Group Title: Journal of Experimental & Clinical Cancer Research 2009, 28:48
Title: The effect of blue light exposure in an ocular melanoma animal model
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Title: The effect of blue light exposure in an ocular melanoma animal model
Series Title: Journal of Experimental & Clinical Cancer Research 2009, 28:48
Physical Description: Archival
Creator: Di Cesare S
Maloney S
Fernandes BF
Martins C
Marshall JC
Antecka E
Odashiro AN
Dawson WW
Burnier MN
Publication Date: 39910
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Bibliographic ID: UF00100287
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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The effect of blue light exposure in an ocular melanoma animal
Sebastian Di Cesare*', Shawn Maloney', Bruno F Fernandes',
Claudia Martins', Jean-Claude Marshall', Emilia Anteckal,
Alexandre N Odashiro', William W Dawson2 and Miguel N Burnier Jr'

Address: 'The Henry C Witelson Ophthalmic Pathology Laboratory and Registry, McGill University Health Center, Montreal, PQ, Canada and
2Department of Ophthalmology, University of Florida, Gainesville, Fl, USA
Email: Sebastian Di Cesare*; Shawn Maloney;
Bruno F Fernandes; Claudia Martins; Jean-
Claude Marshall; Emilia Antecka;
Alexandre N Odashiro; William W Dawson;
Miguel N Bumier
* Corresponding author

Published: 7 April 2009 Received: 6 February 2009
journal of Experimental & Clinical Cancer Research 2009, 28:48 doi: 10.1 186/1756-9966-28-48 Accepted: 7 April 2009
This article is available from:
2009 Di Cesare et al; licensee BioMed Central Ltd.
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 work is properly cited.

Background: Uveal melanoma (UM) cell lines, when exposed to blue light in vitro, show a
significant increase in proliferation. In order to determine if similar effects could be seen in vivo,
we investigated the effect of blue light exposure in a xenograft animal model of UM.
Methods: Twenty New Zealand albino rabbits were injected with 1.0 x 106 human UM cells (92.1)
in the suprachoroidal space of the right eye. Animals were equally divided into two groups; the
experimental group was exposed to blue light, while the control group was protected from blue
light exposure. The eyes were enucleated after sacrifice and the proliferation rates of the re-
cultured tumor cells were assessed using a Sulforhodamine-B assay. Cells were re-cultured for I
passage only in order to maintain any in vivo cellular changes. Furthermore, Proliferating Cell
Nuclear Antigen (PCNA) protein expression was used to ascertain differences in cellular
proliferation between both groups in formalin-fixed, paraffin-embedded eyes (FFPE).
Results: Blue light exposure led to a statistically significant increase in proliferation for cell lines
derived from intraocular tumors (p < 0.01). PCNA expression was significantly higher in the FFPE
blue light treated group when compared to controls (p = 0.0096).
Conclusion: There is an increasing amount of data suggesting that blue light exposure may
influence the progression of UM. Our results support this notion and warrant further studies to
evaluate the ability of blue light filtering lenses to slow disease progression in UM patients.

Background rate for UM ranges from 4.3-10.9 cases per million,
Uveal Melanoma (UM) is the most common primary depending on the specific criteria used to diagnose this
malignant intraocular tumor in adults [1]. The incidence disease [2]. Although it is a relatively uncommon malig-

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Journal of Experimental & Clinical Cancer Research 2009, 28:4E

nancy, approximately 50% of all patients initially diag-
nosed with UM will end up developing liver metastasis
within 10-15 years [3]. Predispositions to this disease
include the presence of choroidal nevi, which occur quite
frequently within the aging population.

With age, the human lens becomes progressively more
yellow. This process is thought to effectively filter more
blue light from passing through the yellowed lens [4,5].
Following cataract surgery, the removal of the aged lens is
accompanied by loss of natural ability to filter blue light
(500-444 nm, The CIE International Diagram for Blue

Further studies have suggested that blue light exposure
may play a role in the malignant transformation of
melanocytes, which can eventually lead to the develop-
ment of melanoma [6]. It has been previously shown that
rats subjected to long-term blue light exposure developed
intraocular masses that were pathologically diagnosed as
ocular melanoma [7]. A recent statistical study has dem-
onstrated an increased risk of developing dysplastic skin
nevi in children previously treated with neonatal blue-
light therapy at birth [8]. Several well-documented risk
factors for the development of UM have been identified,
including age, iris color and skin pigmentation [2]. Even
though sunlight exposure is considered a significant risk
factor by some [9], the relationship between sunlight
exposure and UM development remains controversial

It has been demonstrated in primates that blue light can
mediate the production of reactive oxygen species (ROS)
in the posterior segment of the eye. This ROS production
due to blue light exposure could be responsible for cellu-
lar damage to the retinal pigment epithelial (RPE) cells
[11]. The production of these ROS may therefore play an
important role in the development of age-related macular
degeneration [12].

Our laboratory has previously shown that the prolifera-
tion rates of human uveal melanoma cell lines increase
significantly in vitro after exposure to relatively high
amounts of blue light [6]. We therefore propose to extend
these preliminary in vitro studies to investigate the poten-
tial effects of blue light in an in vivo ocular melanoma ani-
mal model [13].

The animal model was carried out in compliance with the
Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and
Vision Research. The approval of both the Animal Care
Committee and the Ethics Subcommittee at McGill Uni-
versity was obtained prior to all experiments.

Twenty female New Zealand albino rabbits (Charles River
Canada, St-Constant, Quebec) were randomly divided
into two groups, control and experimental, with mean ini-
tial weights of 3.2 + 0.18 kg and 3.2 0.17 kg respectively.
Female animals were used to avoid aggressive conflicts
that can occur when group-housing male animals. The
animals were immunosuppressed daily using intramuscu-
lar injections of cyclosporine A (CsA; Sandimmune 50
mg/ml, Novartis Pharmaceuticals Canada Inc., Dorval,
Quebec, Canada) in order to avoid rejection of the human
cells. CsA administration was maintained throughout the
8-week experiment to prevent tumor regression. The dos-
age schedule recommended in previous studies was
employed: 15 mg/kg/day, 3 days before cell inoculation
and during 4 weeks thereafter, followed by 10 mg/kg/day
during the last 4 weeks of the experiment [13]. CsA doses
were adjusted weekly according to the animal weight to
compensate for any weight loss during the experiment.

Cell line and cell injection procedure
The injection procedure and subsequent animal handling
were carried out as previously described [13]. The 92.1
primary human uveal melanoma cell line [14], kindly
provided by Dr. Antonia Saomil from the Instituto Uni-
versitario de Oftalmobiologia Aplicada (IOBA), Univer-
sity of Valladolid, was used. This selection was based on
previous studies performed in our laboratory where this
cell line demonstrated high proliferative and invasive
potential in vitro [15]. The cells were maintained at 37 C
in a humidified 5% CO2-enriched atmosphere (Thermo
Forma Series II Water Jacketed CO2 Incubator, Fisher Sci-
entific Limited, Ontario, Canada). The cells were cultured
in RPMI-1640 medium (Invitrogen, Burlington, Ontario,
Canada), supplemented with 5% heat inactivated fetal
bovine serum (FBS; Invitrogen), 1% fungizone (Invitro-
gen), and 1% penicillin-streptomycin (Invitrogen). One
million cells (cellular viability greater than 99%) sus-
pended in 0.1 ml of RPMI-1640 media were injected into

Figure I
Gross & histopathological images of an enucleated
rabbit eye. A) Cross section of the right eye (O.D) from a
control group rabbit, displaying a large intraocular mass and
hemorrhage, at week 5 of the experiment. B) Photomicro-
graph of the same rabbit eye (O.D), H&E displaying hemor-
rhage surrounding the tumor cells (200x).

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Journal of Experimental & Clinical Cancer Research 2009, 28:48

1: fl .--
.. -- -.'-'. .

R *. "- ; ** . :
*- .. r.% M- s ; A^.' 6; */";- . -' ". .':

*.. f *.. . .* ;: ; *. 4 -* '1 -- ., . ., .. .. ; -,. .
;.'"-:'-- 'U" t; .. "- ^ ..

k." .' -. .- N' ^ .. '' *.. . .,r. ,

J.. m -. -M .d r ..
^:,' .* -..,. ,* ". t , .: .,.. , ,, ,,a.* '.. . =: .. ., -.
. -_ .. . r.
*', .'. '. *:* ..---.. . ; : if

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S .' 2 4
C * -. *60
.' Opt.

,, 'E,,,, , -. .. *n 1
.;,--. .. *'- 8 = 0 0
P . *. .

: -- *4, Blue Light

Figure 2
', ; '- - 80 p= .0.6.

cells (92.1) from a rabbit in the control group (200x). C) Negative Control (200x). D) Box and Whisker plot depicting the rel-
ative percentage of PCNA positivity between rabbits exposed to blue light, and those not exposed.
-. .. ,'. .- .. (-) (+)

the suprachoroidal space of the right eye of each rabbit
according to a previously described technique [13]. Keta-
mine (35 mg/kg; Vetalar, Vetrepharm Canada Inc., Bel-
leville, Ontario, Canada) and xylazine (5 mg/kg; Anased,
Novopharm Limited, Toronto, Ontario, Canada) were
used as anesthetics during the surgical procedure.

Blue Light Exposure
The 20 rabbits used in this experiment were randomly
divided into two separate groups of 10 rabbits each. The
experimental group was exposed to blue light 8 hours per
day for the duration of the 8-week experiment. The ani-
mals were group-housed in a large pen into which the
blue light-emitting apparatus was placed. The apparatus
consisted of a large metal cage in which twenty-four 6600

k bulbs were suspended, each covered by a sheet of co-
extruded polycarbonate film (Rosco, Color Filter #74
Night Blue) that allowed light only in the blue portion of
the spectrum to pass through. This apparatus was placed
in the middle of the pen, with suspended bulbs reaching
to approximately 6" from the ground to achieve maximal
light exposure at eye level. Additionally, the pen was lined
with 3' high reflective aluminum to ensure adequate blue
light exposure in all areas of the pen. As a rabbit's gaze is
typically 10 to 15 degrees below the horizontal plane, 3'
high reflective aluminum was adequate to ensure contin-
uous blue light exposure in the direction of gaze. All lights
were connected to a timer that turned on at 11 am and
turned off at 7 pm daily. Protective goggles were provided
to all personnel entering the housing area during the

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Journal of Experimental & Clinical Cancer Research 2009, 28:48

Figure 3
Cytospins prepared from re-cultred 92.1 cells from rabbit eyes (OD) stained for HMB-45. A) Cytospin of UM cells
(92.1) isolated from the right eye of a control group rabbit. B) Cytospin of UM cells (92.1) isolated from the right eye of a blue
light treated rabbit. C) Cytospins of CMCs (92. I) isolated from the blood (buffy coat) of a control group rabbit. D) Negative
Control (92.1) (400x).

period of blue light exposure. The control group was in
the adjacent pen, which was covered by a polycarbonate
film (Rosco, Color Filter #15 Deep Straw) that ensured
proper blockage of any light within the blue portion of the
visible spectrum (500-444 nm, CIE International Dia-
gram for blue light ranges) from entering the control pen.

Indirect ophthalmoscopy of dilated pupils using Tropica-
mide (Alcon Canada Inc., Mississauga, Canada; Mydria-
cyl, Alcon Canada Inc.) was performed before cell
inoculation to rule out any existing ocular pathologies,
and weekly after cell inoculation to clinically document
intraocular tumor development.

In order to document the time-course of the disease, par-
ticularly the development of metastasis, one animal per
group was euthanized per week starting at two weeks post-
inoculation of cells into the eye. The selection criterion
was based on the appearance of the animal, signs of CsA
toxicity and veterinary recommendations. The remaining
rabbits (n = 4) were sacrificed at the end of the experi-
ment. The method of euthanasia was exsanguination by
cardiac puncture following anesthesia using intramuscu-
lar ketamine-xylazine (35 mg/kg-5 mg/kg). An autopsy
was performed on every animal that was sacrificed. The
enucleated eyes and other organs with possible metastatic
disease such as lungs, livers and kidneys were collected,

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Journal of Experimental & Clinical Cancer Research 2009, 28:48

A 1.0

c 0.7
0 0.5

Control Group

Blue Light Group

Control Group Blue Light Group

p < 0.0001

Control Group

Blue Light Group

Figure 4
Box and Whisker plots depicting the change in cellular proliferation of re-cultured 92.1 cells from rabbit eyes
(O.D) when exposed to blue light. A) Change in cellular proliferation of primary tumors after 48 h incubation. B) Change
in cellular proliferation of primary tumors after 72 h incubation. C) Change in cellular proliferation of isolated CMCs after 48 h

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p = 0.0112

0 -p = 0.0018






Journal of Experimental & Clinical Cancer Research 2009, 28:4E

macroscopically examined and preserved in 10% phos-
phate buffered formalin. Formalin-fixed, paraffin-embed-
ded sections of the collected specimens were stained with
hematoxylin and eosin for histopathologic assessment.

Re-Culturing of Cells Post-Euthanasia
The right eye of each rabbit was processed prior to for-
malin fixation in order to acquire a fresh tumor sample
from each rabbit. Cells were cultured in a 6-well plate in
5% FBS supplemented RPMI and grown to confluence
before seeding for proliferation assay experiments. All
blood collected from cardiac puncture of rabbits during
euthanasia was processed via the Ficoll-Paque' Plus
Method (Amersham Biosciences) in order to harvest and
culture the buffy coat. This was done in order to capture
and document presence of circulating malignant cells
(CMCs) throughout the duration of the experiment.
CMCs were allowed to adhere to the bottom of the 6-
well plate, while remaining non-adherent white blood
cells were washed off during subsequent media changes.
CMCs were allowed to grow to confluence prior to seed-
ing the proliferation assays. All re-cultured cells (primary
tumors, CMCs) were passage only once in order to
maintain any phenotypic changes these cells may have
acquired in vivo.

Immunohistochemistry was performed using the Ventana
BenchMark fully automated machine. The fully auto-
mated processing of bar code labeled slides included bak-
ing of the slides, solvent-free deparaffinization, and CC1
(Tris/EDTA buffer pH 8.0) antigen retrieval. Slides were
incubated with a mouse monoclonal anti-human Prolifer-
ating Cell Nuclear Antigen (PCNA) antibody (dilution
1:200; Dako Canada Inc., Mississauga, Ontario; Clone
PC10) for 30 min. at 370C, followed by application of
biotinylated secondary antibody (8 min. at 37 C) and an
avidin/streptavidin enzyme conjugate complex (8 min at
37C). Finally, the antibody was detected using the Fast
Red chromogenic substrate and counterstained with
hematoxylin. As positive controls, sections of human
small intestine and colon were used for the PCNA anti-
body. For negative controls the primary antibody was
omitted. Sections were analyzed for PCNA nuclear expres-
sion in tumor samples and surrounding ocular tissues. A
total of 10 rabbit xenograft (92.1) UMs were used for this
analysis. Samples were also independently graded as
either positive or negative for PCNA nuclear expression in
each of the samples by two different pathologists. The per-
centage and intensity of overall tumor positivity were also

Cytopsins of all re-cultured cells (primary tumor, CMCs)
were made using a Cytospin3 machine (Shandon). Cells

from culture were diluted to a concentration of 250,000
cells/ml, and a 300 iL solution at that concentration was
placed in each spin to be evenly distributed on each slide.
All slides were then immunostained with a primary anti-
human mouse monoclonal antibody against Melano-
some (Dako Canada Inc., Mississauga, Ontario; Clone
HMB-45) using the Ventana"T automated immunostain-
ing machine programmed to use a standard Avidin-Biotin
Complex method. HMB-45 is a well-established marker
used by pathologists in order to identify the presence of
uveal melanoma cells [16,17]. These stainings were done
in order to ensure that the re-cultured cells were actually
uveal melanoma cells.

Proliferation Assay
The Sulforhodamine-B based assay kit (TOX-6, Sigma-
Aldrich, St. Louis, Missouri, USA) was performed accord-
ing to the National Cancer Institute protocol [18]. Re-cul-
tured cells obtained from the rabbits (primary tumor,
CMCs) were seeded in a 96-well plate at a concentration
of 2.5 x 103 cells per well, with six wells per cell line from
each group (blue light, control). Cells were allowed to
adhere overnight and incubate for 48 and 72 hours. Fol-
lowing both the 48 and 72 hour incubation periods, cells
were fixed to the bottom of the wells using a solution of
50% Trichloroacetic acid (TCA) for 1 hour at 40C. Plates
were then rinsed with distilled water to remove the TCA
and excess media and were air-dried. The Sulforhodam-
ine-B dye solution was then added to each well and
allowed to stain for 30 minutes. The Sulforhodamine-B
solution was subsequently removed by washing with a
1% acetic acid solution and once more allowed to air dry.
The dye that had become incorporated into the fixed cells
at the bottom of the wells was solubilized in a 10 mM
solution of Tris base solution. The absorbance of the sol-
ute was measured using a microplate reader at a wave-
length of 565 nm.

Statistical Analysis
Results from the proliferation assays for both time points (48
h, 72 h) were analyzed using the Student's t-test. A result was
considered significant when a p-value of < 0.05 was obtained
for each t-test performed. Results from the PCNA staining
were interpreted using a Correlation analysis. A correlation
was drawn by comparing PCNA staining intensity with
exposed or non-exposed rabbits. A result was considered sig-
nificant when a p-value of < 0.05 was obtained.

At the first week timepoint, 2 animals from the control
group and 3 animals from the experimental group had
fundoscopically detectable intraocular masses. By week 3,
the total number of visible tumors was 5 and 4 in the con-

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trol and experimental groups, respectively. These numbers
remained unchanged until the end of the experiment.

Histopathological Studies
Macroscopically detectable intraocular masses were seen
in 6 animals of the control group and 4 animals in the
experimental group (Figure 1). Histopathological evalua-
tion of the enucleated eyes revealed tumors in 7 of the ani-
mals in the control group and in 5 of the experimental

No macroscopic metastatic disease was found in either
group. Serial sections of the animals' lungs revealed met-
astatic disease in 4 animals in the control group and in 4
animals in the experimental group. No liver metastasis
was seen. The differences seen between the two groups
were not statistically significant.

Re-Culturing of Cells Post-Euthanasia
A total of 5 primary tumors from the control group and 4
primary tumors from the experimental group were suc-
cessfully re-cultured (1 passage) for subsequent use in the
cytospin analysis and proliferation assays. In addition, 2
CMC cultures from the control group and 1 from the
experimental group were retrieved for subsequent cyt-
ospin and proliferation assay analysis.

All of the FFPE control rabbit eyes were negative for PCNA
(n = 5). The FFPE blue light treated group had 3 rabbit eyes
that were highly positive (85-100%), and 2 rabbit eyes that
had mild positivity when stained with PCNA (n = 5). A
Correlation analysis was preformed to relate staining inten-
sity and blue light exposure. Statistically significant results
were obtained (n = 10, r = 0.8, p = 0.0096) (Figure 2).

All re-cultured samples (primary tumors, CMCs) stained
positive for the monoclonal mouse anti-human Melano-
some marker (Figure 3). This specific positivity indicates
that all re-cultured cells used in the proliferation assays
were indeed the human uveal melanoma cell line 92.1
that was initially inoculated in the eyes of the rabbits.

Proliferation Assay
Cells from the blue light treated group proliferated signif-
icantly faster than the control group cells at the 48 h (p =
0.0112) and 72 h (p = 0.0018) time points. The CMCs iso-
lated from the blue light group proliferated significantly
faster (48 h) than the cells from the control group (p <
0.0001) (Figure 4).

Current hypotheses indicate that several environmental
and genetic factors may play a role in the progression of

uveal melanoma formation [19-21]. Typical phenotypic
progression of this disease usually begins with the appear-
ance of benign nevi. Later events include the transforma-
tion of the cells within the nevi to a spindle-cell and
eventually epithelioid-cell uveal melanoma. Epithelioid
cells are considered the most aggressive type of uveal
melanoma cells and carry the worst prognosis. This gener-
alized progression towards a more malignant phenotype
may also be influenced by exposure to natural sunlight,
particularly the UV and blue light portions of the electro-
magnetic spectrum [22]. A recent meta-analysis by Shah et
al identified welding, which is a significant source of blue-
light, as a risk-factor for uveal melanoma [20]. Interest-
ingly, ocular melanoma could also be induced by expos-
ing rats to blue-light during an experimental animal
model [7].

The rationale behind a possible relationship between blue
light and tumorigenesis is that visible light of short wave-
lengths can cause DNA damage [11]. The secondary muta-
tion can be transferred to further generations of
transformed cells ultimately generating a malignant
clone. Previous work in our laboratory has shown that
blue light increases the proliferation rate of uveal
melanoma cell lines [6]. These results also indicated that
the use of UV and blue light filtering intra-ocular lenses
(IOLs) conferred a protective effect. These IOLs signifi-
cantly reduced the proliferative effect that blue light
caused in the un-protected uveal melanoma cells. As in
vitro results can not necessarily be extrapolated to under-
stand in vivo effects, we performed the current experiment
using an established animal model of uveal melanoma
[13]. When the re-cultured cells from the experimental
group were compared to the control group, higher prolif-
eration rates were seen. In other words, the blue light was
able to penetrate to the posterior of the eye and induce the
necessary molecular changes that ultimately resulted in
higher proliferation rates of uveal melanoma cells. Simi-
larly, the PCNA staining confirms these findings by being
significantly more expressed in the blue light treated
group when compared to controls.

A question that one may raise is whether or not the
changes secondary to blue-light exposure are permanent.
We have reasons to believe that they are. The fact that even
the CMCs from the experimental group presented with
higher proliferation rates is further evidence that the
changes induced by blue light exposure are not transient.
Whatever molecular changes were induced, the secondary
generations of those cells still exhibited a higher prolifer-
ation profile, even after being in circulation and away
from a blue light source. The number of eyes that devel-
oped tumors, primary tumor size and number of metasta-
sis were not statistically different between groups. We
believe that the difference in proliferation rate was not sig-

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Journal of Experimental & Clinical Cancer Research 2009, 28:4E

nificant enough to cause measurable differences in tumor
size during the time period of the study.

Another important question to be answered is whether
blue light can induce malignant transformation of a nor-
mal melanocyte. The main barrier to get this answer is the
scarcity of established cell lines of normal uveal melano-
cytes. Even if development and availability of such cell
lines were adequate, there would likely be numerous
changes in gene expression profiles after successive pas-
sages and immortalisation, rendering any conclusions
drawn from such a comparison incomplete. However,
there are a number of epidemiological studies on pediat-
ric literature showing clinical evidence that blue light can
indeed affect normal melanocytes. Neonates exposed to
blue light phototherapy as a treatment for jaundice
present with a larger number of dysplastic cutaneous nevi
later in life [23]. Nevi count tends to be higher and the
average nevus size is also larger in the exposed group com-
pared to controls [24]. Considering that dysplastic nevus
is the most important predisposing lesion for cutaneous
melanoma, this is strong evidence that blue-light can
induce the transformation of a normal melanocyte into a
pre-malignant lesion.

The human crystalline lens offers natural protection by fil-
tering UV and blue light. As an individual ages, the ability
of the lens to naturally filter out blue light increases signif-
icantly [4,25]. In patients that undergo cataract surgery,
the protection provided by the naturally yellowing crystal-
line lens is lost. Despite all the controversy about the use
of blue light filtering lenses in humans, there is compel-
ling evidence that visible blue light is potentially hazard-
ous. Considering the projections for increases in life
expectancy, patients are expected to live several years after
cataract surgery and secondary lens implantation. Many
years of cumulative exposure could be potentially danger-
ous especially in eyes harboring uveal nevi. It is estimated
that between five and ten percent of the population have
asymptomatic uveal nevi [26]. Therefore, the use of UV
and blue light filtering IOLs could be considered a pre-
ventative measure against possible blue light induced
malignant transformation of existing uveal nevi.

In summary, we present evidence that blue light exposure
can influence uveal melanoma cells and further substanti-
ate the results of previous in vitro studies. Our data dem-
onstrated a significant increase in uveal melanoma
cellular proliferation after exposure to blue light. This data
warrants further investigation assessing the efficacy of
blue light filtering IOLs to slow the progression of uveal

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
SDC re-cultured the cell lines, ran all proliferation assays,
and wrote the entire manuscript. SM organized the animal
model, and oversaw all technical aspects of the model
over the 8 week period. BFF performed weekly fundo-
scopic examinations, oversaw all gross and clinical his-
topathology for the entire model. CM was responsible for
all blood extractions. JCM was responsible for all Ficoll-
Paque processing throughout the model. EA performed all
the immunohistochemistry. ANC was the second inde-
pendent pathologist who graded all the immunohisto-
chemistry. WWD was responsible for the design of the
blue light setup. MNB Revised the entire manuscript.

We would like to take this opportunity to thank the generous help and sup-
port provided for this animal model by the McGill University Animal
Resource Center. In particular we would like the thank Lori Burgess, Karen
Stone, and Dr. Lynn Matsumiya. We would also like to thank Dr. Martine
Jager for the establishment of the 92.1 cell line. This study was funded by a
grant provided by the Cedars Cancer Institute.

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