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

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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.
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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
derivedfromprimarylungepithelialcellsfromCBL/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
Childrens 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
Childrens 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
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Molarity
1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
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Molarity
1 million
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1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
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1e − 9 1e − 8 1e − 7 1e − 6 1e − 5
0
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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willhavesuperiorclinicalecacy.
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).
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... Light in the NIR range (650-900 nm) has deeper tissues penetration due to less absorption by hemoglobin and water, and thus less autofluorescence by surrounding tissue. 27 In a study by Predina and colleagues, 28 IMI was used for localization of GGOs using the folate-NIR combination intraoperatively. The study subjects were given OTL38 systemically before resection, and no toxicity was observed. ...
... To overcome detection-depth limits, the folate motif was modified to enable conjugation of the NIRF dye, S0456 (indole cyanine-like green), producing OTL38 (MW = 1,414 g/mol; Ex/Em = 776/793 nm; Figure 1) (31). In vivo studies using mouse models and canine patients demonstrated the advantages of imaging in the NIRF range and supported the continued development of OTL38 (32,33). To date, at least 9 clinical trials have begun (Phase I-III) using OTL38 and initial results ( Table 1) are encouraging as described below. ...
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Fluorescence guided surgery (FGS) is an imaging technique that allows the surgeon to visualise different structures and types of tissue during a surgical procedure that may not be as visible under white light conditions. Due to the many potential advantages of fluorescence guided surgery compared to more traditional clinical imaging techniques such as its higher contrast and sensitivity, less subjective use, and ease of instrument operation, the research interest in fluorescence guided surgery continues to grow over various key aspects such as fluorescent probe development and surgical system development as well as its potential clinical applications. This review looks to summarise some of the emerging opportunities and developments that have already been made in fluorescence guided surgery in recent years while highlighting its advantages as well as limitations that need to be overcome in order to utilise the full potential of fluorescence within the surgical environment.
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Multifunctional bioimaging probes based on metal clusters have multiple characteristics of metal clusters and functional conjugates, and their development has broad application prospects in the fields of biomedical imaging and tumor diagnosis. However, current bioconjugation methods on metal clusters are time-consuming and have low reaction efficiency, which hinders the construction of bioimaging probes with multifunctional components. Here, we report a concise and promising design strategy to realize the simple and efficient introduction of functional conjugates through bioorthogonal reactions based on azido-functionalized metal clusters. Based on this strategy, taking the probe FA-CuC@BSA-Cy5 as an example, we demonstrated the design of a copper cluster-based multifunctional near-infrared (NIR) fluorescent probe and its real-time imaging application in vivo. Through the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, the tumor-specific targeting ligand folic acid (FA) and fluorophore (Cy5) can be chemically conjugated to azido-functionalized CuC@BSA-N3 quickly and efficiently under biocompatible conditions. The prepared probe showed numerous advantages of metal clusters, including good stability, ultra-small particle size and low toxicity and rapid renal clearance. At the same time, FA-modified FA-CuC@BSA-Cy5 can specifically target KB cells with high FR expression, and in vivo fluorescence imaging shows higher tumor accumulation. The construction of the azido functional metal cluster platform can be extended to various metal clusters with functional probes and prodrugs, thereby providing more promising candidates for future medical diagnoses.
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Objective To investigate the safety and efficacy of OTL38, a folate-targeted, intraoperative fluorescence agent, in patients undergoing robotic-assisted laparoscopic partial nephrectomy. Methods Patients with proven or suspected localized renal cell carcinoma at a single academic institution were selected from 2016 to 2018. Patients received one dose of OTL38 at 0.025 mg/kg prior to robotic-assisted laparoscopic partial nephrectomy. The da Vinci Fluorescence Imaging Vision System was used to identify the tumor and inspect for residual disease after resection. Immunohistochemistry was performed to quantify folate receptor alpha in both the tumor and surrounding normal parenchyma. Patient follow-up was 1 month. Outcome data included descriptive statistics of the patient cohort and surgeon and pathologist surveys. Results Ten cases were performed. Mean patient age was 62.9 years (range = 50–70). Mean tumor size was 2.45 cm. Pathologic tumor stages ranged from T1a–T3a. Histologic tumor types included clear cell, chromophobe, type 1 papillary renal cell carcinoma and oncocytoma. The tumors did not fluoresce, while the surrounding normal parenchyma did show fluorescence. No adverse reactions were seen. Staining for folate receptor alpha was localized to the proximal renal tubules. Average staining in normal surrounding renal parenchyma was significantly greater than staining observed in tumor tissue (0.2086 vs 0.0467; p = 0.002). The mean difference in staining between tumor tissue and surrounding normal renal parenchyma was 0.1619 (95% CI = 0.0796–0.2442). Conclusions Based on our initial experience, OTL38 shows potential as a safe, effective and easy to use tool to improve visualization and resection of renal tumors.
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Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional-imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high-operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low-cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infrared (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity.
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Fluorescence guided surgery (FGS) is a developing field of surgical and oncologic research. Practically, FGS has shown useful applications in urologic surgery, benign biliary surgery, colorectal cancer liver metastasis resection, and ovarian cancer debulking. Most notably in in cancer surgery, FGS allows for the clear delineation of cancerous tissue from benign tissue. FGS requires the utilization of a fluorescent contrast agent and an intraoperative fluorescence imaging device (IFID). Currently available IFIDs are expensive, unable to work with multiple fluorophores, and can be cumbersome. This study aims to describe the development and utility of a small, cost-efficient, and interchangeable IFID made from commercially available components. Extensive research was done to design and construct a light-weight, portable, and cost-effective IFID. We researched the capabilities, size, and cost of several camera types and eventually decided on a near-infrared (NIR) charged couple device (CCD) camera for its overall profile. The small portable interchangeable imager of fluorescence (SPIIF) is a "scout" IFID system for FGS. The main components of the SPIIF are a NIR CCD camera with an articulating light filter. These components and a LED light source with an attached heat sink are mounted on a small metal platform. The system is connected to a laptop by a USB 2.0 cable. Pixielink (c) software on the laptop runs the system by controlling exposure time, gain, and image capture. After developing the system, we evaluated its utility as an IFID. The system weighs less than two pounds and can cover a large area. Due to its small size, it is easily made sterile by covering it with any sterile plastic sheet. To determine the system's ability to detect fluorescent signal, we used the SPIIF to detect indocyanine green under ex and in-vivo conditions and fluorescein under ex-vivo conditions. We found the SPIIF was able to detect both ICG and fluorescein under different depths of a semi-opaque colloid. Second, we found that a concentration as low as 0.5 g/ml of indocyanine green dissolved in plasma was detectable. Lastly, in a murine and human cancer model, the SPIIF was able to detect indocyanine green signal within tumors and generate a signal-to-background ratio (SBR) of 3.75. This study shows that a low-cost IFID can be made from commercially available parts. Second, this IFID is capable of in and ex-vivo detection of multiple fluorophores without sacrificing its small size or favorable ergonomics.
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A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.
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Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.
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Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.
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With the advent of targeted therapies directed towards folate receptor alpha, with several such agents in late stage clinical development, the sensitive and robust detection of folate receptor alpha in tissues is of importance relative to patient selection and perhaps prognosis and prediction of response. The goal of the present study was to evaluate the expression of folate receptor alpha in non-small cell lung cancer specimens to determine its frequency of expression and its potential for prognosis. The distribution of folate receptor alpha expression in normal tissues as well as its expression and relationship to non-small cell lung cancer subtypes was assessed by immunohistochemistry using tissue microarrays and fine needle aspirates and an optimized manual staining method using the recently developed monoclonal antibody 26B3. The association between folate receptor alpha expression and clinical outcome was also evaluated on a tissue microarray created from formalin fixed paraffin embedded specimens from patients with surgically resected lung adenocarcinoma. Folate receptor alpha expression was shown to have a high discriminatory capacity for lung adenocarcinomas versus squamous cell carcinomas. While 74% of adenocarcinomas were positive for folate receptor alpha expression, our results found that only 13% of squamous cell carcinomas were FRA positive (p<0.0001). In patients with adenocarcinoma that underwent surgical resection, increased folate receptor alpha expression was associated with improved overall survival (Hazard Ratio 0.39, 95% CI 0.18-0.85). These data demonstrate the diagnostic relevance of folate receptor alpha expression in non-small cell lung cancer as determined by immunohistochemistry and suggest that determination of folate receptor alpha expression provides prognostic information in patients with lung adenocarcinoma.
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The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.
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The receptor for folic acid constitutes a useful target for tumor-specific drug delivery, primarily because: (1) it is upregulated in many human cancers, including malignancies of the ovary, brain, kidney, breast, myeloid cells and lung, (2) access to the folate receptor in those normal tissues that express it can be severely limited due to its location on the apical (externally-faring) membrane of polarized epithelia, and (3) folate receptor density appears to increase as the stage/grade of the cancer worsens. Thus, cancers that are most difficult to treat by classical methods may be most easily targeted with folate-linked therapeutics. To exploit these peculiarities of folate receptor expression, folic acid has been linked to both low molecular weight drugs and macromolecular complexes as a means of targeting the attached molecules to malignant cells. Conjugation of folic acid to macromolecules has been shown to enhance their delivery to folate receptor-expressing cancer cells in vitro in almost all situations tested. Folate-mediated macromolecular targeting in vivo has, however, yielded only mixed results, largely because of problems with macromolecule penetration of solid tumors. Nevertheless, prominent examples do exist where folate targeting has significantly improved the outcome of a macromolecule-based therapy, leading to complete cures of established tumors in many cases. This review presents a brief mechanistic background of folate-targeted macromolecular therapeutics and then summarizes the successes and failures observed with each major application of the technology.
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Most cancer drugs are designed to interfere with one or more events in cell proliferation or survival. As healthy cells may also need to proliferate and avoid apoptosis, anticancer agents can be toxic to such cells. To minimize these toxicities, strategies have been developed wherein the therapeutic agent is targeted to tumour cells through conjugation to a tumour-cell-specific small-molecule ligand, thereby reducing delivery to normal cells and the associated collateral toxicity. This Review describes the major principles in the design of ligand-targeted drugs and provides an overview of ligand-drug conjugates and ligand-imaging-agent conjugates that are currently in development.
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Paradigm shifts in surgery arise when surgeons are empowered to perform surgery faster, better and less expensively than current standards. Optical imaging that exploits invisible near-infrared (NIR) fluorescent light (700-900 nm) has the potential to improve cancer surgery outcomes, minimize the time patients are under anaesthesia and lower health-care costs largely by way of its improved contrast and depth of tissue penetration relative to visible light. Accordingly, the past few years have witnessed an explosion of proof-of-concept clinical trials in the field. In this Review, we introduce the concept of NIR fluorescence imaging for cancer surgery, examine the clinical trial literature to date and outline the key issues pertaining to imaging system and contrast agent optimization. Although NIR seems to be superior to many traditional imaging techniques, its incorporation into routine care of patients with cancer depends on rigorous clinical trials and validation studies.