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Topical Molecular Beacons for In Vivo Image-Guided Resection of Oral Carcinoma
by
Laura Allysa Jane Burgess
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Medical Biophysics University of Toronto
© Copyright by Laura Burgess 2014
ii
Topical Molecular Beacons for In Vivo Image-Guided Resection of
Oral Carcinoma
Laura Allysa Jane Burgess
Master of Science
Department of Medical Biophysics
University of Toronto
2014
Abstract
Oral carcinoma has become a major health problem, with nearly 300,000 people diagnosed
worldwide annually. The 5-year survival rate is as low as 30%, mainly attributed to poor
delineation of lesions. Optical imaging approaches to identify oral carcinoma tissue during
surgery are currently in trial. While decreased recurrence rates are shown, high rates of false
positives occur. Expanding upon this, a molecular beacon strategy for oral carcinoma delineation
was devised.
The selected MMP molecular beacon consists of a fluorophore conjugated to a quencher via a
disease-specific linker, activated by MMPs. The activated beacon becomes fluorescent, guiding
resection. MMPs have been associated with oral tumors and several members as highly
upregulated in oral carcinoma, making them an ideal target.
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I investigated, in vivo, the utility of this MMP-cleavable beacon in targeting oral carcinoma. Here
I demonstrate its high tumor specificity and potential for integration into the clinic to improve
patient outcome.
iv
Acknowledgments
To my supervisors, Dr. Gang Zheng and Dr. Alex Vitkin, thank you for supporting me and
allowing this work to be possible. Gang, thank you for your encouragement over these past few
years, and for giving me the opportunity and the room to grow. To my committee members, Dr.
Brian Wilson and Dr. Ming Tsao, thank you for your guidance, I’ve learned so much from you
both.
This work would not have been possible without help from so many. Dr. Tracy Liu, very simply,
you were my first mentor here and you taught me how science should be done, all while being
one of the great friends I made here. Dr. Juan Chen, you’re always there to help for any problem
and I know you have nothing but the best interests of all your students in mind, thank you. Dr.
Nikolaus Wolter, you were amazing to work with and I’ll always appreciate your advice and
encouragement. Dr. Eduardo Moriyama, you’re so good at what you do and you’re always
willing to help everyone around you. Thank you for all the help you gave me. Everyone at ARC,
particularly Sadiya Yousef, who always goes above and beyond to help me, Dr. David Hanwell
and everyone else who worked with my hamsters, thank you. The illness and sacrifice of those
hamsters will never been forgotten. To everyone at the Zheng lab, I’ve learned so much and have
had a great time with all of you. Especially Lizzie, Moj, TD, Luby, Tracy, Neeshma, Ken, Stash,
and Danielle; you helped me when I needed it, listened to me whine, made me laugh, provided
frequent distractions and we had quite a few amazing PNPs. I’ll miss you all and I hope the
memories don’t stop now.
Mom and Dad, any ambition, work ethic, dedication and empathy in me are the result of you.
You always gave me every opportunity and supported all of dreams, from putting me into
engineering and science camps to trips on a whim to see David Beckham play and everything in
between. You have always been and always will be my biggest cheerleaders. Dad, it feels like
it’s been forever since you sat across from me while I did my homework every night, quizzed me
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for every test and annoyed me when I thought I couldn’t figure something out. Simply put, any
accomplishment now or in the future, wouldn’t have been possible without you and your
unwavering support. Samantha, Dayna, and Darrell, you always pushed me to do more and be
better, striving to be more like my big brother and sisters. To you, and Allan, Matt and Monique,
thanks for never treating me like your annoying little sister.
Lastly, Daniel, thank you for putting up with me when grad school literally made me crazy and
for making everything better when it all seemed to be too much. I wouldn’t have completed this
thesis now and be going to medical school in the fall if you hadn’t told me I’d be amazing at it,
until one day I finally believed you.
vi
Table of Contents
Acknowledgments .......................................................................................................................... iv
Table of Contents ........................................................................................................................... vi
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ ix
Chapter 1 Potential Value of Molecular Beacons for Treatment of Oral Carcinoma ..................... 1
1.1 Oral Cancer ......................................................................................................................... 1
1.1.1 Oral Carcinoma ....................................................................................................... 1
1.1.2 Current Treatments ................................................................................................. 4
1.2 Optical Imaging ................................................................................................................ 11
1.2.1 Fluorescence ......................................................................................................... 11
1.2.2 COOLS Trial ......................................................................................................... 14
1.3 Matrix Metalloproteinases ................................................................................................ 17
1.3.1 MMPs in Cancer ................................................................................................... 19
1.4 Molecular Beacons ............................................................................................................ 23
1.4.1 Peptide-Based Molecular Beacons ....................................................................... 25
1.4.2 MMP-Targeted Molecular Beacons ...................................................................... 26
Chapter 2 Topically Applied Molecular Beacons for Fluorescence-Guided Resection of Oral
Carcinoma ................................................................................................................................ 31
2.1 Introduction ....................................................................................................................... 31
2.2 Materials and Methods ...................................................................................................... 33
2.3 Results ............................................................................................................................... 37
2.3.1 In vivo validation of molecular beacon specificity in a murine head and neck
cancer model ......................................................................................................... 37
vii
2.3.2 In vivo activation and specificity of the molecular beacon in a clinically
relevant animal model ........................................................................................... 39
2.3.3 Ex vivo HPLC Studies ........................................................................................... 43
2.4 Discussion ......................................................................................................................... 45
Chapter 3 Future Directions, Research and Development ............................................................ 48
3.1 Introduction ....................................................................................................................... 48
3.2 Immunostain-like Procedure To Identify MMPs Within Hamster Cheek Tissue ............. 49
3.3 Quantification of Fluorescence Microscopy ..................................................................... 51
3.4 Topical Gel or Spray ......................................................................................................... 52
3.5 Fluorescence-Guided Resection ........................................................................................ 54
3.6 Increased Tissue Penetration ............................................................................................. 55
3.7 Conclusions ....................................................................................................................... 57
References ..................................................................................................................................... 59
viii
List of Tables
Table 1: Treatment modalities for oral carcinoma .......................................................................... 5
Table 2: The side effects associated with the different treatment modalities for OSCC 7,13. ....... 9
ix
List of Figures
Figure 1: The different stages of disease progression. .................................................................... 3
Figure 2: Jablonski diagram illustrating absorption, fluorescence and phosphorescence. ........... 12
Figure 3: Patient with an occult recurrent carcinoma identified by the use of the autofluorescence
technique. ...................................................................................................................................... 15
Figure 4: The probability of local recurrence-free survival of the 60 patients enrolled in the
preliminary trial, with this optically-guided approach. ................................................................. 16
Figure 5: The domain structure of MMPs. .................................................................................... 18
Figure 6: The various functions of MMPs in cancer progression. ................................................ 20
Figure 7: Immunohistochemistry was performed on formalin-fixed human oral carcinoma tissue
using mouse monoclonal antibodies demonstrating MMP expression in oral cancer. ................. 23
Figure 8: Molecular beacon structure. .......................................................................................... 24
Figure 9: Validation of the MB's MMP-7 specificity. .................................................................. 27
Figure 10: MMP-7-specific activation validation in vitro with fluorescent and brightfield
confocal images. ........................................................................................................................... 28
Figure 11: In vivo images of a mouse bearing a KB flank tumor post-i.v. injection of MB. ....... 29
Figure 12: The HPLC traces of beacon incubated with various MMPs for 48 hours. .................. 30
Figure 13: Molecular beacon structure in the absence and presence of the specific protease
capable of activating the beacon. .................................................................................................. 32
Figure 14: In-house fluorescence imaging system. ....................................................................... 34
x
Figure 15: The experimental protocol for all hamster cheek studies involved beginning with
tumor-bearing hamster cheeks. ..................................................................................................... 36
Figure 16: Representative xenograft model fluorescence images with PPMMPB and PPMMP
demonstrating tumor-associated fluorescence and whole tongue fluorescence, respectively.. .... 38
Figure 17: Representative hematoxylin and eosin (i) and confocal images (ii) of tumor-bearing
tongues. ......................................................................................................................................... 39
Figure 18: Representative images of a hamster cheek treated with PPMMPB... ............................ 40
Figure 19: Representative images of various control hamster cheeks: A) PPMMP-treated, B)
negative control-treated, C) healthy cheek having undergone surgery and treated with PPMMPB.
....................................................................................................................................................... 41
Figure 20: Representative hematoxylin and eosin and confocal images of cheeks treated with
PPMMPB.. ....................................................................................................................................... 42
Figure 21: Representative hematoxylin and eosin (i) and confocal images (ii) of hamster cheeks
treated with the various controls.. ................................................................................................. 43
Figure 22: Representative HPLC traces of A) beacon extracted from tumor showing that both
intact PPMMPB and PPMMP fragment are present within the tumor, B) beacon applied to the
tumor.. ........................................................................................................................................... 44
Figure 23: Representative images of a hamster cheek treated with PPMMPB and corresponding
PPMMPB cleavage or activation. .................................................................................................... 45
Figure 24: Tumor-bearing hamster cheeks that did not undergo PPMMPB application in vivo were
frozen and tissues slices obtained for the immunostain-like procedure on sequential slices. ...... 50
Figure 25: Confocal images of droplets of A) 1 mM, B) 10 M (n=2). ....................................... 52
Figure 26: Hamster cheek treated with the thermal-transitioning PPMMPB.. ................................ 54
Figure 27: Confocal and transmitted light images of MT-1 cells incubated with (A) pyro, (B)
pyro-5r and (C) pyro-8r. ............................................................................................................... 56
1
Chapter 1 Potential Value of Molecular Beacons for Treatment of Oral
Carcinoma
1.1 Oral Cancer
Oral cancer is the sixth most common malignancy in the world, after lung, stomach, breast, colon
and rectum, and cervix and corpus uteri cancers. Worldwide, those diagnosed with oral cancer
have a 5-year survival rate of only 50% 1,2. Four thousand Canadians are diagnosed with oral
cancer every year 3,4 and even in developed countries like Canada, a third of patients diagnosed
with oral cancer will die as a result of their disease.
1.1.1 Oral Carcinoma
The epidermis, including that within the oral cavity, mainly consists of squamous cells.
Squamous cell carcinoma (SCC) occurs at many different sites in the body (including bladder,
skin, and the digestive tract) 5. While SCC at different sites in the body presents with different
symptoms and these sites are treated differently, SCC is histologically distinct from other forms
of cancer. Epithelial cells multiply uncontrollably and distinct architectural changes ensue,
including epithelial stratification and premature keratinization 6. Oral squamous cell carcinoma
(OSCC) represents more than 95% of all oral cancers 7,8. Most commonly, OSCC arises in the
mucous membrane of the tongue, floor of mouth and vermillion borders of lips and is
characterized by having a much greater linear extent than depth 9,10.
Oral cancer development is multistep and sequential, with oral cancer preceded by premalignant
lesions or dysplasia, often asymptomatic 11. Precancerous lesions consist of morphologically
altered tissues where cancer is more likely to occur, when compared to normal tissues 12.
Dysplastic cells are classified based on a number of changes, including irregular epithelial
stratification, premature keratinization, increased nuclear size and increased number of nucleoli
2
9. Such lesions can be further classified as being either leukoplakia or erythroplakia. These
premalignant or early malignant lesions typically appear as painless white or red patches,
respectively, 11,13.
The most common precancerous lesion is leukoplakia. Leukoplakia is a predominantly white
lesion of the oral mucosa that cannot be classified as any other definable lesion 10,12,15. These
develop in 1-4% of the population and up to 40% of these transform into malignant lesions
within 5 years 10. Leukoplakias can either be smooth, homogeneous (see Figure 1a) or speckled,
pitted, non-homogeneous (see Figure 1b). There is increased risk of malignant transformation
with pitted, non-homogeneous lesions. Additionally, speckled leukoplakia often have
microscopic dysplasia and malignancy 16. Erythroplakias (see Figure 1c) are red and a rarer
precancerous lesion. These are typically thought to be of highest risk for cancer development,
with a 90% malignant transformation rate 10. Typically, dysplasia is graded based on
smoothness of leukoplakias and erythroplakias or based on the depth of architectural changes
within the epithelium.
3
Figure 1: The different stages of disease progression with diseased regions circled: A) a typical
homogeneous leukoplakia, B) a typical speckled leukoplakia, C) a typical erythroplakia and D)
typical late stage oral carcinoma 11, reproduced with permission.
Globally, oral cancer incidence increases with age, with 98% of oral cancer patients in developed
countries being over forty years of age 1. This is because oral cancer occurrence is highly
correlated to a number of risk factors which have a mutagenic effect inducing genetic, epigenetic
and metabolic changes with increased exposure 17. The most significant of these risk factors are
heavy alcohol and tobacco consumption, particularly when used in conjunction; they have a
supermultiplicative effect in the mouth 1,18. Poor diet, exposure to UV light, human papilloma
virus, a slight familial correlation and transplants, where patients are immunosuppressed, are also
thought to be risk factors for oral cancer 19-27.
OSCC originates by “field cancerization”, a process where a carcinogenic agent preconditions an
area of epithelium. By definition, this carcinogenic agent is active with sufficient intensity over
long enough time that a change towards cancer of this preconditioned epithelium is inevitable 10.
Specifically, when examining tumors with a diameter less than 1 cm, separate foci of cancer can
4
be found that later coalesce as they grow. The time between application of the carcinogen and the
development of cancer is varied. The field can break down at multiple points, thus multiple oral
cancers can arise from independent cell clones. This means that there are multifocal areas of
precancer that lead to OSCC 10. The specific mechanism is unclear, but could be due to the
spread of clonal stem cells, saliva inoculation or the presence of undetectable invasiveness. Field
cancerization helps to explain the high local-recurrence rate associated with oral cancer and must
be considered when developing treatments for OSCC.
1.1.2 Current Treatments
The goal when treating OSCC is complete tumor eradication, while either restoring or preserving
both anatomy and function and limiting recurrence or a second primary tumor 13,28. While
overall survival and disease-free survival are typical measures of success of cancer treatment,
patient quality of life is of great concern, particularly in a region of such functional and cosmetic
importance 29. Functionally, the oral cavity performs a variety of complex tasks, including
speech, chewing, tasting, swallowing and salivation 13,29. Aesthetically speaking, reconstruction
is very common, but can still lead to significant disfigurement.
The mainstay of OSCC treatment is surgical excision, but radiation therapy and chemotherapy
are also used in its treatment, either alone or in combination 5,13,30. Single modalities are
generally used in early stages of the disease, typically surgery or radiation therapy, and
combination therapies are used with more advanced disease 5,13,30. The type and the extent of
treatment are determined by tumor characteristics, side effects and co-morbidities, as described
in greater detail in Table 1.
5
Table 1: Treatment modalities for oral carcinoma
Treatment
type
Applicable stage of
disease
Stand alone or
combination therapy
Method of action
Surgery Early stage and late
stage
Primary treatment modality Remove malignant tissue,
leaving remaining normal tissue
functionally intact
Radiation
therapy
Early stage and late
stage
Sometimes used as stand
alone, but most commonly
combination
Destroy DNA of malignant cells
in localized region, impacting
target and adjacent normal
tissue
Chemotherapy Late stage Combination therapy Inhibit rapidly dividing cells,
managing spread and metastasis
1.1.2.1 Surgery
Surgery, the most common form of treatment, attempts to remove all abnormal tissue while
leaving histologically normal tumor margins and trying to preserve tissue anatomy and function
31-34.
Negative margins are crucial in effectively treating the disease. Patients having positive margins
are expected to have double the rate of local recurrence, compared to patients with pathologically
negative margins 35. Consequently, one to two centimeter margins of tissue that appears normal
should be removed to prevent microscopic disease from being left behind 13. As the margins are
of the greatest concern, and of high prognostic significance, in surgical treatment of OSCC,
intraoperative frozen tissue assessment of both mucosal and deep margins is often performed 36.
However, accurately identifying any positive surgical margins has been a source of treatment
failure; in a study of 25 patients, all of whom were diagnosed with pathologically negative
6
surgical margins during intraoperative frozen tissue assessment, five of the patients had biopsy-
proven local recurrences within 7 months of their initial surgical resection 37. Many studies are
looking at molecular staging, for example, detecting p53 mutations common in many head and
neck cancers, and preliminary clinical results show promising results 37,38.
The surgical technique varies based on lesion size and access. With smaller tumors, surgeons can
often remove the tumors from within the oral cavity, but if access is limited or the tumor is very
large, the surgeon might have to approach from outside the cavity; removing soft tissue and bone
7. Cheek flaps are often removed to access the floor of the mouth and the mandible or they may
approach from under the eye to access the maxilla 34. In fact, often the mandible and/or maxilla
need to be fully excised, facilitating sufficient access to the oral cavity 34,39. More advanced
stages of disease are often associated with lymph node invasion and may require radical neck
dissection 28,31,34,39. Like treatment of the primary site, the extent of neck dissection is related
to the extent of lymph node invasion: size, site, number 28,34,39.
Following surgical excision, reconstruction surgery may be required to restore loss of functions
and/or to repair cosmetic defects 13,34. Small defects are often covered with split-thickness
grafts and larger defects with forearm grafts 40. Mandibular reconstructions, following removal
of a portion of the mandible, are most commonly performed with bone from the patient’s fibula
13.
In addition to possible neck dissection, advanced OSCC is also typically treated with
combination therapy: surgery and radiotherapy or surgery, radiotherapy and chemotherapy 11,13.
7
1.1.2.2 Radiation Therapy
Radiation therapy (RT) aims to damage the DNA in malignant cells within a small localized
region while preserving both function and anatomy of normal tissue in adjacent regions 28,30. In
early stage OSCC, RT, specifically external beam radiation therapy possibly with brachytherapy,
is offered as an alternative to surgical resection, when surgical access proves difficult or the
patient refuses surgery 30,40. In early stages of the disease, RT has similar 5-year survival rates
and a similar 37% rate of local recurrence when compared to surgery 41,42. That being said, it is
rarely used as a primary treatment modality, with its main purposes being prevention of
recurrences and eliminating remnant tumor following an incomplete resection 11.
Patients with large tumors and those with positive margins post-resection are commonly treated
with postoperative RT. RT is typically administered postoperatively so as to minimize risk of
infection and maximize postoperative healing 13,28. The treatment is planned to maximize dose
in the region surrounding the tumor, where there is the greatest likelihood of genetic, epigenetic
and metabolic changes that could lead to a secondary malignancy 30. The standard RT protocol
involves administration of about 60 Gy total given as 2 Gy daily from Monday to Friday over 6
weeks 13. Avoidance of breaks is critical as several studies have shown there to be a significant
correlation between timing of treatment without breaks, and locoregional control and overall
survival rates 43.
Recent studies show that the use of altered fractionated treatments and combination
chemotherapy both improve survival, although neither have yet been widely adopted 44-46. The
most recent alteration to RT protocol involves the addition of concurrent chemotherapy to the
treatment protocol. Meant to improve radiosensitization, it is currently being explored in the
hopes that it will improve malignant cell sensitivity to RT compared to surrounding healthy
tissue 47,48. However, for patients with low risk of post-surgery recurrence, concurrent
chemotherapy is of no benefit 49.
8
1.1.2.3 Chemotherapy
Chemotherapy (chemo), systemic anticancer drug therapy, is not a curative single modality for
OSCC. Chemo was previously thought to mainly be of use as a palliative treatment, however, it
is becoming an important adjunctive therapy in the battle against OSCC 40. Chemo targets
malignant, abnormal and rapidly dividing cells in the hopes of preventing spread and targeting
any metastases 50.
While chemo can be used before surgery, concurrently with postoperative RT, or after the
completion of both surgery and RT, the most promising results are associated with concurrent
RT 51-53. The combination of RT and chemo has proved to increase RT efficacy leading to
better locoregional tumor control, reduced mortality and improved survival 47,54,55.
1.1.2.4 Treatment Side Effects
OSCC patients can have a wide variety of side effects from the various treatment options and
morbidities must be considered when determining the treatment protocol for each patient, with
common side effects summarized in Table 2.
9
Table 2: The side effects associated with the different treatment modalities for OSCC 7,13.
Treatment modality Short-term side effects Long-term side effects
Surgery Difficulty swallowing and speaking
Wound infection
Fistula
Functional problems
Cosmetic defects
Tissue and bone loss
Nerve pain
Radiation therapy Loss of energy
Dermatitis
Mucositis
Xerostomia
Altered taste
trismus
Persistent xerostomia
Fibrosis
Osteoradionecrosis
Increased risk of tooth decay and
periodontal disease
Hypothyroidism
Laryngaeal edema
Chemotherapy Nausea and vomiting
Diarrhea
Stomatitis
Bone marrow suppression
Neuropathy
OSCC, and its subsequent treatment, has profound impact on patient quality of life, in the long-
term and short-term, both physically and psychologically. Like other cancer survivors, patients
who have undergone OSCC treatment have a risk of recurrence, of secondary primary tumors
and can suffer from pulmonary, renal, cerebrovascular and cardiovascular complications 56,57.
Additionally, patients who have undergone surgery may suffer from impaired swallowing, eating
and speaking as a result of their disease or treatment, which may become permanent. It is critical
that patients undergo rehabilitation to improve swallowing, and speech, and have dental
rehabilitation. This can either begin preoperatively or postoperatively, but is crucial in
minimizing any long-term deficits. Following treatment, nerve pain and loss of sensation can
10
occur 58,59. Additionally, the anatomical changes associated with surgery can lead to cosmetic
defects requiring extensive reconstruction and even with reconstruction, permanent
disfigurement, limited movement and loss of function can result 60.
RT has both acute and late side effects. In the short term, RT results in mucositis in more than
50% of patients, can lead to a lack of energy, a loss of taste, radiation-induced dermatitis and
burn, xerostomia (dry mouth caused by a change in saliva function or damage to the salivary
glands) and trismus (muscle spasm leading to limited mouth opening) 50,61. For more than 60%
of patients, xerostomia is caused by radiation-induced damage to the salivary glands and remains
permanent, significantly increasing the risk of tooth decay and periodontal disease 13,61. In
addition to permanent xerostomia, RT can also lead to long-term side effects including
permanent mucositis, laryngeal edema and hypothyroidism (like xerostomia, induced by a
significant radiation dose to the larynx and thyroid, respectively), difficulty swallowing, tissue
fibrosis and osteoradionecrosis 40. Osteoradionecrosis is a non- or slow-healing bone damage
that can result soon after, or many years post-completion of RT and results from radiation-
induced hypoxia or hypovascularity to the bone 62,63. The mandible is particularly susceptible
to osteoradionecrosis. RT destroys malignant cells, but also neighbouring healthy tissue, to a
certain extent, and can damage normal cells, limiting blood supply to the bone. Eventually, the
mucosa overlying the bone falls away and a necrotic bone is left exposed. Additionally, RT is
also associated with the possibility of radiation-induced secondary cancers 13.
While surgery and RT are associated with localized side effects, chemotherapy involves the
systemic administration of anticancer agents and is, thus, associated with side effects across the
whole body. Many chemotherapy agents are associated with nausea, vomiting, diarrhea,
mucositis and suppression of the bone marrow, leaving patient susceptible to infections 61 and
others are associated with renal problems and neuropathy 64,65. Additionally, the
cardiovascular, cerebrovascular, pulmonary and renal complications that most cancer patients
suffer from are generally associated with chemotherapy agents 57. With chemo, most side effects
are dose-dependent and dependent on patient age and overall health, and all these factors must be
considered when the patient is treated 50. There are a number of potential avenues to reduce the
11
treatment-associated side effects, namely better identification of tumor boundaries during
surgery, which would facilitate the complete and accurate removal of diseased tissue and,
requiring less use of RT and chemo.
1.2 Optical Imaging
While there have been significant improvements to the therapeutic management of OSCC, there
hasn’t been any significant improvement in the prognosis of the disease over the last few
decades, with 5-year survival rates ranging between 30 and 60% depending on the region of the
world 66. Additionally, there is a very high rate of recurrence of OSCC at the primary site, in
fact, one of the highest rates of recurrence across all cancers 67-69. As discussed, the primary
treatment modality of OSCC is surgical excision where physicians estimate tumor boundaries
based on tissue texture and their own experience. This is relatively inaccurate, with high rates of
failure to achieve negative margins, contributing to the poor survival rates and high rates of
recurrence. Conversely, excessive tissue removal leads to unnecessary functional and cosmetic
damage, detrimental to patient quality of life. There is a need for real-time visualization of tumor
boundaries during surgery, facilitating complete removal of diseased tissue, while sparing
healthy oral tissue. A relatively new approach to facilitate the detection of disease boundaries
involves the use of tissue optics and fluorescence.
1.2.1 Fluorescence
Photoluminescence is the process of absorption and subsequent re-emission of photons that
occurs in certain molecules upon excitation by light from their ground state 70. This absorption
of light raises the molecule from its ground state to its excited state. Its subsequent return to
ground state is accompanied by the spontaneous emission of light. The Jablonski diagram, in
Figure 2, is convenient for visualizing the various processes that are possible with
photoluminescence, most commonly fluorescence and phosphorescence 70.
12
Figure 2: Jablonski diagram illustrating absorption, fluorescence and phosphorescence. Straight
arrows represent radiative processes and wavy arrows represent non-radiative processes and S
represents singlet states and T triplet states 70, reproduced with permission.
Upon energy absorption, an electron of a fluorescent molecule, a fluorophore, is excited into the
first excited singlet state (S1), a higher vibrational level of this singlet state, or into a higher
singlet state. With excitation into higher states, some of the absorbed energy is frequently shed
through processes including vibrational relaxation and internal conversion (IC) causing the
electron to fall into the first excited singlet state’s lowest vibrational energy level. From the first
excited singlet state’s lowest vibrational energy level, fluorescence (S1 S0) may occur, as
shown in Figure 2 70,71. Alternatively, from the excited singlet state’s lowest vibrational energy,
the electron may undergo intersystem crossing (ISC) to produce a triplet state, see Figure 2,
potentially leading to phosphorescence 72.
13
Fluorescence involves short-lived emission, within nanoseconds of absorption, from the lowest
vibrational level of a singlet state and involving spin-paired electrons 70. Fluorescence is made
very powerful, and has become a popular technique for the diagnostics and image-guidance in
cancer treatment, due to the Stokes shift: light emitted upon return to a molecule’s ground state
has a longer wavelength than the light used to excited the molecule 70. This allows for the
visualization of fluorescence, by blocking out exciting light with filters.
Excited state fluorophores don’t always have to undergo fluorescence, they may proceed through
ISC, changing from an excited singlet state to an excited triplet state by a change in the
electron’s spin 70. Emission of light from this triplet state down to ground state is
phosphorescence (T1 S0). But phosphorescence often requires microseconds up to tens of
seconds to occur.
1.2.1.1 Quenching
Quenching refers to the reversible loss of a fluorescent signal due to intramolecular interactions
between a fluorophore and its environment, or a quencher molecule 71. The specific mechanism
through which quenching occurs varies with quencher and fluorophore and some examples
include electron transfer, self-quenching and contact quenching.
Of particular interest, Forster resonance energy transfer (FRET) is a quantum mechanical process
involving nonradiative energy transfer from an excited state fluorophore, known as the donor,
and another chromophore, known as the acceptor 73,74. Generally, the extent of FRET between
the donor and the acceptor is determined by the proximity of the donor to the acceptor and the
extent of spectral overlap in the emission spectrum of the donor and the absorption spectrum of
the acceptor 72. When FRET occurs, the donor’s excited state energy is transferred to the
14
acceptor, should they be in close proximity, resulting in an increase in fluorescence intensity of
the acceptor and a decrease in the fluorescence intensity of the donor. That is, the donor becomes
optically silent 75.
1.2.2 COOLS Trial
Recently, there has been a change in the way carcinogenesis is viewed with respect to OSCC.
With the emergence of the field cancerization model, surgeons try to target at risk fields of
tissue. Traditionally, surgeons have removed lesions with abnormal tissue texture and
appearance, but altered tissue extends beyond these borders, that is, all diseased tissue is not
clinically visible. The high rates of recurrence associated with OSCC may be attributed to the
inability to accurately delineate the boundaries of diseased or altered tissue. Autofluorescence
imaging of oral tissue may provide a means to identify the abnormal tissue that cannot currently
be seen clinically.
Autofluorescence imaging of the oral cavity consists of application of higher-energy light to
excite specific fluorophores within the tissue, which then emit lower-energy light. In some
countries, autofluorescence imaging is considered the standard of care for detection of early
stage lung cancer. Additionally, it is being used in various stages of clinical trials for cervical,
skin, esophageal, bladder and colon cancer 76-79. Autofluorescence by endogenous fluorophores
can be used in the detection of malignancy because their optical properties can be correlated with
disease progression 80-82. Normal oral tissue will autofluoresce a pale green colour when
excited by blue light (400-450 nm), but autofluorescence decreases with malignancy and tissue
appears brown or black 83, illustrated in Figure 3.
15
Figure 3: Patient with an occult recurrent carcinoma identified by the use of the
autofluorescence technique; A) under white light no lesion is visible on the tongue, B) When
irradiated with blue light, a region with decreased autofluorescence can be seen (note the arrows
in A and B indicate the correlation between the images), C) A biopsy from the anterior portion of
the tongue showed dysplasia and epithelial thickening, while from the posterior portion of the
tongue there was carcinoma in situ D) Moderate inflammation can be identified by increased
stromal activity, E) Severe inflammation can be identified direct visualization 84, reproduced
with permission.
The loss of autofluorescence is the direct result of both fluorophore alterations and structural or
morphological changes in tissue associated with the transition from normal tissue to malignant
tissue. When the oral cavity is illuminated with blue light, the resulting fluorescence in normal
tissue mainly originates from collagen, its crosslinks and elastin. These are located in both the
stroma and the basement membrane 80,84,85. In addition to changes in these components,
progression towards malignancy is also associated with changes to nuclear morphology,
epithelial thickness and tissue vascularization, all impacting absorption and scattering of light
and contributing to the fluorescence properties of diseased tissue 85.
16
This technology was used in a 20-patient study where patients underwent surgical removal of
their tumors. The study found that autofluorescence can identify lesions that cannot be seen
under white light conditions 84. Additionally, all tumors displayed a loss of autofluorescence and
in 19 of the 20 tumors, this autofluorescence loss extended between 4mm and 25 mm beyond the
clinically visible lesion 86. From these promising preliminary results, a longitudinal study
involving 60 patients was performed. Patients were either treated with white light surgical
resection or with this optically-guided approach to surgery. At least twelve months after surgery,
none of the patients who had this optically-guided approach to surgery showed any signs of
recurrence. Seven of the 28 patients in the white light surgery group, however, have shown
recurrence with severe dysplasia or malignancy 87. The probability of local recurrence-free
survival is shown in Figure 4. This has led to a multi-center clinical trial called the Canadian
Optically-Guided Approach to Oral Lesions Surgical Trial, COOLS, currently underway.
Figure 4: The probability of local recurrence-free survival of the 60 patients enrolled in the
preliminary trial, with this optically-guided approach. There is a significant decrease in the
probability of local recurrence using this optically-guided approach when compared to white
light visualization alone 87, reproduced with permission.
Despite the very positive results associated with this autofluorescence-based technique, there are
still some potential issues with this technique. Several studies have shown that autofluorescence
is unable to differentiate between benign lesions, early stage disease and malignancy. This could
17
lead to high rates of false positives and unnecessary removal of functionally and cosmetically
important tissue; an unacceptable morbidity.
1.3 Matrix Metalloproteinases
One of the defining characteristics of malignant lesions is their ability to invade tissues and
subsequently establish metastases 88. Such tumor invasion is a dynamic and complex process
where tumor cells must detach from their point of origin, traverse both the extracellular
membranes and basement membranes and enter into lymphovascular channels 89. Thus, the
extracellular matrix (ECM) of tumors and the non-malignant tumor-associated stroma cells are
critical in tumor progression 90. Proteolytic degradation of basement membrane and ECM
components requires specific proteases, including matrix metalloproteinases (MMPs). This
makes MMPs crucial to the regulation of tumor microenvironment. In fact, there is increased
MMP expression in most human cancers, when compared to normal tissue 91.
MMPs are a family of zinc-dependent endopeptidases, with more than 21 members of human
MMPs, collectively capable of cleaving almost any ECM component 89,91. They are either
secreted or anchored to the plasma membrane and are generally divided into five distinct groups:
collagenases, gelatinases, stromelysins, matrilysins and membrane-type MMPs 89,91. There is a
high degree of similarity between the enzymes within each group (roughly 80%) and between
enzymes in different groups (roughly 50%) 92. MMPs consist of an N-terminal propetide
domain, a catalytic domain, a hinge region and a C-terminal haemopexin-like domain, as seen in
Figure 5 93.
18
Figure 5: The domain structure of MMPs where S is signal peptide; Pro is propeptide domain;
Cat is catalytic domain; Zn is zinc active site; Hpx is haemopexin-like domain 93, reproduced
with permission.
Under normal physiological conditions, MMP activities are tightly regulated. But a loss of MMP
regulation can lead to any number of diseases, including cancer, arthritis, atherosclerosis,
nephrititis and fibrosis 94. MMP regulation occurs through both transcriptional and post-
transcriptional mechanisms 95. This regulation is influenced by several factors including
hormones, growth factors, oncogenes and cytokines 89. Additionally, MMPs are synthesized as
zymogens, an inactive form, requiring extracellular activation. They remain inactive due to the
interaction between the zinc ion bound to the catalytic domain and a cysteine-sulphydryl group
in the propeptide domain. Activation occurs through proteolytic removal of the propeptide
domain 96. Most MMPs are activated extracellularly either by other activated MMPs or by serine
proteinases. There are exceptions, for example MMP-11, MMP-28 and MT-MMPs can be
activated intracellularly 96. Several inhibitors, including endogenous inhibitors, also affect MMP
activity. For example, in tissue fluids the main inhibitor of MMPs is 2-macroglobulin, a plasma
protein 97. Upon binding to MMPs, a 2-macroglobulin-MMP complex forms which then binds
to a scavenger receptor, facilitating clearance by endocytosis. Similarly, thrombospondin-2
complexes with MMP-2 leading to clearance by the same mechanism 98. The most well-studied
19
endogenous MMP inhibitors, though, are tissue inhibitors of metalloproteinases (TIMPs) -1, -2, -
3 and -4. TIMPs reversibly inhibit MMPs and each TIMP displays different tissue-specific
expression and MMP inhibition 99.
1.3.1 MMPs in Cancer
Cancer progression is associated with several distinct changes to normal cell physiology, known
as the hallmarks of cancer: self-supported growth through its own growth signals; insensitivity to
signals inhibitory to growth; ability to escape apoptosis; ability to replicate infinitely;
angiogenesis; and tissue invasion and metastasis 88. Initially, it was thought that MMPs were
important exclusively in invasion and metastasis, but it has been found that MMPs play a pivotal
role in many of these hallmarks of cancer, summarized in Figure 6.
20
Figure 6: The various functions of MMPs in cancer progression. Various MMPs play critical
roles in cancer cell growth and survival, in angiogenesis, in invasion, in differentiation and in the
body’s immune response to cancer 91, reproduced with permission.
MMPs have been shown to promote cancer cell proliferation through three different mechanisms,
illustrated in Figure 6a. MMPs release precursors of various growth factors including TGF-
100, MMPs allow any growth factors within the ECM to become bioavailable, for example
insulin growth factor (IGF) is released when MMPs cleave insulin growth factor binding protein
101,102, and MMPs can indirectly regulate proliferative signals through integrins 103.
MMPs also play a role in apoptosis, a process critical for cancer cells to avoid so they can
survive even with significant genetic instability. Specifically MMP-3, -7, -9, and -11 regulate
apoptosis and MMPs have both apoptotic and anti-apoptotic roles, illustrated in Figure 6b. When
21
MMP-3 is overexpressed in mammary epithelial cells, it induces apoptosis, likely through
degradation of laminin 104,105. MMP-7 possesses both apoptotic and anti-apoptotic functions; it
releases membrane-bound FASL, a stimulator of death receptor FAS. Once released, FASL
either induces apoptosis of surrounding cells or decreases cancer cell apoptosis 106,107.
Conversely, MMP-7 also inhibits apoptosis by cleaving pro-heparin-binding epidermal growth
factor (pro-HB-EGF), producing HB-EGF, a promoter of cell survival 108. MMP-11 inhibits
cancer cell apoptosis, with its overexpression decreasing spontaneous apoptosis in xenograft
models 109. It has been hypothesized that MMP-11 inhibits apoptosis through the release of
IGFs, which act as survival factors 102,110.
MMPs appear to be important contributors to tumor angiogenesis, blood vessels sprouting from
previously existing blood vessels to support tumor tissue, with MMP inhibitors decreasing tumor
angiogenesis in various animal experiments 111-113. It is possible that MMPs contribute to
tumor angiogenesis purely through degradation of ECM, enabling endothelial cells to invade
tumor stroma. It has in fact been shown that cleavage of collagen type I is necessary for
endothelial cells to invade the ECM and for blood vessels to subsequently form 114. Specific
MMPs shown to regulate angiogenesis include MMP-2, -9, -14 and -19 114-117. Additionally,
MMPs produce anti-angiogenesis fragments, shown in Figure 6c. MMP-2, -3, -7, -9 and -12 can
cleave plasminogen to produce angiostatin 118,119 and MMP-3, -9, -12, -13 and -20 may
produce endostatin 120, both of which reduce endothelial cell proliferation 121,122.
In order for a cancer cell to metastasize, it must first cross the epithelial basement membrane,
invade tumor-associated stroma, enter blood vessels and/or lymphatics, extravasate and establish
proliferating metastases 91. The role of MMPs in this process has been investigated in vitro
through invasion assays and in vivo through xenograft metastasis assays. The overexpression of
MMP-2, -3, -13 and -14 or inhibition of TIMPS all promote the invasion of cell lines through
collagen type I 123-126. In a metastasis assay, the downregulation of MMP-9 in mice led to
significantly reduced numbers of colonies formed in mice lungs 127. Another important step in
invasion is migration and MMP-2 and -14 cleavage of laminin-5 reveals an important site that
triggers motility, shown in Figure 6d, 128,129. This cleaved form of laminin-5 is found in
22
experimental tumors, and MMP-14 and laminin-5 are co-localized in human cancers 128,129.
For migration, a cancer cell must detach from its surrounding matrix and its neighbouring cells.
CD44 is cleaved by MMP-14, leading to the release of the extracellular domain, but when the
cleavage site is mutated, cell migration is inhibited, illustrating the importance of MMP
activation for tumor cell motility 130. MMPs, specifically MMP-3 and -7, also play a critical role
in the downregulation of cell-cell adhesion, freeing the cells to migrate, and in the transition from
epithelial cells to mesenchymal cells, separated by the basement membrane (see Figure 6e),
associated with a more aggressive malignancy 131,132.
1.3.1.1 MMPs in Oral Carcinoma
OSCC is a highly invasive malignancy that begins in the oral cavity epithelium and progresses
by degradation of both the ECM and the basement membrane. MMPs are critical to such ECM
and basement membrane remodelling, so it is expected that they play a role in OSCC. In fact,
there are many studies that have shown MMP-1, -2, -3, -9, -10, 11, and -13 to be expressed in
oral carcinoma and to be critical in the oral tumor progression 133-139.
MMP-1 mRNA has been found to be expressed in epithelial cells within tumor nests and in
surrounding tumor-associated connective tissue 133,134. MMP-2 has been found in in vivo
OSCC, within small tumor islands 135, but recently there has been evidence that it is derived
from tumor-associated stroma, mainly fibroblasts, rather than the cancer cells themselves
140,141. Additionally, MMP-2 expression in OSCC has been positively correlated to lymph
node metastases, illustrating the critical role that tumor microenvironment plays in tumor
invasion. Kusukawa et al. 136 found many oral carcinoma tumors that expressed MMP-3 at their
invasive margin and they later showed a positive correlation between MMP-3 expression and
tumor size, tumor thickness and invasion. MMP-9 has been shown to be expressed at the tumor-
stroma interface of malignant keratinocytes 142,143. The overexpression of MMP-10 and -11
has been correlated with tumor differentiation and overall invasiveness 141. MMP-13 is
expressed both by fibroblasts in the stromal region and by the invading tumor front 139,144.
23
A recent study investigated the MMPs present in various patient oral tissue samples, including
samples with OSCC 145. The patient tissue samples were tested for expression of various
MMPs, including some (MMP-7, 19 and -26) never or rarely tested in OSCC. The expression of
most MMPs tested, MMP-2, -3, -7, -9, -10, -12, -13, -26, were upregulated in OSCC samples and
in 90% of the samples MMP-7, -9, -10, and -13 were expressed on the cancer cells, while
expression of MMP-2, -3 and -12 was mostly on the stromal cells 145, see Figure 7.
Figure 7: Immunohistochemistry was performed on formalin-fixed human oral carcinoma tissue
using mouse monoclonal antibodies demonstrating: A) MMP-7 expression is high in cancer cells
in OSCC samples, with occasional expression in stromal cells, B) MMP-13 is highly expressed
in invasive OSCC, C) MMP-9 is expressed in highly invasive OSCC 145, reproduced with
permission.
It is clear that MMPs play a critical role in OSCC and any number of them could provide a target
for specific treatment of OSCC.
1.4 Molecular Beacons
In the post-genomic era, there has been great focus on developing agents that can specifically
target particular genomic sequences or proteomic sequences overexpressed in diseased tissue,
including disease-associated enzymes like MMPs 146-148. These agents would facilitate specific
24
treatment of diseased tissue, sparing healthy tissue. Molecular beacons are one such class of
probes with high sensitivity and selectivity biomolecular recognition 146.
Conventional molecular beacons (MBs) consist of a stable stem-loop oligonucleotide, forming a
hairpin, with a fluorophore on one end, typically the 5’-end, and a quencher on the other end, as
seen in Figure 8a 146,149. In the hairpin conformation, the MBs show no fluorescence, as the
close proximity of the quencher to the fluorophore facilitates efficient energy transfer from the
fluorophore to the quencher, quenching fluorescence 146. The MB is designed such that the
hairpin’s loop is complementary to the target of interest, that is, the genomic sequence
overexpressed in the diseased tissue of interest. In the presence of the target, the MB undergoes a
conformational change and the structure from hairpin to linear sequence. In this new linear
conformation, the fluorophore and quencher are too far from one another for efficient FRET and
the fluorophore is free to fluoresce. Consequently, the conformational change is characterized by
a sharp increase in fluorescence, illustrated in Figure 8b.
Figure 8: A) The conventional molecular beacon with a hairpin stem formed by complementary
sequences and when the probe hybridizes with its target, the stem loop structure cannot exist,
resulting in a conformational change and B) MB specificity is illustrated with the MB incubated
with complementary target (a) leading to a strong increase in fluorescence intensity, whereas a
single mismatch (b) and a single deletion (c) led to no significant fluorescence increase 146,
reproduced with permission.
25
Conventional MBs display high specificity, capable of differentiating between two DNA
sequence targets with as little as one differing nucleotide, as demonstrated in Figure 8b
146,150,151. Additionally, with the appropriate selection of fluorophores and quenchers, very
high signal-to-background ratios (with 200-fold increases often occurring under optimal
conditions) are possible, as they are non-fluorescent in their native state 152. Both of these have
led to MBs being used for a number of bioanalysis applications including DNA arrays and
monitoring of real-time polymerase chain reaction 151,153,154.
1.4.1 Peptide-Based Molecular Beacons
While MBs were originally designed to specifically recognize nuclei acid sequences, they have
also been designed to be specifically activated by certain protein sequences 148. These can be
useful for imaging purposes, with MB activation and fluorescence confined to tissues
overexpressing the protein target of interest and remaining quenched in all other tissues. These
peptide-based MBs are cleaved by proteases, catalytic enzymes that hydrolyze peptide bonds
155,156. MBs have been widely explored for cancer imaging, with their increased protease
expression, and typically fall into one of three categories: classic peptide probes; polymer-based
peptide beacons; nanoparticle-based peptide beacons.
Classic peptide beacons consist of a fluorophore and quencher molecule conjugated to a short
protease-cleavable linker. Due to the close proximity of the fluorophore and quencher molecules,
there is no fluorescence when the linker remains intact. Enzymatic cleavage of the MB by a
protease leads to an increase in fluorescence by the fluorophore. These classic MBs have been
used in vivo for imaging of tumors (providing image guidance and the ability to discern healthy
and tumor tissue), imaging of tumor-associated protease activity, assessment of protease inhibitor
therapies and to provide greater understanding of the role proteases play in tumorigenesis,
angiogenesis and metastasis 158,159.
26
However, there are a few issues associated with these classic peptide MBs. Small peptides tend
to have poor pharmacokinetic properties, with the main concern being poor tumor accumulation
caused by their short plasma half-life and limited tumor penetration 160. Consequently, a number
of strategies were developed to try and improve peptide-based MBs for in vivo applications,
namely conjugation to a polymer and encapsulation in nanoparticles 158,160-162.
1.4.2 MMP-Targeted Molecular Beacons
With heightened MMP expression in almost every human cancer and given its expression is
correlated with advanced tumor stage, increased invasion and metastasis, and shorter survival 91,
it seems only natural that MMPs have become a protease target for molecular beacons.
Our lab previously designed a MB to be cleaved in the presence of MMP-7 163, targeted because
of its epithelial origin and high expression in a number of malignancies 164,165. The
fluorophore, pyropheophorbide (pyro), was selected for its near infrared emission and high tumor
affinity 166. Near-infrared (650-900 nm) fluorescence imaging is considered optimal due to the
greater tissue penetration, as a result of low tissue absorption, and low autofluorescence 167,168.
Pyro was separated from the quencher, black hole quencher 3 (BHQ3), via a short MMP-7-
cleavable peptide sequence, GPLGLARK; specifically cleaved between G and L, indicated in
italics 166. We hypothesized that upon entrance into MMP-7-expressing cells, MMP-7 would
specifically cleave the peptide sequence, separating pyro from BHQ3, thus restoring pyro’s
fluorescence.
MMP-7-triggered MB activation was first validated in solution studies. The MB was incubated at
37C, with MMP-7, with MMP-7 and its inhibitor, with MMP-2 and an uncleavable MB was
incubated with MMP-7 and the fluorescence intensity was monitored over time, see Figure 9. An
immediate increase in fluorescence intensity was observed in the MB incubated with MMP-7,
but no fluorescence increase was observed in the other samples. Samples were subsequently
27
analyzed by high-performance liquid chromatography (HPLC) and this confirmed that MMP-7
was able to specifically cleave the MB at the known cleavage site between G and L 169,170.
Figure 9: Validation of the MB's MMP-7 specificity: the fluorescence intensity was monitored
over time with MMP-7, MMP-7 and an inhibitor, MMP-2 and of an uncleavable MB, C-PPB,
and MMP-7 163, reproduced with permission.
MMP-7 activation of the MB was then assessed in cancer cells in vitro. Confocal fluorescence
microscopy studies were performed using KB cells (MMP-7 positive) and BT20 cells (MMP-7
negative) 171 incubated with either the MB, or its uncleavable counterpart. The confocal images
show a strong fluorescence signal in KB cells incubated with the MB (see Figure 10a 2), but
minimal fluorescence is observed in BT20 cells incubated with the MB or either cell line
incubated with the uncleavable beacon (see Figure 10a 3,5,6).
28
Figure 10: MMP-7-specific activation validation in vitro with fluorescent and brightfield
confocal images of 1) KB cells alone, 2) KB cells with 60 uM MB, 3) KB cells plus 60 uM
uncleavable beacon, 4) BT20 cells alone, 5) BT20 cells with 60 uM MB, 6) BT20 cells with 60
uM uncleavable beacon 163, reproduced with permission.
In vivo validation was also performed. A mouse bearing a KB tumor had a tail vein injection of
80 nmol of MMP-7-specific MB and its fluorescence intensity was monitored over time, as
shown in Figure 11. There is no fluorescent signal prior to injection and there is a strong increase
in fluorescence in the tumor following MB injection, reaching maximum intensity by 3 hours
post-injection, as seen in Figure 11, illustrating MMP-7 activation of the beacon in vivo.
Subsequent studies have been performed with other cell lines and more clinically relevant animal
models, including vertebral metastases and femur metastases, all showing this MB to be highly
effective and specific in identifying and combating malignancy 172,173.
29
Figure 11: In vivo images of a mouse bearing a KB flank tumor: a) prescan image, b) 10 min
post-i.v. injection, c) 3 hrs post-i.v. injection, d) 5 hrs post-i.v. injection 163, reproduced with
permission.
Additional studies were also performed examining the specificity of the beacon to MMP-7,
compared to other MMPs expressed in various malignancies, as MMPs have overlapping
cleavage sites and often MMP-specific peptide sequences can be specifically cleaved by multiple
MMPs. The beacon was incubated in solution with MMP-9, -8, -7, -3, -1, -13, -12, -11, -10,
respectively, for 48 hours and then analyzed with the HPLC. Based on the HPLC traces, shown
in Figure 12, MMP-3, -7, -9, -10 and -12 all show specific beacon cleavage, all of which are
implicated in OSCC.
30
Figure 12: The HPLC traces of beacon incubated with various MMPs for 48 hours; the traces
show that MMP-3, -7, -9, -10, and -12 all specifically cleave the beacon 173, reproduced with
permission.
31
Chapter 2 Topically Applied Molecular Beacons for Fluorescence-Guided
Resection of Oral Carcinoma
2.1 Introduction
Optical imaging-based approaches, specifically using autofluorescence, have been widely
investigated for the identification of a variety of malignant diseases including lung, skin, cervical
and esophageal cancers 76-78. This technique has also recently been evaluated for use in the
detection of oral carcinoma. The 5-year survival rate for patients with oral carcinoma is as low as
30% 174. Additionally, it has one of the highest rates of recurrence of all cancers, mainly
attributed to the inability to accurately identify tumor boundaries. An ongoing clinical trial, the
COOLS study, uses autofluorescence to aid in the identification of oral carcinoma during
surgical resection. Preliminary results from this trial show decreased recurrence rates using this
optically-guided approach when compared to white light surgery, the standard of care for
patients with oral carcinoma, alone 84. However, autofluorescence cannot discern malignant
lesions from benign lesions or very early stage premalignant lesions, potentially leading to high
rates of false positives and the unnecessary disfiguration of patients. As an alternative, optical
imaging with exogenous contrast agents, offer promise for improvement in outcome.
Our group had previously reported molecular beacons for specific fluorescence imaging of
malignant lesions 163. These beacons consist of a fluorophore conjugated to a quencher
molecule by a short, disease-specific peptide linker (Figure 13). The beacons were designed for
the peptide linker to be specifically cleaved by enzymes highly expressed in diseased tissue, with
only minimal expression in healthy tissue. Consequently, in healthy tissue the linker would
remain intact and should the beacon be irradiated with excitation light, there would be no
fluorescence, as the fluorophore would be quenched. In diseased tissue, though, the enzymes
32
capable of cleaving the linker were prevalent. They cleaved the linker and the fluorophore was
unquenched. Thus, there was fluorescence when beacons in diseased tissue were illuminated
with excitation light.
Figure 13: Molecular beacons consist of a fluorophore (F) conjugated to quencher (Q) via a
short disease-specific linker. In healthy tissue, the linker remains intact and no fluorescence is
observed, but in diseased tissue, the linker is specifically cleaved, activating the beacon and
allowing for fluorescence.
The success of other optical imaging strategies combined with the high specificity associated
with molecular beacons, led to the concept of using molecular beacons to effectively identify
oral carcinoma for the purposes of real-time surgical guidance. The beacons had been used to
effectively identify regions of disease in other malignancies 173,175, but its ability to identify
regions of oral carcinoma is dependent upon its linker being specifically cleaved by enzymes
much more highly expressed in oral carcinoma than in surrounding healthy oral tissue. MMPs
are proteolytic enzymes that have long been associated with various stages of tumor progression
and have more recently been associated with oral carcinoma 136,141. With the overexpression of
MMPs in oral carcinoma, it was thought that a beacon that was specifically cleaved by MMPs
could serve as an effective probe for the detection of oral carcinoma at the molecular level,
33
providing faster, more effective delineation of oral carcinoma boundaries. This could facilitate
and complete and accurate removal of diseased tissue, while sparing healthy tissue.
The potential of an MMP-specific molecular beacon as an image-guided approach to surgery is
evaluated herein. The molecular beacon used consists of pyropheophorbide (pyro) is linked to its
quencher molecule black hole quencher 3 (BHQ3) via an MMP-cleavable peptide sequence,
GPLGLARK and is referred to as PPMMPB. The ability of PPMMPB to effectively and specifically
activate and fluoresce in oral carcinoma is evaluated, providing the first step towards the
implementation of PPMMPB as a strategy to identify oral carcinoma for the purposes of real-time
surgical guidance
2.2 Materials and Methods
PPMMPB Synthesis: The PPMMPB consists of pyro conjugated to black hole quencher 3 (BHQ3)
via an MMP-cleavable peptide sequence, GPLGLARK, with the cleavage site is indicated in
italics. This PPMMPB was synthesized as described previously 163. The positive control PPMMP
was synthesized by the same standard protocol, but without the addition of BHQ3.
Cell Line: UM-SCC-1, a human oral carcinoma cell line, was kindly provided by Dr. Thomas
Carey at the University of Michigan, USA 176. The cells were grown and maintained in
Dulbecco’s Modified Eagles’ Medium (D-MEM) supplemented with 10% fetal bovine serum,
100 U/ml penicillin, 100 g/ml streptomycin, 100 M non-essential amino acids and 2 mM L-
glutamine, at 37C in a humidified incubator with 5% CO2.
34
In vivo Xenograft Model: All animal studies were carried out with institutional approval
(University Healthy Network, Toronto, Canada). Adult male athymic nude mice (athymic,
Charles River, Wilmington, USA) were inoculated with 1x105 UM-SCC-1 cells in 20 L of
media at the tip of the tongue. All animals were maintained in pathogen-free conditions in
autoclaved microisolator cages. Two weeks after injection, the tumors reached 2-3mm in
diameter. A 50 nmol dose of either PPMMPB or PPMMP was formulated in 20 L of aqueous
solution with 5% dimethyl sulfoxide (DMSO, Sigma Aldrich) and 1.5% Tween-80. Under
general inhalation anaesthesia (isofluorane in oxygen), the tongues were imaged using an in-
house fluorescence imaging endoscopy system (650 20 nm excitation, 700 25 nm detection,
various integration times, set manually), similar to those described previously 177 and illustrated
in Figure 14. The PPMMPB or PPMMP was then injected into the tumor and surrounding tongue
tissue using a 31G needle. 15 minutes post-injection the tongues were imaged again, using the
same imaging system.
Figure 14: In-house fluorescence imaging system; A) the various components to the
fluorescence imaging system, adapted from 177 with permission, B) The imaging set up with an
animal under the endoscope, in position for imaging.
35
Ex vivo Xenograft Studies: Tongues were harvested from the mice immediately following
fluorescence imaging. They were snap-frozen and stored at -80C. Frozen sections were cut on a
cryostat (6m slices). The slices were immersed in phosphate buffered saline (PBS) for 5
minutes, dried and 5 L of mounting solution with DAPI (4’,6-diamidino-2-phenylindole from
Vector Laboratories Inc.) was added as a nuclear stain. The sections were covered with a
coverslip and imaged by confocal microscopy (Olympus, 460 nm excitation, 690 40 nm
emission).
In vivo Hamster Model: Male 6-8 week old hamsters (Syrian, Harlan, Indianapolis, USA) were
used as a model to mimic oral carcinoma in humans. Under general inhalation anaesthesia, 0.5%
DMBA (7,12-dimethylbenz(a)anthracene) in DMSO was applied to both sides of the hamster
cheeks three times every week over the course of 16-20 weeks. KimWipes (Kimberly-Clark)
were packed into the cheek pouch prior to application, to minimize any spills down the throat,
and a non-absorbent material (5mm in diameter) was used to apply the DMBA to their cheeks for
6 seconds at a time. The KimWipes were removed after the application was completed. After 16-
20 weeks of application, the tumors reached 5-10 mm in size. A 50 nmol dose of PPMMPB or
PPMMP was formulated in 100 L of aqueous solution with 5% DMSO and 1.5% Tween-80.
Under general injectable anaesthesia (80 mg/kg ketamine and 5 mg/kg xylazine), a 90-95%
resection of the tumor of interest was performed (shown in Figure 15), to mimic the clinical
scenario. After this initial resection, the cheek was imaged using the in-house fluorescence
imaging system (prescan). Then either the PPMMPB or PPMMP solution was applied topically to
the tumors and surrounding healthy cheek tissue for 15 minutes. After 15 minutes, the solution
was removed and the cheek was washed with sterile saline solution and imaged again with the
in-house imaging system. Hamsters were euthanized by pentobarbital overdose (Euthanyl®
Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada).
36
Figure 15: The experimental protocol for all hamster cheek studies involved beginning with
tumor-bearing hamster cheeks (A) and 90-95% of the tumor would be resected (B) leaving
remnant tumor tissue, illustrated by the arrows in B, and fluorescence imaging would be used to
try and detect this tumor tissue.
Ex vivo HPLC studies: HPLC studies to confirm the specificity of PPMMPB activation were
performed on hamster cheek pouch tumors. Whole tumors were either topically incubated in
PPMMPB solution in vivo (while still in the hamster cheek) or ex vivo (immediately following
surgical removal of the tumor) for 15 minutes. 200 L of DMSO was added to the tumors
(enough to ensure they were completely submerged) to extract any PPMMPB for 2 hours. The
extracted solution was then run through the HPLC-MS.
Ex vivo Hamster Cheek Studies: Whole cheeks were harvested from the hamsters immediately
following fluorescence imaging. They were snap-frozen and stored at -80C. Frozen sections
were cut on a cryostat (6m slices). The slices were immersed in PBS for 5 minutes, dried and
30 L of mounting solution with DAPI (4’,6-diamidino-2-phenylindole from Vector
Laboratories Inc.) was added as a nuclear stain. The sections were covered with a coverslip and
imaged by confocal microscopy (Olympus, 460 nm excitation, 690 40 nm emission).
37
2.3 Results
2.3.1 In vivo validation of molecular beacon specificity in a murine head and neck cancer model
The PPMMPB activation in a murine head and neck cancer model was examined. Following
injection of 50 nmol of PPMMPB into the tongue tumors and surrounding healthy tongue tissue,
fluorescence signal was detected within the tumor but not in surrounding healthy tissue,
indicating selective PPMMPB activation by cancerous tissue (Figure 16Av). To further validate
the specificity, the same was performed with PPMMP. Tongues treated with PPMMP became
fluorescent in tongue tumor as well as in healthy tongue within 15 minutes of injection (Figure
16Bv). This was anticipated as, with no quencher present, no specificity should be seen with
PPMMP. When tongue tumors were treated with a negative control, no pyro was present, no
fluorescence observed over the 15 minutes. Additionally, no fluorescence was observed when
PPMMPB was injected into healthy tongues.
38
Figure 16: Representative xenograft model fluorescence images with PPMMPB and PPMMP
demonstrating tumor-associated fluorescence and whole tongue fluorescence, respectively. A)
Tongues injected with PPMMPB: i) pre-injection colour white light image, ii) pre-injection
monochrome white light image, iii) pre-injection fluorescence image, iv) 15 min post-injection
monochrome white light image, v) 15 min post-injection image showing fluorescence localized
to the tip of the tongue, tumor tissue. A) Tongues injected with PPMMP: i) pre-injection colour
white light image, ii) pre-injection monochrome white light image, iii) pre-injection fluorescence
image, iv) 15 min post-injection monochrome white light image, v) 15 min post-injection image
showing fluorescence throughout tongue, in healthy and tumor tissue (n=3 animals).
The fluorescence images obtained within 15 minutes post-tongue injections suggested that
specific PPMMPB activation was obtained within the tongue tumors, but this needed to be
validated with histology and fluorescence microscopy. Confocal images of the frozen sections of
harvested tongues confirmed that the PPMMPB activation and fluorescence was confined within
cancerous tissue (Figure 17A). No activation was observed in healthy tissue, whereas in tongues
treated with PPMMP, there was fluorescence in both tumor and healthy tissue alike (Figure 17B).
39
Figure 17: Representative hematoxylin and eosin (i) and confocal images (ii) where red is
activated beacon fluorescence and blue is Dapi stain. A) tongue injected with PPMMPB, validating
that PPMMPB activation leading to fluorescence is confined to cancerous tissues, B) tongue
injected with PPMMP, confirming that injection of PPMMP leads to fluorescence within cancerous
and healthy tissue alike, C) tongue injected with negative control, confirming that no
fluorescence is due to the aqueous solution in which PPMMPB is brought up (n=3 animals).
These data confirm the specific activation of PPMMPB and subsequent fluorescence within tumor
tissue in vivo in a murine head and neck cancer model
2.3.2 In vivo activation and specificity of the molecular beacon in a clinically relevant animal model
While there was great promise to the in vivo xenograft model data, in order to validate the
effectiveness of MMP-specific molecular beacons for this clinical application, a study in a more
clinically relevant animal model was required. Hamster cheek pouch oral tumors are the model
of choice when studying oral carcinoma as the disease progression closely mimics that in
humans 178, and so were used to evaluate MMP-specific molecular beacons. Clinically, I
envision that surgeons would first remove all oral tissue they believe to be diseased under white
light conditions before the MMP-specific molecular beacon would be topically applied to
identify any remnant disease. With this in mind, once the oral tumors were induced by DMBA
(over the 16-20 week period), 90-95% of the tumors were resected. After this resection, PPMMPB
was topically applied to the cheeks for 15 minutes and the cheeks imaged. There was some
40
background autofluorescence observed prior to PPMMPB application, but it does not allow
diseased and healthy tissue to be discerned. Following the topical application, there was a
significant increase in tumor-associated fluorescence. The resection area, intentionally left
remnant tumor, and an additional tumor also left intentionally could all be easily identified
(Figure 18). Diseased tissue could be visualized by fluorescence, whereas the surrounding
healthy tissue showed no fluorescent signal.
Figure 18: Representative images of a hamster cheek treated with PPMMPB. A) Hamster cheek
following 90-95% resection and before any PPMMPB is applied: i) colour white light image, ii)
monochrome white light image, iii) fluorescence image showing some background fluorescence
is detected, but not sufficient to accurately identify lesions. B) Hamster cheek following 15 min
topical application of PPMMPB: i) colour white light image, ii) monochrome white light image,
iii) fluorescence image showing that the resected area, the remnant tumor and an additional
tumor intentionally left behind can all be easily visualized (n=5 animals).
To examine the beacon’s tumor specificity more closely, tumor-bearing hamsters were also
treated with PPMMP or with a negative control, containing no fluorophore. Additionally healthy
hamster cheeks underwent the same surgery as tumor-bearing cheeks would and were similarly
treated with PPMMPB. Similarly to the xenograft results, hamster cheeks treated with PPMMP
41
showed no specific fluorescence: healthy and tumor tissue alike fluoresced after the 15 minute
application (Figure 19A). There was no observed fluorescence in the tumor-bearing cheeks
treated with the negative control (Figure 19B) or in the healthy cheeks treated with PPMMPB
(Figure 19C).
Figure 19: Representative images of various control hamster cheeks: A) PPMMP-treated, B)
negative control-treated, C) healthy cheek having undergone surgery and treated with PPMMPB
where i) prescan colour white light image, ii) prescan monochrome white light image, iii)
prescan fluorescence white light image, iv) post-application colour white light image, v) post-
application monochrome white light image, vi) post-application fluorescence image. Looking at
Avi) it is clear that both healthy and cancerous tissue is fluorescent post-PPMMP application. In
Bvi) and Cvi) it can be seen that there is no fluorescence associated with the negative control or
with PPMMPB applied to surgically-treated healthy tissue, respectively (n=5 animals).
To ensure that the observed fluorescence in tumor-bearing cheeks treated with PPMMPB was
indeed the result of tumor-specific PPMMPB activation, the cheeks were imaged ex vivo. Using
confocal microscopy, activated PPMMPB is confined to tumor tissue (Figure 20); healthy
surrounding tissue does not activate PPMMPB. Consequently, fluorescence is confined and
specific to oral carcinoma tissue. It is also clear, though, that PPMMPB displays limited depth
penetration: it is only present at the outermost layers of the tumor tissue (Figure 20B and D).
This does not prevent identification of the boundaries of the disease, critical for complete
42
surgical removal of the disease. Additionally, oral carcinoma is characterized as being much
greater in linear extent than depth 10,174, meaning identification of lateral boundaries is the
most critical metric of successful surgical resection.
Figure 20: Representative hematoxylin and eosin and confocal images, where red is activated
beacon fluorescence and blue is Dapi stain. A) hematoxylin and eosin image of a depth-wise
slice through the tissue and B) corresponding confocal image showing activated PPMMPB signal
only in tumor tissue, at the outermost layer. C) hematoxylin and eosin image of an en-face slice
of tissue and D) corresponding confocal image showing activated PPMMPB at the outer layer of
tumor tissue (n=5 animals).
Similarly, the PPMMP and negative control treated tissue, as well as the surgery-treated healthy
cheeks were also imaged using confocal microscopy, again validating the in vivo fluorescence
data. PPMMP-treated cheeks show no specificity: PPMMP is equally distributed within healthy and
tumor tissue alike (Figure 21A). There is also no activated beacon signal within tissue treated
with the fluorophore-lacking negative control (Figure 21C) or the surgery-treated healthy cheek
treated with PPMMPB (Figure 21B).
43
Figure 21: Representative hematoxylin and eosin (i) and confocal images (ii), where red is
activated beacon fluorescence and blue is Dapi stain. A) Tissue treated with PPMMP shows non-
specific fluorescence in healthy and cancerous tissue with limited penetration. B) Healthy cheek
treated that underwent surgery and were subsequently treated with PPMMPB shows no PPMMPB
activation or fluorescence, further validating PPMMPB’s tumor specificity. C) Tissue treated with
negative control shows no activated PPMMPB or fluorescence, confirming that fluorescence is not
the result of the aqueous solution in which PPMMPB is brought up (n=3 animals).
Combined, the fluorescence microscopy data confirms the tumor-specific PPMMPB activation
following a 15-minute topical application, which may facilitate the complete and accurate
identification of oral carcinoma in vivo.
2.3.3 Ex vivo HPLC Studies
Tumor-specific PPMMPB activation was confirmed by in vivo fluorescence and subsequent
fluorescence microscopy. In an attempt to further validate this and also examine whether the
44
activation was in fact the result of MMP-specific cleavage, HPLC-MS of the PPMMPB extracted
from the hamster cheek pouch tumors was performed. Topical application of PPMMPB to the
tumors occurred both ex vivo and in vivo (but not jointly for the same sample). For the ex vivo
application, whole tumors were removed and incubated in PPMMPB and all beacon was extracted.
The corresponding HPLC traces confirmed tumor-specific cleavage: both intact PPMMPB and the
cleaved fragment (pyro-GPLG), a cleavage site characteristic of MMP 179, were observed in all
treated tumors (Figure 22A). The PPMMPB solution that had been applied to these tumors only
had intact PPMMPB (Figure 22B), confirming that PPMMPB cleavage and activation occurred in
the tumor.
Figure 22: Representative HPLC traces of A) beacon extracted from tumor showing that both
intact PPMMPB and PPMMP fragment are present within the tumor, B) beacon applied to the tumor,
where only intact PPMMPB is present, confirming PPMMPB activation occurs in the tumor (n=3
animals).
For the in vivo application, hamster cheeks were imaged prior to tumor removal, facilitating the
direct comparison between in vivo fluorescence images and PPMMPB activation. All tumors
showed some level of fluorescence and PPMMPB activation (Figure 23), with cleavage of the
peptide sequence occurring at the site characteristic of MMP-mediated activation 179. However,
the extent of activation varied between tumors, even within the same cheek (with every tumor
displaying 10-60% cleavage). The variation in activation can be attributed to any number of
reasons. The most likely explanations are that this variation is the result of tumor heterogeneity
or the tumors may not all have been fully submerged in PPMMPB solution, due to difficulty in
applying an aqueous solution to all of the tumors. In addition to the tumors applied with PPMMPB,
45
surrounding tissue that had been immersed in PPMMPB was also analyzed and showed no
activated PPMMPB, although intact PPMMPB was present. This corresponds well with the
fluorescence images (Figure 23) and provides further support for tumor-specific molecular
beacon activation and its utility in the detection of oral carcinoma.
Figure 23: Representative images of a hamster cheek treated with PPMMPB and corresponding
PPMMPB cleavage or activation. A) Hamster cheek before any PPMMPB is applied: i) colour white
light image, ii) monochrome white light image, iii) fluorescence image showing some
background fluorescence, mainly caused by bacteria. B) Hamster cheek following 15 min topical
application of PPMMPB: i) colour white light image, ii) monochrome white light image, iii)
fluorescence image showing each of the three tumors become fluorescent, although to different
extents (n=3 animals).
2.4 Discussion
These data demonstrate the specific activation of PPMMPB, its enzymatic cleavage, potentially by
MMP, resulting in unquenching and subsequent fluorescence, in oral carcinoma tissue. Specific
PPMMPB activation was validated in a xenograft model for oral carcinoma (Figure 16 and Figure
17). Its activation was then validated with topical application in a hamster cheek pouch model: a
model that closely mimics the disease in patients (Figure 18 - Figure 21). HPLC was used to
confirm that oral carcinoma was in fact activating PPMMPB at a site characteristic of MMP-
46
mediated cleavage (Figure 22 and Figure 23). The lateral boundaries of oral carcinoma can be
effectively identified in a very short time scale following topical application, allowing for ease of
integration into current clinical practices; and it would not significantly delay surgical resection.
The combination of PPMMPB’s high tumor specificity combined with this fast activation
following topical application can see PPMMPB fluorescence-guided resection improving treatment
of oral carcinoma; in combination with current clinical methods, improving survival and
recurrence rates.
That being said, there are limitations to this molecular beacon image-guided resection approach
to oral carcinoma treatment, as well as critical studies left to be performed. MMP expression is
heterogeneous across not only different oral carcinoma tumors, but also the various cells within a
given tumor. This differential expression will lead to varying fluorescence across tumors and
tumor cells. Should a tumor or certain cells within the tumor not express any of the MMPs
capable of activating the molecular beacon, they will not fluoresce or be identified by this
approach. This could lead to diseased tissue going undetected and consequently being left
behind. Additionally, it is critical to know the minimum concentration of activated beacon to see
fluorescence and the number of tumor cells required to activate the beacon.
On the opposite side of things, this MMP-specific molecular being is able to be cleaved by five
different MMPs (MMP-3, -7, -9, -10, -12) located within both tumor cells and stromal cells,
alike. This can lead to more than just tumor tissue being identified by this fluorescence-based
approach and, like the COOLS study, to some unnecessary loss of functionally and cosmetically
important tissue.
All things being considered, it is likely that this MMP-specific molecular beacon image-guided
treatment may need to be considered with other approaches. For example, image endogeneous
fluorophores characteristic with the disease using the COOLS autofluorescence-based approach
and then exogenous fluorophores with the MMP-specific molecular beacon fluorescence. They
47
each provide different information about the tissue that can be combined to establish optimal
treatment protocol.
48
Chapter 3 Future Directions, Research and Development
3.1 Introduction
Based upon the successes of optical imaging-based approaches for the identification of a number
of malignancies 77,78 and the high specificity provided by peptide-cleavable molecular beacons,
a molecular beacon approach to the detection and delineation of oral carcinoma for the purposes
of real-time surgical guidance was devised. Several members of the MMP family are highly
upregulated in oral carcinoma 135,136,141, and so an MMP-cleavable molecular beacon,
PPMMPB, was thought to be appropriate. This PPMMPB was designed to remain quenched in
healthy tissue, but MMP would specifically cleave its peptide linker in oral carcinoma, activating
PPMMPB, producing fluorescence. Thus, it has the potential to guide surgical resection by
specifically fluorescing in and, therefore, identifying oral carcinoma
This PPMMPB was first validated in vivo in a murine head and neck cancer model: PPMMPB
activation was found to be tumor-specific, with fluorescence confined to cancerous tissue.
Moving into a more clinically relevant model, better representing oral carcinoma in patients,
tumor tissue from incomplete resections and whole tumors were readily identified based on the
tumor-specific activation and subsequent fluorescence of PPMMPB following a short topical
application. To further examine PPMMPB’s specificity with respect to oral carcinoma, HPLC-MS
was used to identify beacon, whether intact or activated, in healthy and cancerous hamster cheek
tissue. This confirmed that PPMMPB is activated in oral carcinoma and remains fully quenched,
producing no fluorescent signal, in surrounding healthy tissue. Additionally, the site of cleavage
on PPMMPB was characteristic of MMP-mediated cleavage, suggesting that tumor-specific
activation could be MMP-mediated. The potential of an MMP-specific molecular beacon as an
49
image-guided approach to surgery has been demonstrated, but additional steps must be taken
prior to any clinical integration. Some of the next critical studies are described herein.
3.2 Immunostain-like Procedure To Identify MMPs Within Hamster Cheek Tissue
In vivo fluorescence imaging with the in-house fluorescence endoscopy system, combined with
fluorescence microscopy and histological validation, confirmed that PPMMPB was specifically
activated (enzymatically cleaved and unquenched, producing fluorescence) in oral carcinoma
(Figure 18 - Figure 21). Additionally, HPLC-MS confirmed that PPMMPB activation occurred
through cleavage of the peptide sequence at a site that is characteristic for MMP-mediated
cleavage (Figure 22 and Figure 23). However, immunostaining could not be performed to
identify MMPs within the tissue, due to a lack of hamster MMP antibodies, to confirm that
PPMMPB activation was in fact MMP-mediated. Ideally, the fluorescence microscopy data could
be overlaid with histology and tissue slices immunostained, illustrating any MMPs and
potentially attributing PPMMPB activation to the MMPs.
In an attempt to mimic immunostaining procedures and elucidate the MMPs within the cheek
tissue, a beacon activation approach to tissue staining was conceived. In this pilot study, whole
cheeks were harvested from the hamsters immediately following fluorescence imaging. They
were snap-frozen and stored at -80C. Frozen sections were cut on a cryostat (6m slices). The
slices were immersed in PBS for 5 minutes, and dried. Then 50 M of PPMMPB or PPMMP in
aqueous solution with 5% DMSO and 1.5% Tween-80 was applied to the tissue for 15 minutes.
The solution was washed off with PBS, and 30 L of mounting solution with DAPI (4’,6-
diamidino-2-phenylindole from Vector Laboratories. Inc.) was added as a nuclear stain. The
sections were covered with a coverslip and imaged by confocal microscopy (Olympus, 460 nm
excitation, 690 40 nm emission).
50
Following the application of PPMMPB to the frozen tissue slice, there was activation and
associated fluorescence within certain structures within the cheek tissue (Figure 24Aii). This can
be compared to PPMMP stained frozen tissue, which has non-specific fluorescence to the point of
saturation in all tissue structures (Figure 24Aiii). Additionally, unstained frozen tissue and tissue
stained with negative control (no fluorophore) exhibited no fluorescence (Figure 24Ai and Figure
24Aiv, respectively). Looking more closely at the frozen tissue stained with PPMMPB, it appears
that PPMMPB is being activated within tumor tissue and, potentially, normal epithelial tissue
(Figure 24Bi), but the fluorescence is fairly minimal, making accurate differentiation between
regions of activated PPMMPB and intact PPMMPB challenging.
Figure 24: Tumor-bearing hamster cheeks that did not undergo PPMMPB application in vivo were
frozen and tissues slices obtained for the immunostain-like procedure on sequential slices at
different magnifications (A and B). A) i) confocal image of an unstained tissue slice showing no
fluorescence, ii) confocal image of a slice stained with 50 M PPMMPB for 15 min with
activation and fluorescence in some parts of the tissue, but difficult to discern, iii) confocal
image of a slice stained with 50 M PPMMP for 15 min with non-specific fluorescence, iv)
51
confocal image of a slice stained with negative control for 15 min, confirming no staining is
accomplished by the DMSO and Tween-80 used to solubilize PPMMPB, v) hematoxylin and eosin
image representative of the tissue in the confocal images. B) Looking more closely at the tumor
region in the slice stained with 50 M PPMMPB, i) confocal image of the tumor region shows
activation and fluorescence are localized to tumor and epithelial cells, but the fluorescence is not
strong, ii) corresponding hematoxylin and eosin image showing the tissue in the tumor region of
interest (n=3).
Attempting to potentially increase the signal difference between regions of activated PPMMPB
and intact PPMMPB, the staining protocol was modified: various protocols with increased PPMMPB
concentration and/or increased staining time were tested. However, it appeared that both of these
led to increased non-specific PPMMPB activation. This suggests a difference in MMP activity or
localization is possible in the frozen tissue. Future directions will investigate the activity and
localization of the MMPs in the frozen tissue and optimize the staining concentration and
staining incubation accordingly. Additionally, studies using MMP inhibitors in vivo can be
performed to assess MMP-specific activity.
3.3 Quantification of Fluorescence Microscopy
The fluorescence microscopy data of the xenograft tongues and hamster cheeks demonstrate
specific PPMMPB activation within oral carcinoma (Figure 17 and Figure 20, respectively). The
extent of activation within specific structures and regions can be deduced by comparing the
relative fluorescence intensity within these images, under the same imaging conditions.
Additionally, the extent of activation was quantified in various hamster cheek tumors with
HPLC-MS (Figure 22 and Figure 23). It is desirable, though, to be able to quantify the
concentration of activated PPMMPB within the various tissues. This is possible by quantifying the
fluorescence intensity within the microscopy images.
52
Activated PPMMPB behaves as PPMMP. Consequently, PPMMP was used for a pilot study
investigating the potential to quantify fluorescence microscopy images. A series of 1 L droplets
of PPMMP was formulated in aqueous solution with 5% DMSO and 1.5% Tween-80 and with
varying concentrations: 1 mM, 100 M, 10 M and 1 M. The droplets were covered with a
coverslip and imaged by confocal microscopy (Olympus, 460 nm excitation, 690 40 nm
emission).
The fluorescence intensity can be correlated to different concentrations of PPMMP (Figure 25).
However, more concentrations need to be assessed, particularly between 1 mM and 10 M
(Figure 25) to truly quantify the activated PPMMPB within the different regions of oral tissues.
These investigations are currently underway.
Figure 25: Confocal images of droplets of A) 1 mM, B) 10 M (n=2).
3.4 Topical Gel or Spray
Topical application is the safest and most desirable method for treatment of epithelial lesions and
it also requires fewer approval guidelines, aiding in clinical translatability. PPMMPB is currently
applied topically as an aqueous solution and there are a number of drawbacks associated with the
53
application of a liquid. A liquid provides minimal control over the location of topical application,
as it can leak away from regions of interest and down the patient’s throat. Additionally, it is not
feasible to apply a liquid to all structures within the oral cavity. For example, a large percentage
of oral carcinoma patients have lesions on their tongues 64, but a liquid cannot easily be applied
to this region, limiting the usefulness of PPMMPB for fluorescence-guided resection of oral
carcinoma.
With this in mind, a thermal-transitioning PPMMPB gel was created. The formulation was
identical to the liquid PPMMPB, but also had 20% mass/volume Pluronics™, creating a PPMMPB
that was liquid at 4C, but a gel at 37C. This allowed for the PPMMPB to be applied as a liquid to
the region of interest within the oral cavity, where it would immediately gel and stay in place
(Figure 26A). This thermal transitioning PPMMPB was used to identify remaining diseased tissue
following an initial resection.
The tumor left intentionally can be better visualized following the administration of the topical
thermal-transitioning PPMMPB (Figure 26C). However, a more significant increase in
fluorescence is preferred to ease identification of diseased tissue and a stronger fluorescence is
associated with liquid PPMMPB (Figure 18). It is possible that the gel prevents migration of
PPMMPB outside of the gel, limiting the amount exposed to the hamster cheek tissue. Future
directions should optimize the gel with respect to PPMMPB migration into tissue, while still
forming a solution that can be specifically applied to regions of interest and stay in place over the
course of desired application. Alternatively, a spray, a cream or other substance could be created
and subsequently optimized.
54
Figure 26: Hamster cheek treated with the thermal-transitioning PPMMPB. A) The appearance of
the thermal-transitioning PPMMPB, which stays in place as soon as applied to the tissue. B)
Images of the hamster cheek before thermal-transitioning PPMMPB was applied: i) colour white
light image, ii) monochrome white light image, iii) fluorescence white light image showing
minimal background fluorescence. B) Images of the hamster cheek after 15 min application of
thermal-transitioning PPMMPB: i) colour white light image, ii) monochrome white light image,
iii) fluorescence white light image showing a small increase in fluorescence and an easier
identification of resection area and remnant lesions. However, the fluorescence increase is not
significant, which may be attributed to the complete tumor resection or the inability of the
PPMMPB to penetrate into the tissue when in this gel.
3.5 Fluorescence-Guided Resection
The PPMMPB has been shown to specifically elucidate regions of oral carcinoma in vivo,
demonstrating its potential clinical utility in facilitating complete surgical resections. However,
PPMMPB fluorescence-guided resection has yet to be performed. The studies need to be expanded
to include, not only the identification of oral carcinoma in hamster cheek pouches, but also the
subsequent surgical resection of all fluorescent tissue, which would contain the activated
PPMMPB. Post-resection, whole cheeks could be harvested and analyzed to see if PPMMPB
fluorescence facilitates complete removal of oral carcinoma, a critical next step in the evaluation
of this strategy to improve treatment of oral carcinoma.
55
3.6 Increased Tissue Penetration
Throughout the studies performed to date, PPMMPB has been shown to only penetrate into the
outer layers of tissue (Figure 20 and Figure 21). This should not hinder the ability of PPMMPB to
delineate the lateral boundaries of oral carcinoma, critical to the complete and accurate removal
of diseased oral tissue. Oral carcinoma has a much greater linear extent than depth 10,174 and
current clinical practices involve taking 1-2 cm margins of healthy tissue at the boundaries of
disease. Consequently, PPMMPB-guided surgical resection could have a much greater immediate
clinical impact on the amount of healthy surrounding tissue that is removed laterally from the
lesions, rather than depth-wise. That being said, identification of the lower boundaries of disease
could spare healthy tissue and also provides a possibility for surgical bed clean up, rather than
additional resections.
One strategy to improve PPMMPB penetration that has been investigated involves the use of a
zipper molecular beacon (ZMB). This ZMB forms through electrostatic attraction between a
polycationic peptide and polyanionic peptide, separated by the same MMP-cleavable peptide
linker that was used for all studies. At the end of the polyanion is the BHQ3 and of the
polycation is the pyro. The polycation increases cellular uptake of the fluorophore 158,180,181,
thus polycation-fluorophore complexes are referred to as cell penetrating peptides, which has the
potential to significantly improve penetration of PPMMPB within oral carcinoma.
The ZMB was optimized to maximize cellular uptake. It was believed that longer polycation
sequences would be more easily internalized than their shorter counterparts, as it is reported in
literature that cell internalization increases with increasing length of cationic arm, until a certain
point where a maximum is reached and cellular toxicity begins to increase 182. The cell
penetrating peptide was tested with pyro conjugated to five (pyro-5r) and eight (pyro-8r) arginine
(Arg) residues, respectively. These were analyzed by confocal microscopy and pyro-5r was
56
internalized just as effectively as its eight-Arg counterpart and both much more effectively than
pyro alone (Figure 27).
Figure 27: Confocal and transmitted light images of MT-1 cells incubated with (A) pyro, (B)
pyro-5r and (C) pyro-8r for (i) 30 minutes and (ii) 1 hour. Both pyro-5r and pyro-8r show
significantly enhanced cell penetration compared to pyro alone.
An enhanced cell penetrating ability was demonstrated using this arginine-conjugated pyro,
when compared to pyro alone as used in PPMMPB for all studies described in Chapter 2. This
suggests that greater delivery of activated PPMMPB might be possible using this ZMB, rather than
the classical PPMMPB. This should be further investigated, potentially in conjunction with a spray
or gel to improve topical application and tissue penetration. This could allow for surgical bed
clean up to be used, rather than additional surgical resection. For example, photodynamic therapy
(PDT) could be used. In addition to being a fluorophore, pyro is also a photosensitizer (PS),
57
meaning it induces cytotoxicity due to a series of photooxidative reactions, specifically through
the generation of singlet oxygen. PDT has generated significant interest because it allows for the
destruction of tumors with limited and reversible damage to surrounding healthy tissue 183. It is
minimally invasion, does not generate heat and has minimal impact on connective tissue 184.
The feasibility of using PDT for treatment of oral carcinoma is well established, with superficial
lesions and accessible light delivery 183. Should improved penetration of PPMMPB into tumors be
achieved, PDT should be investigated for surgical bed clean up, further minimizing any damage
to healthy tissue.
3.7 Conclusions
Every year, almost 300,000 individuals worldwide, including four thousand Canadians, are
diagnosed with oral cancer, more than 90% of these being oral carcinoma 4,185. The standard
treatment for these patients is surgical resection. However, accurate delineation of these tumor
boundaries is the greatest challenge facing complete and accurate removal of diseased tissue.
Currently, tissue texture is used to identify tumor boundaries, but this is fairly inaccurate and is
attributed as a cause of one of the highest rates of recurrence among cancers 42,174. In an
attempt to improve the likelihood of negative margins, surgeons aim for 1-2 cm margins when
removing oral carcinoma, leading to a huge loss of functionally and cosmetically important
tissue, often leading to permanent disfigurement, even with reconstructive surgery. Even with
these measures, positive surgical margins are common. For the patients with positive surgical
margins, radiation therapy is required and chemotherapy may also be necessary. These have a
long list of side effects including xerostomia, dermatitis, and nausea and vomiting, in the short
term. More permanent side effects include osteoradionecrosis, fibrosis, tooth decay and
neuropathy (Table 2). Even after suffering through surgery, potential disfigurement and the side
effects of radiation therapy and chemotherapy, the disease may very likely recur, with a 5-year
rate of recurrence of 30% 3.
58
The ability to accurately identify oral carcinoma boundaries during surgery could significantly
improve patient prognosis. Negative surgical margins could be more easily achieved, potentially
facilitating the removal of smaller margins of healthy tissue, minimizing the debilitating effects
of surgery on quality of life. Additionally, it could minimize or even remove the need for
radiation therapy and chemotherapy, minimizing treatment procedures and leaving patients with
fewer side effects. That is, the development and clinical integration of PPMMPB fluorescence-
guided resection of oral carcinoma could change the prognosis and quality of life of patients with
oral carcinoma.
If instead of current clinical practices, surgeons could first remove all oral tissue they believe to
be diseased and then have PPMMPB fluorescence confirm the accuracy of the resections, patient
prognosis and quality of life could be improved. Following that initial white light resection,
PPMMPB could be topically applied for 15 minutes and subsequent fluorescence imaging could
effectively identify any remaining oral carcinoma. This could then be surgically removed, and if
need be, another PPMMPB topical application, followed by fluorescence imaging, given until we
could be confident in negative surgical margins. Patients wouldn’t have to suffer through
additional therapy and the associated side effects. Importantly, the rate of recurrence would be
expected to decrease significantly. This also has the potential to impact survival rates for patients
with oral carcinoma.
The work outlined involves the first steps, and the next steps, towards this goal. Disease-specific
PPMMPB activation facilitates the identification of oral carcinoma, and with continued work, we
may see integration of this molecule into the clinic, improving patient treatment and quality of
life.
59
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