Casulla, Lawrence S.

ENG10-T1R
2012-48773

Chapter II: Review of Related

Literature

EARLY DETECTION OF EYE CANCER IN HUMANS THROUGH

BIOLUMINESCENCE IMAGING USING FIREFLY LUCIFERASE

LITERATURE REVIEW

The knowledge of possible extraction and transfer of bioluminescent

properties from a bioluminescent organism (source) to a non-bioluminescent one
has led the scientific world to aim for an advancement using the said property in
many areas of research. Some of the most prolific areas whereas bioluminescence
is being highly regarded are the industry, medicine, exterior design and animal
science. In this particular paper, the possible effect of using the bioluminescent
property in human cells to detect eye cancer in its early stages using
Bioluminescence Imaging (BI) is investigated. It is hypothesized that given the
adequate evidences about its high success rate, less harm given to man and more
thorough definition of the process, the society will accept bioluminescence
imaging. The following six literature reviews attempt to give enough explanations
to support the given hypothesis.
Starting with the basic experiments on bioluminescence imaging, a study by
Thomulka (1995) showed how the bioluminescence phenomena can be an
important aspect in the fields of science, specifically in detecting and finding the
toxicity of drinking water by the presence of a light. The main focus of the study
was to introduce the use of bioluminescence imaging to the world since at that
particular time, the concept is still new and unknown to the society. Another main
reason for the commencement of the study was to aid the students in detecting
1

potentially biohazardous materials in the needed potable water by observing the

reduction of bacterial bioluminescence. In this method, they used Vibrio fischeri as
the indicator of toxicity. The principle followed in the study is that certain strains of
bacteria emit light when they are healthy, and when they are exposed to toxic
substances, the amount of light emitted reduces. Given that specific idea, the
hypothesis made was that the higher the reduction of light, the higher the number
of hazardous wastes present in the water.
The result in this study supported the earlier hypothesis that there is a
direct relationship between the reduction of light and number of hazardous wastes
in water. As time goes by, after two different samples of varying amounts of toxic
substances has been reacted with Vibrio fischeri, the sample with the highest
amount of wastes had the most obvious change in the emission of light. It was
greatly reduced upon stoppage of the said experiment. This shows that the
bioluminescent property and characteristics of certain organism can be used to
detect a needed sample within a larger population (Thomulka, 1995).
The limitation of this study is it does not involve a study in vivo. Another is
the fact that it did not specify as to what conditions or factors may cause the
experiment to be nulled, such as temperature, amount of oxygen, or water volume.
Next, in a study conducted by Christoph et al. (2013), they investigated on
the bioluminescence imaging using firefly luciferase in monoclonal (single cell
clone) and polyclonal (multi cell clone) populations of leukemia cells, another type
of cancer, in vitro (in glass; in the laboratory) and in vivo (in living organisms)
using mouse. The main target of the study was to know what specific cell type
must be used for the bioluminescence to take place in a foreign organism not
naturally exhibiting the property. This study also aimed to increase the rate of light
emission by selecting the most appropriate cell clone type that will react with lightemitting enzymes for positive expression of bioluminescence. In the initial
hypothesis, it was predicted that there will be no difference in light emission and
intensity whether the monoclonal or the polyclonal cell populations will be used.
After hypothesis testing, results showed that bioluminescence signal
intensity was unstable in polyclonal populations and decreased significantly with
2

repeated passage in culture. The decreasing signal observed may be due to

survival and growth advantages for the non-transduced cells within the polyclonal
cell population (Christopht et al., 2013). On the other hand, light activity and
intensity was stable in monoclonal luciferase-expressing leukemia cell lines. The
results indicate that the bioluminescence signal intensity and the dynamics of
luciferase activity in vitro were cell line dependent. This is contrary to what is
hypothesized and believed that there will be no difference of results in the
monoclonal or polyclonal cell populations. Though ground-breaking, there are still
some limitations in the experiment. One is the fact that cell populations may vary
from one organism into another. Another is there is no further information as to
what specific equipment may be used in order to perform or show the result
constantly without changing.
The third study focused about the use of bioluminescence for optical cell
tracking approach. In a clinical study made by Aswendt et al. (2013), they
established a new bioluminescence imaging protocol dedicated for neuroimaging,
which increases sensitivity especially for non-invasive tracking of brain cell grafts
called NSCs. The idea that reliable and quantitative bioluminescence of
transplanted cells in brain may be highly challenging because of all the
connections within was also considered (Aswendt et al., 2013).The main focus of
the study is to quantitatively compare the advanced and the standard imaging
protocol. The hypothesis is that using the advanced protocol, there will be higher
detection of transplanted NSCs in mice brain.
The outcome of the study showed that the standard or conventional protocol
was not able to detect 3,000 and 6,000 transplanted NSCs, while the advanced or
novel protocol efficiently and clearly detected even 3,000 transplanted NSCs. This
finding confirms the hypothesis that the advanced protocol will give higher
detection rate of transplanted NSCs in the brain. This may be due to the fact that
the advanced protocol was initially designed to improve and further add the
aspects needed for detection, which the standard or conventional protocol lacks. It
was

also

observed

that

even

bioluminescence may be possible.

in

vivo,

cell

number

estimation

through

This quantitative analysis of the luciferase

kinetics in living organism and the ratio of signal-to-light presence can serve as the
3

premise for related studies in the near future. The limitation of the study is that it
did not define how the NSCs will be grafted in the brain and what specific type of
luciferase from a source will be used.
Next, in a clinical study led by Maguire et al. (2013), a team of researchers
investigated on the probability of using a novelty source different from the
mainstream sources which will yield the same percentage of success rate. The
main focus was to figure out an alternative source of bioluminescent properties
aside from the mainstream firefly luciferase and Renilla luciferase. To address the
issue, they characterized codon-optimized luciferase from Vargula hilgendorfii as a
reporter for mammalian gene expression. Another target was to present a triple
luciferase reporter system for in vivo bioluminescence imaging, and be able to
multiplex Vargula hildendorfii luciferase with those of firefly (Photinus pyralis) and
Renilla. It is predicted that since the three different bioluminescent sources have
specific target substrates, complications may arise resulting to either one nonparticipating source or inhibition of one adjusting source by another.
The results in this study showed that luciferase activity from the three
sources did not contradict the activity of each other while undergoing the imaging
process. Actually, the presence of each other helped increase the possibility of
having triple bioluminescent imaging, or multimodal imaging. This system was
able to incorporate different technologies such as fluorescence, bioluminescence,
positron emission tomography, and magnetic resonance imaging (Maguire et al.,
2013). These results contradict the initial hypothesis that the differences in activity
of each bioluminescent source will only

hamper the production of a multiple

bioluminescence imaging system. Because each of the luciferase is specific to its

own substrate, they can be multiplexed together to monitor three distinct
biological events in the same biological system. According to Maguire et al. (2013),
this reporter system could be extended to different fields, such as immunology,
oncology, virology, and neuroscience, where simultaneous monitoring of multiple
parameters (from a cell population) is actually needed and required. The limitation
of the study is the lack of evidences that each luciferase was able to monitor three
distinct biological events and the fact that the study was done under strict

conditions such that environmental factors like temperature and amount of oxygen
was not really considered.
Next, an investigation on non-invasive visualization and interrogation of
biological processes in living animals was made through in vivo Bioluminescent
Imaging. In a clinical study made by Close et al. (2010), they thoroughly studied
bioluminescence imaging and its possible medical significance in the near future.
The main focus of the study was to review the various bioreporter/biosensor
integrations of bioluminescence imaging and discuss how it is being applied
towards a new and better visual understanding of biological processes within the
living organism (Close et al., 2010). It is predicted that bioluminescence can be
expressed in an organism not naturally exhibiting the said phenomena, and this
will help in many areas of research such as tumorigenesis (production of tumor),
cancer treatment, and disease regression. The study was done in vitro and in vivo
using mice as the living organism.
The results showed positive as bioluminescent properties were easily
expressed and exhibited in the experimental dummy (mice) upon reaction with the
injected metastatic tumor cell. Results also demonstrated that the luciferase
source can be used as a proxy for overall tumor burden.

This supports the

hypothesis of possible use of bioluminescent imaging in the near future,

specifically in its use for detection of tumors and diseases. One small limitation to
the study is the resolution problem associated with imaging small metastatic tumor
formation in living tissues.

Despite the imperfection of the currently available

luciferase systems, they can be adapted to give information that would previously
remain hidden from view, and advances in many fields of improving early detection
of disease will make bioluminescence imaging in the future possible (Close et al,
2010)
Finally, in a research article written by Williams (2009), a more clear and
extensive research on the possible use of bioluminescence imaging for detection of
eye cancer in a living organism was investigate using mice again as the
experimental dummy. In this method, a human eye cancer tumor cell was injected
to a mice and was subjected to bioluminescence imaging using the NightOwl LB

981 Molecular Imaging System to monitor the growth and succession of these
created tumors. The main objective of the study is to create an ideal test through
mice in detecting human eye cancer without doing any harm to the test subject.
This experiment was also done in order to have an alternative for biopsy analysis to
determine the progression of eye cancer. Through bioluminescence imaging, it is
believed that doctors will now be able to detect tumors earlier and choose a
method of treatment that does not necessarily involve eye surgery. The hypothesis
is that the tumor cells injected in mice will react with the luciferase enzyme, and
this will show positive in the detection of eye cancer. The study was conducted in
the province of Shanghai in China headed by Dr. Qian Huang M.D.
The results of the experiment indicate that eye cancer in humans can be
detected through bioluminescence imaging even if the cell was transplanted to an
experimental dummy (mice).

Doctor Huang said that Bioluminescence allowed

sensitive and quantitative localization and monitoring of intraocular and metastatic

tumor growth in vivo, and also believed that this medical technology might be a
useful tool to study cancer biology as well as anti-cancer therapies. Since eye
cancer is the most common and aggressive form of cancer, locating the tumors
during early stages stages of the disease is key. This concept will definitely lessen
eye removal surgery for large tumors, and this early detection will help in the
preservation of vision, eye retention and even survival (Huang, 2009). These
results definitely support the hypothesis that this advancement will help in the
improvement of the conventional method (biopsy analysis) for the early detection
of eye cancer in humans. This knowledge is groundbreaking as it is believed that
through this, even cancer of different body parts may be treated given a more in
depth study about bioluminescence imagings mode of action. The results also give
us a better picture of how accurate the results are when subjected to
bioluminescence imaging. More importantly, it gave us the advantages of the
process over biopsy analysis, such as in vivo monitoring, higher sensitivity, easier
use and an overall more accurate correlation between cell numbers detected and
tumor growth (Williams, 2009). The limitation of the study is the question of
safeness of the experiment if subjected to humans, and if it may cause pain or
further discomfort to the patient. Also, it was not raised whether there are possible

side effects of bioluminescence imaging which can cause harm to the patient
undergoing possible treatment.
Taken

together

as

whole,

the

results

obviously

indicate

that

bioluminescence advancements, specifically, the Bioluminescence Imaging, may be

a possible medical advancement that will help in detection of terminal diseases
which may lead to its early cure or much better, prevention (Aswend et al., 2013;
Close et al., 2010; Christoph et al., 2013; Maguire, 2013; Thomulka, 1995;
Williams,

2009).

These

results

are

mere

evidences

of

the

promise

that

bioluminescence imaging may bring in the near future.

Through these six literature revies, we can safely assume that bioluminescence
imaging will be an effective method in tracing a target cell. However, despite all
the promises it brings, more parallel studies that involve humans or even species
of close relatedness to human must be considered in order to provide a more
close and accurate result if the experiments are actually done to a human
patient. Also, researchers and scientist must be able to disseminate and expound
the information and whole process, equipment to be used and possible side effects
of bioluminescence imaging for better understanding and knowledge. This way,
cancer patients, specifically eye cancer patients will be more open to the idea of
treatment using this new method with hopes of reviving them instead of fear that
the process may cause them more harm than good.

REFERENCES
Aswendt, M., Adamczak, J., Despres, S. (2013). Boosting Bioluminescence
Neuroimaging: An Optimized Protocol for Brain Studies. Retrieved October
18, 2014, from http://www.plosone.org/article
Close, D., Xu, T., Sayler, & G., Ripp, S. (2010). In Vivo Bioluminescent Imaging
(BLI): Noninvasive Visualization and Interrogation of Biological Processes in
Living Animals. Retrieved November 1, 2014, from sensors-11-00180.pdf

Christoph, S., Schlegel, J., Calderon, F., Kim., Y., Brandao, L., Deryckere, D., &
Graham, D. (2013). Bioluminescence imaging of leukemia cell lines in
vitro and in mouse xenografts: effects of monoclonal and polyclonal cell
populations on intensity and kinetics of photon emission. Retrieved October
30, 2014, from http://www.jhoonline.org/content/6/1/10

Maguire, C., Bovenberg, S. (2013). Triple Bioluminescence Imaging for In Vivo

Monitoring of Cellular Processes. Retrieved October 30, 2014, from
http://www.nature.com/mtna/journal/v2/n6/full/mtna201325a. html
Thomulka, K., (1995). Use of Bioluminescence in Detecting Biohazardous Wastes in
Water.
Retrieved
October
30,
2014,
from
http://www.ableweb.org/volumes/vol-16/16-thomulka.pdf
Williams, J. (2009). Bioluminescence Imaging of Reporter Mice for Studies of
Infection and Inflammation. Retrieved October 18, 2014, from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863000/