily for palpable tumors. the U87MG (Figure 1(a)) and the RAJI (data not shown) tumor types grew consistently in NOD/SCID mice prior to 2.4. Enzastaurin Administration. Enzastaurin treatment was drug treatment and [ F]FDG PET imaging reliably detected initiated when the tumors reached a volume of at least glucose uptake in xenografts as determined by Standardized 150 mm . Mice with similar tumor sizes were matched in Uptake Value (SUV) (see Materials and Methods). In these the control and enzastaurin treated groups. Enzastaurin was feasibility studies, we also found that SUV correlated with suspended in 10% acacia (Fisher Scientific, Fair Lawn, NJ, U87MG tumor size as measured by CT (Figure 1(b)). USA) in water and dosed by gavage twice daily at 75 mg/kg Based on these preliminary observations of initial tumor based upon weekly body measurements for each treated growth, we elected to use both tumor cell lines to evalu- group. Control groups were treated only with vehicle. ate enzastaurin-induced metabolic changes as detected by [ F]FDG uptake. 2.5. PET and CT Imaging. Each animal was anesthetized Enzastaurin-induced metabolic changes were evaluated with acepromazine (1-2 mg/kg i.m.) and torbugesic (2 mg/kg using [ F]FDG uptake in two independent experiments i.m.) and placed on a custom bed for imaging. Animals for each tumor cell line, U87MG and RAJI, respectively were administered 0.5–1 mCi of [ F]FDG via a tail vein (Table 1). Consistent with previous studies in nude mice injection. A static 15-minute PET study was performed using [12], enzastaurin induced tumor growth delay in NOD/SCID the IndyPET II scanner [14, 15] at 45 minutes posttracer mice implanted with U87MG and RAJI (Figures 2(a) and injection. Following the PET study, the animal bed was 2(b)). U87MG cells grew slower than RAJI cells, and moved to and mounted on an EVS RS9 microCT scanner, enzastaurin had a tumor growth delay mainly in the U87MG and a volumetric image that encompassed the tumor volume tumor cell model (Figures 2(a) and 2(b)). A significant was imaged at approximately 90 micron spatial resolution. tumor growth delay was seen in U87MG after treatment with enzastaurin over the period of 3 weeks (Figure 2(a)). In the FDG Utilization Estimates. FDG uptake estimates are gen- RAJI xenograft model, there was a trend in the tumor growth erated by placing ROIs on PET images acquired over the delay, but not a statistically significant difference between Journal of Oncology 3 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 100 200 300 400 500 600 700 800 0 50 100 150 200 250 Caliper Tumor volume (CT) (a) (b) Figure 1: Correlation of Standard Caliper Measurements to FDG-PET uptake measured by SUV (Panel (a)) and Correlation of SUV with CT- based Tumor Size (Panel (b)): U87MG xenograft tumor cell growth was assessed over a period of 3 weeks using standard caliper measurements (mm ), serial FDG-PET uptake, and tumor size by CT scan (see Material and Methods). Correlation between the three different tumor growth measurements was plotted after 2 weeks (open symbols) and after 3 weeks (closed symbols) of tumor growth. Correlation between standard caliper measurements and standardized uptake value (SUV) was plotted (Panel (a)), and SUV was correlated with tumor size determined by CT scan (Panel (b)) (correlation coefficient 0.8, P = .001). (Correlation excludes one mouse with necrotic intratumoral tissue at Week 3 indicated by .) vehicle and enzastaurin-treated groups (Figure 2(b)). Con- (Figures 2(g) and 2(h)). While the collective analysis of all comitant with caliper measurements, [ F]FDG PET was animals from 2 independent experiments did not reveal a performed. [ F]FDG PET imaging was evaluated using SUV clear distinction between enzastaurin- and vehicle-treated (Figures 2(c) and 2(d)) and tumor/muscle ratio (Figures animals, there were some animals that did show a metabolic 2(e) and 2(f )). Enzastaurin administration did not alter change after enzastaurin treatment. In these cases we saw a tumor glucose metabolism as measured by SUV changes decline only after two weeks of treatment that was essentially in U87MG xenografts (Figure 2(c)). Compared to RAJI, undetectable after the third week of treatment (Figures the SUV changes in U87MG xenografts occurred at smaller 3(a)–3(f ) and Figures 4(a)–4(f )). The changes observed in signal intensities, and thus contributed to an overlap of SUV U87MG were different from changes observed in RAJI. In measurements between vehicle- and enzastaurin-treated U87MG xenografts, [ F]FDG uptake declined after the 2nd mice (compare Figures 2(c) and 2(d)). In RAJI, SUV uptake week of treatment. The RAJI tumors were fast growing and appeared to be increased at weeks 2 and 3 in enzastaurin- had large areas of necrotic tissue and some areas with newly treated compared to vehicle-treated mice (P< .10 at increased [ F]FDG uptake (Figure 5). Thegrowthpattern weeks 2 and 3; Figure 2(d)). It is possible that metabolic of these tumors is partly responsible for the intertumoral heterogeneity of the tumor glucose metabolism in different heterogeneity seen in this study and may contribute to the areas of the tumor may have caused this difference in SUV lack of detecting enzastaurin-induced changes in the tumor. uptake. In addition to SUV, we also used tumor/muscle ratio to determine the metabolic effect of enzastaurin in tumors. The tumor/muscle ratio uses muscle tissue with its 4. Discussion low metabolic rate to normalize the tumor tissue [ F]FDG uptake. Based on this analysis there was no clear evidence In this study we used a specialized PET imaging approach of a metabolic change induced by enzastaurin compared which was developed to assess activity of anti-cancer agents to vehicle treatment (Figures 2(e) and 2(f )). However, for in small animals [14]. Changes in [ F]FDG uptake were U87MG there was a trend in FDG uptake in enzastaurin- generally found to correlate with reduction in tumor size treated mice (P< .10 at week 3), which was not observed as determined by caliper measurements [17, 18]. Our in RAJI xenografts (Figures 2(e) and 2(f ),resp.). Next,we study uses a metabolic kinase inhibitor to compare drug- determined tumor volume by using standard volumetric mediated tumor growth reduction with metabolic alterations measurements based on CT. After enzastaurin treatment, in vivo. While previous imaging studies evaluated anti-tumor we observed a trend in tumor size reduction for RAJI activity of cytotoxic agents, it is not clear how [ F]FDG xenografts but not for U87MG xenografts at week 2 (P< .10) PET imaging can help determine antitumor activity of kinase SUV SUV 4 Journal of Oncology U87MG RAJI 200 1000 0 500 −100 −200 0 12 3 12 3 Week Week (a) (b) 0.3 0.6 U87MG RAJI 0.5 0.2 0.4 0.1 0.3 0.2 0.1 −0.1 −0.2 −0.1 −0.3 −0.2 −0.4 −0.3 12 3 12 3 Week Week (c) (d) 3 1.5 U87MG RAJI 2 1 1 0.5 0 0 −1 −0.5 −2 −1 12 3 12 3 Week Week (e) (f ) U87MG RAJI 100 700 50 500 0 300 −50 100 −100 −100 12 3 123 Week Week (g) (h) Figure 2: Tumor assessments of Xenografts U87MG and RAJI at 2 and 3 weeks after enzastaurin treatment. Standard caliper measurements show a tumor growth delay for U87MG (P< .05 at week 3), but not for RAJI (Panels (a) and (b)). [ F]FDG-PET imaging was performed at the same time as standard caliper measurements (Panels (c)-(d)). Tumor glucose metabolism changes were measured by SUV (Panels (c), (d)) and tumor/muscle ratio (Panels (e), (f )) in U87MG and RAJI xenografts. Only in RAJI xenografts enzastaurin treatment showed a trend in increased SUV (Panel (d); P< .10 at weeks 2 and 3). Using tumor/muscle ratio indicated a trend for FDG uptake in U87MG (P< .10 at week 3) (Panel (e)), but not in RAJI xenografts (Panel (f )). The tumor size assessment based on CT scan (Panels (g) and (h)) indicated a trend for detecting reduced tumor size only in RAJI xenografts after enzastaurin treatment at week 2 (P< .10). Mice treated with vehicle alone are shown in green; mice treated with enzastaurin are shown in red, representation of 2 independent experiments. Table 1: Evaluable sample sizes for caliper measures (CT/PET measures in parentheses). U87MG (n)RAJI(n) Week Enzastaurin Vehicle Enzastaurin Vehicle 2 19 (12) 15 (8) 7 (7) 7 (5) 3 15 (11) 12 (6) 7 (7) 6 (4) Caliper Tumor volume (CT) SUV Tumor/muscle Tumor/muscle SUV Caliper Tumor volume (CT) Journal of Oncology 5 Baseline Week 2 Week 3 3 3 3 359 mm 410 mm 218 mm (a) (b) (c) 3 3 3 256 mm 164 mm 293 mm (d) (e) (f) Figure 3: SUV in NOD/SCID mice implanted with U87MG. Representative mouse treated with vehicle (Panel (a)–(c)) and enzastaurin (Panel (d)–(f )) at baseline ((a),(d)) and after 2 weeks ((b), (e)) and 3 weeks ((c), (f )) of treatment. Green circle represents the region of interest (ROI) to assess changes in FDG uptake. Pictures are a fused image of the CT and PET. Each panel contains the caliper measurements (bottom right). inhibitors. For instance, imatinib activity was evaluated in with other studies which reported on the need of specific mice with limited success [19]. In addition to its cost, the use tumor sizes for scanner assessment [21]. The subsequent of PET imaging may be limited due to the spatial resolution studies with enzastaurin treatment did not provide clear of current PET scanners [20]. Thus, few studies have been evidence of enzastaurin-induced metabolic changes in either published which evaluate the use of small animal imaging in of the two tumor types examined. However, there are drug discovery. several possibilities why enzastaurin-induced changes were For the first time, we assessed the anti-tumor activity not detected by [ F]FDG PET imaging. of the serine/threonine kinase inhibitor enzastaurin by First, it is possible that enzastaurin may not have a [ F]FDG uptake in mice. Because we used NOD/SCID homogenous impact on the metabolic rate in the tumor instead of conventional nude mice, we first confirmed that tissue. Although PKC isoenzymes have been implicated [ F]FDG PET images could reproducibly be obtained in in cell proliferation [9], their specific role during glucose xenografts of glioblastoma and lymphoma. This feasibility metabolism is still not understood. On one hand, glucose assessment was important to establish the tumor size at can induce PKC-β expression [22], and on the other hand, which [ F]FDG uptake is detectable in mice. Tumors had overexpression of PKC-β reduces glucose uptake in cells [23]. to be at least 150 mm in volume to be visualized by the Hence, selective PKC-β inhibitors have been investigated scanner, and the best assessment was observed in tumors as potential treatments for diabetes [24]. Whether such a that were more than 400 mm (Figure 1). This is consistent PKC-β-dependent glucose regulation exists in tumor cells U87MG Enzastaurin Vehicle 6 Journal of Oncology Baseline Week 2 Week 3 3 3 3 397 mm 900 mm 1468 mm (a) (b) (c) 3 3 3 389 mm 447 mm 1064 mm (d) (e) (f) Figure 4: SUV in NOD/SCID mice implanted with RAJI. Representative mouse treated with vehicle (Panel (a)–(c)) and enzastaurin (Panel (d)–(f )) at baseline ((a), (d)) and after 2 weeks ((b), (e)) and 3 weeks ((c), (f )) of treatment. Green circle represents the region of interest (ROI) to assess changes in FDG uptake. Pictures are a fused image of the CT and PET. Each panel contains the caliper measurements (bottom right). is not known [25]. Considering the observation of this the caliper measurements can be used to demonstrate study, in which tumor growth delay and glucose metabolism anti-tumor effects while the imaging of the deeper tissue are not correlated with enzastaurin activity in xenograft by [ F]FDG or CT is not able to detect differences of tumors, it is possible that enzastaurin is not able to modulate treatment effect. Because of the inability to delineate clearly the complex glucose regulation in the tumor cells [26]. the borders of the infiltrating tumor tissue, the current Recently, the antiangiogenic kinase inhibitor AZD2171 was study may underestimate the metabolic change and thus also evaluated in a small animal study for its metabolic lead to false-negative imaging results. In some animals, change in tumors. Compared to [ F]FDG PET imaging, we used contrast dye to delineate better the tumor border only [ F] fluoromethane proved as a useful tool to assess the in mice. In these instances, caliper and CT scans showed anti-tumor activity of AZD2171 [27]. Therefore, it appears comparable results. Hence, future studies will need to be that only some kinase inhibitors will have metabolic changes conducted with contrast imaging techniques to approximate in tumors, which can be assessed by [ F]FDG PET imaging. the anatomical borders of the tumor. Tumor weight did not Secondly, the s.c. implanted tumors tend to grow initially correlate with caliper measurements, because CT scans or along the skin surface and infiltrate the underlying tissue to caliper measurements were taken during the course of the a lesser extent. This observation might explain why perhaps study, while tumor weight was collected only at the end of the RAJI Enzastaurin Vehicle Journal of Oncology 7 RAJI xenograft at week 3 RAJI xenograft at week 3 RAJI xenograft at week 3 3 3 3 1392 mm 1047 mm 1257 mm Mouse 1 Mouse 2 Mouse 3 (a) (b) (c) Figure 5: SUV in NOD/SCID mice implanted with RAJI. 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In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography

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Hindawi Publishing Corporation
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Copyright © 2009 Karen E. Pollok et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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1687-8450
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DOI
10.1155/2009/596560
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Hindawi Publishing Corporation Journal of Oncology Volume 2009, Article ID 596560, 8 pages doi:10.1155/2009/596560 Research Article In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography 1 2 3 4 Karen E. Pollok, Michael Lahn, Nathan Enas, Ann McNulty, 4 1 1 1 Jeremy Graff, Shanbao Cai, Jennifer R. Hartwell, Aaron Ernstberger, 2 2 5 Donald Thornton, Les Brail, and Gary Hutchins Section of Pediatric Hematology & Oncology, Herman B Wells Center for Pediatric Research, Indiana University Simon Cancer Center, 1044 West Walnut Street R4 321, Indianapolis, IN 46202, USA Therapeutic Area Oncology, Lilly Research Laboratories, Indianapolis, IN 46285, USA Program Phase Statistics, Lilly Research Laboratories, Indianapolis, IN 46285, USA Oncology Discovery, Lilly Research Laboratories, Indianapolis, IN 46285, USA Department of Radiology, Indiana University Cancer Center, Indianapolis, IN 46202, USA Correspondence should be addressed to Michael Lahn, mlahn@lilly.com Received 21 July 2008; Accepted 13 March 2009 Recommended by Bruce Baguley 18 18 Background. The use of 2-[ F]fluoro-2-deoxy-D-glucose ([ F]FDG) may help to establish the antitumor activity of enzastaurin, a novel protein kinase C-beta II (PKC-βII) inhibitor, in mouse xenografts. Methods. The hematologic cell line RAJI and the solid tumor cell line U87MG were each implanted in NOD/SCID mice. Standard tumor growth measurements and [ F]FDG PET imaging were performed weekly for up to three weeks after tumor implantation and growth. Results. Concomitant with caliper measurements, [ F]FDG PET imaging was performed to monitor glucose metabolism. Heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment. This heterogeneity may limit the use of [ F]FDG PET imaging to measure enzastaurin-associated changes in xenograft tumors. Conclusion.[ F]FDG PET imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin. Future studies are needed to determine the use of [ F]FDG PET imaging in preclinical models. Copyright © 2009 Karen E. Pollok et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction [2], non-Hodgkin lymphoma [3], and glioblastoma [4]. Because of this increased utility in oncology [5–7], [ F]FDG Imaging techniques play an important role in the diagnosis, PET is being evaluated as a tool to assess antitumor effects of staging, and follow-up of cancer patients. While standard standard or novel anticancer drugs in both human subjects imaging techniques, such as computed tomography (CT), and in animal models of cancer [8]. PET imaging may are based on differences in the anatomical structure of the provide evidence of biological responses of novel anticancer compounds, which, in turn, can facilitate the transition of tissues, positron emission tomography (PET) uses radiola- beled molecular probes to assess differences in biological or compounds from preclinical to clinical investigation. One biochemical properties of tissues [1]. The most commonly such novel compound is enzastaurin, which was developed used tracer for PET is the glucose analogue 2-[ F]fluoro- as a Protein kinase C-beta (PKC-β) inhibitor. 2-deoxy-D-glucose ([ F]FDG), which provides an estimate The family of Protein Kinase C (PKC) has been impli- of tissue glucose utilization. Today, [ F]FDG PET is widely cated in processes that control tumor growth, survival, and used in clinical diagnoses of cancer, including lung cancer progression [9]. In particular, PKC-β activation has been 2 Journal of Oncology recognized as an important contributor to malignant growth time period from 45–60 minutes posttracer administration. in diffuse large cell B cell lymphoma [10] and in glioblastoma Indices of tumor FDG uptake were generated by calculating [11]. Recently, enzastaurin has shown antitumor activity in a ratio of the tumor to muscle uptake and by calculating the xenograft models of the colorectal cancer cell line HCT116 standardized uptake value (SUV): and in the glioblastoma cell line U87MG [12]. While enzastaurin was designed as a selective PKC-β inhibitor, C (t)dt SUV = . (1) recent studies suggest that its antitumor activity is modulated FDG ρ × ID /M by activation of GSK-3β and the PI3K/AKT pathway [12]. In this study we evaluated the extent to which [ F]FDG PET In (1) C (t) is the concentration of [F-18] in the tumor imaging can accurately characterize the antitumor activity of from the PET image, M is the mass of the animal, ID is the enzastaurin in two different mouse xenograft tumor models. dose of the tracer injected into the animal, and ρ is tissue density. [ F]FDG was prepared by the Hamacher method 2. Material and Methods using a commercial synthesis unit provided by PETNET/CTI (Knoxville, Tenn, USA) [16]. 2.1. Animal Model. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were raised and cared 2.6. Statistical Analyses. Endpoints included tumor volume for by the Indiana University Simon Cancer Center Trans- via calipers, tumor volume via CT, PET-determined mea- plant and Xenograft Core following the institutional guide- sures for tumor, muscle, tumor/muscle ratio, and a stan- lines of animal care. The study was conducted from October dardized measure of uptake. Changes from baseline for each 2004 to July 2005. endpoint were analyzed for each cell line (U87MG, RAJI) and each time point (Week 2, Week 3) using nonparametric 2.2. Cell Lines. The glioblastoma cell line, U87MG, and statistical methods (Wilcoxon rank-sum test) to compare Burkitt’s lymphoma cell line, RAJI, were obtained from the treated versus control animals. Due to the exploratory nature American Type Culture Collection (ATCC, Manassas, Va, of the analyses, P-values were used to indicate trends and USA) and cultured as recommended by ATCC. As previously potential future research hypotheses, rather than to test and published, U87MG and RAJI cells express high levels of PKC- confirm prespecified hypotheses. β [12, 13]. 3. Results 2.3. Xenograft Tumor Studies. Prior to subcutaneous (s.c.) injection, cells were resuspended in a 1 : 1 ratio of tumor We first investigated whether two tumor cell lines provided cells : matrigel (BD Biosciences, Bedford, Mass, USA) and acceptable tumor growth in NOD/SCID mice to allow Burkitt’s lymphoma cell line, RAJI was resuspended in a 1 : 2 reproducible imaging with [ F]FDG PET (Figure 1). We ratio of tumor cells : matrigel. Each mouse was injected s.c. used U87MG cells as a representative cell line for solid in the right flank with 5 × 10 cells. Mice were monitored tumors, and RAJI as a model for hematologic cancers. Both daily for palpable tumors. the U87MG (Figure 1(a)) and the RAJI (data not shown) tumor types grew consistently in NOD/SCID mice prior to 2.4. Enzastaurin Administration. Enzastaurin treatment was drug treatment and [ F]FDG PET imaging reliably detected initiated when the tumors reached a volume of at least glucose uptake in xenografts as determined by Standardized 150 mm . Mice with similar tumor sizes were matched in Uptake Value (SUV) (see Materials and Methods). In these the control and enzastaurin treated groups. Enzastaurin was feasibility studies, we also found that SUV correlated with suspended in 10% acacia (Fisher Scientific, Fair Lawn, NJ, U87MG tumor size as measured by CT (Figure 1(b)). USA) in water and dosed by gavage twice daily at 75 mg/kg Based on these preliminary observations of initial tumor based upon weekly body measurements for each treated growth, we elected to use both tumor cell lines to evalu- group. Control groups were treated only with vehicle. ate enzastaurin-induced metabolic changes as detected by [ F]FDG uptake. 2.5. PET and CT Imaging. Each animal was anesthetized Enzastaurin-induced metabolic changes were evaluated with acepromazine (1-2 mg/kg i.m.) and torbugesic (2 mg/kg using [ F]FDG uptake in two independent experiments i.m.) and placed on a custom bed for imaging. Animals for each tumor cell line, U87MG and RAJI, respectively were administered 0.5–1 mCi of [ F]FDG via a tail vein (Table 1). Consistent with previous studies in nude mice injection. A static 15-minute PET study was performed using [12], enzastaurin induced tumor growth delay in NOD/SCID the IndyPET II scanner [14, 15] at 45 minutes posttracer mice implanted with U87MG and RAJI (Figures 2(a) and injection. Following the PET study, the animal bed was 2(b)). U87MG cells grew slower than RAJI cells, and moved to and mounted on an EVS RS9 microCT scanner, enzastaurin had a tumor growth delay mainly in the U87MG and a volumetric image that encompassed the tumor volume tumor cell model (Figures 2(a) and 2(b)). A significant was imaged at approximately 90 micron spatial resolution. tumor growth delay was seen in U87MG after treatment with enzastaurin over the period of 3 weeks (Figure 2(a)). In the FDG Utilization Estimates. FDG uptake estimates are gen- RAJI xenograft model, there was a trend in the tumor growth erated by placing ROIs on PET images acquired over the delay, but not a statistically significant difference between Journal of Oncology 3 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 100 200 300 400 500 600 700 800 0 50 100 150 200 250 Caliper Tumor volume (CT) (a) (b) Figure 1: Correlation of Standard Caliper Measurements to FDG-PET uptake measured by SUV (Panel (a)) and Correlation of SUV with CT- based Tumor Size (Panel (b)): U87MG xenograft tumor cell growth was assessed over a period of 3 weeks using standard caliper measurements (mm ), serial FDG-PET uptake, and tumor size by CT scan (see Material and Methods). Correlation between the three different tumor growth measurements was plotted after 2 weeks (open symbols) and after 3 weeks (closed symbols) of tumor growth. Correlation between standard caliper measurements and standardized uptake value (SUV) was plotted (Panel (a)), and SUV was correlated with tumor size determined by CT scan (Panel (b)) (correlation coefficient 0.8, P = .001). (Correlation excludes one mouse with necrotic intratumoral tissue at Week 3 indicated by .) vehicle and enzastaurin-treated groups (Figure 2(b)). Con- (Figures 2(g) and 2(h)). While the collective analysis of all comitant with caliper measurements, [ F]FDG PET was animals from 2 independent experiments did not reveal a performed. [ F]FDG PET imaging was evaluated using SUV clear distinction between enzastaurin- and vehicle-treated (Figures 2(c) and 2(d)) and tumor/muscle ratio (Figures animals, there were some animals that did show a metabolic 2(e) and 2(f )). Enzastaurin administration did not alter change after enzastaurin treatment. In these cases we saw a tumor glucose metabolism as measured by SUV changes decline only after two weeks of treatment that was essentially in U87MG xenografts (Figure 2(c)). Compared to RAJI, undetectable after the third week of treatment (Figures the SUV changes in U87MG xenografts occurred at smaller 3(a)–3(f ) and Figures 4(a)–4(f )). The changes observed in signal intensities, and thus contributed to an overlap of SUV U87MG were different from changes observed in RAJI. In measurements between vehicle- and enzastaurin-treated U87MG xenografts, [ F]FDG uptake declined after the 2nd mice (compare Figures 2(c) and 2(d)). In RAJI, SUV uptake week of treatment. The RAJI tumors were fast growing and appeared to be increased at weeks 2 and 3 in enzastaurin- had large areas of necrotic tissue and some areas with newly treated compared to vehicle-treated mice (P< .10 at increased [ F]FDG uptake (Figure 5). Thegrowthpattern weeks 2 and 3; Figure 2(d)). It is possible that metabolic of these tumors is partly responsible for the intertumoral heterogeneity of the tumor glucose metabolism in different heterogeneity seen in this study and may contribute to the areas of the tumor may have caused this difference in SUV lack of detecting enzastaurin-induced changes in the tumor. uptake. In addition to SUV, we also used tumor/muscle ratio to determine the metabolic effect of enzastaurin in tumors. The tumor/muscle ratio uses muscle tissue with its 4. Discussion low metabolic rate to normalize the tumor tissue [ F]FDG uptake. Based on this analysis there was no clear evidence In this study we used a specialized PET imaging approach of a metabolic change induced by enzastaurin compared which was developed to assess activity of anti-cancer agents to vehicle treatment (Figures 2(e) and 2(f )). However, for in small animals [14]. Changes in [ F]FDG uptake were U87MG there was a trend in FDG uptake in enzastaurin- generally found to correlate with reduction in tumor size treated mice (P< .10 at week 3), which was not observed as determined by caliper measurements [17, 18]. Our in RAJI xenografts (Figures 2(e) and 2(f ),resp.). Next,we study uses a metabolic kinase inhibitor to compare drug- determined tumor volume by using standard volumetric mediated tumor growth reduction with metabolic alterations measurements based on CT. After enzastaurin treatment, in vivo. While previous imaging studies evaluated anti-tumor we observed a trend in tumor size reduction for RAJI activity of cytotoxic agents, it is not clear how [ F]FDG xenografts but not for U87MG xenografts at week 2 (P< .10) PET imaging can help determine antitumor activity of kinase SUV SUV 4 Journal of Oncology U87MG RAJI 200 1000 0 500 −100 −200 0 12 3 12 3 Week Week (a) (b) 0.3 0.6 U87MG RAJI 0.5 0.2 0.4 0.1 0.3 0.2 0.1 −0.1 −0.2 −0.1 −0.3 −0.2 −0.4 −0.3 12 3 12 3 Week Week (c) (d) 3 1.5 U87MG RAJI 2 1 1 0.5 0 0 −1 −0.5 −2 −1 12 3 12 3 Week Week (e) (f ) U87MG RAJI 100 700 50 500 0 300 −50 100 −100 −100 12 3 123 Week Week (g) (h) Figure 2: Tumor assessments of Xenografts U87MG and RAJI at 2 and 3 weeks after enzastaurin treatment. Standard caliper measurements show a tumor growth delay for U87MG (P< .05 at week 3), but not for RAJI (Panels (a) and (b)). [ F]FDG-PET imaging was performed at the same time as standard caliper measurements (Panels (c)-(d)). Tumor glucose metabolism changes were measured by SUV (Panels (c), (d)) and tumor/muscle ratio (Panels (e), (f )) in U87MG and RAJI xenografts. Only in RAJI xenografts enzastaurin treatment showed a trend in increased SUV (Panel (d); P< .10 at weeks 2 and 3). Using tumor/muscle ratio indicated a trend for FDG uptake in U87MG (P< .10 at week 3) (Panel (e)), but not in RAJI xenografts (Panel (f )). The tumor size assessment based on CT scan (Panels (g) and (h)) indicated a trend for detecting reduced tumor size only in RAJI xenografts after enzastaurin treatment at week 2 (P< .10). Mice treated with vehicle alone are shown in green; mice treated with enzastaurin are shown in red, representation of 2 independent experiments. Table 1: Evaluable sample sizes for caliper measures (CT/PET measures in parentheses). U87MG (n)RAJI(n) Week Enzastaurin Vehicle Enzastaurin Vehicle 2 19 (12) 15 (8) 7 (7) 7 (5) 3 15 (11) 12 (6) 7 (7) 6 (4) Caliper Tumor volume (CT) SUV Tumor/muscle Tumor/muscle SUV Caliper Tumor volume (CT) Journal of Oncology 5 Baseline Week 2 Week 3 3 3 3 359 mm 410 mm 218 mm (a) (b) (c) 3 3 3 256 mm 164 mm 293 mm (d) (e) (f) Figure 3: SUV in NOD/SCID mice implanted with U87MG. Representative mouse treated with vehicle (Panel (a)–(c)) and enzastaurin (Panel (d)–(f )) at baseline ((a),(d)) and after 2 weeks ((b), (e)) and 3 weeks ((c), (f )) of treatment. Green circle represents the region of interest (ROI) to assess changes in FDG uptake. Pictures are a fused image of the CT and PET. Each panel contains the caliper measurements (bottom right). inhibitors. For instance, imatinib activity was evaluated in with other studies which reported on the need of specific mice with limited success [19]. In addition to its cost, the use tumor sizes for scanner assessment [21]. The subsequent of PET imaging may be limited due to the spatial resolution studies with enzastaurin treatment did not provide clear of current PET scanners [20]. Thus, few studies have been evidence of enzastaurin-induced metabolic changes in either published which evaluate the use of small animal imaging in of the two tumor types examined. However, there are drug discovery. several possibilities why enzastaurin-induced changes were For the first time, we assessed the anti-tumor activity not detected by [ F]FDG PET imaging. of the serine/threonine kinase inhibitor enzastaurin by First, it is possible that enzastaurin may not have a [ F]FDG uptake in mice. Because we used NOD/SCID homogenous impact on the metabolic rate in the tumor instead of conventional nude mice, we first confirmed that tissue. Although PKC isoenzymes have been implicated [ F]FDG PET images could reproducibly be obtained in in cell proliferation [9], their specific role during glucose xenografts of glioblastoma and lymphoma. This feasibility metabolism is still not understood. On one hand, glucose assessment was important to establish the tumor size at can induce PKC-β expression [22], and on the other hand, which [ F]FDG uptake is detectable in mice. Tumors had overexpression of PKC-β reduces glucose uptake in cells [23]. to be at least 150 mm in volume to be visualized by the Hence, selective PKC-β inhibitors have been investigated scanner, and the best assessment was observed in tumors as potential treatments for diabetes [24]. Whether such a that were more than 400 mm (Figure 1). This is consistent PKC-β-dependent glucose regulation exists in tumor cells U87MG Enzastaurin Vehicle 6 Journal of Oncology Baseline Week 2 Week 3 3 3 3 397 mm 900 mm 1468 mm (a) (b) (c) 3 3 3 389 mm 447 mm 1064 mm (d) (e) (f) Figure 4: SUV in NOD/SCID mice implanted with RAJI. Representative mouse treated with vehicle (Panel (a)–(c)) and enzastaurin (Panel (d)–(f )) at baseline ((a), (d)) and after 2 weeks ((b), (e)) and 3 weeks ((c), (f )) of treatment. Green circle represents the region of interest (ROI) to assess changes in FDG uptake. Pictures are a fused image of the CT and PET. Each panel contains the caliper measurements (bottom right). is not known [25]. Considering the observation of this the caliper measurements can be used to demonstrate study, in which tumor growth delay and glucose metabolism anti-tumor effects while the imaging of the deeper tissue are not correlated with enzastaurin activity in xenograft by [ F]FDG or CT is not able to detect differences of tumors, it is possible that enzastaurin is not able to modulate treatment effect. Because of the inability to delineate clearly the complex glucose regulation in the tumor cells [26]. the borders of the infiltrating tumor tissue, the current Recently, the antiangiogenic kinase inhibitor AZD2171 was study may underestimate the metabolic change and thus also evaluated in a small animal study for its metabolic lead to false-negative imaging results. In some animals, change in tumors. Compared to [ F]FDG PET imaging, we used contrast dye to delineate better the tumor border only [ F] fluoromethane proved as a useful tool to assess the in mice. In these instances, caliper and CT scans showed anti-tumor activity of AZD2171 [27]. Therefore, it appears comparable results. Hence, future studies will need to be that only some kinase inhibitors will have metabolic changes conducted with contrast imaging techniques to approximate in tumors, which can be assessed by [ F]FDG PET imaging. the anatomical borders of the tumor. Tumor weight did not Secondly, the s.c. implanted tumors tend to grow initially correlate with caliper measurements, because CT scans or along the skin surface and infiltrate the underlying tissue to caliper measurements were taken during the course of the a lesser extent. This observation might explain why perhaps study, while tumor weight was collected only at the end of the RAJI Enzastaurin Vehicle Journal of Oncology 7 RAJI xenograft at week 3 RAJI xenograft at week 3 RAJI xenograft at week 3 3 3 3 1392 mm 1047 mm 1257 mm Mouse 1 Mouse 2 Mouse 3 (a) (b) (c) Figure 5: SUV in NOD/SCID mice implanted with RAJI. 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