Original Article

A Real-Time Near-Infrared Fluorescence
Imaging Method for the Detection of Oral
Cancers in Mice Using an Indocyanine
Green–Labeled Podoplanin Antibody

Technology in Cancer Research &Treatment
Volume 17: 1-11
ª The Author(s) 2018
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DOI: 10.1177/1533033818767936
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Akihiro Ito, BA1,2, Mitsuhiko Ohta, DDS3,4, Yukinari Kato, MD, PhD5,
Shunko Inada, PhD6, Toshio Kato, MD7, Susumu Nakata, MD, PhD8,
Yasushi Yatabe, MD, PhD2, Mitsuo Goto, DDS3,
Norio Kaneda, PhD1, Kenichi Kurita, DDS3,
Hayao Nakanishi, MD, PhD2,9, and Kenji Yoshida, DDS3

Abstract
Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucialrole in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here,
we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)–labeled anti-podoplaninantibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma–xenografted
tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld
device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin
expression–dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using ahandheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging
methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors
than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method
using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative,
and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection andsubsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.
Keywords
podoplanin, oral squamous cell carcinoma, near-infrared fluorescence imaging, indocyanine green, NZ-1 antibody

1Graduate School of Pharmaceutical Sciences, Meijo University, Nagoya, Japan
Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Central Hospital, Nagoya, Japan
3
The First Department of Oral and Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan
4
Department of Oral and Maxillofacial Surgery, Daiyukai General Hospital, Ichinomiya, Japan
5
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan
6
Information and Communications Headquarters/Graduate School of Information Science, Nagoya University, Nagoya, Japan
7
Department of Diagnostic Pathology, Daiyukai General Hospital, Ichinomiya, Japan
8
Department of Clinical Oncology, Kyoto Pharmaceutical University, Kyoto, Japan
9
Laboratory of Pathology and Clinical Research, Aichi Cancer Center Aichi Hospital, Okazaki, Japan
2

Corresponding Author:
Hayao Nakanishi, MD, PhD, Laboratory of Pathology and Clinical Research, Aichi Cancer Center Aichi Hospital, Kuriyado, Okazaki, Aichi 444-0011, Japan.
Email: hnakanis@aichi-cc.jp
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License(http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission
provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Technology in Cancer Research & Treatment

Abbreviations
EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; ICG, indocyanine green; IRS, immunoreactivescore; PDE, photodynamic eye; mAb, monoclonal antibody; NIR, near-infrared; OSCC, oral squamous cell carcinoma; ROI, regionof interest.
Received: September 24, 2017; Revised: December 22, 2017; Accepted: February 16, 2018.

Introduction
Oral cancer is the sixth most common cancer worldwide.
Although diagnostic and therapeutic modalities have
improved, the prognosis of patients with advanced oral squamous cell carcinoma (OSCC) still remains relatively poor, with
5-year survival rates of approximately 50%.1 The poor prognosis is mainly due to the lack of early detection of OSCC, thehigh incidence of metastasis/recurrence after surgery, and thechemoresistance of OSCC.2,3 A new optical imaging method
capable of sensitively and specifically detecting OSCC must be
developed to improve the prognosis of OSCC.4
Small-sized OSCC is frequently overlooked by conventional examinations under white light but gross inspection is
the still a gold standard for the screening and diagnosis of oral
neoplasms. Recently, a light-induced tissue autofluorescencebased examination has been introduced to assist in screening
for OSCC.5 Upon illumination by blue light (436 nm), specificcomponents of fluorophores present in the mucosa, including
flavin adenine dinucleotide (FAD) and nicotinamide adenine
dinucleotide (NAD) in the cross-linked collagen/elastin fibers,
emit low-energy light, which is visualized as an autofluorescence image of the mucosa. Malignant transformation is usually associated with an increase in the epithelial cell density,
which reduces the excitation of collagen fibers, leading to adecrease in the blue–green autofluorescence and a darker
appearance than the uninvolved mucosa. This convenient andsensitive autofluorescence visual imaging technique is a widelyused and safe method approved by the US Food and Drug
Administration (FDA), but the differentiation of malignant
neoplasms from premalignant or benign lesions still remains
to be evaluated.6,7
Alternative imaging methods other than autofluorescencebased imaging include imaging of fluorophores synthesized intissues after the administration of a precursor drug, such as 5aminolevulinic acid (5-ALA),8 and fluorophores injected as
exogenous drugs, such as a fluorescent dye–labeled antibody
raised against the target molecule.9 The latter method has
advantages over the former in terms of fewer toxic side effects,
such as photo hypersensitivity disorder, as well as higher specificity, leading to its potential utility for molecular targetingtherapy. The selection of target molecules for optical imaging
is important for the sensitive, specific, and safe detection oforal SCC. Epidermal growth factor receptor (EGFR) is
expressed in *90% of OSCC, and cetuximab, a chimerichuman mouse EGFR antibody, is FDA-approved for clinicaluse in patients with OSCC, making it a potential target formolecular imaging previously.10 Another candidate target
molecule for molecular imaging of OSCC is podoplanin. Podoplanin is a transmembrane O-glycoprotein that binds to C-type
lectin-like receptor 2 and shows several important physiological functions11-13 and unique expression patterns in several
tumors, such as squamous cell carcinomas, testicular tumors,
mesothelioma, and brain tumors.14-17 In OSCC, podoplanin is
known to be overexpressed in at least 70% to 80% of cases.18
Podoplanin expression in OSCC was recently shown to be
associated with tumor invasion and lymph node metastasis,1921
and podoplanin also possesses prometastatic activities, suchas platelet-aggregating activity.22 Furthermore, podoplanin
reportedly serves as a molecular marker of cancer stem cells
in OSCC.23 Therefore, podoplanin represents a potential target
molecule for molecular imaging and therapy. As reported previously, an anti-podoplanin antibody might be a potential tool
for therapy targeting malignant mesothelioma and malignant
brain tumors.24-26 To our knowledge, however, molecular imaging techniques that target podoplanin as a fluorescence probein OSCC have not yet been reported. Indocyanine green (ICG),a near-infrared (NIR) fluorophore that has been approved by
the FDA for clinical use as a liver function test, has been widelyused for sentinel lymph node navigation surgery and real-time
identification of liver cancers.27,28 Additionally, ICG has beenused to label antibodies for molecular imaging,29 raising thepossibility of using an ICG-labeled podoplanin antibody for
NIR imaging of OSCC in clinical settings.
In the present study, we developed a real-time NIR imaging
method that consists of an ICG-labeled podoplanin antibody
and a handheld ICG detection system and compared NIR imaging methods with IVIS detection systems and a non-NIR
autofluorescence-based method in xenograft models. The preclinical and potential clinical applications of this NIR imaging
method using an ICG-labeled anti-podoplanin antibody for
detecting premalignant and malignant oral neoplasms are
discussed.

Materials and Methods
Reagents
A rat monoclonal antibody (mAb) to human podoplanin (NZ-1)
was produced previously by Kato et al.26 The NZ-1 antibody

Ito et al
was used for molecular imaging, flow cytometry, and immunohistochemistry in mouse tumors. Mouse mAbs against
human podoplanin (D2-40) and human EGFR (PharmDX
staining kit; DakoCytomation, Carpinteria, California) wereused for immunohistochemical staining of clinical specimens
because of clear staining pattern. Rat immunoglobulin G (IgG)
was purchased from Beckman Coulter (Fullerton, California).

Patients and Ethics Statement
Patients with fibroma, papilloma, leukoplakia, and OSCC (5-7
patients with each type of lesion) were examined in this study.
These patients underwent an operation at the Department ofOral and Maxillofacial Surgery at Daiyukai General Hospitalfrom 2011 to 2015. Tumors were removed with either excisional biopsy or partial glossectomy without neck dissection.
The tumor stage and histology of OSCC were stage 1 andmoderately differentiated SCC, based on the UICC criteria.
The use of resected specimens from patients was approvedby the Human Ethical Review Committee of the Daiyukai
General Hospital, and written informed consent was provided
by each participant, with the approval of the institutional
review board.

Cell Lines
HSC-3 and Ca9-22 cells were obtained from the RIKEN CELLBANK (Tsukuba, Japan). UMSCC-81 cells were kindly provided by the University of Michigan. These cells were cultured
in Dulbecco’s Modified Eagle’s Medium ( Nissui Pharmaceutical Company, Tokyo, Japan) supplemented with 10% fetal
bovine serum (Gibco, Grand Island, New York), 100 U/mLof penicillin, and a 100 mg/mL streptomycin solution (Invitrogen Corp, Carlsbad, California) and incubated at 37 C in a 5%
CO2 atmosphere.

Flow Cytometry and Immunofluorescence Staining
Tumor cells were harvested with trypsin/EDTA, washed twicewith FACS buffer (5 mM EDTA and 5 mg/mL BSA in PBS)
and incubated with a primary rat antihuman podoplanin mAb(NZ-1, 1 mg/mL) for 30 minutes on ice. After washing twicewith fluorescence activated cell sorting (FACS) buffer; cells
were then incubated with an Alexa Fluor 488-conjugated goat
antirat IgG secondary antibody (Invitrogen, Eugene, Oregon)
on ice for an additional 30 minutes. The labeled cells were thenwashed, and the fluorescence intensity was evaluated using a
FACS Calibur flow cytometer (BD Biosciences, San Diego,
California).

Quantitative Reverse Transcription Polymerase Chain
Reaction Analysis
Podoplanin expression was examined by quantitative reverse
transcription polymerase chain reaction. Cells were rinsed with
PBS and dissolved in ISOGEN (Nippon Gene, Tokyo, Japan).

3
Total RNA was extracted, and cDNAs were synthesized using
SuperScript II RNase H-reverse transcriptase (Invitrogen,
Carlsbad, California). Using the resulting first-strand cDNA
templates, single-step real-time RT-PCR was performed using
the Universal ProbeLibrary system (Roche Diagnostic, Mannheim, Germany), specific oligonucleotide primer pairs, andTaqman probes on the LightCycler instrument. The sequences(50 -30 ) of the podoplanin primer pairs and Taqman probe number (#) used in this study were described in a previous study.21
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
analyzed as an internal control. Podoplanin expression in the
3 cell lines was measured in triplicate and comparedstatistically.

Immunohistochemistry of Paraffin-Embedded Preclinicaland Clinical Specimens
Surgically resected tumors from patients (n ¼ 5-7) and xenografts removed from nude mice (n ¼ 3) were fixed with 10%
buffered formalin for 24 hours, embedded in paraffin and sectioned to a 5-mm thickness. Immunohistochemical staining ofclinical specimens was performed using the following antibodies: a mousemonoclonal antihuman podoplanin mAb (D2-40)
and a mouse monoclonal antihuman EGFR mAb (PharmDX
staining kit, DakoCytomation, Carpinteria, California) becauseof clear staining pattern. For antigen retrieval, the sections weremicrowaved at 98 C for 15 minutes for the D2-40 antibody andtreated with a protease K solution for 5 minutes at room temperature for the EGFR antibody. After blocking nonspecificreactions with normal serum for 30 minutes, these sections
were incubated with primary antibodies overnight at 4 C, thoroughly washed with PBS, incubated with secondary antibody(biotinylated rabbit antibody to mouse IgG) and subsequently
incubated with a streptavidin-peroxidase complex (Vector
Laboratories, Burlingame, California) for 60 minutes. For
EGFR staining, sections were incubated with secondary antibody labeled by horseradish peroxidase conjugated to a common dextran polymer backbone. The sites of peroxidasebinding were visualized using 0.01% diaminobenzidine.
Podoplanin expression was interpreted by visualizing theimmunostaining of the specimens and scored using a scoring
system based on the quantity and staining intensity of only
squamous epithelial tumor cells. The immunoreactive score(IRS; range: 0-15) of each specimen was calculated by multiplying the quantity score (range: 0-5) and staining intensity
score (range: 0-3)30,31 and was used for the quantitative assessment by statistical analysis (n ¼ 5-7).

Animals
Five- to 6-week-old male athymic nude mice of the KSN strain
were purchased from Japan SLC (Hamamatsu, Japan). These
mice were housed in specific pathogen-free conditions at Aichi
Cancer Center Research Institute. Chlorinated water and foodthat had been autoclaved for 5 minutes were provided ad libitum. Animal health, including body weight and skin conditions,4
was monitored twice weekly. Tumor size was small, with amaximal diameter of 1 cm, and ulceration, a reduction in animal mobility and weight loss, was not observed during theexperiment. After imaging experiments, mice were immediately euthanized by cervical dislocation to prevent animals
from developing large tumors or weight loss. All experiments
were conducted with the approval of the Institutional EthicalCommittee for Animal Experiments of Aichi Cancer Center
Research Institute (Permission No., ACC 27-2) and met thestandards defined by international guidelines.32

Near-Infrared Imaging of OSCC Xenografts in Mice
With an ICG-Labeled Podoplanin Antibody
Three OSCC cell lines (UMSCC-81, HSC-3, and Ca9-22 cells)
were harvested with trypsin/EDTA, washed with PBS, and 3-5
 106 cells/0.2 mL PBS were intracutaneously and subcutaneously injected into the upper and lower back, respectively. In
some instances, cells (1  106 cells/0.05 mL PBS and 3-5 
106 cells/0.3 mL PBS) were injected into the tip of mouse
tongue and mouse peritoneal cavity, respectively. For in vivo
NIR imaging of xenografted tumors in nude mice (n ¼ 4), an
antipodoplanin antibody (NZ-1; or rat IgG as anisotype control) was labeled with ICG using the ICG-Labeling Kit-NH2(Dojindo Laboratories, Kumamoto, Japan), according to the
manufacturer’s protocol.29 In this system, labeling efficiency
of ICG to podoplanin antibody (ICG: antibody ratio) was calculated to be average 0.9 (range: 0.8-1.0) based on the following formula: A800147,000 O (A280-A800  0.075)/E(¼ molar
absorption coefficient) (A: absorption). In addition, on the stability, this ICG-labeled antipodoplanin antibody was stable atleast 1 week and 1 month in vivo (in xenograft) and in vitro,
respectively. Each mouse was intraperitoneally injected with50 mg of the ICG-labeled podoplanin antibody plus 250 mL of
PBS. Indocyanine green fluorescence imaging for intracutaneous, subcutaneous, tongue, and intraperitoneal tumors was
performed on mice under isoflurane anesthesia 24 to 48 hours
after the injection using an IVIS Lumina II device (Xenogen,
Alameda, California) with an excitation filter of 745 nm andthe emission filter of approximately 820 nm for ICG. Fluorescence intensity was quantitated as an average radiance(photons/sec/cm2/sr) using “living image” software and theregion of interest (ROI) tool, according to the manufacturer’s
protocol. Alternatively, a handheld ICG imaging device comprising an near-infrared (NIR) light emitting diode (LED) (760
nm, 5 mW/cm2) light source, and ICG camera (830 nm), withhigh-sensitivity NIR image sensor (768  494 pixel), a photodynamic eye (PDE; Hamamatsu Photonics, Hamamatsu, Japan)
was used for the real-time imaging of xenografted tumors inmice (n ¼ 4) under 2, 2, 2-tribromethanol anesthesia using thesame settings for the exposure time and black balance
described in a previous study.27 Fluorescence intensity of the
ICG spectrum image of the tumor at 830 nm was quantitatedwith ImageJ software using ROI after normalization (subtraction) to the background and baseline intensity.

Technology in Cancer Research & Treatment

Autofluorescence-Based Imaging of OSCC Xenografts
in Mice
Autofluorescence-based imaging of xenografted tumors innude mice (n ¼ 3) under 2, 2, 2-tribromoethanol anesthesia
was performed using a VELscope VX device (LED Dental Inc,
Burnaby, British Columbia, Canada), which consists of a120-W metal-halide arc lamp and a filtersystem optimized for
producing blue light with 430 (400-460) nm according to the
manufacturer’s protocol.6 Apple green fluorescence images
were captured by digital camera with exposure time of 0.1 sec(Sony, Tokyo, Japan).

Statistical Analysis
The statistical significance of differences in RT-PCR data
between cell lines was determined by applying Student t tests.
Differences in ICG fluorescence imaging data between xenograft tumors in nude mice were determined by Mann-whitney
U test. The difference in the podoplanin expression status basedon IRS among benign, premalignant, and malignant neoplasms
in patients was analyzed using 1-way analysis of variance. A P
value <.05 was considered significant.

Results
Podoplanin and EGFR Expression in Clinical Specimensof Oral Benign, Premalignant, and Malignant Tumors
Based on the immunohistochemical staining of normal tongue
epithelium, podoplanin was weakly expressed and restricted tothe basal cell layer of the squamous epithelium. Fibroma in the
submucosadid not express podoplanin, with the exception oflymphatic vessels surrounding the tumor. Similarly, podoplanin was weakly expressed in the basal layer of papilloma specimens. In leukoplakia, a premalignant lesion, weak-to-moderate
podoplanin expression was observed in the basal cell layer andthin suprabasal layer of the epithelium. In contrast, squamouscell carcinoma exhibited moderate-to-strong podoplanin
expression in both basal cells and the thick suprabasal layers(Figure 1A). The quantitative analysis using the IRS scoring
system clearly showed that podoplanin expression significantly
increased with the grade of pathology in the order of normal(fibroma) < benign (papilloma) < premalignant (leukoplakia) <malignant neoplasm (SCC, P < .01; Figure 1C).
On the other hand, EGFR, another potential target for molecular imaging, was expressed at high levels in the basal andupper spinosum layers of normal squamous epithelium and inalmost all cell layers of the papilloma and leukoplakia specimens, with the exception of the superficial keratinizing layer,
similar to OSCC (Figure 1B). Based on the results of the quantitative analysis, no significant difference in EGFR expression
was observed among normal, benign, premalignant, and malignant neoplasms (Figure 1D), indicating that podoplanin is distinctively overexpressed in premalignant and malignant oral
neoplasms compared with EGFR.

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5

Figure 1. Podoplanin and EGFR expression in clinical specimens from patients with oral neoplasms, as evaluated by immunohistochemistry.
A, Podoplanin expression in clinical specimens, including fibroma (normal epithelium), papilloma, leukoplakia, and SCC. Bars ¼ 500 mm.
B, EGFR expression in the clinical specimens described above. Insets indicate higher magnification view. C, Quantitative analysis of theintensity of podoplanin staining in normal, papilloma, leukoplakia, and SCC tissues (n ¼ 5-7) based on the immunoreactivity scores.
D, Quantitative analysis of the intensity of EGFR staining in normal, papilloma, leukoplakia, and SCC tissues (n ¼ 5-7) based on the
immunoreactivity scores. Bar ¼ standard error.*P < .01 compared with normal tissues, EGFR indicates epidermal growth factor receptor;
NS, not significant; SCC, squamous cell carcinoma.

Podoplanin Expression in OSCC Cell Lines
Podoplanin expression was examined in 3 OSCC cell lines,
including UMSCC-81 (poorly differentiated SCC), HSC-3(moderately differentiated SCC), and Ca9-22 cells (welldifferentiated SCC), in vitro and in vivo. According to theresults of the flow cytometry analysis, podoplanin was
expressed in these cell lines in the following order:
UMSCC-81 > HSC-3 > Ca9-22 cells (Figure 2A). Quantitative RT-PCR analysis of cultured cells confirmed that podoplanin expression was significantly different (P < .01) among
these cell lines in the following order: UMSCC-81 > HSC-3 >Ca9-22 cells (Figure 2B). An immunohistochemical analysis
of xenografted tumor in nude mice showed that podoplanin
was expressed at higher levels in these cell lines in the following order: UMSCC-81 > HSC-3 > Ca9-22 cells. UMSCC81 and HSC-3 cells expressed high and moderate levels of
podoplaninin the majority of tumor cells, respectively,

whereas Ca9-22 cells only expressed low levels of podoplanin(Figure 2C).

Near-Infrared Imaging Using an ICG-Labeled AntiPodoplanin Antibody and an IVIS Detection System
As mentioned above, podoplanin expression was more distinctive for malignant oral neoplasms than EGFR expression.
Therefore, in this study, we focused on the utility of an ICGlabeled anti-podoplanin antibody as a probe for NIR fluorescence imaging of OSCC tumors in nude mice. Time-course
experiment using intraperitoneal metastasis model of
UMSCC-81 cells showed that the labeled antibody accumulated in the intraperitoneal tumors and the unbound residual
antibody was first up-taken by the liver and then excreted fromthe body through intestine in approximately 1-day postinjection of labeled antibody. Near-infrared fluorescence images

6Technology in Cancer Research & Treatment

Figure 2. Podoplanin expression in 3 OSCC cell lines in vitro and in vivo. A, Flow cytometry analysis of 3 OSCC cell lines with different levelsof podoplanin expression. Levels of podoplanin expression: UMSCC-81 > HSC-3 > Ca9-22. Blue and green indicates fluorescence staining withisotype control and antipodoplanin antibody, respectively. B, Quantitative RT-PCR analysis of the podoplanin mRNA in 3 OSCC cell lines. *P <.01 compared with UMSCC-81 cells. Bar ¼ standard error. C, Immunohistochemical staining of 3 OSCC subcutaneous tumors in nude mice.
Strong, moderate, and very weak podoplanin expression was observed in UMSCC-81, HSC-3, and Ca9-22 tumors, respectively. Specimens werecounterstained with hematoxylin. Bars ¼ 100 mm. mRNA indicates messenger RNA; OSCC, oral squamous cell carcinoma; RT-PCR, reverse
transcriptase polymerase chain reaction.

were available for 1 to 7 days, with the peak fluorescenceobserved 1 to 3 days after injection (Supplemental Figure A).
The NIR fluorescence of intraperitoneal tumor observed using
the ICG-labeled antipodoplanin antibody was abolished by the
coinjection of an excess amount (10-fold) of unlabeled podoplanin antibody in the blocking experiment (Supplemental Figure B), confirming the specificity of NIR imaging using the
ICG-labeled antipodoplanin antibody.
The sensitivity of NIR fluorescent imaging with the ICGlabeled antipodoplanin antibody was then examined using 3
OSCC cell lines with different levels of podoplanin expression.
Subcutaneous and tongue tumors generated by UMSCC-81
cells (high expression) and HSC-3 (moderate expression) cells
were clearly visible in nude mice using the IVIS imaging system. In contrast, Ca9-22 (very low expression) subcutaneousand tongue tumors were virtually invisible. In addition, no
significant fluorescent signal was detected in the SCC xenografts after the intraperitoneal injection of the ICG-labeled rat
IgG as a negative control (Figure 3A). The size of xenografted
tumors detected with this imaging system was more than 3 mm
in diameter. According to the results of the statistical analysis,
the ICG fluorescence intensity (photons/sec/cm2/sr) of subcutaneous tumors was increased in the following order: UMSCC-

81 > HSC-3 > Ca9-22 (n ¼ 4 mice each, NS; UMSCC-81 vs
HSC-3; P < .05, UMSCC-81 vs Ca9-22; Figure 3B). Thus, theICG fluorescence intensity of tumors in mice was well correlated with the podoplanin expression in tumor cells in vitro.

Real-Time NIR Imaging using a Handheld Detection
Device (PDE)
We next tested a handheld detection device (PDE) to examine
whether PDE detects OSCC tumors in mice in real time after theadministration of the ICG-labeled anti-podoplanin antibody(Figure 4A). As shown in the bright and dark field images, PDE
can visualize UMSCC-81 and HSC-3 tumors with high-tomoderate podoplanin expression, respectively, in nude mice inreal time, but not Ca9-22 tumors with very low podoplanin expression similar to NIR imaging using the ICG-labeled control antibody. Furthermore, PDE detected ICG fluorescence from both
intracutaneous tumors and subcutaneous tumors (Figure 4B).
We quantitatively compared the ICG fluorescence intensityof UMSCC-81, HSC-3, and Ca9-22 tumors (n ¼ 4) by PDE
detection device and image analysis with ImageJ software (version 1.45) (Figure 5A). Both intracutaneous and subcutaneoustumors were quantitatively detected by PDE in real time, with

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7

Figure 3. Near-infrared imaging of OSCC xenografted tumors in mice using the ICG-labeled antipodoplanin antibody and IVIS detection
device. A, Brightfield (left panel) and dark field images (right panel, pseudocolor image) of subcutaneous tumors (black arrows) and a dark field
image of a tongue tumor (white arrows) approximately 1 week after the injection of 3 OSCC cell lines. UMSCC-81 and HSC-3 subcutaneoustumors with high and moderate podoplanin expression, respectively, are clearly visible, whereas Ca9-22 tumors with weak podoplanin
expression are virtually invisible. No significant ICG fluorescent signal was observed in the UMSCC-81 tongue tumor using NIR imaging withan ICG-labeled rat IgG as a negative control (upper right panel). B, Quantitative analysis of NIR fluorescent intensity of UMSCC-81, HSC-3,
and Ca9-22 subcutaneous tumors in mice (n ¼ 4). Bar ¼ standard deviation.*P < .05 compared with UMSCC-81 tumors. ICG indicates
indocyanine green; IgG, immunoglobulin G; NIR, near-infrared; NS, not significant; OSCC, oral squamous cell carcinoma.

the following order of fluorescence intensity: UMSCC-81 >HSC-3 > Ca9-22 (n ¼ 4 mice each, NS; UMSCC-81 vs HSC3; P < .05, UMSCC-81 vs Ca9-22; Figure 5B). These findings
are consistent with the podoplanin expression in subcutaneoustumors measured by NIR imaging using the IVIS detection system. Indocyanine green fluorescence at low level in the liver,
irrespective of tumor-bearing mice is observed, indicating transient accumulation in the liver as an excretory organ for ICG.
The autofluorescence-based method using the VELscope system also detected both intracutaneous and subcutaneous tumors
as “punched-out” lesions, but the latter images are sometimes
weaker and less clear than NIR images using the ICG-labeledanti-podoplanin antibody, due to the reduced penetration of blue
light for excitation through the skin (Figure 6).

Discussion
In the present study, we developed a real-time NIR imaging
method for OSCC using an ICG-labeled anti-podoplaninantibody in combination with a handheld ICG detection device(PDE). This imaging system has the following advantages overthe conventional NIR imaging method using an IVIS detection
device and the non-NIR autofluorescence-based imaging
method. (1) This system allows positive and clear images ofsmall intracutaneous, subcutaneous, and tongue OSCC tumors
with a diameter greater than 3 mm. (2) This system allows thesemiquantitative detection of podoplanin-positive OSCC tumors
in mice in a podoplanin expression–dependent manner. (3) Thissystem also allows the real-time detection of OSCC tumors
compared with imaging methods using the desktop detection
device (IVIS). Photodynamic eye is a convenient and smallsized device that fairly comparable to the autofluorescencebased method.27,28 These findings suggest that this NIR imaging
method using the ICG-podoplanin antibody and a handheld
detection device (PDE) would be an useful tool for real-time
imaging of OSCC in preclinical and in some clinical studies.
On the other hand, an inherent problem with this imaging
system using the ICG-labeled antipodoplanin antibody exists:

8Technology in Cancer Research & Treatment

Figure 4. Real-time NIR imaging of OSCC-xenografted tumors in mice using the ICG-labeled anti-podoplanin antibody and a handheld ICG
detection device (PDE). A, Overview of real-time NIR imaging using PDE. Bright field (upper panel) and dark field images (lower panel).
B, Representative real-time NIR images of UMSCC-81, HSC-3, and Ca9-22-xenografted tumors. No significant ICG fluorescent signal wasobserved in the UMSCC-81 tumor using the ICG-labeled rat IgG as a negative control (upper right panel). Short arrows and long arrows indicate
intracutaneous and subcutaneous tumors, respectively. Arrowheads indicate the liver. ICG indicates indocyanine green; IgG, immunoglobulin
G; NIR, near-infrared; OSCC, oral squamous cell carcinoma; PDE, photodynamic eye.

the potential risk of missing 20% to 30% of podoplaninnegative OSCCs and some premalignant lesions. Other disadvantages of this ICG-based imaging system for the detection of
OSCC include minimal invasiveness, possible antibody-relatedside effects, and some cost due to the intravenous injection of
the ICG-labeled antibody before imaging compared with theautofluorescence-based method without any requirement for a
pretreatment. Furthermore, the safety of NH2-reactive ICG for
antibody labeling, which includes an active(succinimidyl) ester
group in the ICG molecule, remains to be elucidated, although
the use of ICG itself has been well established in clinical practice. Unlike computed tomography (CT)/magnetic resonanceimaging (MRI)/positron emission tomography (PET), themajor limitation of optical imaging, including our NIR imaging
system and non-NIR autofluorescence-based method, is thedifficulty in applying these technique to the whole-body imaging of tumors located in the deep organs because of the low
tissue penetration of the light even if NIR light. However,regarding oral cancer, this is not a limiting factor in clinicalsettings because of the superficial location of oral tumors.
This NIR imaging method using an ICG-antipodoplaninantibody and a handheld device (PDE) is not only applicable
to mouse preclinical models but also applicable to the detectionof podoplanin-positive OSCC in some clinical settings. Several
potential clinical applications should be considered. (1) The
first application of this ICG imaging method is image-guided
surgery, an option for minimizing the risk of residual tumors
and resection volume of the healthy tissue by visualizing thetumor margin. (2) The second application of this imaging
method is the prediction and monitoring of efficacy of future
molecular-targeting therapy using the antipodoplanin antibody.33 The present finding that the ICG-labeled antipodoplanin antibody efficiently accumulated in OSCC xenografts inmice for a prolonged period (at least 1 week) suggests its
potential therapeutic efficacy for OSCC. Podoplanin wasrecently reported to be a good target for molecular therapy inpodoplanin-positive malignant mesothelioma and glioma. As
shown in the study by Abe et al, the podoplanin antibody (NZ8/NZ-1) significantly reduces the growth of subcutaneoustumors generated by malignant mesothelioma cells throughnatural killer (NK) cell–mediated, antibody-dependent cellularcytotoxicity (ADCC).24 Chandramohan et al also reported theantitumor effect of a recombinant antipodoplanin (NZ-1)
immunotoxin on malignant brain tumors, such as glioblastoma
and medulloblastoma.25 Although the antitumor effect of the
podoplanin antibody on OSCC remains to be elucidated, these

Ito et al

9Figure 5. Comparison of real-time NIR images of 3 OSCC-xenografted tumors in mice using the ICG-labeled anti-podoplanin antibody and a
handheld detection device (PDE). A, Real-time NIR images (dark field) of 3 OSCC intracutaneous (short arrows) and subcutaneous tumors (longarrows) in mice (n ¼ 4). Arrowheads indicate the liver. B, Quantitative analysis of NIR fluorescence intensity of UMSCC-81, HSC-3, and Ca922 intracutaneous and subcutaneous tumors. Bar ¼ standard deviation. * P < .05 compared with the UMSCC-81 tumors. AU indicates arbitrary
units; NIR, near-infrared; NS, not significant; PDE, photodynamic eye; OSCC, oral squamous cell carcinoma.

Figure 6. Real-time non-NIR imaging of OSCC-xenografted tumors in mice using the autofluorescence-based method (VELscope). Bright field(left) and dark field (right) images of 3 OSCC intracutaneous (white arrow) and subcutaneous tumors (black arrow in HSC-3 tumors). In the dark
field images, UMSCC-81 and HSC-3 intracutaneous tumors (white arrows) are clearly visible as punched-out lesions. Ca9-22 intracutaneoustumors (white arrows) and HSC-3 subcutaneous tumor (black arrow) are only weakly visible. NIR indicates near-infrared; OSCC, oral squamouscell carcinoma.

10findings suggest the possibility that real-time NIR imaging
with an ICG-labeled antipodoplanin antibody and PDE device
may be a useful tool for predicting the sensitivity of moleculartargeting therapy using an antipodoplanin antibody in the
future clinical settings. (3) The third potential application ofthis imaging is its utility as an adjunct to the diagnosis of OSCC
and premalignant lesions, such as leukoplakia and erythroplakia based on the capability for semiquantitative assessment of
podoplanin expression in oral neoplasms. According to deVicente et al, 38% of patients with leukoplakia are classified
as podoplanin positive, and the remaining 62% lesions are
podoplanin negative.34 Podoplanin expression is significantly
correlated with the grade of dysplasia and the risk of progression to OSCC. Similar results were reported in erythroplakia.35
Based on these findings, the semiquantitative evaluation of
podoplanin expression using the present ICG imaging method
maybe an useful tool to assist the correct diagnosis of premalignant oral lesions and OSCC.

Conclusions
In this study, we developed a new ICG imaging system using an
ICG-labeled anti-podoplanin antibody and an ICG detection
device (PDE) and showed that this system could clearly visualize
OSCC in mice in real time. Although the sensitivity and usabilityof real-time ICG imaging with a handheld PDE device may be
still somewhat insufficient for early diagnosis of OSCC at present,a brighter LED light source, more sensitive/smaller ICG detection
camera and a real-time pseudocolor image processor would offer
an even greater potential for the real-time diagnosis of OSCC in
preclinical and future some clinical settings.
Acknowledgments
The authors would like to thank Ms. M. Yoshimura and N. Saito for
their expert technical assistance.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect tothe research, authorship and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the
research, authorship, and/or publication of this article: The study was
supported by a Grant-in-Aid for Scientific Research from the Ministryof Education, Science, Sports, Culture and Technology (MEXT), Japan,
a Grant-in-Aid for Priority Research Project from Knowledge Hub
Aichi, Japan and a Grant-in-Aid for the Platform for Drug Discovery,
Informatics, and Structural Life Science (PDIS) from Japan Agency for
Medical Research and development (AMED), Japan.

Supplemental Material
Supplementary material for this article is available online.

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