Near Infrared Imaging of EGFR of Oral Squamous Cell
Carcinoma in Mice Administered Arsenic Trioxide
Lingbo Zhang1., Kezheng Wang2., Falin Zhao3, Weiping Hu1, Junjie Chen4, Gregory M. Lanza4,
Baozhong Shen2*, Bin Zhang1*
1Stomatology Department, Institute of Hard Tissue Development and Regeneration, 2nd Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China,
2Radiology Department and Molecular Imaging Center, 4th Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China, 3School of Health Management,
Hangzhou Normal University, Hangzhou, Zhejiang, China, 4Division of Cardiology and C-TRAIN, Washington University School of Medicine, St. Louis, Missouri, United
States of America
Background: The effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR)
expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was
studied in mice.
Material and Methods: A431 OSCC in vitro were exposed to 0 mM, 0.5 mM, 2.5 mM, or 5 mM of As2O3for 0 h, 24 h, 48 h and
72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related
signal decline with As2O3treatment. Next, mice with pharynx-implanted A431 cells received As2O3i.p. every 48 h at 0.0, 0.5,
2.5, or 5 mg/kg/day (n=6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on
days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.
Results: In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p,0.05) with increasing As2O3
dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p,0.05) in As2O3treated groups versus
controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while
body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was
reduced by As2O3treatment in a dose responsive pattern.
Conclusion: This study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of
the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As2O3treatment.
Citation: Zhang L, Wang K, Zhao F, Hu W, Chen J, et al. (2012) Near Infrared Imaging of EGFR of Oral Squamous Cell Carcinoma in Mice Administered Arsenic
Trioxide. PLoS ONE 7(9): e46255. doi:10.1371/journal.pone.0046255
Editor: Martin W. Brechbiel, National Institute of Health, United States of America
Received July 1, 2012; Accepted August 31, 2012; Published September 28, 2012
Copyright: ? 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge grant support from the National Natural Science Foundation of China (81170960, 81130028, 30970807 and 30570527) (for
BZ, BS), the International Cooperation and Exchanges Program of the National Ministry of Science and Technology (2009DFB30040) (for BS), Foundation of
Department of Science and Technology of Heilongjiang Province (GC12C303-2) (for BZ), the National Natural Science Foundation for Young Scholars of China
(81101086) (for KW), China Postdoctoral Science Foundation (20100471020) (for KW) and Medical Scientific Research Foundation of Heilongjiang Province Health
Department (2010-156) (for KW), and the National Institutes of Health (CA119342, CA154737, HL094470, HL073646, HL078631, HL113392, AR056468, NS073457)
and DOD (CA100623) (for GML). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
National Natural Science Foundation of China: http://www.nsfc.gov.cn/e_nsfc/desktop/zn/0101.htm; The International Cooperation and Exchanges Program of
the National Ministry of Science and Technology http://www.most.gov.cn/eng/cooperation/; Foundation of Department of Science and Technology of
Heilongjiang Province: http://220.127.116.11/kjt/tztg/200902/t20090210_98480.htm; National Natural Science Foundation for Young Scholars of China: http://
www.nsfc.gov.cn/e_nsfc/desktop/zn/0106.htm; China Postdoctoral Science Foundation: http://www.chinapostdoctor.org.cn/V3/Program/Main.aspx; Medical
Scientific Research Foundation of Heilongjiang Province Health Department: www.hljwst.gov.cn/; NIH: http://www.nih.gov/; DOD: http://www.defense.gov/.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (BZ); firstname.lastname@example.org (BS)
. These authors contributed equally to this work.
Oral squamous cell carcinoma (OSCC) is one of the ten most
common cancers , and is by far the most common malignant
neoplasm in the oral cavity . Despite advances in diagnosis and
therapy over the last three decades, the prognosis of OSCC
remains unsatisfying, with increasing high rates of relapse and
lymph node metastases. The overall five-year relative survival rate
remains less than 60% . Surgical therapy is the primary
treatment for OSCC. However, only a minority of patients benefit
from curative surgery, since most post-surgical patients succumb to
locally advanced or metastatic disease and many suffer from
marked facial disfigurement. Effective medical therapy to better
debulk or cure OSCC would be preferred, particularly for patients
not suitable for surgical resection. Unfortunately, OSCCs are
resistant to most conventional chemotherapeutic drugs.
Arsenic trioxide (As2O3, TRISENOX) is the most widely used
and studied arsenic-based anticancer drug . It is an effective
chemotherapeutic agent for treating relapsed or refractory acute
promyelocytic leukemia (APL) [5–7]. Abundant preclinical
evidence has shown that As2O3is also effective on solid tumors
PLOS ONE | www.plosone.org1 September 2012 | Volume 7 | Issue 9 | e46255
in liver , lung , ovary , gastric system , prostate ,
nasopharynx , as well as the oral cavity . However, the
anticancer mechanisms of As2O3 for inhibiting growth and
triggering apoptosis of cancer cells are not fully understood .
Recently, As2O3was reported to inhibit epidermal growth factor
receptor (EGFR) expression on the surface of OSCC cells in
EGFR is an important biomarker and useful prognostic
indicator in oral cancer , being widely overexpressed in
dysplasia and OSCC [18,19]. In OSCC patients, high EGFR
expression is usually associated with poor prognosis [1,18].
However, the in vivo therapeutic effect of As2O3 on EGFR
expression of OSCC tumor xenografts has not been reported.
Rapid and direct interrogation of biomarkers to risk stratify and
guide OSCC in patients would have significant clinical advantage
. Currently, immunohistochemistry (IHC)  and mRNA
expression  are commonly used to assess EGFR expression at
protein and gene levels in biopsy samples. Unfortunately, these ex
vivo methods cannot provide quantitative and spatial information
about OSCC EGFR expression in situ . Noninvasive molecular
imaging techniques for quantitatively assessing tumor biochemical
status  based on quantitative EGFR imaging would facilitate
initial in vivo selection of therapeutic carepaths and provide a tool
for longitudinal monitoring of early recurrence . Near infrared
(NIR) fluorescence imaging of EGFR is well suited to evaluation of
oral cavity lesions [25,26], given the superficial nature of oral
epithelial and submusosal cancers.
The goal of this study was to explore the feasibility of
noninvasively monitoring the therapeutic effect of arsenic trioxide
on EGFR expression of OSCC with NIR optical imaging in vivo.
In vitro results with confocal microscopy,
immunohistochemistry and flow cytometry
Fluorescence microscopy of targeted A-431 tumor cells
(Figure 1, A–D) confirmed probe uptake into the cell membrane
and cytoplasm. Less intense cellular fluorescence signal was
observed in all As2O3treated cells when compared with control
cells. Additionally, fluorescent signal intensity decreased in the
cells receiving higher As2O3concentrations (2.5 mM or 5.0 mM)
compared with those treated with 0.5 mM. The change in optical
contrast was corroborated with the levels of EGFR expression
(Figure 1, E–H) appreciated with immunohistochemistry micros-
Using flow cytometry, the EGFR expression in treatment group
decreased from 0 h, 24 h, 48 h, to 72 h while in the control group,
EGFR expression increased over the same time course (Figure 2).
Before treatment, cellular EGFR expression of 0.0 mM 0.5 mM,
2.5 mM and 5.0 mM groups were 70.461.3%, 73.461.0%,
71.861.5%, and 70.961.7% (n=3, p.0.05 for all comparisons).
At 72 h post treatment, cellular EGFR expression was 57.363.2%
(p,0.05), 29.962.2% (p,0.01), and 10.762.4% (p,0.01) in cells
treated with 0.5 mM, 2.5 mM, and 5.0 mM As2O3, respectively,
which were significant lower than the control group (74.461.8%, *
p,0.05, ** p,0.01).
In vivo NIR imaging of tumor response to As2O3
In vivo NIR fluorescent imaging of the tumor region of interest
(ROI) was performed dynamically and fluorescence luminosity
(signal intensity, SI) was measured before treatment (day 0) and 4, 8,
12 days aftertitrated As2O3treatment (Figure 3A). On day0 (before
As2O3treatment), the SI of the tumor ROIs among four groups
(0.0 mg/kg/day,0.5 mg/kg/day,2.5 mg/kg/day,5.0 mg/kg/day)
were not different: (2.2060.54)6104a.u., (1.8760.53)6104a.u.,
(2.1060.66)6104a.u. and (2.0160.44)6104a.u., respectively
(Figure 3B, p.0.05). From days 4 to 12, tumor SI increased
progressively in the control group but gradually decreased in As2O3
groups. On day 12, the tumor SI was decreased inversely with each
As2O3dosage (1.2160.35)6104a.u., (0.6560.16)6104a.u., and
(0.3560.14)6104a.u., respectively, versus the control group
(3.1860.63)6104a.u. (p,0.05, Figure 3C).
Inhibition of tumor growth by arsenic trioxide
At baseline, tumor volume did not differ among the treatment
groups (p.0.05, Figure 4A). Tumor volume growth rate on day
12, similar to tumor SI, was decreased (p,0.01) by 17.1%, 41.3%
or 56.4% with As2O3dosages of 0.5 mg/kg/day, 2.5 mg/kg/day
or 5.0 mg/kg/day, compared with control group, respectfully. In
contradistinction to the effects on tumor volume, serial As2O3
treatment did not affect body weight (p.0.05), suggesting no
grossly apparent toxicity.
EGFR immunohistochemical analysis
Immunohistochemical assays were carried out to correlate the
magnitude of tumor uptake (signal brightness) with the tumor
receptor density distribution. Tumor tissue sections from the
control group exhibited the highest grade of EGFR positive
staining (3+) compared with high dose treatment groups (5 mg/
kg/day group, 1+; 2.5 mg/kg/day group=1+,2+, p,0.05,
Figure 5), but no significant difference was noted versus the
0.5 mg/kg/day group (2+,3+, p.0.05). The grade of EGFR
positive stained tissue decreased as the dose of As2O3increased,
which were consistent with the in vitro and in vivo fluorescence
To our best knowledge, this is the first proof of concept report to
demonstrate the feasibility of using near infrared optical imaging
methodology to monitor the in vivo therapeutic effects of As2O3on
inhibiting of OSCC cells EGFR expression. The results presented
here indicated that the As2O3is active against EGFR of OSCC in
vivo and in vitro and the anti-EGFR effect of As2O3was dependent
on dose and duration of exposure, which is in agreement with
previous work .
Enthusiasm for promoting the clinical application of As2O3
(TRISENOX) has motivated the use of As2O3in treatment of
solid tumors, even though As2O3was initially approved as an
effective chemotherapeutic drug for acute promyelocytic leukemia
(APL) . The current widely acceptable anticancer mechanisms
of As2O3activity suggest that it induces apoptosis and influences
distinct signaling pathways, including mitogen-activated protein
kinases (MAPK), p53, activator protein-1 or nuclear factor kappa
B . Recently, other researchers have indicated that As2O3also
inhibits EGFR expression in cancer cells through p21 activation
leading to cell death via the EGFR-Ras-Raf-ERK1/2 pathway
based on in vitro or ex vivo methods [27,30], however, the in vivo
therapeutic effects of As2O3on pharyngeal OSCC tumors and the
relationship of this response to EGFR expression detected
noninvasively with NIR imaging has not been explored.
As we previously demonstrated that EGF-Cy5.5 uptake into the
OSCC cells was mediated by EGFR, the fluorescence signal
intensity was proportional to EGFR expression of tumor cells .
Less intense cellular fluorescence signal was observed in treated
cells compared with control cells, the fluorescent signal intensity
was inversely related to As2O3concentration (0.5 mM, 2.5 mM, or
5 mM), agreeing with IHC detection of less EGFR expression in
NIR In Vivo Imaging of EGFR in As2O3Treated OSCC
PLOS ONE | www.plosone.org2September 2012 | Volume 7 | Issue 9 | e46255
these treated cells (Figure 1). Of note, cell numbers decreased as
the arsenic trioxide concentration increased from 0 mM to 5 mM
in both fluorescent and immunostained images, indicating that
As2O3 induced a dose-dependent inhibition on tumor cell
proliferation, consistent with previous reports . In addition,
EGFR expression dynamically decreased in As2O3treatment cell
groups assayed by flow cytometry, indicating the inhibiting effect
of As2O3 on EGFR in dose and duration dependent manner
(Figure 2). A similar phenomenon was also shown in vivo NIR
imaging (Figure 3), which agreed with ex vivo EGFR expression
(Figure 5). The in vivo imaging results were correlated with the
results of in vivo inhibition of tumor growth by As2O3(Figure 4A),
further indicating that feasibility of using NIR optical imaging
method to noninvasively monitor the therapeutic effect and the
inhibiting effect of As2O3on tumor EGFR expression in vivo was
also dose and time dependent, consistent with previous reported
During the course of treatment, the maximum fluorescence
intensity of tumors was achieved 4 h post injection of EGF-Cy5.5,
which agreed with previous reports [24,32]. Some investigators
have found that fluorescence signal persisted for 4 to 5 days post
injection of fluorescent agent [33,34], but in current experiment,
EGF-Cy5.5 signal was barely detectable in the tumor site 96 h
post injection, with no indication of signal accumulation with serial
use. This likely reflects the very low dose of contrast administered
(1 nmol/kg) combined with the rapid tumor growth observed in
These results have several clinical implications. Firstly, near
infrared (NIR) optical molecular imaging of EGFR expression, as
demonstrated in this study, can noninvasively and sensitively
identify pharyngeal OSCC and can be used to longitudinally
monitor and guide As2O3treatment. While the diagnostic value of
NIR fluorescent probes is frequently challenged by tissue
penetration depth, despite lower background absorption, superfi-
cial neoplasms like oral squamous cell carcinoma are highly
accessible to interrogation of the lesion in situ, providing clear
advantages over biopsy and IHC for early tumor detection and for
therapeutic management. Secondly, direct optical molecular
imaging should provide at least semi-quantitative information
regarding the spatial and temporal expression of EGFR in vivo.
The opportunity to standardize EGFR expression level measure-
ments would accommodate the development of improved,
evidenced-based guidelines for the assessment and management
of oral OSCC. In particular, noninvasive targeting imaging of
early EGFR responses to medical therapy could indicate
effectiveness, whereas particularly when tumor volume shrinkage
is often delayed response to therapy using traditional extracellular
space contrast agents .
Moreover, the accessibility of OSCC for direct topical or
subcutaneous injection, may facilitate the use of EGF peptide–
based contrast agents at lower the dosage requirements, with
Figure 1. Effects of As2O3on A431 cell EGFR expression. Cells were exposed to different concentrations of As2O3. At 48 h post-treatment, cells
were assessed by fluorescence microscopy for visualization of the intake of EGF-Cy5.5 and cell immunohistochemistry for assay of EGFR expression.
A–D, representative fluorescence images of different groups (Scale bar=100 mm), Cy5.5 was pseudo-colored red, DAPI was pseudo-colored blue; E-H,
representative images of cellular EGFR Immunohistochemistry assay (Scale bar=100 mm), diaminobenzidine (DAB) showed as brown color
represented EGFR expression and hematoxylin showed as blue color indicated the cellular nuclear. Interestingly, cell numbers decreased as the
arsenic trioxide concentration increased from 0 mM to 5 mM in both fluorescent and immunostained images, indicating that As2O3induced a dose-
dependent inhibition on tumor cell proliferation as previously reported .
Figure 2. The cellular EGFR expression percentage of different
treatment groups in vitro assessed by flow cytometry. The
dynamic EGFR cellular expression percentage after treatment of
different concentrations of As2O3(0 mM, 0.5 mM, 2.5 mM, 5.0 mM) varied
with time (*p,0.05, **p,0.01). All experiments were carried out in
triplicate; each point represents the mean 6 standard error values.
NIR In Vivo Imaging of EGFR in As2O3Treated OSCC
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accelerate time to peak signal, and reduced residual contrast
washout. In fact, one might envision very short interval between
contrast administration and follow-up NIR examination that
would readily be accommodated in the workflow patterns of
dentists and oral maxillary surgeons. Finally, although no body
weight loss due to As2O3was determined in current experiment,
the issue of potential toxicities with systematic administration of
As2O3remains. The oral superficial nature of OSCC suggests that
direct local low dose therapy, applied topically (e.g., oral rinse) or
with subcutaneous injection under image guidance would be
feasible and perhaps optimal.
This study presented a proof-of-concept that noninvasive optical
imaging could be used to evaluate the therapeutic effect of As2O3
by quantifying EGFR expression, likely As2O3 inhibits EGFR
expression through p21 activation leading to cell death via the
EGFR-Ras-Raf-ERK1/2 pathway [27,30]. However, the linearity
of the EGFR expression by tumor cells response to As2O3therapy
is not clear and will require further study to elucidate. Although
useful for preliminary preclinical research, the use of the
prototypical EGF-Cy5.5 agent in this study may require modifi-
cation for translation. Specifically, the selection of an EGF
receptor antagonist as homing ligand may be preferred to avoid
activation of downstream EGFR signaling . The substitution
of the fluorophore Cy5.5 with a higher wavelength NIR dye would
improve tissue penetration and ideally would be regulatory agency
approved or approvable in order to expedite clinical experimen-
tation . Furthermore, more appropriate modeling methods
accounting for variability attributable to nonspecific binding and
contrast delivery efficiency (e.g., blood flow, vascular permeability,
blood vessel density and hydrostatic pressure, etc) on in vivo
receptor imaging accuracy is desirable .
Figure 3. In vivo dynamic near-infrared fluorescent imaging of A-431 tumor models. A: The representative fluorescence images of the
tumor regions in mice were acquired at 4 h post injection of EGF-Cy5.5. Fluorescence signal from Cy5.5 was pseudo-colored red. B: The dynamic
measurement comparison of fluorescence intensity of tumor in different groups. It was demonstrated that the fluorescence intensity in the tumor
regions were changed with time (p,0.05). On day 0 (before As2O3treatment), there was no significant difference of signal intensity of tumors
between treatment and control groups (p.0.05). On day 4, 8, 12 (after As2O3treatment), the signal intensity of EGF-Cy5.5 uptake by control group
(0 mg/kg/day As2O3) gradually increased, while the intensities in other three groups with different concentrations (0.5 mg/kg/day, 2.5 mg/kg/day,
5.0 mg/kg/day) of As2O3treatment gradually decreased (p,0.05). C: The in vivo fluorescence intensity was compared between post-treatment (on
day 12) in four different groups compared with respective pre-treatment (on day 0). All plots are representative of results from groups of mice treated
under the same experimental conditions. Each point represents the mean values (n=6/group, *p,0.05, **p,0.01).
NIR In Vivo Imaging of EGFR in As2O3Treated OSCC
PLOS ONE | www.plosone.org4 September 2012 | Volume 7 | Issue 9 | e46255
This study demonstrated that the response of EGFR expression
by oral squamous carcinoma implanted within the mouse pharynx
can be treated effectively with As2O3 and the response to
treatment can be noninvasively assessed with EGF-Cy5.5 and
NIR molecular imaging techniques. These results suggest that oral
NIR molecular imaging with EGF-Cy5.5 based probes could
enhance early detection as well as facilitate image based guidance
for effective chemotherapeutic treatment of OSCC with As2O3.
Materials and Methods
Fluorochrome probe generation
The EGFR specific targeting NIR fluorescent agent, EGF-
Cy5.5, was developed by coupling EGF (ImClone Systems,
Branchburg, N.J., USA) to cyanine dye 5.5 (Cy5.5) molecules
through a monofunctional N-hydroxysuccinimide (NHS) ester
(Cy5.5-NHS, GE Healthcare, Piscataway, N.J., USA) according to
our previously reported protocol . Briefly, EGF (35 mg,
233.45 nmol, ImClone Systems, Branchburg, N.J., USA) was
mixed with Cy5.5-NHS (4.2 mg, 1242.5 nmol, GE Healthcare,
Piscataway, N.J., USA) in H2O (3.0 ml) in darkness at 4uC for 2 h,
then the reaction was quenched by adding 3.0 ml of 5% acetic
acid (HOAc). The EGF-Cy5.5 was isolated using a PD-10
disposable column (GE Healthcare, Piscataway, N.J., USA),
lyophilized, and resuspended in saline at a concentration of
1 mg/ml, then stored at 220uC in darkness until use.
In vitro cell studies
Human epidermoid carcinoma A431 cells (Institute of Bio-
chemistry and Cell Biology at the Chinese Academy of Sciences,
Shanghai, China) that constitutively express a high levels of EGFR
 were obtained and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen Corp., Carlsbad, CA, USA)
supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco
BRL, Cleveland, Ohio, USA). In these series of experiments, all
cells were incubated under humidified atmosphere of air/CO2
(19:1) at 37uC. A431 cells were plated in flat bottomed 24-well
microtiter plates on coverslips at a density of 16105cells/well.
After 24 h, cells were washed three times with phosphate buffered
saline (PBS, pH 7.2), then treated with 0.0 mM, 0.5 mM, 2.5 mM,
5.0 mM As2O3(Sigma Chemical Co., St. Louis, MO, USA) in
calcium- 0.9% sodium chloride solution for either 0 h, 24 h, 48 h,
or 72 h. After treatment, all cells were washed three times with
Figure 4. Monitoring tumor growth and body weight of tumor-bearing mice during treatment. Tumor growth (A) and body weight (B) of
tumor-bearing mice treated with saline (as untreated control group), arsenic trioxide (ATO) at 0.5 mg/kg, 2.5 mg/kg or 5.0 mg/kg daily for 12 days.
Six mice were used in each group. The tumor volume and body weight of all four groups were also measured every two days. Tumor volume was
calculated according to the formula V=(a6b2)/2 where a and b represent the length and width of the tumor. Measurements were continued to 12th
day. p,0.05 is a significant difference between control and treatment groups.
Figure 5. EGFR immunohistochemical assay of the tumor sections from different groups. A: 0 mg/kg As2O3(control). The strongest red-
brownish membrane-bound immunostaining on the A431 tumor tissue slice reflected the abundant over expression of EGFR (+++). B: 0.5 mg/kg
As2O3treatment group. Strong brownish membrane staining indicated plentiful of EGFR expression (++,+++). C: 2.5 mg/kg As2O3treatment group
demonstrated moderate to low EGFR expression (+,++). D: 5.0 mg/kg As2O3 treatment group showed weak EGFR expression (+). (Scale
NIR In Vivo Imaging of EGFR in As2O3Treated OSCC
PLOS ONE | www.plosone.org5September 2012 | Volume 7 | Issue 9 | e46255
Cell immunohistochemistry microscopy
Cellular EGFR expression was evaluated by immunohisto-
chemistry. Briefly, cells were fixed with 4% paraformaldehyde in
4uC PBS for 25 min then washed three times with PBS. After
blocking to reduce nonspecific antibody binding for 30 min,
monoclonal rabbit anti-human EGFR antibody (1:200, COOH
terminus; Santa Cruz Biotechnology, Santa Cruz, Calif., USA)
was incubated with the cells at 37uC for 2 h, then the unbound
ligand was removed in three washes of PBS. Next, the cells were
treated with a biotinylated goat anti-rabbit IgG (Southern
Biotechnology Associates, Birmingham, Ala., USA), followed with
a streptoavidin-biotin peroxidase reagent (Histofine kit; Nichirei
Biosciences Inc., Tokyo, Japan). Finally, diaminobenzidine (DAB)
and 1% hydrogen peroxidase were applied as chromogen, and the
cells were counterstained with hematoxylin. Imaging was per-
formed with a Nikon E800 microscope using a Nikon DXM 1200
digital camera (Nikon, Tokyo, Japan).
Fluorescent microscopy assay
The treated cells were incubated with 500 ml EGF-Cy5.5
(20 nM final concentration) for 30 minutes at 37uC in darkness.
After incubation, all the cells were washed three times with PBS.
Fluorescence microscopy (with an Olympus microscope outfitted
with NIR diode sources and filters) of tumor cells was performed
for visual confirmation of EGF-Cy5.5 uptake. Diamidino-phenyl-
indole (DAPI) was used to stain cell nuclei. In the microscopic
images EGF-Cy5.5 was pseudo-colored red (emission at 680–
710 nm), while DAPI was pseudo colored blue (emission at
Flow cytometry assay
After incubation with EGF-Cy5.5, A431 cells were also
suspended with trypsin solution and centrifugal elutriation twice
in PBS. Quantification of fluorescent intensity of EGF-Cy5.5
binding to EGFR was assessed using flow cytometry (FACSort,
Becton Dickinson, Franklin Lakes, N.J., USA). All experiments
were replicated in triplicate.
All experimental protocols were pre-approved by the Experi-
mental Animal Ethic Committee of Harbin Medical University,
China (Animal Experimental Ethical Inspection Protocol No. -
HAYIWEIDONGSHENZi 2010035). Use of animals was con-
firmed with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH
Publication No. 85–23, revised 1996).
In vivo mouse studies
Athymic nude mice (half male and half female, BALB/c-nu/nu,
4–6 weeks old, 18–22 g) from Vital River Laboratory Animal
Technology Co. Ltd (National Science Institute, Beijing, China)
were housed five per cage and provided with UV-sterilized pellet
chow and autoclaved distilled water. Animals were maintained in
a pathogen-free mouse colony at Harbin Medical University
(Harbin, China). A431 cells (56106) in 200 ml were slowly injected
into the floor of the mouth of anesthetized mice with isoflurane
. When the tumors reached 0.4 to 0.6 cm in diameter (1–2
weeks after inoculation), the tumor-bearing mice were subjected to
in vivo NIR imaging.
In vivo NIR optical imaging
To characterize EGFR expression in A431 cells in vivo, an
eXplore Optix time-domain fluorescence imaging system (ART/
GE Healthcare, Saint-Laurent, Quebec, Canada) which featured a
667-nm excitation pulse laser, with a 710-nm emission bandpass
filter was used to image the tumor-bearing mice. The eXplore
OPTX-optView software installed on the imaging system was used
for data acquisition and processing.
The tumor-bearing mice were divided into a single control and
three As2O3 treatment groups (0 mg/kg/day, 0.5 mg/kg/day,
2.5 mg/kg/day, 5.0 mg/kg/day, 6 mice/group). As2O3 was
injected intraperitoneally (i.p.) on days 0, 2, 4, 6, 8, and 10. The
control group received an equal volume of saline under the
identical conditions. For in vivo characterization of EGFR, mice
were sedated with isoflurane and intravenously (i.v.) injected with
1 nmol/kg EGF-Cy5.5 diluted in 0.3 ml saline via the tail vein
before As2O3treatment (on day 0) and on days 4, 8, 12 thereafter
(Figure S1). Fluorescence images were acquired at 4 h post
injection of EGFR targeting agent, near the peak intensity of the
fluorescence signal [24,32]. Tumor volumes and body weights of
all animals were measured every 2 days, which were used as
indicators of efficiency and systemic toxicity of the treatment,
respectively. Tumor volume was calculated according to the
formula V=(a6b2)/2 where a and b represent the length and
width of the tumor . Measurements were continued to the
12th day of study.
Mice were euthanized at the termination of the study. Tumors
were harvested and fixed with formalin and embedded in paraffin
for immunohistochemical analysis. Tissues were sectioned at
8 mm-thickness de-paraffinized, microwave pretreated, and then
incubated with 0.3% hydrogen peroxide for 30 min. EGFR
labeling and analysis were accomplished by the same two-step
method as described above in cell IHC methods. The immuno-
reactivity in tumor cells were classified and scored as follows: the
intensity of staining was scored as 0, no staining (,10%); 1+, weak
(10–25%); 2+, moderate (26–50%); 3+, strong (51–100%), which
included at least 1000cells per sample within 5 regions of interest
(ROI, 200 cells/ROI) [2,18]. Tumor receptor density distribution
analyzed by IHC was correlated the magnitude of tumor contrast
uptake (i.e., signal intensity).
Data were presented as mean 6 standard deviation. The effect
of time and As2O3 dose on cellular EGFR expression was
statistically analyzed using a factorial design ANOVA. Fluores-
cence intensity was defined as total photon counts/pixel within
manually inscribed region of interest (ROI) area divided by the
laser pulse time (ms) and unit time . The repeated-measure
analysis with covariates was used to assess the effects of time and
As2O3dose on fluorescence intensity, tumor size, body weights
adjusted for their baseline values, respectively. Subanalysis of
specific paired comparisons utilized student’s t test. Nonparametric
tests (Mann-Whitney) were used to compare the difference of IHC
results. Analyses were performed using the SPSS statistical
software package (SPSS 18.0; SPSS, Inc., Chicago, Ill., USA).
P,0.05 was considered as statistically significant.
imaging. When the tumors reached 0.4 to 0.6 cm in diameter
(1–2 weeks after inoculation), the tumor-bearing mice were divided
into a single control and three As2O3treatment groups (0 mg/kg/
day, 0.5 mg/kg/day, 2.5 mg/kg/day, 5.0 mg/kg/day, 6 mice/
group). As2O3was injected intraperitoneally (i.p.) on days 0, 2, 4,
The protocol of As2O3 treatment and NIR
NIR In Vivo Imaging of EGFR in As2O3Treated OSCC
PLOS ONE | www.plosone.org6 September 2012 | Volume 7 | Issue 9 | e46255
6, 8, 10 (m). The control group was injected with an equal volume
of saline under the identical conditions. For in vivo NIR imaging,
mice were sedated with ketamine/xylazine and intravenously (i.v.)
injected with 1 nmol/kg EGF-Cy5.5 diluted in 0.3 ml saline via
the tail vein before As2O3treatment (on day 0) and on days 4, 8,
12 (&) after As2O3treatment. (The arrow indicated the tumor
We thank Jon N. Marsh Ph.D. from Washington University School of
Medicine for assistance in preparing the manuscript.
Conceived and designed the experiments: LZ KW JC GL BS BZ.
Performed the experiments: LZ KW FZ JC. Analyzed the data: LZ FZ
KW WH. Contributed reagents/materials/analysis tools: LZ KW WH GL
BS BZ. Wrote the paper: LZ KW GL BZ.
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