studies in living animals are critical to oncology research , and many experimental models have been exploited for drug development and basic studies [ 1 , 2 ] .
fast - growing tumors can be generated in mice by orthotopic or ectopic implantation of tumor cell lines .
however , models exhibiting spontaneous oncogenesis better mimic human disease therefore , oncogene - driven or chemically induced tumor models have come into use more recently [ 3 , 4 ] . in both spontaneous and transplanted tumor models ,
the most common readouts are primary tumor growth and metastatic spread , but accurate measurement of these parameters is challenging . unlike necropsy , noninvasive imaging techniques could offer an ideal solution as they allow measurement of tumor burden in the whole body without the need to sacrifice the animal .
this makes longitudinal studies possible , simultaneously reducing the number of animals required and producing more robust data .
these technologies are also sensitive and accurate enough to detect microscopic nodules , whose importance in human disease prognosis is increasingly recognized [ 5 , 6 ] .   several imaging techniques have recently become available for small animals .
these include 2-deoxy-2-[18f]fluoro - d - glucose positron emission tomography ( fdg - pet ) , t2-weighted magnetic resonance imaging ( t2w - mri ) , and optical imaging , encompassing bioluminescence imaging ( bli )   and fluorescence imaging ( fli ) .
both fdg - pet and t2w - mri are used clinically in humans , whereas optical imaging is specifically used for research and preclinical studies .
while each method has its own advantages , a detailed side - by - side comparison of their use for tumor imaging has yet to be carried out .
the purpose of the present study is to compare practicality and performance of these four imaging techniques in the context of mouse tumor studies . specifically , we assessed four different parameters , namely , practicality , performance for small tumor detection , performance for tumor burden measurement , and performance for tumor identification .
the performance for tumor burden measurement was conducted specifically for optical methods , since they are well adapted for this purpose .
conversely , the performance for tumor identification was compared only between mri and fdg - pet , since optical methods can not be applied for this purpose in current tumor models .
the b16 transplanted tumor model is well - defined and offers a high level of flexibility .
b16 cells can be modified to express the transgenes required for detection by optical imaging , followed by injection of these cells into the animal by different routes to produce either subcutaneous or pulmonary lesions .
tumor onset is predictable , so nodules can be tracked from their microscopic stage , making this model ideal to assess the practicality and performance of each technique for small tumor detection and tumor burden measurement .
in contrast to transplanted tumor models , retaad tumors may arise at any location in the skin ( cutaneous melanoma tumors ) and internal organs ( visceral metastases ) .
this model is , therefore , particularly suited for the assessment of the performance of imaging techniques in tumor identification , as it makes it possible to calculate the specificity , the sensitivity , and the positive predictive value for tumor detection .
tumors in spontaneous models usually do not express reporter genes and are , therefore , not suited for optical imaging technologies .
therefore , we have used this model to compare fdg - pet and t2w - mri .
b16-f10-luc cells ( xenogen , alameda , calif , usa ) express firefly luciferase ( sequence from pgl3 , promega ) under the control of the sv40 promoter .
b16-f10-rfp cells express dsred2 under the control of the cmv promoter [ 11 , 12 ] .
all studies were approved by the institutional animal care and use committee of the biological resource center and of singhealth .
two perpendicular diameters ( d1  d2 ) of the tumor were then measured using caliper , and were used to calculate the tumor volume ( v ) ( v = 4/3    d1  d2/8 ) . prior to necropsy , the investigator was unaware of the results of the imaging scans , rendering the two analyses independent .
animals injected intravenously with b16 cells were examined for their lungs and peritoneal cavity as described .
retaad mice develop tumors spontaneously with widespread metastases , so they were subjected to more extensive necropsy . for each mouse
, a necropsy diagram was filled to document the location , size , and morphology of nodules .
four mice subcutaneously injected with b16 cells were used to determine the smallest detectable tumor .
ten retaad mice were used to assess performance in tumor identification .   after fasting overnight ,
mice were prewarmed to 37c , and approximately 5.5  mbq of fdg ( 0.6  mm ) ( department of nuclear medicine , singapore general hospital ) was administered intraperitoneally .
micro - pet imaging was performed using a r4 micropet scanner ( concordes microsystems inc . ) with a ring diameter of 26  cm , 7.8  cm axial field of view and an average intrinsic spatial resolution of 1.75  mm . under isoflurane anesthesia
, mice were subjected to 15 minutes of acquisition . for image reconstruction , an energy window of 350700  kev and a coincidence timing window of 6ns were used .
two - dimensional histograms by fourier rebinning and image reconstruction by filtered backprojection were used .
the image data were corrected for nonuniformity of the scanner response , dead time count losses , and physical decay to the time of injection .
no correction was applied for attenuation , scatter , or partial - volume averaging , as these parameters are not critical for mouse models . in the reconstructed images ,
tumors were identified as regions of high uptake in study animals that were absent from images of control mice . to allow quantitative image analysis ,
regions of interest ( roi ) were manually drawn over areas of high uptake . within these regions ,
counting rates were converted to standardized uptake values ( suvs ) using a system calibration factor derived from the imaging of a mouse - size water - equivalent phantom containing f. four mice subcutaneously injected with b16 cells were used to determine the smallest detectable tumor .
data were acquired at the singapore bioimaging consortium on a 9.4 t mri scanner ( varian , palo alto , calif , usa ) using a transmit - receive volume rf coil .
a multislice 2d fast spin echo with periodically rotated parallel lines with enhanced reconstruction ( propeller ) pulse sequence   was used to give high image quality and robustness to motion .
brain scans were conducted using the following parameters : repetition time ( tr ) = 4000  ms ; effective echo time ( te ) = 51  ms ; echo spacing ( esp ) = 6.4  ms ; echo train length ( etl ) = 16 ; field - of - view = 25.6  25.6  mm ; blade matrix = 256  16 ; number of blades = 32 ; reconstructed matrix = 256  256 ; slice thickness = 1  mm ; slice gap = 0.5  mm ; slices = 5 ; averages = 1 ; orientation = axial ; readout bandwidth = 208  khz and acquisition time = 2  min 16  s. for the abdomen , several changes were made to accommodate the shorter t2 so that , te = 20  ms ; esp = 5.0  ms ; etl = 8 ; blade matrix = 128  8 ; reconstructed matrix = 128    128 ; slice gap = 0.2  mm . the resulting data were used to reconstruct images as described .
tumors were identified as highly contrasted masses or nodules that were present in study animals but absent from control mice . for all bli experiments ,
studies to determine the smallest detectable tumors , accuracy for tumor burden measurement , and tissue attenuation used four , four , and six mice , respectively , each subcutaneously injected with b16 cells . to further demonstrate the possibility to use bli for accurate followup of tumor growth ( as shown in figure 5 ) , 4 unshaved mice were injected subcutaneously with b16 cells and another 4 were injected intravenously . for other experiments ,
1525 minutes before imaging , mice were injected intraperitoneally with 200  l of d - luciferin ( 15  mg / ml in pbs ) as described   and then anesthetized using isoflurane . for in vitro imaging , cells were plated in pbs in flat - bottomed 96 well plates before d - luciferin was added to a final concentration of 1.5  mg / ml . immediately after necropsy , some tumors were excised and imaged ex vivo on tissue culture plates .
these plates were scanned for 5 to 40 seconds , whereas mice were scanned for 30 to 60 seconds using the ivis spectrum photon - counting device optical imaging system ( xenogen , alameda , calif , usa ) .
bioluminescence signal was reported as total light emission within the region of interest ( photon / s ) .
specific signal was calculated as the ratio of bioluminescent signal in the region of interest to the bioluminescent signal in a background region containing no cells or tumors .
a signal was defined as positive when it was greater than the sum of the mean background signal plus 2 standard deviations of the background signal . for all fli experiments ,
studies to determine the smallest detectable tumors , accuracy for tumor burden measurement , and tissue attenuation used four , four , and six mice , respectively , each subcutaneously injected with b16 cells .   for in vivo imaging
, animals were anesthetized using isoflurane and some mice were shaved as indicated in the figure legends . for in vitro imaging , cells were plated in pbs in flat - bottomed 96-well plates . immediately after in vivo imaging
, some tumors were excised at necropsy and imaged ex vivo on tissue culture plates .
plates or mice were scanned for 0.1 to 1 seconds using the ivis spectrum photon - counting device optical imaging system ( xenogen , alameda , ca ) with filters for red fluorescence ( excitation 535  nm , emission 600  nm ) and background fluorescence ( excitation 465  nm , emission 600  nm ) . regions of interest were drawn and quantified using living image software version 2.5 .
specific signal was reported as the ratio of the fluorescence signal in the region of interest to the fluorescence signal in a background region containing no cells or tumors .
a signal was defined as positive when it was greater than the sum of the mean background signal plus 2 standard deviations of the background signal .
bli and fli specific signals ( signal - to - noise ratio ) for single time point experiments were compared using the mann - whitney test .  
the specificity was defined as ( number of sites where no tumor was found)/(total number of sites without tumor confirmed at necropsy ) , where the total number of possible sites for tumor growth in each retaad mouse was 14 ( cheeks , neck , genitals , flanks , forelimbs , hind limbs , and peritoneum , each time on the left or right side ) .
the positive predictive value was defined as ( number of tumors detected by imaging and confirmed at necropsy)/(total number of tumors detected by imaging ) .
the sensitivity for tumor identification was defined as follows :   ( number of tumors detected by imaging and confirmed at necropsy)/(total number of tumors observed at necropsy ) .
three parameters were taken into account : animal preparation , time for analysis , and ease of access to the technology ( table 1 ) . all techniques required anesthesia of the animal by isoflurane inhalation , taking approximately 4 minutes per mouse .
the additional tracer injection and preincubation time for fdg - pet resulted in at least 3-fold longer preparation time per animal compared to the other techniques .
shaving requires 10 minutes per animal , and while there has been debate on whether it can be omitted for optical imaging , we found it to be dispensable for bli ( see below ) .
because devices differ , we selected widely used platforms for comparison : ivis spectrum ( xenogen ) for optical imaging , r4 micropet ( concordes microsystem ) for fdg - pet , and 9.4 t mri ( varian ) for t2w - mri .
the main differences in practicality between technologies were highlighted by the ease of scale - up to larger groups of animals .
for example , the ivis spectrum allows parallel imaging of up to 5 mice , taking as little as 2 minutes to complete all the scans .
in contrast , both fdg - pet and t2w - mri can typically image only one mouse at a time ( maximum 2 in some settings ) , taking at least 15 minutes per scan .
overall , this meant that optical scanning of 10 mice could be completed in 1 - 2 hours , whereas fdg - pet or t2w - mri would take 1330 hours .  
each technique has low reagent and consumable costs of around five us dollars per scan per animal .
fdg can usually be obtained as surplus material from nuclear medicine departments or comes at a low cost compared to other pet reagents .
equipment for optical imaging is accessible in many research institutes and costs less than five hundred thousand us dollars , with the pet scanner costing around six hundred thousand us dollars .
overall bli and fli are the most practical techniques and are particularly suitable for large studies requiring high throughput imaging
. the b16 melanoma model allowed us to assess the smallest tumors that could be accurately detected by the various techniques .
four mice per technique were injected subcutaneously with 10 b16 cells , resulting in tumor growth at the injection site .
these tumors were clinically undetectable up to day 10 after injection , but were evident at necropsy .
after day 10 , tumors become macroscopic ( 2  mm diameter and above ) and were measured in living animal with a caliper . bli and fdg - pet detected nonpalpable tumors ( < 1  mm ) , whereas the smallest tumors detected by t2w - mri and fli were 1  mm and 2  mm diameter , respectively ( figure 1 ) .
bli detected microscopic tumors as early as 1 day after subcutaneous injection ( figure 1(c ) ) when the nodules were too small to be detected even at necropsy . while this means that their presence could not be confirmed either visually or histologically , these tumors were actively growing , increasing their bli signal and
overall , bli and fdg - pet are applicable for in vivo detection of microscopic tumors , whereas t2w - mri and fli are only applicable to palpable tumors . both optical imaging techniques can be used to follow transplanted tumor growth . to carry out a detailed comparison , we selected two b16-luc and b16-rfp clones showing equivalent performance for in vitro imaging ( figure 2(a ) ) with similar growth rates in vitro ( not shown ) and in vivo ( figure 2(b ) ) . at day 1 , 2 , 3 and 5 after injection , only bli detected nonpalpable tumors ( figure 2(c ) ) . at further time points ,
the tumor - specific signal detected by bli was significantly greater than that seen by fli for tumors of equivalent size ( figure 2(c ) ) .
both techniques may , therefore , be used to follow small macroscopic tumors , but only bli provides data at the microscopic stage .
we investigated the higher sensitivity observed for bli compared to fli when performed in vivo .
attenuation occurs when tissues around the tumor absorb some of the imaging excitation and emission signal , autofluoresce , leading to a reduction in the signal to noise ratio . to test this hypothesis
, we measured signal reduction by comparing ex vivo ( figure 3(a ) ) and in vivo ( figure 3(b ) ) signals after tumor excision . the signal measured in vivo on shaved mice
was reduced 3-fold for bli and 14-fold for fli compared to the signal of tumors ex vivo after excision ( figure 3(b ) ) , confirming the hypothesis that fli is more prone to tissue attenuation . to address the contribution of mouse hair to further signal attenuation
again , the fli signal ( over than 400 fold reduced ) was more prone to attenuation than the bli signal ( 70 fold reduction ) ( figure 3(c ) ) . in summary ,
tissue and hair surrounding the tumor significantly reduced the ability of fli to detect small tumors in vivo .
we compared tumor volumes estimated in vivo by bli and fli , to those calculated by caliper measurements and found a good correlation between these two techniques ( figure 4 ) .
bli has demonstrated an ability to detect microscopic tumors and to estimate tumor volumes in vivo with good accuracy . to further explore the power of the technique we injected unshaved mice either subcutaneously or intravenously with b16-luc cells and imaged them repeatedly .
as expected , bli detected subcutaneous tumors earlier than clinical examination ( figure 5(a ) ) .
moreover , the bioluminescent signal follows a characteristic gompertzian curve as expected for tumor growth , therefore more accurately reflecting the biology of the tumor compared to caliper measurements .
bli detected a signal following intravenous injection of b16-luc only after a few minutes , which likely reflects the initial trapping of the injected cells in the lung . by day 4
most of these cells were cleared and the bli signal dropped , only to increase again as tumor growth occurred in the lungs , peritoneal cavity and at the point of injection ( figure 5(b ) ) .
bli can , therefore , be used in shaved or unshaved mice for quantitative followup of tumor growth at both cutaneous and internal sites .
spontaneous or carcinogen - induced tumor models are increasingly used for cancer research . in these animals ,
a variable number of tumors arise in a range of locations over a less predictable time course .
the need to sacrifice the animal for information is a disadvantage of such models , but to replace necropsy by in vivo imaging , three criteria must be met .
firstly , the technique must correctly predict the absence of tumors at normal sites and for nontumor bearing mice ( specificity ) .
secondly , the technique must identify tumors accurately , with a low rate of false positives ( high predictive value ) .
thirdly , the technique must be sufficiently sensitive to detect all the tumors that necropsy currently does .
we compared the specificity , predictive value and sensitivity of fdg - pet and t2w - mri in the retaad spontaneous melanoma model . during the course of disease ,
these mice develop tumors of various sizes in wide - ranging anatomical locations , making the model an ideal test for the performance of these techniques .  
some background is evident in the bladder , heart , and eye regions , but this was expected due to the excretion and circulation of the probe , and the presence of the harderian glands . the same effect was seen in control mice , and these regions were accordingly excluded from analysis .
a total of 10 retaad mice and 4 control mice were independently analyzed by fdg - pet and necropsy .
of the 28 tumors identified by fdg - pet , 24 were confirmed by necropsy or histology , making the positive predictive value of the fdg - pet 86% .
the 4 tumors that were not confirmed at necropsy were embedded in the muscles of the back and the limbs , sites for which histological analysis could not be carried out .
an additional ten tumors were found at necropsy but not by fdg - pet , most likely because of low metabolic activity .
only 4 tumors were predicted by fdg - pet but could not be confirmed at necropsy or histology .
taking into account 14 possible sites for tumor growth for each of 14 mice analyzed , the specificity of fdg - pet was 98% . 

figure 7 shows a typical t2w - mri scan of a tumor - bearing retaad mouse .
a total of 6 retaad mice and 4 control mice were independently analyzed by t2w - mri and necropsy .
twenty - two tumors were identified by t2w - mri , of which 21 were confirmed at necropsy , making the predictive value of t2w - mri 95% .
five small tumors ( 4 out of 5 were < 1  mm diameter ) were observed at necropsy but not detected by imaging .
taking into account 14 possible sites for tumor growth for each of 10 mice analyzed , the specificity of t2w - mri was 99% .
overall , both fdg - pet and t2w - mri allow precise 3d visualization of tumors with good specificity , sensitivity , and accuracy and are , therefore , highly recommended for any study aiming at identifying tumors .
interestingly , fdg - pet shows slightly lower sensitivity for tumor detection than mri , probably due to the fact that some tumors lack the minimal metabolic activity required for detection .
therefore , fdg - pet would be the preferred choice if the assessment of metabolic activity is desired ; otherwise , mri is recommended ( table 2 ) .
to compare bli and fli in vivo , we used tumors originating from two different b16 cell lines expressing firefly luciferase and dsred2 , respectively .
the two prototypical reporter genes have been chosen among the most commonly used and most efficient markers at the time of writing .
results obtained might change in the future when new reporters are developed . with current reporter genes , both bli and
fli detected their respective cell lines equally well in vitro , but when the cells were injected into mice and allowed to form tumors , only bli was able to image microscopic nodules .
we showed that this difference was due to the tissues surrounding the tumor during in vivo imaging , a phenomenon known as tissue attenuation .
this is especially relevant for fli as the tissue can absorb and scatter fluorescent light at both the excitation and emission level . for bli
these experiments used subcutaneous tumors , but for internal tumors the differences between the techniques could only be expected to be magnified due to the increased optical path through the tissues .   despite fli being less sensitive than bli with the instrumentation we used
, it has been used successfully for whole - body imaging in other studies , even with very low cell numbers .
for example , labeled proteins have enabled fluorescent imaging of tumor cell mobility , invasion and angiogenesis ( reviewed in ) .
hirakawa et al . successfully used fli to monitor the dissemination of very small numbers of gfp - labeled skin tumor cells to the proximal lymph nodes of mice in vivo .
importantly , fli is so far the only imaging technology to give single - cell resolution   or even subcellular resolution in vivo [ 23 , 24 ] .  
while each technique tested is state of the art , imaging technologies are constantly being improved .
for example , optical techniques are being modified to permit three - dimensional reconstruction of the bioluminescent source and tumor localization .
it is even becoming possible to combine imaging modalities using multiple fusion reporter genes within the same animal ( see , e.g. , ) . 

table 3 summarizes the main features of the imaging methods , with their primary advantages and disadvantages .   estimating the real cost of the different technologies is difficult , and largely depends on equipment availability .
however , on the basis of equipment costs , operating expenses , and the level of training required , optical imaging is normally less costly than t2w - mri and fdg - pet .  
while the design of the current study did not involve the comparison of all techniques using a single tumor - bearing animal , the b16 model is reproducible enough to carry out a fair comparison . by using groups of at least 4 mice to perform statistical analyses
, we were able to detect some major differences between the various imaging modalities investigated .
further studies that include larger number of animals could be performed in order to detect even more subtle differences between these various imaging techniques .
in fact , doing so requires generation of transgenic mice expressing the reporter gene in the cell lineage of interest , followed by either carcinogen treatment or intercrossing with an oncogene driven transgenic mouse line .
for example , vooijs et al . expressed luciferase under a pituitary gland - specific promoter in a model of spontaneous pituitary cancer .
similarly , lyons et al . constructed an oncogen - driven prostate cancer model with luciferase expression in the prostate .
consistent with our findings , both authors successfully monitored tumor growth in vivo using bioluminescence .
therefore , optical imaging is a valid strategy , but it is time consuming for spontaneous tumor models .
in contrast to optical imaging techniques , t2w - mri and fdg - pet can be applied to any tumor - bearing mice , including spontaneous tumor models . using a b16 mouse tumor model
, we showed that t2w - mri and fdg - pet scans allow early detection of tumors and exhibit good sensitivity and positive predictive value when compared to necropsy .
results obtained with pet scanning are related to tumor metabolism and glucose uptake by the tumors ; hence , they could vary from one tumor cell line to the other .
however , published data have shown that pet sensitivity is high in other tumor models ( see , e.g .
t2w - mri performed slightly better , presumably because its basis is anatomical rather than requiring tumor metabolism , as in the case of fdg - pet .
indeed , in treated cancer patients , some responsive tumors simply lose their metabolic activity while the tumor mass is unchanged .
this typically translates into a tumor mass anatomically identified by mri or ct scan but fdg - pet negative .
in addition , mri and fdg - pet are less affected than optical imaging by attenuation due to the depth of the tumor , and both have the significant advantage of providing precise locations of even small nodules . as mri and fdg - pet are used in the clinical setting , their application in preclinical research may help translate basic findings into clinical studies . in this context , if whole - body metabolic imaging is required , fdg - pet is the best option for longitudinal followup of tumor burden and can be combined with ct which we did not address here .
t2w - mri is better used for specific body sections or to monitor the development of a particular tumor over time , as it may provide contrast and anatomical information related to location , volume , vascularization , and invasion .
for example , diffusion - weighted imaging was successfully used to detect glioma tumors in rats , and more recently used for whole - body imaging .
overall , this study has highlighted how various imaging techniques can be best used in different types of tumor models or to assess particular readouts .
bli in particular offers fast , sensitive whole - body tumor imaging , even detecting microscopic tumors .
bli could replace traditional caliper measurements , as it is able and well suited to determine tumor burden in longitudinal studies . however , the main disadvantage of optical techniques is the requirement for tumor cells to express a reporter gene .
this has so far largely limited the use of bli and fli to transplanted tumor models . however , with time and resources , spontaneous tumor models that also express reporter genes will become increasingly available .   in the meantime , however , this means that nonoptical methods are preferable for tumor detection in spontaneous models .
both available techniques have specific advantages and disadvantages ; t2w - mri accurately reflects tumor volume and morphology , but it is more time consuming , whereas fdg - pet uniquely measures metabolic activity . in summary , each technique represents a valuable tool to study tumor - bearing animals , but the careful selection of the most appropriate method will be critical to maximize the benefit of their use .
 
 introduction and purpose . monitoring solid tumor growth and metastasis in small animals is important for cancer research . 
 noninvasive techniques make longitudinal studies possible , require fewer animals , and have greater statistical power . 
 such techniques include fdg positron emission tomography ( fdg - pet ) , magnetic resonance imaging ( mri ) , and optical imaging , comprising bioluminescence imaging ( bli ) and fluorescence imaging ( fli ) . 
 this study compared the performance and usability of these methods in the context of mouse tumor studies . 
 methods . 
 b16 tumor - bearing mice ( n = 4 for each study ) were used to compare practicality , performance for small tumor detection and tumor burden measurement . using retaad mice , which develop spontaneous melanomas 
 , we examined the performance of mri ( n = 6 mice ) and fdg - pet ( n = 10 mice ) for tumor identification . 
 results . 
 overall , bli and fli were the most practical techniques tested . 
 both bli and fdg - pet identified small nonpalpable tumors , whereas mri and fli only detected macroscopic , clinically evident tumors . 
 fdg - pet and mri performed well in the identification of tumors in terms of specificity , sensitivity , and positive predictive value . 
 conclusion . 
 each of the four methods has different strengths that must be understood before selecting them for use . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion