Characterization of preclinical models of prostate cancer using PET-based molecular imaging

ORIGINAL ARTICLE

Characterization of preclinical models of prostate cancerusing PET-based molecular imaging

Sara Belloli & Elena Jachetti & Rosa M. Moresco & Maria Picchio & Michela Lecchi &Silvia Valtorta & Massimo Freschi & Rodrigo Hess Michelini & Matteo Bellone &

Ferruccio Fazio

Received: 6 November 2008 /Accepted: 30 January 2009 /Published online: 11 March 2009# Springer-Verlag 2009

AbstractPurpose Transgenic adenocarcinoma of the mouse prostate(TRAMP) mice spontaneously develop hormone-dependentand hormone-independent prostate cancer (PC) that poten-tially resembles the human pathological condition. The aimof the study was to validate PET imaging as a reliable toolfor in vivo assessment of disease biology and progressionin TRAMP mice using radioligands routinely applied inclinical practice: [18F]FDG and [11C]choline.Methods Six TRAMP mice were longitudinally evaluatedstarting at week 11 of age to visualize PC development andprogression. The time frame and imaging pattern of PClesions were subsequently confirmed on an additionalgroup of five mice.Results PET and [18F]FDG allowed detection of PClesions starting from 23 weeks of age. [11C]Choline wasclearly taken up only by TRAMP mice carrying neuroen-

docrine lesions, as revealed by post-mortem histologicalevaluation.Conclusion PET-based molecular imaging represents astate-of-the-art tool for the in vivo monitoring andmetabolic characterization of PC development, progressionand differentiation in the TRAMP model.

Keywords Prostate cancer . TRAMPmouse . PET.

[18F]FDG . [11C]Choline

Introduction

Prostate cancer (PC) is one of the most common malignan-cies among men, accounting for approximately one third ofall cancer diagnoses in well-developed countries. Thistumour is a slow-growing hormone-dependent disease with

Eur J Nucl Med Mol Imaging (2009) 36:1245–1255DOI 10.1007/s00259-009-1091-3

S. Belloli :R. M. Moresco (*) :M. Picchio : S. Valtorta :F. FazioDepartment of Nuclear Medicine,Scientific Institute H San Raffaele,Via Olgettina 60,20132 Milan, Italye-mail: [email protected]

S. Belloli :R. M. Moresco :M. PicchioIBFM, CNR,Milan, Italy

S. Belloli :R. M. Moresco :M. Picchio : F. FazioUniversity of Milano-Bicocca,Milan, Italy

E. Jachetti :R. H. Michelini :M. BelloneCancer Immunotherapy and Gene Therapy Program,San Raffaele Scientific Institute,Milan, Italy

M. FreschiPathological Anatomy Unit,San Raffaele Scientific Institute,Milan, Italy

M. LecchiInstitute of Radiological Sciences,University of Milan,Milan, Italy

E. JachettiFellowship of the Doctorate School of Cellularand Molecular Medicine, University of Vita-Salute,San Raffaele Institute,Milan, Italy

S. ValtortaFellowship of the Doctorate School of Molecular Medicine,University of Milan,Milan, Italy

a natural progression from localized to hormone-refractorysystemic disease [1].

Imaging is an indispensable tool for cancer diagnosis,patient’s stratification and follow-up after treatment. Posi-tron emission tomography (PET) is a non-invasive imagingtechnique that provides in vivo information on biochemicaland physiological processes in normal and pathologicaltissues depending on the tracer used [2]. Both [18F]FDGand [11C]choline are used in diagnostic oncology withuptake values regulated by the activation of differentbiochemical pathways. [18F]FDG uptake is regulated byglucose transporters and hexokinase activity, whereas [11C]choline retention is related to the presence of cholinetransporters and choline kinase activity [3]. This pathway,although less well characterized than glycolysis, is currentlythe focus of intense research. In a recent study, theactivation of choline kinase has been proposed to have arole in oncogene-mediated transformation and in generationof tumours [4]. [11C]Choline or its derivative [18F]cholineare in clinical use for the detection of PC recurrence, inconcomitance with increased prostate-specific antigen(PSA) levels [5–12].

On the contrary, physiological urinary secretion of [18F]FDG, which interferes with imaging of the pelvic region,and its low and highly heterogeneous levels of uptake limitthe use of [18F]FDG PET in PC patients [3, 13, 14].Nonetheless, [18F]FDG PET has been recently indicated asa possible means to assess the biological activity of bonemetastases and for the evaluation of a specific subgroup ofpatients carrying lesions with an aggressive phenotype [13].

Because the social relevance of PC and the interest in thedevelopment of new diagnostic and therapeutic strategies,several mouse models of PC have been recently developedand investigated for preclinical studies [15]. Among thevariousmodelsproposed, the transgenicadenocarcinomaof themouse prostate (TRAMP) is particularly promising [16].TRAMP mice are transgenic for the SV40 early genes (Tand t antigens; Tag) under the control of the rat probasinregulatory element and express Tag at puberty selectively onthe prostate tissue [17]. In particular, TRAMP miceprogressively develop mouse prostate intraepithelial neopla-sia (mPIN; weeks 6–12), adenocarcinoma (weeks 12–18),seminal vesicles, lymph node and lung metastases (weeks18–30). Cancer development [18], androgen sensitivity [19],fine aspects of neo-angiogenesis [20] and several metabolicpathways [21, 22] have been characterized in TRAMP mice,showing similarity with human PC [23]. Indeed, TRAMPmice, both spontaneously or upon androgen withdrawal[18, 20], may develop androgen-independent adenocarci-noma [24] and an aggressive neuroendocrine variant,whose origin is still under investigation [25]. The presenceof focal neuroendocrine differentiations, although a matterof debate, may occur also in human tumours characterized

by aggressiveness and resistance to hormone therapy[26–28]. Hence TRAMP mice are suitable for investigat-ing PC biology and for the in vivo validation of noveltherapeutic approaches.

At present, pathological examination is the method ofchoice for the evaluation of disease progression in TRAMPmice, but it requires that the mice be sacrificed at differenttime windows. Although the method presents limitations,abdominal palpation is commonly used to monitor thepresence of advanced lesions in the genitourinary tract ofliving animals. Magnetic resonance imaging (MRI) hasbeen successfully used in TRAMP mice for the in vivomonitoring of tumour disease onset and progression [29].However, to better understand the biological features of theprostate tumour in TRAMP mice and to evaluate itscomparative significance for human PC, the evaluation ofthe potential use of PET is of particular relevance. Inparticular, the recent development of dedicated smallanimal PET allows the quantitative assessment of selectivebiochemical pathways that characterize tumour growth inliving animals.

In the present study, [18F]FDG and [11C]choline havebeen investigated to validate PET modality as a reliable toolfor the in vivo assessment of disease biology in TRAMPmice at different stages of cancer progression. The imagingprotocols for the analysis of TRAMP mice were set up onthe syngeneic mouse model based on the subcutaneousinjection of TRAMP-C1 cells, derived from a hormone-independent TRAMP lesion [24].

Materials and methods

Animals

In vivo and ex vivo experiments were approved by theEthics Committee for Animal Care (IACUC) of the SanRaffaele Hospital and carried out in accordance with theItalian and EEC recommendations for the care and use oflaboratory animals.

Male TRAMP mice were derived by breeding heterozy-gous transgenic C57BL/6 female mice to non-transgenicC57BL/6 male mice (Charles River, Italy) to obtain non-transgenic and transgenic [C57BL/6 TRAMP x C57BL/6]F1 males. Animals were typed for Tag expression bypolymerase chain reaction (PCR)-based screening assay, asdescribed in www.jax.org. Isolation of mouse tail genomicDNA was performed as previously described [17].

Feasibility experiments, designed to verify whether [18F]FDG and [11C]choline were able to trace in vivo TRAMPlesions, were conducted in wild-type C57BL/6 male micesubcutaneously implanted in the flank with 2.5×106

TRAMP-C1 cells, [24]. Animals were monitored twice a

1246 Eur J Nucl Med Mol Imaging (2009) 36:1245–1255

http://www.jax.org

week for weight and tumour growth (by measuring twoperpendicular diameters by a caliper) and sacrificed whenthe tumour reached an average dimension of ≥15 mm indiameter. In vivo and ex vivo experiments were conductedwhen lesions reached an area of approximately 50–80 mm2.The kinetic parameters obtained from ex vivo and in vivobiodistribution studies in TRAMP-C1 mice were subse-quently applied for the PET imaging of the transgenicmodel.

Study design

A first group of six TRAMP mice was evaluated in vivowith [18F]FDG and [11C]choline every 2 weeks, startingfrom 11 weeks until a maximum of 33 weeks of age,depending on illness severity. On the basis of the resultsobtained from the first group of animals, an additionalgroup of five 23-week-old TRAMP mice was evaluatedonce with [18F]FDG and [11C]choline PET and thensacrificed (see Fig. 1 for a schematic representation).

Histology and immunohistochemistry

After the animals’ death, the urogenital apparatus wascollected, photographed, weighed and fixed in 4% formalinfor 6 h, then embedded and included in paraffin wax.Haematoxylin and eosin (H&E) and Tag staining of 5-mm-thick sections was performed as previously described [30].A pathologist in a blind fashion evaluated the macroscopicand microscopic specimens. Histology sections were scoredas previously described [30], with partial modifications: +/−,scattered (<20%) Tag+ cells with mild increase of nucleus tocytoplasm ratio; +, ≥20% Tag+ cells with increasing nucleusto cytoplasm ratio, nuclear hyperchromasia, stratification andmicropapillary projections; ++, presence of all the featuresdescribed in + and cribriform structures with mild enlarge-

ment of acini; +++, presence of all the features describedin ++ and either proliferation of the epithelial andstromal cells of the seminal vesicles or mild proliferationof smooth muscle stromal cells of prostatic acini withacinar enlargement; ++++, presence of all the featuresdescribed in +++ and marked proliferation of smoothmuscle stromal cells with penetration of malignant Tag+

cells through the basement membrane of mPIN-involvedglands into the surrounding stroma; Adeno = invasiveadenocarcinoma; NE= androgen-independent neuroendo-crine tumour characterized by small cells with total loss ofglandular structure.

TRAMP-C1 cells uptake studies

[18F]FDG is routinely prepared in our facility for clinicaluse (European Pharmacopoeia V ed.). [11C]Choline radio-labelling was performed as previously described [31]. Bothradiotracers were injected with a radiochemical puritygreater than 99%.

TRAMP-C1 cells were maintained in DMEM supple-mented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin. Cells wereroutinely subcultivated when 75% confluent, plated in75-cm2 flasks and incubated in an atmosphere of 5%CO2, 95% air at 37°C. The uptake assay was performed asdescribed [32]. Briefly: cells were seeded in 6-well multi-well plates at the concentration of 1×105 cells/ml 24 hbefore the experiments. The day of the experiment, the me-dium was removed and replaced with a glucose-freemedium containing [18F]FDG (0.04 MBq/ml) or a completemedium containing [11C]choline (0.15 MBq/ml) and incu-bated at 37°C for 60 and 40 min, respectively. After thesetimes, the media were removed and counted in a gammacounter and the cells washed twice with ice-cold phosphate-buffered saline (PBS). These time frames were selected on

11

WEEKS

GROUP 1: longitudinal PET evaluation with [18F]FDG and [11C]choline(n=6)

2 WEEKS

GROUP 2: single point PET evaluation with [18F]FDG and [11C]choline (n=5)

FIRST

PET

LAST

PET

33

WEEKS

23 WEEKS

(mean time of [18F]FDG and/or [11C]choline signal comparison)

Fig. 1 Schematic representationof the study design

Eur J Nucl Med Mol Imaging (2009) 36:1245–1255 1247

the basis of previous studies performed on PC cells [33, 34]and on personal observations on the kinetics of [18F]FDGand [11C]choline uptake on TRAMP-C1 cells (data notshown). Cells were trypsinized and once detached, thereaction was neutralized adding DMEM glucose-free orDMEM complete medium for [18F]FDG or [11C]cholinecellular uptake determination in the gamma counter, respec-tively. Radioactivity uptake was expressed as % uptake=(radioactivity in the cells/total radioactivity added) × 100.

Ex vivo and in vivo PET studies in wild-type C57BL/6mice bearing TRAMP-C1 prostate cancer

For ex vivo studies, 3.7±0.8 MBq of [18F]FDG were injectedintravenously in male C57BL/6 mice bearing subcutaneousTRAMP-C1 tumours in the flank. Tumoural uptake wasevaluated at 30, 60, 90 and 120 min after tracer injection(three animals at each time point). At the indicated times,mice were sacrificed by decapitation under light anaesthesia(ether). Samples of blood, tumour and muscle were collectedand placed in pre-weighed tubes. Radioactivity was countedin a gamma counter and its concentration calculated as thepercentage of injected dose per gram of tissue (%ID/g). Twoadditional animals underwent a 30-min-long PET scanstarting 60 min after tracer injection. In vivo imaging studieswere performed using a small animal tomograph: the YAP-(S)PET II (I.S.E. s.r.l., Pisa, Italy) scanner [35]. Ten minutesbefore radioligand administration, mice were administered1.7% tribromoethanol solution (10 μl/g of weight, i.p.) andmaintained anaesthetized until the end of PET study. At theend of PET acquisition animals were sacrificed and processedfor tissue sampling analysis. [11C]Choline biodistribution wasalso evaluated ex vivo and in vivo. For ex vivo studies,radioactivity distribution was assayed at 10, 30 and 60 min(three animals for a time frame) after the i.v. injection of 1.3±0.1 MBq of radioligand. Blood, tumour and muscle sampleswere collected and placed in pre-weighed tubes and radio-activity concentration calculated as the %ID/g. As for the [18F]FDG study, two additional mice were evaluated in vivo withPET and acquired for 30 min starting from tracer injection.

In order to check for possible confounding effectsderiving from abdominal and kidney background, in vivovisualization of [18F]FDG and [11C]choline uptake wasperformed also in mice bearing TRAMP-C1 lesion in theright hind leg. [18F]FDG and [11C]choline PET data wereacquired in list mode using the full axial acceptance angleof the scanner (3-D mode) and then reconstructed with theexpectation maximization (EM) algorithm [36]. Aftercalibration of PET images and correction for the isotopehalf-life, radioactivity concentration was calculated intumour and muscle and expressed as tumour to muscleratios using region of interest (ROI) analysis. Circular ROIs(area=15 mm2) were centred on the tumour lesion or on a

leg muscle. At the end of PET studies animals weresacrificed and processed for tissue sampling analysis, asdescribed above.

In vivo PET studies in TRAMP mice

A group of six TRAMP mice were evaluated in vivo with[18F]FDG and [11C]choline PET approximately every2 weeks starting from the age of 11 weeks. Imagingprotocols were defined on the basis of the ex vivo resultsobtained from syngeneic mice. [18F]FDG studies wereperformed as followed: animals were injected in a tail veinwith 4.2±0.4 MBq of the tracer. Ten minutes beforeradioligand administration, animals were anaesthetized, asdescribed above. Immediately before image acquisition,mice were positioned supine on the tomograph bed andtheir abdomen centred in the tomograph field of view. PETscans started at 60 min after tracer injection and lasted for30 min. Images were than processed as described in theprevious paragraph. The day after [18F]FDG study, micewere evaluated with [11C]choline; animals were anaesthe-tized as for [18F]FDG, positioned supine on the bedtomograph and injected in a tail vein with 4.1 ± 1.3 MBqof radioligand. PET acquisitions started immediately after[11C]choline injection and continued for the following 30min. Based on the results of longitudinal studies, anadditional group of five TRAMP mice was evaluated with[18F]FDG and [11C]choline PET at 23 weeks of age. Theday of the study, animals were anaesthetized as mentionedabove, i.v. injected with 4.3±0.2 MBq of [18F]FDG andPET images were acquired for 30 min starting 60 min aftertracer injection. The day after, animals received 4.8±0.8 MBq of [11C]choline intravenously and PET imageswere acquired immediately after, for 30 min. Animals werethen sacrificed for histological and immunohistochemicalevaluations. Acquisition protocols, reconstruction and post-processing images analysis were performed as indicated inthe previous paragraph.

Statistical analysis was performed using Student’s t testfor independent samples.

Results

In order to set up the imaging protocols for the analysis ofTRAMP mice we took advantage of TRAMP-C1 cells.TRAMP-C1 is a hormone-refractory PC cell line establishedfrom a heterogeneous 32-week tumour that spontaneouslydeveloped in a TRAMPmouse. Upon subcutaneous injection,TRAMP-C1 cells develop into poorly differentiated and well-vascularized tumours, which express cytokeratine, indicatingthat tumours are epithelial in origin. TRAMP-C1 cells expressalso E-cadherin and AR, but not synaptophysin [24, 37],


therefore excluding their neuroendocrine differentiation.Another interesting characteristic of TRAMP-C1 cells istheir rather indolent growth in vivo with a doubling time of11.3 days, a trait common to human PC.

TRAMP-C1 cells uptake studies

Firstly, we conducted in vitro experiments to investigatewhether TRAMP-C1 cells had the potential to acquire andretain [18F]FDG and [11C]choline by measuring tracer after60 and 40 min of incubation, respectively. Uptake of bothtracers was evident in TRAMP-C1 cells, although the cellsshowed a markedly higher retention of [18F]FDG incomparison to [11C]choline. The percentages of uptake of[18F]FDG and [11C]choline were 48.43±1.63 and 4.94±1.28, respectively.

Subcutaneous TRAMP-C1 tumours are well definedby [18F]FDG PET imaging

The in vitro results prompted us to investigate whether [18F]FDG and [11C]choline were useful tracers to image TRAMP-C1 tumours in vivo. Results of the kinetics of radioactivityaccumulation measured ex vivo in tumour, muscle and bloodafter [18F]FDG and [11C]choline injection are presented inFig. 2, panels a and b, respectively. High levels ofradioactivity were observed in tumour lesions confirmingthe results obtained in vitro. [18F]FDG accumulated alreadyat 30 min after injection and maximum uptake was observedat 90 min (11.17±0.26%ID/g), reaching at 60 min a tumourto muscle ratio of 15.99±1.24 (Fig. 2a).

[11C]Choline uptake showed faster kinetics (Fig. 2b).Indeed, radioactivity concentration rapidly increased up to4.55±0.62%ID/g 10 min after [11C]choline injection andremained stable thereafter. At 30 min the tumour to muscleratio displayed maximum values of 2.71±0.53, thereforeconfirming that overall [11C]choline uptake was lower thanthat of [18F]FDG. On the basis of these results, PET studieswere performed between 0 and 30 min and between 60 and90 min for [11C]choline and [18F]FDG, respectively. Theseacquisition protocols were applied to visualize TRAMP-C1

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 10 20 30 40 50 60 70

time (min)

time (min)

%ID

/g

muscle

tumor

blood

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 15 30 45 60 75 90 105 120 135

%ID

/ga

b

Fig. 2 Peripheral [18F]FDG (a) and [11C]choline (b) distribution inTRAMP-C1 mice i.v. injected with 3.7±0.8 MBq or 1.3±0.1 MBq,respectively. Radioactivity concentration is calculated as percentage ofinjected dose per gram of tissue (%ID/g). Values are expressed asmean ± SD of three mice for each time point

a b

[18

F]FDG [ [18

F]FDG [11

C]Choline11

C]Choline

c d

Fig. 3 YAP-(S)PET II images of four TRAMP-C1 mice with tumourimplanted in the flank (a and b) or in the leg (c and d). Animals wereanalysed after the i.v. injection of 4.35 MBq of [18F]FDG (a and c)and 3.34 MBq of [11C]choline (b and d). At the top of each panel, the

coronal view of the 3-D rendering reconstruction with the tumourallesion indicated by an arrow; at the bottom, a transaxial slice taken inthe middle of the tumour, where the lesion is indicated in a dashedcircle


tumours. Three-dimensional rendering images obtainedafter [18F]FDG or [11C]choline PET scans are presented inFig. 3. As previously observed in ex vivo studies, TRAMP-C1 tumours were better visualized using [18F]FDG thanwith [11C]choline independently on the side of TRAMP-C1cell inoculation (flank or leg). ROIs analysis performed onthe animals inoculated in the flanks revealed uptake values

comparable with those obtained ex vivo. In particular, inthe two mice evaluated with [18F]FDG, we observed uptakevalues of 13.35%ID/g and 7.01%ID/g and tumour tomuscle ratios of 7.53 and 6.84, whereas in the two animalsinjected with [11C]choline the uptake was of 4.33%ID/gand 2.46%ID/g and tumour to muscle ratios were 2.31 and1.58. No significant differences in radioligand uptake wereobserved in animals with tumour implanted in the leg.

[18F]FDG and [11C]choline PET visualize spontaneous PClesions in TRAMP mice

The results obtained in the TRAMP-C1 model suggestedthat [18F]FDG and [11C]choline PET might be used tovisualize prostate lesions spontaneously occurring inTRAMP mice. Hence, we set up a longitudinal study in agroup of six TRAMP mice. PET imaging was performedapproximately every 2 weeks starting from the age of11 weeks (Fig. 1).

One animal died at 16 weeks of age, after complicationsdue to intestinal inflammation; the other five animals diedfrom causes related to tumour development at 28.4±5.5 weeks. Results of H&E and anti-Tag (Tag+) stainingon prostate tissues collected post-mortem are shown inTable 1 (disease score), together with the results of PETquantifications expressed as tumour to muscle ratio (T/M)and with the urogenital (UGA) apparatus weight. All theanimals were positive for Tag. Two of the six animals (micea and e) developed a neuroendocrine PC (NE), while theother four developed mPIN and adenocarcinoma.

Despite its high accumulation in the bladder, [18F]FDGallowed visualization and monitoring of primary prostatelesions in the whole group of animals starting from 23.6±3.3 weeks of age. Figure 4 shows an example of diseaseprogression monitored with [18F]FDG (mouse a). Neuro-endocrine lesions displayed higher in vivo uptake of both

16 weeks 20 weeks 22 weeks Fig. 4 YAP-(S)PET II imagesof the same TRAMP mouse(corresponding to mouse a ofTable 1) after the i.v. injection of4.2±0.4 MBq of [18F]FDG atdifferent stages of disease.Example of disease progressionmonitored with PET. At the topof each panel, the coronal viewof the 3-D rendering recon-struction; at the bottom, a coro-nal slice taken in the prostatearea with tumour growth indi-cated in a dashed circle

Table 1 Longitudinal evaluation and post-mortem histological anal-ysis of six TRAMP mice

Mouse PET prostate quantification(T/M)

Disease score(Tag+/H&E)

UGAweight (g)

[18F]FDG [11C]Choline

a 4.55 3.61 NE 1.6

b 3.51 1.87 ++++/Adeno 2.5

c nd nd ++/+++ 1.2

d 2.94 1.80 ++++/Adeno 1.3

e 4.33 3.21 NE 4.2

f 3.12 2.24 ++++/Adeno 1.3

Summary of [18 F]FDG and [11 C]choline PET and immunohistochem-istry evaluation of six TRAMP mice (a–f) in the longitudinal study.Histology sections were scored as followed: +/−, scattered (<20%)Tag+ cells with mild increase of nucleus to cytoplasm ratio; +, ≥20%Tag+ cells with increasing nucleus to cytoplasm ratio, nuclearhyperchromasia, stratification and micropapillary projections; ++,presence of all the features described in + and cribriform structureswith mild enlargement of acini; +++, presence of all the featuresdescribed in ++ and either proliferation of the epithelial and stromalcells of the seminal vesicles or mild proliferation of smooth musclestromal cells of prostatic acini with acinar enlargement; ++++,presence of all the features described in +++ and marked proliferationof smooth muscle stromal cells with penetration of malignant Tag+

cells through the basement membrane of mPIN-involved glands intothe surrounding stroma; Adeno invasive adenocarcinoma, NE neuro-endocrine tumour, androgen independent, small cells with total loss ofglandular structure


[18F]FDG and [11C]choline (Table 1 and Fig. 5) whencompared with prostate lesions of the other four animals,which were classified as mouse intraepithelial neoplasia(mPIN= ++/+++) or adenocarcinoma (++++/Adeno)(Table 1). The animal classified as mPIN had no detectableuptake of either [18F]FDG or [11C]choline in the prostate.Examples of [18F]FDG (on the left) and [11C]choline PET(on the right) images related to disease score classificationare presented in Fig. 5a, b and c. T/M values obtained fromTRAMP mice after [18F]FDG and [11C]choline injectionwere comparable with those measured in the TRAMP-C1model, as presented in the previous paragraph. In addition,animals carrying NE lesions displayed T/M values defi-nitely higher than those classified as adenocarcinoma (41and 73% higher [18F]FDG and [11C]choline, respectively).

To confirm our findings, a second group of TRAMPmice was investigated at 23 weeks of age. Results of in

vivo [18F]FDG and [11C]choline PET quantification (T/M),together with anatomical and immunohistochemistry (IHC)observations for the second group of TRAMP mice, arereported in Table 2 and in Fig. 6. As observed for the firstgroup of TRAMP mice, the in vivo uptake of both [18F]FDG and [11C]choline was higher in NE lesions than inadenocarcinoma.

Considering the whole group of mice and excludingthe animal that died at 16 weeks of age, three of tenlesions were defined as neuroendocrine and in theseanimals the uptake of [11C]choline and [18F]FDG wassignificantly higher (unpaired t test) than in those with lesionsdefined as adenocarcinoma (+62 and +67%, respectively;Fig. 7). In particular, mean [18F]FDG tumour to muscleratios were 4.93±0.87 and 2.95±0.50 (p=0.0015 forneuroendocrine and adenocarcinoma, respectively), whereasthose obtained with [11C]choline were 3.32±0.25 and 2.05±

NE

a

[18

F]FDG [11

C]Choline

++++/Adeno

[18

F]FDG [11

C]Choline

++/+++

[18

F]FDG [11

C]Choline

22 weeks 33 weeks 16 weeks

b c

Fig. 5 YAP-(S)PET II, anatomy and histology images of threeTRAMP mice selected from the six animals followed in thelongitudinal study (a–c). Top, PET images after the i.v. injection of4.2±0.4 MBq of [18F]FDG (left part) and of 4.5±1.3 MBq of [11C]choline (right part). Bottom, anatomy (left) of urogenital organs and

H&E staining (right) of the dorsolateral prostate gland. The area oftumour growth is indicated by a dashed circle. Corresponding valuesof PET image quantification and IHC observations of each animal arepresented in Table 1 (animals a–c, respectively)

Mouse PET prostate quantification (T/M) Disease score (Tag+/H&E) UGA weight (g)

[18F]FDG [11C]Choline

g 2.92 2.55 ++++/Adeno 1.42

h 5.93 3.15 NE 4.33

i 2.86 2.35 ++++/Adeno 1.21

j 1.96 2.05 ++++/Adeno 1.37

k 3.35 1.47 ++++/Adeno 1.41

Table 2 PET evaluation andpost-mortem histological analy-sis of five 23-week-old TRAMPmice

Summary of [18 F]FDG and[11 C]choline PET and immuno-histochemistry observation offive TRAMP mice (g–k) ana-lysed at 23 weeks of age


0.36 (p=0.0006). No correlation between tumour size and[18F]FDG or [11C]choline uptake was observed.

Discussion

In the present study, [18F]FDG and [11C]choline PETimaging have been used to validate the TRAMP model ofPC and to set up a procedure able to non-invasivelymonitor illness appearance and progression in TRAMPmice.

Overall, our results indicate that both [18F]FDG and[11C]choline are useful tracers to visualize and quantifylesions in mouse models of subcutaneous and orthotopicPC. In both TRAMP-C1 and TRAMP models, differentlyfrom what is generally observed in clinical practice or inhuman cell lines, PC tumours were better visualized by[18F]FDG than [11C]choline. The highest uptake observed

using [18F]FDG cannot be explained by an artificialincrease in radioactivity concentration caused by a spillovereffect deriving from the urinary bladder. In fact, lowervalues of [11C]choline uptake in comparison with [18F]FDGwere also observed in vitro during cell line experiments andin vivo in the TRAMP-C1 model. However, the presence ofthe urinary bladder near to the lesions may reduce thesensitivity of [18F]FDG in detecting small PC lesionspresent at early times.

Considering the capability of the two PET tracers in thedetection of PC, these results seem to be at odds with datacollected in patients where PET with [18F]FDG has a lowsensitivity in the detection of primary lesions in themajority of PC patients [14]. However, it has been reportedthat [18F]FDG uptake in PC is significantly increased intumours of high histological grade or advanced clinicalstage with metastases [38]. In addition, according to someauthors, patients with a high [18F]FDG uptake in prostatecancer presented a significantly worse prognosis than thosewith a low [18F]FDG uptake, suggesting a prognostic valueof [18F]FDG PET [38, 39].

The use of two different PET tracers evaluating differentmetabolic pathways is particularly useful to better under-stand the behaviour of PC which is a heterogeneous tumourcharacterized by multifocal distribution of variously differ-entiated cancer cells. Indeed, the Gleason system, which isthe most common grading system in use to stratify patientsby describing the PC cells’ differentiation, is commonlyreported as the combination of the two most commonneoplastic patterns within the tumour. Tumour lesions,whose structure is well differentiated, present a biologicalbehaviour less aggressive than those characterized bypoorly differentiated cells. In particular, it has been reportedthat a multifocal neuroendocrine feature may occur in PCbeing characterized by high aggressiveness and resistance

[11

C]Choline [18

F]FDG [11

C]Choline

a b

[18

F]FDG

Fig. 6 YAP-(S)PET II images of two 23-week-old TRAMP mice(a, b) acquired after the i.v. injection of 4.3 and 4.5 MBq of [18F]FDG,respectively (left part of each panel), and of 5.6 and 4.9 MBq of [11C]choline, respectively (right part of each panel). The area of tumour

growth is indicated by a dashed circle. Corresponding values of PETimage quantification and IHC observations of the two animals arepresented in Table 2 (animals g and h, respectively)

0

1

2

3

4

5

6

7

8

Adeno

NE

Tu

mo

r to

mu

sc

le r

ati

o

**

*

[11C]Choline [18F]FDG

Fig. 7 Average of tumour to muscle radioactivity concentration ratiosof [11C]choline and [18F]FDG uptake calculated in TRAMP micecarrying a neuroendocrine (NE) or an adenocarcinoma (Adeno) lesion(n=3 and 7, respectively). Statistical analysis was performed usingStudent’s t test for unpaired data. *p<0.01; **p<0.001


to therapy [26–28]. The heterogeneous behaviour of PCresults also from preclinical studies on xenograft mice. Infact, if [18F]FDG may be efficiently used for the evaluationof CWR22 or PC-3 cells mouse models, this is not the casefor LNCaP xenograft mice, where the lesions are bettervisualize using [11C]choline or its derivatives [40, 41].

In TRAMP mice, [18F]FDG and [11C]choline uptakedisplayed a bimodal behaviour, being in both casessignificantly higher in mice classified as carrying a neuroen-docrine PC. The lower signal to noise ratios observed inmice with adenocarcinoma, particularly in the case of [11C]choline, might be of help in differentiating adenocarcinomafrom neuroendocrine lesions. Hence, PET imaging is aversatile non-invasive tool that allows not only the longitu-dinal analysis of single TRAMP mice, thus substitutingabdominal palpation, but also the differentiation betweenadenocarcinoma and neuroendocrine prostate lesions. Thelatter is particularly relevant for preclinical studies, consid-ering the relatively high frequency and aggressiveness ofneuroendocrine tumours in TRAMP mice [42].

Indeed, whilst primary neuroendocrine tumours arerather uncommon in human prostates [43], in TRAMPmice, foci of neuroendocrine-like cells may be present atearly time points, but they progress to rapidly expandingtumours later in the life of the mice. In our experience, largeneuroendocrine tumours in C57BL/6 TRAMP mice are rarebefore week 20 and account for approximately 20–25% ofthe tumours arising in mice older than 24 weeks (ourunpublished observations).

The metabolic behaviour observed in neuroendocrinelesions is in agreement with the more aggressive phenotypeof these cells [25]. Our findings are of potential interest alsofor the interpretation of the results obtained in clinicalpractice. In humans an extensive and multifocal neuroen-docrine feature occurs in less than 10% of all prostaticmalignancies. However, a heterogeneous number of neuro-endocrine cells is frequently present in PC lesions [44, 45].Although the specific role that neuroendocrine cells play inthe prostate and their relationship to PC pathogenesis is stilluncertain, it has been suggested that they are involved inthe regulation of tumour growth [46]. Because of the lackof information on the association between neuroendocrinedifferentiation and [18F]FDG/[11C]choline uptake inhumans, a comparative analysis between human andexperimental PC cannot be conducted at this time.

None the less, our results suggest that the presence ofneuroendocrine cells could influence [11C]choline uptakeand participate in the heterogeneous behaviour of this tracerin primary lesions [46, 47]. Consequently, the potential useof [11C]choline for the identification of neuroendocrine fociwithin PC lesions deserves further studies.

The higher accumulation of [18F]FDG than [11C]cholinein tumour-bearing TRAMP mice suggests that this model

cannot represent the totality of PC patients, but only aspecific subgroup of those carrying lesions characterized byan increased metabolic activity in the glycolytic pathway.

Finally, we did not detect distant metastases in TRAMPmice both by PETand post-mortem dissection. In the TRAMPmodel, distant metastases are reported to appear in a highpercentage of animals between 24 and 30 weeks [48, 18].Hence, this specific issue will be verified in a dedicatedstudy performed on older mice.

In this study we have developed a procedure able tomonitor in vivo the progression of PC in TRAMP mice and todifferentiate a neuroendocrine lesion from adenocarcinoma.PET molecular imaging is playing an increasingly significantrole in clinical oncology being able to provide translationalinformation by validating a non-invasive diagnostic tool in apreclinical model to be transferred to patients as well astransferring clinical evidence to preclinical models to betterunderstand the biological behaviour of diseases. The tightconnection between preclinical and clinical PET molecularstudies might be particularly useful in the development of newimaging modalities mainly focused on proper patient selectionand monitoring.

In addition, although a larger number of experiments areneeded to further support this thesis, our results suggest thatTRAMP PC model presents some differences with respectto human pathology.

Conclusion

In conclusion, results of the present study show that: (a)PET with [18F]FDG, in contrast to PET with [11C]choline,is a reliable tool for detection and monitoring prostatelesions in TRAMP mice and (b) the combined use of both[18F]FDG and [11C]choline allows one to distinguishbetween neuroendocrine and adenocarcinoma lesions.

Further studies should be performed in TRAMP mice toevaluate whether [18F]FDG and [11C]choline PET tracersmay allow detection of distant metastases.

Acknowledgments We thank Pasquale Simonelli for technicalassistance in animal preparation and imaging experiments andDr. Maria Grazia Minotti for radiochemical production and qualitycontrols. Supported by grants from: the Italian Ministero della Saluteand Ministero dell’Università e della Ricerca and by EMIL (EuropeanMolecular Imaging Laboratory), Sixth European Program, Project No:LSHC-CT-2004-503569.

References

1. Gittes RF. Carcinoma of the prostate. N Engl J Med 1991;324(4):236–45.

2. Margolis DJ, Hoffman JM, Herfkens RJ, Jeffrey RB, Quon A,Gambhir SS. Molecular imaging techniques in body imaging.Radiology 2007;245(2):333–56.


3. Lawrentschuk N, Davis ID, Bolton DM, Scott AM. Positronemission tomography and molecular imaging of the prostate: anupdate. BJU Int 2006;97(5):923–31.

4. Ramirez de Molina A, Sarmentero-Estrada J, Belda-Iniesta C,Taron M, Ramirez de Molina V, Cejas P, et al. Expression ofcholine kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study. LancetOncol 2007;8(10):889–97.

5. de Jong IJ, Pruim J, Elsinga PH, Vaalburg W, Mensink HJ. 11C-choline positron emission tomography for the evaluation aftertreatment of localized prostate cancer. Eur Urol 2003;44(1):32–8.discussion 8–9.

6. Picchio M, Messa C, Landoni C, Gianolli L, Sironi S, Brioschi M,et al. Value of [11C]choline-positron emission tomography for re-staging prostate cancer: a comparison with [18F]fluorodeoxyglu-cose-positron emission tomography. J Urol 2003;169(4):1337–40.

7. Yoshida S, Nakagomi K, Goto S, Futatsubashi M, Torizuka T. 11C-choline positron emission tomography in prostate cancer: primarystaging and recurrent site staging. Urol Int 2005;74(3):214–20.

8. Schmid DT, John H, Zweifel R, Cservenyak T, Westera G,Goerres GW, et al. Fluorocholine PET/CT in patients with prostatecancer: initial experience. Radiology 2005;235(2):623–8.

9. Heinisch M, Dirisamer A, Loidl W, Stoiber F, Gruy B, Haim S,et al. Positron emission tomography/computed tomography withF-18-fluorocholine for restaging of prostate cancer patients:meaningful at PSA <5 ng/ml? Mol Imaging Biol 2006;8(1):43–8.

10. Scattoni V, Picchio M, Suardi N, Messa C, Freschi M, RoscignoM, et al. Detection of lymph-node metastases with integrated[11C]choline PET/CT in patients with PSA failure after radicalretropubic prostatectomy: results confirmed by open pelvic-retroperitoneal lymphadenectomy. Eur Urol 2007;52(2):423–9.

11. Husarik DB, Miralbell R, Dubs M, John H, Giger OT, Gelet A, etal. Evaluation of [(18)F]-choline PET/CT for staging and restag-ing of prostate cancer. Eur J Nucl Med Mol Imaging 2008;35(2):253–63.

12. Krause BJ, Souvatzoglou M, Tuncel M, Herrmann K, Buck AK,Praus C, et al. The detection rate of [(11)C]Choline-PET/CTdepends on the serum PSA-value in patients with biochemicalrecurrence of prostate cancer. Eur J Nucl Med Mol Imaging2008;35(1):18–23.

13. Schoder H, Larson SM. Positron emission tomography for prostate,bladder, and renal cancer. Semin Nucl Med 2004;34(4):274–92.

14. Hara T. 11C-choline and 2-deoxy-2-[18F]fluoro-D-glucose intumor imaging with positron emission tomography. Mol ImagingBiol 2002;4(4):267–73.

15. Abate-Shen C, Shen MM. Mouse models of prostate carcinogenesis.Trends Genet 2002;18(5):S1–S5.

16. Chiang CF, Son EL, Wu GJ. Oral treatment of the TRAMP micewith doxazosin suppresses prostate tumor growth and metastasis.Prostate 2005;64(4):408–18.

17. Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD,Aspinall JO, et al. Prostate cancer in a transgenic mouse. ProcNatl Acad Sci U S A 1995;92(8):3439–43.

18. Kaplan-Lefko PJ, Chen TM, Ittmann MM, Barrios RJ, Ayala GE,Huss WJ, et al. Pathobiology of autochthonous prostate cancer in apre-clinical transgenic mouse model. Prostate 2003;55(3):219–37.

19. Han G, Foster BA, Mistry S, Buchanan G, Harris JM, Tilley WD,et al. Hormone status selects for spontaneous somatic androgenreceptor variants that demonstrate specific ligand and cofactordependent activities in autochthonous prostate cancer. J BiolChem 2001;276(14):11204–13.

20. Huss WJ, Hanrahan CF, Barrios RJ, Simons JW, Greenberg NM.Angiogenesis and prostate cancer: identification of a molecularprogression switch. Cancer Res 2001;61(6):2736–43.

21. Narayanan BA, Narayanan NK, Pittman B, Reddy BS. Regressionof mouse prostatic intraepithelial neoplasia by nonsteroidal anti-

inflammatory drugs in the transgenic adenocarcinoma mouseprostate model. Clin Cancer Res 2004;10(22):7727–37.

22. Kaplan PJ, Mohan S, Cohen P, Foster BA, Greenberg NM. Theinsulin-like growth factor axis and prostate cancer: lessons fromthe transgenic adenocarcinoma of mouse prostate (TRAMP)model. Cancer Res 1999;59(9):2203–9.

23. Shappell SB, Thomas GV, Roberts RL, Herbert R, Ittmann MM,Rubin MA, et al. Prostate pathology of genetically engineered mice:definitions and classification. The consensus report from the BarHarbor meeting of the Mouse Models of Human Cancer ConsortiumProstate Pathology Committee. Cancer Res 2004;64(6):2270–305.

24. Foster BA, Gingrich JR, Kwon ED, Madias C, Greenberg NM.Characterization of prostatic epithelial cell lines derived fromtransgenic adenocarcinoma of the mouse prostate (TRAMP)model. Cancer Res 1997;57(16):3325–30.

25. Huss WJ, Gray DR, Tavakoli K, Marmillion ME, Durham LE,Johnson MA, et al. Origin of androgen-insensitive poorlydifferentiated tumors in the transgenic adenocarcinoma of mouseprostate model. Neoplasia 2007;9(11):938–50.

26. Vashchenko N, Abrahamsson PA. Neuroendocrine differentiationin prostate cancer: implications for new treatment modalities. EurUrol 2005;47(2):147–55.

27. Hansson J, Abrahamsson PA. Neuroendocrine differentiation inprostatic carcinoma. Scand J Urol Nephrol Suppl. 2003;(212):28–36.

28. Ather MH, Abbas F. Prognostic significance of neuroendocrinedifferentiation in prostate cancer. Eur Urol 2000;38(5):535–42.

29. Degrassi A, Russo M, Scanziani E, Giusti A, Ceruti R, Texido G,et al. Magnetic resonance imaging and histopathological charac-terization of prostate tumors in TRAMP mice as model for pre-clinical trials. Prostate 2007;67(4):396–404.

30. Degl’Innocenti E, Grioni M, Boni A, Camporeale A, BertilaccioMT, Freschi M, et al. Peripheral T cell tolerance occurs early duringspontaneous prostate cancer development and can be rescued bydendritic cell immunization. Eur J Immunol 2005;35(1):66–75.

31. Hara T, Kosaka N, Kishi H. PET imaging of prostate cancer usingcarbon-11-choline. J Nucl Med 1998;39(6):990–5.

32. Smith TA, Sharma RI, Thompson AM, Paulin FE. Tumor 18F-FDG incorporation is enhanced by attenuation of P53 function inbreast cancer cells in vitro. J Nucl Med 2006;47(9):1525–30.

33. Davoodpour P, Bergström M, Landström M. Effects of 2-methoxyestradiol on proliferation, apoptosis and PET-traceruptake in human prostate cancer cell aggregates. Nucl Med Biol2004;31(7):867–74.

34. Hara T, Bansal A, DeGrado TR. Effect of hypoxia on the uptakeof [methyl-3H]choline, [1-14C] acetate and [18F]FDG in culturedprostate cancer cells. Nucl Med Biol 2006;33(8):977–84.

35. Del Guerra A, Belcari N. Advances in animal PET scanners. Q JNucl Med 2002;46(1):35–47.

36. Motta A, Damiani C, Del Guerra A, Di Domenico G, Zavattini G.Use of a fast EM algorithm for 3D image reconstruction with theYAP-PET tomograph. Comput Med Imaging Graph 2002;26(5):293–302.

37. Bertilaccio MT, Grioni M, Sutherland BW, Degl’Innocenti E,Freschi M, Jachetti E, et al. Vasculature-targeted tumor necrosisfactor-alpha increases the therapeutic index of doxorubicin againstprostate cancer. Prostate 2008;68(10):1105–15.

38. Oyama N, Akino H, Suzuki Y, Kanamaru H, Sadato N, YonekuraY, et al. The increased accumulation of [18F]fluorodeoxyglucosein untreated prostate cancer. Jpn J Clin Oncol 1999;29(12):623–9.

39. Oyama N, Akino H, Suzuki Y, Kanamaru H, Miwa Y, Tsuka H, etal. Prognostic value of 2-deoxy-2-[F-18]fluoro-D-glucose positronemission tomography imaging for patients with prostate cancer.Mol Imaging Biol 2002;4(1):99–104.

40. Zhang Y, Saylor M, Wen S, Silva MD, Rolfe M, Bolen J, et al.Longitudinally quantitative 2-deoxy-2-[18F]fluoro-D-glucose mi-cro positron emission tomography imaging for efficacy of new


anticancer drugs: a case study with bortezomib in prostate cancermurine model. Mol Imaging Biol 2006;8(5):300–8.

41. Price DT, Coleman RE, Liao RP, Robertson CN, Polascik TJ,DeGrado TR. Comparison of [18F]fluorocholine and [18 F]fluorodeoxyglucose for positron emission tomography of andro-gen dependent and androgen independent prostate cancer. J Urol2002;168(1):273–80.

42. Evangelou AI, Winter SF, Huss WJ, Bok RA, Greenberg NM.Steroid hormones, polypeptide growth factors, hormone refractoryprostate cancer, and the neuroendocrine phenotype. J CellBiochem 2004;91(4):671–83.

43. Freschi M, Colombo R, Naspro R, Rigatti P. Primary and pureneuroendocrine tumor of the prostate. Eur Urol 2004;45(2):166–9.discussion 9–70.

44. Sciarra A, Cardi A, Dattilo C,Mariotti G, DiMonaco F, Di Silverio F.New perspective in the management of neuroendocrine differentia-tion in prostate adenocarcinoma. Int J Clin Pract 2006;60(4):462–70.

45. Shimizu S, Kumagai J, Eishi Y, Uehara T, Kawakami S, TakizawaT, et al. Frequency and number of neuroendocrine tumor cells inprostate cancer: no difference between radical prostatectomyspecimens from patients with and without neoadjuvant hormonaltherapy. Prostate 2007;67(6):645–52.

46. Giovacchini G, Picchio M, Coradeschi E, Scattoni V, Bettinardi V,Cozzarini C, et al. [(11)C]Choline uptake with PET/CT for theinitial diagnosis of prostate cancer: relation to PSA levels, tumourstage and anti-androgenic therapy. Eur J Nucl Med Mol Imaging2008;35(6):1065–73.

47. Farsad M, Schiavina R, Castellucci P, Nanni C, Corti B,Martorana G, et al. Detection and localization of prostate cancer:correlation of (11)C-choline PET/CT with histopathologic step-section analysis. J Nucl Med 2005;46(10):1642–9.

48. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ,Finegold MJ, et al. Metastatic prostate cancer in a transgenicmouse. Cancer Res 1996;56(18):4096–102.


Characterization of preclinical models of prostate cancer using PET-based molecular imaging

Documents

Transcript of Characterization of preclinical models of prostate cancer using PET-based molecular imaging