imaging    techniques play an important role in the diagnosis , staging , and follow - up of cancer patients .   while standard imaging techniques , such as computed tomography ( ct ) , are based on differences in the anatomical structure of the tissues , positron emission tomography ( pet ) uses radiolabeled molecular probes to assess differences in biological or biochemical properties of tissues . the most commonly used tracer for pet is the glucose analogue 2-[f]fluoro-2-deoxy - d - glucose ( [ f]fdg ) , which provides an estimate of tissue glucose utilization . today , [ f]fdg pet is widely used in clinical diagnoses of cancer , including lung cancer , non - hodgkin lymphoma , and glioblastoma . because of this increased utility in oncology [ 57 ] , [ f]fdg pet is being evaluated as a tool to assess antitumor effects of standard or novel anticancer drugs in both human subjects and in animal models of cancer . pet imaging may provide evidence of biological responses of novel anticancer compounds , which , in turn , can facilitate the transition of compounds from preclinical to clinical investigation . one such novel compound is enzastaurin , which was developed as a protein kinase c - beta ( pkc- ) inhibitor . the family of protein kinase c ( pkc ) has been implicated in processes that control tumor growth , survival , and progression . in particular , pkc- activation has been recognized as an important contributor to malignant growth in diffuse large cell b cell lymphoma   and in glioblastoma . recently , enzastaurin has shown antitumor activity in xenograft models of the colorectal cancer cell line hct116 and in the glioblastoma cell line u87 mg .   while enzastaurin was designed as a selective pkc-   inhibitor , recent studies suggest that its antitumor activity is modulated by activation of gsk-3 and the pi3k / akt pathway . in this study we evaluated the extent to which [ f]fdg pet imaging can accurately characterize the antitumor activity of enzastaurin in two different mouse xenograft tumor models . nonobese diabetic / severe combined immunodeficient ( nod / scid ) mice were raised and cared for by the indiana university simon cancer center transplant and xenograft core following the institutional guidelines of animal care . the glioblastoma cell line , u87 mg , and burkitt 's lymphoma cell line , raji , were obtained from the american type culture collection ( atcc , manassas , va , usa ) and cultured as recommended by atcc . as previously published , u87 mg and raji cells express high levels of pkc- [ 12 , 13 ] . injection , cells were resuspended in a 1 : 1 ratio of tumor cells : matrigel ( bd biosciences , bedford , mass , usa ) and burkitt 's lymphoma cell line , raji was resuspended in a 1 : 2 ratio of tumor cells : matrigel . each mouse was injected s.c . in the right flank with 5  10 cells . enzastaurin treatment was initiated when the tumors reached a volume of at least 150 mm . mice with similar tumor sizes were matched in the control and enzastaurin treated groups . enzastaurin was suspended in 10% acacia ( fisher scientific , fair lawn , nj , usa ) in water and dosed by gavage twice daily at 75 mg / kg based upon weekly body measurements for each treated group . each animal was anesthetized with acepromazine ( 1 - 2 mg / kg i.m . ) and torbugesic ( 2 mg / kg i.m . ) and placed on a custom bed for imaging . a static 15-minute pet study was performed using the indypet ii scanner [ 14 , 15 ] at 45 minutes posttracer injection .   following the pet study , the animal bed was moved to and mounted on an evs rs9 microct scanner , and a volumetric image that encompassed the tumor volume was imaged at approximately 90 micron spatial resolution . fdg utilization estimatesfdg uptake estimates are generated by placing rois on pet images acquired over the time period from 4560 minutes posttracer administration . indices of tumor fdg uptake were generated by calculating a ratio of the tumor to muscle uptake and by calculating the standardized uptake value ( suv ) : 
									 ( 1)suvfdg=[4560ct(t)dt](id)/m . 
								 in ( 1 ) ct(t ) is the concentration of [ f-18 ] in the tumor from the pet image , m is the mass of the animal , i d is the dose of the tracer injected into the animal , and  is tissue density . [ f]fdg was prepared by the hamacher method using a commercial synthesis unit provided by petnet / cti ( knoxville , tenn , usa ) . fdg uptake estimates are generated by placing rois on pet images acquired over the time period from 4560 minutes posttracer administration . indices of tumor fdg uptake were generated by calculating a ratio of the tumor to muscle uptake and by calculating the standardized uptake value ( suv ) : 
									 ( 1)suvfdg=[4560ct(t)dt](id)/m . 
								 in ( 1 ) ct(t ) is the concentration of [ f-18 ] in the tumor from the pet image , m is the mass of the animal , i d is the dose of the tracer injected into the animal , and  is tissue density . [ f]fdg was prepared by the hamacher method using a commercial synthesis unit provided by petnet / cti ( knoxville , tenn , usa ) . endpoints included tumor volume via calipers , tumor volume via ct , pet - determined measures for tumor , muscle , tumor / muscle ratio ,   and a standardized measure of uptake . changes from baseline for each endpoint were analyzed for each cell line ( u87 mg , raji ) and each time point ( week 2 , week 3 ) using nonparametric statistical methods ( wilcoxon rank - sum test ) to compare treated versus control animals .   due to the exploratory nature of the analyses , p - values were used to indicate trends and potential future research hypotheses , rather than to test and confirm prespecified hypotheses . we first investigated whether two tumor cell lines provided acceptable tumor growth in nod / scid mice to allow reproducible imaging with [ f]fdg pet ( figure 1 ) . we used u87 mg cells as a representative cell line for solid tumors , and raji as a model for hematologic cancers . both the u87 mg ( figure 1(a ) ) and the raji ( data not shown ) tumor types grew consistently in nod / scid mice prior to drug treatment and [ f]fdg pet imaging reliably detected glucose uptake in xenografts as determined by standardized uptake value ( suv ) ( see materials and methods ) . in these feasibility studies , we also found that suv correlated with u87 mg tumor size as measured by ct ( figure 1(b ) ) .   based on these preliminary observations of initial tumor growth , we elected to use both tumor cell lines to evaluate enzastaurin - induced metabolic changes as detected by [ f]fdg uptake . enzastaurin - induced metabolic changes were evaluated using [ f]fdg uptake in two independent experiments for each tumor cell line , u87 mg and raji , respectively ( table 1 ) . consistent with previous studies in nude mice , enzastaurin induced tumor growth delay in nod / scid mice implanted with u87 mg and raji ( figures 2(a ) and 2(b ) ) . u87 mg cells grew slower than raji cells , and enzastaurin had a tumor growth delay mainly in the u87 mg tumor cell model ( figures 2(a ) and 2(b ) ) . a significant tumor growth delay was seen in u87 mg after treatment with enzastaurin over the period of 3 weeks ( figure 2(a ) ) .   in the raji xenograft model , there was a trend in the tumor growth delay , but not a statistically significant difference between vehicle and enzastaurin - treated groups ( figure 2(b ) ) . [ f]fdg pet   imaging was evaluated using suv ( figures 2(c ) and 2(d ) ) and tumor / muscle ratio ( figures 2(e ) and 2(f ) ) . enzastaurin administration did not alter tumor glucose metabolism as measured by suv changes in u87 mg xenografts ( figure 2(c ) ) . compared to raji , the suv changes in u87 mg xenografts occurred at smaller signal intensities , and thus contributed to an overlap of suv measurements between vehicle- and enzastaurin - treated mice ( compare figures 2(c ) and 2(d ) ) .   in raji , suv uptake appeared to be increased at weeks 2 and 3 in enzastaurin - treated compared to vehicle - treated mice ( p < .10 at weeks 2 and 3 ; figure 2(d ) ) . it is possible that metabolic heterogeneity of the tumor glucose metabolism in different areas of the tumor may have caused this difference in suv 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 low metabolic rate to normalize the tumor tissue [ f]fdg uptake .   based on this analysis there was no clear evidence of a metabolic change induced by enzastaurin compared to vehicle treatment ( figures 2(e ) and 2(f ) ) . however , for u87 mg there was a trend in fdg uptake in enzastaurin - treated mice ( p < .10 at week 3 ) , which was not observed in raji xenografts ( figures 2(e ) and 2(f ) , resp . ) . after enzastaurin treatment , we observed a trend in tumor size reduction for raji xenografts but not for u87 mg xenografts at week 2 ( p while the collective analysis of all animals from 2 independent experiments did not reveal a clear distinction between enzastaurin- and vehicle - treated animals , there were some animals that did show a metabolic change after enzastaurin treatment . in these cases we saw a decline only after two weeks of treatment that was essentially undetectable after the third week of treatment ( figures 3(a)3(f ) and figures 4(a)4(f ) ) . the changes observed in u87 mg were different from changes observed in raji .   in u87 mg xenografts , [ f]fdg uptake declined after the 2nd week of treatment . the raji tumors were fast growing and had large areas of necrotic tissue and some areas with newly increased [ f]fdg uptake ( figure 5 ) . the growth pattern of these tumors is partly responsible for the intertumoral heterogeneity seen in this study and may contribute to the lack of detecting enzastaurin - induced changes in the tumor . in this study we   used a specialized pet imaging approach which was developed to assess activity of anti - cancer agents in small animals . changes in [ f]fdg uptake were generally found to correlate with reduction in tumor size as determined by caliper measurements [ 17 , 18 ] . our study uses a metabolic kinase inhibitor to compare drug - mediated tumor growth reduction with metabolic alterations in vivo . while previous imaging studies evaluated anti - tumor activity of cytotoxic agents , it is not clear how [ f]fdg pet imaging can help determine antitumor activity of kinase inhibitors . in addition to its cost , the use of pet imaging may be limited due to the spatial resolution of current pet scanners . thus , few studies have been published which evaluate the use of small animal imaging in drug discovery . for the first time , we assessed the anti - tumor activity of the serine / threonine kinase inhibitor enzastaurin by [ f]fdg uptake in mice .   because we used nod / scid instead of conventional nude mice , we first confirmed that [ f]fdg pet images could reproducibly be obtained in xenografts of glioblastoma and lymphoma . this feasibility assessment was important to establish the tumor size at which [ f]fdg uptake is detectable in mice . tumors had to be at least 150 mm in volume to be visualized by the scanner , and the best assessment was observed in tumors that were more than 400 mm ( figure 1 ) . this is consistent with other studies which reported on the need of specific tumor sizes for scanner assessment . the subsequent studies with enzastaurin treatment did not provide clear evidence of enzastaurin - induced metabolic changes in either of the two tumor types examined . however , there are several possibilities why enzastaurin - induced changes were not detected by [ f]fdg pet imaging . first , it is possible that enzastaurin may not have a homogenous impact on the metabolic rate in the tumor tissue . although pkc isoenzymes have been implicated in cell proliferation , their specific role during glucose metabolism is still not understood . on one hand , glucose can induce pkc- expression , and on the other hand , overexpression of pkc- reduces glucose uptake in cells .   hence , selective pkc- inhibitors have been investigated as potential treatments for diabetes .   whether such a pkc--dependent glucose regulation exists in tumor cells is not known . considering the observation of this study , in which tumor growth delay and glucose metabolism are not correlated with enzastaurin activity in xenograft tumors , it is possible that enzastaurin is not able to modulate the complex glucose regulation in the tumor cells . recently , the antiangiogenic kinase inhibitor azd2171 was also evaluated in a small animal study for its metabolic change in tumors . compared to [ f]fdg pet imaging , only [ f ] fluoromethane proved as a useful tool to assess the anti - tumor activity of azd2171 . therefore , it appears that only some kinase inhibitors will have metabolic changes in tumors , which can be assessed by [ f]fdg pet imaging . implanted tumors tend to grow initially along the skin surface and infiltrate the underlying tissue to a lesser extent . this observation might explain why perhaps the caliper measurements can be used to demonstrate anti - tumor effects while the imaging of the deeper tissue by [ f]fdg or ct is not able to detect differences of treatment effect .   because of the inability to delineate clearly the borders of the infiltrating tumor tissue , the current study may underestimate the metabolic change and thus lead to false - negative imaging results .   in some animals , we used contrast dye to delineate better the tumor border in mice . , future studies will need to be conducted with contrast imaging techniques to approximate the anatomical borders of the tumor . tumor weight did not correlate with caliper measurements , because ct scans or caliper measurements were taken during the course of the study , while tumor weight was collected only at the end of the study , when animals were sacrificed , and tumors had become heterogeneous . finally , the observed [ f]fdg uptake signals are considerably lower relative to what is measured in humans . the low baseline level in the present xenograft study negatively impacted the ability to assess early changes in metabolism associated with treatment effect . other factors influencing the low uptake signal are heterogeneity of glucose uptake in tumors , possible emergence of drug - resistant tumor cells , variability of pharmacokinetic exposure of the inhibitor , and the diffusion of tracer to the tumor site .   in summary , our data are consistent with the hypothesis that shrinking tumor size ( assessed by caliper measurements ) does not equate to reduction in overall tumor metabolism . in fact ,  pockets  of drug - resistant tumor cells with increased glucose uptake and growth kinetics may become prevalent in certain areas of the tumor , while glucose reduction will be present in other areas . additional longitudinal pet imaging studies will have to examine in detail the variables which affect the accurate measurement of tumor response to drug treatment . our study implies that [ f]fdg pet imaging will be useful only in a select type of tumors and perhaps detect antitumor activity only for a limited number of drugs .
background . 
 the use of 2-[18f]fluoro-2-deoxy - d - glucose ( [ 18f]fdg ) may help to establish the antitumor activity of enzastaurin , a novel protein kinase c - beta ii ( pkc-ii ) inhibitor , in mouse xenografts . 
 methods . 
 the hematologic cell line raji and the solid tumor cell line u87 mg were each implanted in nod / scid mice . 
 standard tumor growth measurements and [ 18f]fdg pet imaging were performed weekly for up to three weeks after tumor implantation and growth . 
 results . 
 concomitant with caliper measurements , [ 18f]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 [ 18f]fdg pet imaging to measure enzastaurin - associated changes in xenograft tumors . conclusion . 
 [ 18f]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 [ 18f]fdg pet imaging in preclinical models .