Comparison of Folate Receptor Targeted Optical Contrast Agents for Intraoperative Molecular Imaging

Abstract

Background. Intraoperative imaging can identify cancer cells in order to improve resection; thus fluorescent contrast agents have emerged. Our objective was to do a preclinical comparison of two fluorescent dyes, EC17 and OTL38, which both target folate receptor but have different fluorochromes. Materials. HeLa and KB cells lines were used for in vitro and in vivo comparisons of EC17 and OTL38 brightness, sensitivity, pharmacokinetics, and biodistribution. In vivo experiments were then performed in mice. Results. The peak excitation and emission wavelengths of EC17 and OTL38 were 470/520 nm and 774/794 nm, respectively. In vitro, OTL38 required increased incubation time compared to EC17 for maximum fluorescence; however, peak signal-to-background ratio (SBR) was 1.4-fold higher compared to EC17 within 60 minutes (p < 0.001). Additionally, the SBR for detecting smaller quantity of cells was improved with OTL38. In vivo, the mean improvement in SBR of tumors visualized using OTL38 compared to EC17 was 3.3 fold (range 1.48-5.43). Neither dye caused noticeable toxicity in animal studies. Conclusions. In preclinical testing, OTL38 appears to have superior sensitivity and brightness compared to EC17. This coincides with the accepted belief that near infrared (NIR) dyes tend to have less autofluorescence and scattering issues than visible wavelength fluorochromes.

Full text

Research Article
Comparison of Folate Receptor Targeted Optical Contrast
Agents for Intraoperative Molecular Imaging
Elizabeth De Jesus,1Jane J. Keating,1Sumith A. Kularatne,2Jack Jiang,1Ryan Judy,1
Jarrod Predina,1Shuming Nie,3Philip Low,4and Sunil Singhal1
1Division of oracic Surgery, Department of Surgery, University of Pennsylvania and Philadelphia VA Medical Center,
Philadelphia, PA 19104, USA
2On Target Laboratories, Inc., West Lafayette, IN 47906, USA
3Departments of Biomedical Engineering and Chemistry, Emory University, Atlanta, GA 30322, USA
4Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA
Correspondence should be addressed to Sunil Singhal; sunil.singhal@uphs.upenn.edu
Received  June ; Accepted  August 
Academic Editor: Irene J. Virgolini
Copyright ©  Elizabeth De Jesus et al. is 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.
Background. Intraoperative imaging can identify cancer cells in order to improve resection; thus uorescent contrast agents have
emerged. Our objective was to do a preclinical comparison of two uorescent dyes, EC and OTL, which both target folate
receptor but have dierent uorochromes. Materials. HeLa and KB cells lines were used for in vitro and in vivo comparisons of
EC and OTL brightness, sensitivity, pharmacokinetics, and biodistribution. In vivo experiments were then performed in mice.
Results. e pea k excitation and emission wavelengths of EC and OTL were / nm and / nm, respectively. In vitro,
OTL required increased incubation time compared to EC for maximum uorescence; however, peak signal-to-background
ratio (SBR) was .-fold higher compared to EC within  minutes (𝑝 < 0.001). Additionally, the SBR for detecting smaller
quantity of cells was improved with OTL. In vivo, the mean improvement in SBR of tumors visualized using OTL compared
to EC was . fold (range .–.). Neither dye caused noticeable toxicity in animal studies. Conclusions. In preclinical testing,
OTL appears to have superior sensitivity and brightness compared to EC. is coincides with the accepted belief that near
infrared (NIR) dyes tend to have less autouorescence and scattering issues than visible wavelength uorochromes.
1. Introduction
Complete surgical resection of malignant tissue is the single
most eective method for managing a patient with a solid
tumor []. However, failure to obtain complete disease clear-
ance due to an incomplete resection such as positive tumor
margins or metastatic cancer cells in lymph nodes is a major
challenge and can occur in –% of cancer operations
[]. Intraoperative molecular imaging has emerged as an
innovative approach to overcome this problem [–]. Tools
such as uorescence guided imaging surgery, which utilize
uorescent probes and sensitive optical imaging devices,
provide real-time information to surgeons about potentially
malignant tissue to improve disease clearance.
Recently, two contrast agents, EC and OTL, have
been proposed to image ovarian and lung adenocarcinomas
during surgery [, ]. ese agents are similar in that they tar-
get the folate receptor alpha (FR𝛼)viaafolateligand.FR𝛼is a
useful target for intraoperative molecular imaging of ovarian
and lung adenocarcinomas. Folate, a B vitamin (molecular
weight ), plays a key role in metabolic processes involved
in DNA and RNA synthesis, epigenetic processes, cellular
proliferation, and survival of lung adenocarcinomas [].
ere are  members of the folate receptor family, though
only FR𝛼and FR𝛽bind folate with high anity. FR𝛼
is naturally expressed at the luminal surface of polarized
epithelial cells; thus these cells do not bind serum folate [–
]. On the other hand, lung adenocarcinomas express FR𝛼
Hindawi Publishing Corporation
International Journal of Molecular Imaging
Volume 2015, Article ID 469047, 10 pages
http://dx.doi.org/10.1155/2015/469047

International Journal of Molecular Imaging
(– million receptors/cancer cell) and bind serum folate 3–
4times more avidly than normal pulmonary epithelial cells
[–]. us, FR𝛼provides a reasonable molecular target on
pulmonary adenocarcinomas for diagnostic purposes.
AlthoughECandOTLhavethesameligand,they
have two dierent uorochromes: EC contains a uorescein
dye and OTL contains a cyanine dye. Fluorescein is in
the visible wavelength and the cyanine is in the NIR range.
ere are biological advantages to NIR imaging due to the
decreased autouorescence and less rejected scattering that
occurs with visible uorophores. However, uorescein has
been well tested for several decades and has a low toxicity
prole, whereas other NIR dyes (except for indocyanine
green) are relatively untested in humans. For these reasons,
the goal of this study was to generate preclinical data to
compare two optical contrast agents, EC and OTL, both
of which target the same receptor, FR𝛼.
2. Materials and Methods
2.1. Cell Lines. e murine lung cancer cell line, TC, was
derivedfromprimarylungepithelialcellsfromCBL/mice
and transformed with the c-Ha-ras oncogene [, ]. HeLa
is the oldest and most commonly used human cancer cell
line. It was established from human cervical papillomavirus
(HPV ) related carcinoma. KB is a human carcinoma
cell line originally believed to be of oropharyngeal origin
but was eventually found to be a derivative of HeLa [].
TC, KB, and HeLa cell lines were cultured, maintained,
and passaged in RPMI (RPMI  folate decient Medium,
Gibco Life Technologies) supplemented with % fetal bovine
serum (FBS; Hyclone), % penicillin/% streptomycin, and %
glutamine. Cell lines were regularly tested and maintained
negative for Mycoplasma and were maintained in % CO2at
∘C, in a humidied incubator.
2.2. Mice. Female CBL/ mice were purchased from Jack-
son Laboratories and female NOD.Cg-Prkdcscid Il2rg tm1Wjl /SzJ
mice were bred at the CHOP Barrier at the Colket Trans-
lational Research Building at the Children’s Hospital of
Philadelphia.emiceweremaintainedinconditions
approvedbytheAnimalCareandUseCommitteesofthe
Children’s Hospital of Philadelphia and the University of
Pennsylvania and in agreement with the Guide for the Care
and Use of Laboratory Animals.
2.3. Reagents. EC was synthesized by a folate (vitamin B)
and uorescein isothiocyanate (FITC) conjugated through
an ethylenediamine spacer to produce folate-FITC, with
a molecular weight of kDa. FITC is a derivative of
uorescein functionalized with an isothiocyanate reactive
group. e folate-FITC conjugate forms a negatively charged
uorescent molecule that specically targets cell-surface FR𝛼
and is subsequently internalized into the cytoplasm [, ].
OTL was synthesized by a folate ligand as well and a cya-
nine backbone dye with a molecular weight of . kDa.
All vials of EC and OTL were supplied by On Target
Laboratories, Inc. (West Lafayette, IN).
2.4. Near-Infrared and Fluorescence Imaging Platforms. e
GloMax Multi Detection System (Promega, Madison, WI)
was used in uorimeter operation mode to quantify EC
and OTL uorescence from samples placed into -
well microplates. Wavelength matched LEDs provide the
excitation light. A PiN-photodiode top-reads the amount
of emission. e SpectraMax M Multi-Mode Microplate
Reader (Molecular Devices, Sunnyvale, CA) was used to
quantify NIR uorescence. is uorimeter uses a -watt
xenon light source and has a wavelength range from  to
 nm. A photomultiplier top-reads the emission intensity.
e “Flocam” is a home built digital imaging system
based on a dual CCD camera system previously described
[] (BioVision Technologies Inc., Exeter, PA). e system
uses two QIClick digital CCD cameras from QImaging
(British Columbia, Canada), one for white brighteld and
one for uorescence overlay. e cameras have  ×
pixel resolution and have a uorescence exposure time of
– ms. Each camera runs on  W supplied through a
Firewire interface. e light source is a Spectra ×Light
Engine (Lumencor, Inc., Beaverton, OR). Six special-order
NIR bandpass lters are employed to produce the excitation
light. Using ImageJ, ROI measurements of the tumor and
normal muscle were quantied and a signal-to-background
ratio (SBR) was calculated. Positive and negative controls
were used for all images.
2.5. In Vitro Models. KB,HeLa,andTCcellswereplatedon
a cell culture treated -well plate (Corning Costar cell culture
plates) and incubated for  hours. Once conuent, EC was
added to one plate of cells, while OTL was added to another.
ecellswereincubatedandsealedinalight-protected
environment for  minutes. Cells were then washed  times
with PBS and plated and underwent uorescence microscopy.
2.6. Murine Flank Tumor Model. Allmicewereupheldin
pathogen-free environments that were maintained on a -
hour light/dark cycle with normal access to food and water.
Experiments were conducted at  weeks of age or older.
All experimental procedures were maintained and were in
compliance with protocols approved by the Animal Care and
Use Committee at the University of Pennsylvania and the
Children’s Hospital of Philadelphia. A total of  NOD/scid
mice were used in order to test both EC and OTL. Mice
were injected subcutaneously in the ank with . ×6
TC cells (CBL/ mice), . ×6HeLa cells (NOD.Cg-
Prkdcscid Il2rgtm1Wjl/SzJ mice), or . ×6KB cells (NOD.Cg-
Prkdcscid Il2rgtm1Wjl/SzJ mice). Tumor cells for subcutaneous
ank injections were suspended in  𝜇LofPBSandinjected
under an IACUC approved protocol. Tumor volume was cal-
culated using the formula (. ×long-axis ×short-axis2)/.
Once tumor volume reached approximately  mm3half
ofthemicewereinjectedwith.mg/kgofECandthe
otherhalfwith.mg/kgofOTLviatailvein.ree
hours later, the uorescence of tumors was measured using
Flocam. Of note, mice were fed an exclusively folate decient
chow (Harlan Laboratories) on the day of inoculation until
endpoint.

International Journal of Molecular Imaging 
O
HN
N
HN
O
N
N
N
OO
H
N
H
N
N
Na
NaO3S
NaO3S
O3
S
ONa
3
S
H2N
H2N
CO2
Na+− O2C
Na+− O2C
N
NN
H
NH
H
S
N
O
O
N
H
N
H
O
OH
O
N
OTL38 is a folate analog ligand conjugated with an indole cyanine green. e chemical formula is C61H63 N9Na44
O17S , with a molecular mass of 1414.42.
Folate-FITC is a conjugate between folate and uorescein isothiocyanate. e chemical formula is C42 H36N10 Na2O10 Swith a molecular mass of 917.
(a)
EC17 OTL38
Peak: 490 nm Peak: 774 nm
0
0.2
0.4
Optical density (OD)
Optical density (OD)
0.6
0.8
1
350 400 450 500 550
Wavelength (nm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
600 650 750700 350 400 450 500 550
Wavelength (nm)
600 650 750700 850800
A1 A1
(b)
OTL38EC17
(c)
F : Continued.

International Journal of Molecular Imaging
0
5
10
15
20
25
30
35
40
SBR
Molarity
OTL38
EC17
4.88E − 09
1.96E−08
1.56E−07
3.13E−07
9.75E − 09
7.81E − 08
6.25E − 07
1.25E − 06
2.50E − 06
5.00E − 06
1.00E − 05
3.90E − 08
(d)
F : (a) e chemical structure of EC and OTL; (b) biophysical properties of EC and OTL including excitation spectra; EC
peak 𝜆ex  nm and OTL peak 𝜆ex nm; (c) light intensity measured with Flocam; and (d) the luminometer of serial dilutions of EC
and OTL.
2.7. Biodistribution Studies. micewithanktumors(KB
and HeLa) (total 𝑁=5) were given . mg/kg of EC
or OTL via tail vein injection. Twenty-four hours later,
mice were euthanized by inhalation of CO2followed by
cervical dislocation. In order to assess the distribution of
each reagent, the tumor, heart, lung, stomach, liver, spleen,
pancreas, small bowel, large bowel, kidneys, bone, fat, and
muscle were harvested and then imaged in 𝜆520 or 𝜆820.
2.8. Immunohistochemical Staining. Tissue specimens were
harvested and bisected with one-half placed either in Tissue-
Tek OC T a n d s t o r e d a t −∘C or in formalin for paran
sectioning. Frozen tumor sections were prepared as previ-
ously described []. To detect FR𝛼, the monoclonal mouse
antibody Mab (Morphotek Inc., PA) was used.
2.9. Fluorescence Microscopy. Fluorescence microscopy was
performed using an Olympus IX uorescent microscope
equipped with a uorescein specic lter set (Chroma ).
Fluorescence microscopy for OTL was performed using an
Olympus IX motorized inverted microscope equipped with
an excitation lter of  nm and an emission wavelength of
 nm (Chroma ). Image capture was achieved using
a PixeLink NIR CCD camera (PL-BEU). Background
readings were taken from adjacent muscle and subcutaneous
tissue in order to generate a signal-to-background ratio
(SBR). All readings were done in quadruplicate.
2.10. Data Analysis. In order to quantitate the tissue uores-
cence, we used region-of-interest soware and HeatMap plu-
gin within ImageJ (http://rsb.info.nih.gov/ij/; public domain
free soware developed by National Institutes of Health). A
background reading was taken from adjacent normal muscle
tissueinmice(e.g.,glutealmuscle)inordertogenerate
a background value (SBR). For experiments comparing
dierences between  groups, unpaired Student’s 𝑡-tests were
used. For studies comparing more than  groups, ANOVA
was implemented. Dierences were considered signicant
when 𝑃 < 0.05. Data are presented as mean (SE), unless
otherwise noted. For purposes of consistency, we set data
acquisition times to  milliseconds on all imaging devices.
3. Results
3.1. Evaluation of Optical Properties of Folate-Targeted Flu-
orophores. In order to conrm the excitation and emission
spectra of EC and OTL (Figure (a)),  nM aliquots
were measured with a luminometer. e peak excitation (𝜆ex)
andemission(𝜆em ) wavelengths of EC and OTL were
/nm(Stokesshinm)and/nm(Stokes
shi,  nm), respectively (Figure (b)). EC had superior
Stokes shi that can allow for better discrimination and
less overlap in developing a camera ltering system. Next,
seven serial dilutions of  𝜇L aliquots of EC and OTL
(ranging from  M to . ×−6 M) were imaged rst with
Flocam (Figure (c)) and then measured in a luminometer in
order to detect signal intensity (Figure (d)). Sterile water was
used for background measurements. Signal-to-background
ratios (SBR) were calculated for each sample by dividing the
luminometer intensity of each reagent concentration divided
by the intensity of sterile water. We found that the SBR of both
reagents increased as the concentration was increased on a
linear logarithmic scale. e SBR of EC ranged from .
to ., while the SBR of OTL was signicantly higher and
ranged from . to ..
3.2. In Vitro Signal-to-Background Measurements. To con-
rm our imaging systems were calibrated and capable of
identifying uorescence, we placed a  𝜇Ltestdropof𝜇M

International Journal of Molecular Imaging 
KB HeLa TC1
FR𝛼staining
(a)
EC17
KB HeLa TC1
(b)
OTL38
KB HeLa TC1
(c)
F : KB, HeLa, and TC cells folate receptor 𝛼staining (a) followed by KB, HeLa, and TC cells cocultured with uorescent probes and
imaged at x using a confocal microscope (b) and (c). Fluorescence was demonstrated in the viable cancer cells. (a) FR𝛼staining (from le
to right) of KB cells positive for folate receptor 𝛼.HeLaFR𝛼staining also positive for folate receptors. TC cells staining negative for FR𝛼.(b)
Fluorescent imaging (bottom le) highlights KB cells uorescing with EC uorescence dye at .𝜇M, followed by uorescent imaging of
HeLa cells with EC. Imaging (top right) exhibits negative coculture for TC. (c) Near-infrared uorescent imaging (bottom le) displays KB
cells uorescing with OTL uorescence dye at .𝜇M. e following image demonstrates HeLa cells uorescing with EC. TC negative
for OTL uorescence.
EC and OTL on a paralm laboratory plastic lm and
recorded uorescence over various integration times  ms,
 ms, and  ms. e uorescence was readily detectable at
each time point with clear borders (data not shown).
Next, in order to determine which agent had the highest
signal-to-background ratio (SBR), several lung cancer cell
lines (KB, HeLa, and TC) were rst identied. Cytospins
were prepared and immunostained for FR𝛼. Staining demon-
strated strong (+) positivity of KB and HeLa and no FR𝛼
expression in TC (Figure (a)); thus TC was chosen as a
negative control.
en, e6KB cells were incubated with ., ., ., .,
or . 𝜇M of EC and OTL. Cells were cocultured for ,
,,,,,andminutesandthenrinsedwithPBS

International Journal of Molecular Imaging
0.0
2.0
4.0
6.0
8.0
10.0
12.0
EC17
0 40 80 120 160 200 240
SBR
Time (min)
0.1 𝜇M
1.0 𝜇M
2.5 𝜇M
5.0 𝜇M
10.0 𝜇M
(a)
Time (min)
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
0 40 80 120
OTL38
160 200 240
0.1 𝜇M
1.0 𝜇M
2.5 𝜇M
5.0 𝜇M
10.0 𝜇M
SBR
(b)
F : Time kinetics and dose titration of EC and OTL in representative KB cell line showing peak uorescence in vitro at  minutes
to  hours. OTL had a higher signal-to-background ratio compared to EC by  minutes.
and imaged (Figures (b) and (c)). Background uorescence
was measured using cells that had not been incubated with
the uorophores. Also, background readings were taken
from TC cells (e.g., FR𝛼negative). ere was negligible
autouorescence from the TC background readings. e
mean background readings for TC were 231 ± 57.
We found that the SBR for EC was less than . (range
.–.) for the rst  minutes aer coculturing. However, by
 minutes, the SBR was >. (mean .; range .–.)
for all dilutions except . 𝜇M. e SBR was not signicantly
dierent (𝑃 > 0.2) from  minutes to  minutes; however
it was markedly reduced by  minutes (Figure (a)).
Similar experiments were then repeated with OTL.
Again, we found the SBR for OTL was less than . (range
.–.) for the rst  minutes aer coculturing. Aer 
minutes, the SBR was above . (mean .; range .–
.) (Figure (b)). No dilution had any signicant dierence
(𝑃 > 0.3) aer  minutes at all designated concentrations.
us, we concluded that although OTL required
increased incubation time for maximum uorescence, its
peak SBR was markedly increased (mean increase .-fold)
compared to EC by  minutes (𝑃 < 0.001).
3.3. In Vitro Sensitivity. During intraoperative imaging in
humans, the goal is to identify the smallest quantity of disease.
In practical intraoperative applications, this translates to the
highest SBR for the least number of tumor cells. In order to
compare the sensitivity of EC and OTL for tumor cells,
we seeded  at-bottom well plates ( mm2) with either KB
or HeLa cells. e cells were allowed to adhere for  hours,
and then they were cocultured with EC and OTL at either
., ., ., ., or . 𝜇M.CellswerewashedwithPBSand
then imaged aer  minutes (Figure ).
For HeLa cancer cells, OTL was more sensitive than
EC. e SBR of EC for HeLa cells ranged from . to .
depending on the molarity and concentration of cancer cells.
ForOTL,theSBRrangedfrom.to..erangeof
improved SBR of OTL compared to EC was from . to
., with a mean improvement in SBR from EC to OTL
being .-fold (𝑃 < 0.002). For KB cancer cells, OTL was
also more sensitive than EC. e SBR of OTL for KB cells
ranged from . to ., whereas, for OTL, the SBR ranged
from . to .. e range of improved SBR of OTL for
KB cells compared to EC ranged from . to ., with a
mean improvement in SBR of .-fold (𝑃 < 0.004).
Importantly, for both HeLa and KB models, SBR was
signicantly better with OTL when there was lower con-
centration of cancer cells. For example, for HeLa, the SBR
for . cells/mm2ranged from . to . for EC, and the
SBR for . cells/mm2ranged from . to . for OTL.
Similarly for KB, the SBR for . cells/m2ranged from .
to.forEC,andtheSBRfor.cells/mm
2ranged from
. to .. us, the SBR for detecting smaller quantity of
cells was signicantly improved with OTL, and this eect
was less pronounced (though present) at larger quantities of
cancer cells.
3.4. In Vivo Optical Imaging. In order to determine which
uorophore would be superior for in vivo imaging, we tested
each contrast agent on a small animal model. We used e6
KB and HeLa cells in NOD/scid mice. Animal anks were
subcutaneously injected with tumor cells, and then they

International Journal of Molecular Imaging 
0
5
10
15
20
25
SBR
Molarity
1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
0
5
10
15
20
25
SBR
Molarity
1 million
100000
10000
1000
100
10
1
1 million
100000
10000
1000
100
10
1
1 million
100000
10000
1000
100
10
1
1 million
100000
10000
1000
100
10
1
1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
0
5
10
15
20
25
SBR
Molarity
1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
0
5
10
15
20
25
SBR
Molarity
1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
EC17 KBEC17 HeLa
OTL38 KBOTL38 HeLa
F : HeLa and KB cells were cultured with EC and OTL to measure sensitivity for small quantities of tumor cells. For both cell lines,
OTL had a higher SBR than EC, particularly at the smaller quantities of cells and with lower dilutions of the uorochromes.
were monitored until they reached  mm3.Oncethey
reached the designated volume, the animals were injected
with . mg/kg EC or OTL via tail vein (Figure ). We
found the mean uorescence signal from the animals injected
with EC to be , ±, au and the uorescence
signal from the OTL to be , ±,. e background
signal from the gluteus muscle in both cohorts with EC and
OTL was negligible (mean <,). However, EC had
signicant technical issues that required image processing
because of the natural autouorescence from the white fur
of NOD/scid mice. Once this issue was accounted for, the
mean improvement in SBR of tumors visualized using OTL
compared to EC was .-fold (range .–.).
We next studied the dye distribution and the systemic
toxicity of EC and OTL in mice. Animals with ank
tumors were euthanized  hours aer EC and OTL,
and the internal organs were harvested for uorescence
imaging. As shown in Figure , the tumor and peritumoral
tissues, including but not limited to a lobe of the lung, heart,
kidney,spleen,liver,bone,fat,andmuscle,wereisolated,
harvested, and imaged. e majority of the EC and OTL
accumulatedinthedigestivesystem,mostlylocalizedin
the stomach, small intestines, and large intestines as shown.
ere was signicant uorescence in the ank tumors of
bothmice.Nosignalwasfoundinthelung,heart,spleen,
muscle, bone, fat, or liver. e OTL was uorescent in

International Journal of Molecular Imaging
OTL38EC17
KB
Tumo r
(a)
HeLa
OTL38EC17
(b)
F : Comparison of in vivo tumor imaging in murine model. (a) OTL uorescence imaging of mouse followed by EC uorescence
imaging, both with KB tumor burden. (b) OTL uorescence imaging of mouse followed by EC uorescence imaging, both with HeLa
tumor burden.
the kidneys, whereas the EC was not. All animals survived
all studies. ere were no signs of acute toxicity in any of the
animals.
4. Discussion
Molecular imaging of ovarian and lung adenocarcinomas
has emerged as a new technology to identify tumors during
surgery. Two contrast agents, EC and OTL, are being
developed to image these tumors types in humans. Both
agentsbindthesametarget(e.g.,FR𝛼)viathefolatelig-
and; however, their uorochromes are vastly dierent. In
preliminary studies from the producers of these tracers, On
Target Laboratories, Inc. (West Lafayette, IN), OTL seems
to have a higher binding anity for folate receptor; however
ongoing tests are currently comparing the pharmacokinetic
and photophysical proles of EC and OTL. EC contains
the uorescein uorochrome and has a spectral wavelength
of–nm,whereasOTLhasacyaninedyebackbone
that emits in the region of – nm. Our goal was to
present some comparative aspects of these dyes in prepara-
tion for clinical use. Based on our data, we postulate that
OTLwillhavesuperiorclinicalecacy.
Fluorophores in the uorescein spectral range have
decreased tissue penetration and increased autouorescence.
Infrared dyes, however, have deeper tissue penetration, a
signicant reduction in scattering, and less autouorescence.
Infrared dyes have optimal imaging properties due to the
fact that wavelength is not in the normal bioluminescence
of surrounding tissue. Collagen and elastin are known to
uoresce in the ultraviolet range (nm– nm) and visible
spectrum (– nm) []. erefore, there is a window of
opportunity from  to  nm where tumor uorescence
can be detected in vivo with decreased background signal and

International Journal of Molecular Imaging 
White light
Tu Lu He Ki S p Mu B o Ad Li Lin Sin St
EC17
OTL38
(a)
(b)
(c)
F : Biodistribution for clearance of tracer from 𝑁=2animals. Representative brighteld (a), uorescent OTL (b), and uorescent
EC (c) imaging of resected vital tissue excised  hours aer intravenous (i.v) injection. Tu, tumor; Lu, lungs; He, heart; Ki, kidneys; Sp,
spleen; Mu, muscle; Bo, bone; Ad, adipose; Li, liver; Sin, small intestine; Lin, large intestine; St, stomach.
increased tissue penetration. Our murine data conrms these
ndings.
We also found that OTL had superior uorescence
ex vivo when background signal was controlled. We used
similar models; thus the receptor frequency was controlled.
is suggests that OTL has superior brightness and/or our
imaging system was more sensitive to NIR signals. We used
luminometry in an attempt to overcome bias in our device
design, and still OTL showed superior signal emission in
this setting as well. We did nd that the OTL incubation
time was slightly longer in vitro. However, we do not believe
this has any signicant practical implications because the
infusion into the patient would occur prior to surgery. us,
short time interval dierences will unlikely aect the natural
ow of an operation.
ese results are promising; however, the translation of
preclinical murine data to humans has been controversial
[, ]. In our experiences, we have performed four clinical
trials in intraoperative imaging using indocyanine green,
which is a nontargeted agent; however we have been dis-
appointed with the correlation of murine data to human
data. Specically, tumor uorescence of murine models is
more reliable than we have found in our human data.
is inconsistency may be due in large part to the lack of
targeted specic tracer binding. Additionally, we also believe
the challenge lies with the dierences in pharmacokinetics,
liver metabolism, and body compartment sizes. Although
murine models are mammalian in origin, the tumors tend
to be relatively heterogeneous. Human tumors are more
chronic and inammatory in nature; thus they tend to have
more genetic variability. Based on our experiences with the
variance of success between murine and human imaging, we
may need to consider both EC and OTL for human trial,
even though the data is strongly in favor of OTL.
Conflict of Interests
Dr. Nie discloses a relationship as a consultant for Spec-
tropath Inc. and Dr. Low is a consultant and stakeholder
in On Target Laboratories LLC. Additionally, Dr. Low is a
ProfessorofChemistryatPurdueUniversity,whichholdsa
patent for OTL.
Acknowledgment
is work was supported by the National Institutes of Health
R CA- (Sunil Singhal and Shuming Nie).
References
[] L. A. Aliperti, J. D. Predina, A. Vachani, and S. Singhal, “Local
and systemic recurrence is the achilles heel of cancer surgery,”
Annals of Surgical Oncology,vol.,no.,pp.–,.
[] S.Singhal,S.Nie,andM.D.Wang,“Nanotechnologyapplica-
tions in surgical oncology,” Annual Review of Medicine,vol.,
pp. –, .
[] S. B. Mondal, S. Gao, N. Zhu, R. Liang, V. Gruev, and
S. Achilefu, “Real-time uorescence image-guided oncologic
surgery,” Advances in Cancer Research,vol.,pp.–,.
[]A.L.Vahrmeijer,M.Hutteman,J.R.vanderVorst,C.J.
H. van de Velde, and J. V. Frangioni, “Image-guided cancer
surgery using near-infrared uorescence,” Nature Reviews Clin-
ical Oncology,vol.,no.,pp.–,.
[] G. M. van Dam, G. emelis, L. M. A. Crane et al., “Intraop-
erative tumor-specic uorescence imaging in ovarian cancer
by folate receptor-𝛼targeting: rst in-human results,” Nature
Medicine, vol. , no. , pp. –, .
[] M. Srinivasarao, C. V. Galliford, and P. S. Low, “Principles in
the design of ligand-targeted cancer therapeutics and imaging

 International Journal of Molecular Imaging
agents,” Nature Reviews Drug Discovery,vol.,no.,pp.–
, .
[] L. E. Kelemen, “e role of folate receptor 𝛼in cancer devel-
opment, progression and treatment: cause, consequence or
innocent bystander?” International Journal of Cancer,vol.,
no. , pp. –, .
[] P. S. Low and A. C. Antony, “Folate receptor-targeted drugs
for cancer and inammatory diseases,” Advanced Drug Delivery
Reviews, vol. , no. , pp. –, .
[] P.S.Low,W.A.Henne,andD.D.Doorneweerd,“Discoveryand
development of folic-acid-based receptor targeting for imaging
and therapy of cancer and inammatory diseases,” Accounts of
Chemical Research,vol.,no.,pp.–,.
[] P. S. Low and S. A. Kularatne, “Folate-targeted therapeutic
and imaging agents for cancer,” Current Opinion in Chemical
Biology, vol. , no. , pp. –, .
[] Y. Lu, E. Sega, and P. S. Low, “Folate receptor-targeted
immunotherapy: induction of humoral and cellular immunity
against hapten-decorated cancer cells,” International Journal of
Cancer,vol.,no.,pp.–,.
[] Y. Lu, L.-C. Xu, N. Parker et al., “Preclinical pharma-
cokinetics, tissue distribution, and antitumor activity of a
folate-hapten conjugate-targeted immunotherapy in hapten-
immunized mice,” Molecular Cancer erapeutics,vol.,no.,
pp. –, .
[]D.J.O’Shannessy,G.Yu,R.Smaleetal.,“Folatereceptor
alpha expression in lung cancer: diagnostic and prognostic
signicance,” Oncotarget,vol.,no.,pp.–,.
[] J. Predina, E. Eruslanov, B. Judy et al., “Changes in the local
tumor microenvironment in recurrent cancers may explain the
failure of vaccines aer surgery,” Proceedings of the National
Academy of Sciences of the United States of America,vol.,no.
, pp. E–E, .
[] K.-Y.Lin,F.G.Guarnieri,K.F.Staveley-O’Carrolletal.,“Treat-
ment of established tumors with a novel vaccine that enhances
major histocompatibility class II presentation of tumor antigen,”
Cancer Research,vol.,no.,pp.–,.
[] H. Eagle, “Propagation in a uid medium of a human epi-
dermoid carcinoma, strain KB,” Proceedings of the Society for
Experimental Biology and Medicine,vol.,no.,pp.–,
.
[] Y. Lu and P. S. Low, “Folate-mediated delivery of macromolec-
ular anticancer therapeutic agents,” Advanced Drug Delivery
Reviews,vol.,no.,pp.–,.
[] OT. Okusanya, B. Madajewski, E. Segal et al., “Small portable
interchangeable imager of uorescence for uorescence guided
surgery and research,” Technology in Cancer Research & Treat-
ment,vol.,no.,pp.–,.
[]B.F.Judy,L.A.Aliperti,J.D.Predinaetal.,“Vascu-
lar endothelial-targeted therapy combined with cytotoxic
chemotherapy induces inammatory intratumoral inltrates
and inhibits tumor relapses aer surgery,” Neoplasia,vol.,no.
, pp. –, .
[] L.I.Laifer,K.M.O’Brien,M.L.Stetz,G.R.Gindi,T.J.Garrand,
and L. I. Deckelbaum, “Biochemical basis for the dierence
between normal and atherosclerotic arterial uorescence,” Cir-
culation,vol.,no.,pp.–,.
[] J. Seok, H. S. Warren, A. G. Cuenca et al., “Genomic responses
in mouse models poorly mimic human inammatory diseases,”
Proceedings of the National Academy of Sciences of the United
States of America,vol.,no.,pp.–,.
[] M. O. Lasaro and H. C. J. Ertl, “Targeting inhibitory pathways in
cancer immunotherapy,” Current Opinion in Immunology,vol.
,no.,pp.–,.