INVESTIGATING THE ABILITY OF HUMAN PAPILLOMAVIRUS INFECTIONS TO SENSITIZE HEAD AND NECK SQUAMOUS CELL CARCINOMA TO ONCOLYTIC

VIRAL THERAPY

Nicholas Kim

Research report submitted to the Department of Biochemistry, Microbiology and Immunology

In partial fulfillment of the requirements For the course BCH 4040

University of OttawaOttawa, Ontario, Canada

April 2015

© April 2015, Nicholas Kim

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the 6th most prevalent form of

cancer. It is recognized for its high morbidity and mortality rates in addition to its metastatic

capacity. The current treatment options for the advanced form of this cancer are limited to

chemoradiotherapy, which is associated with several adverse side effects and limited efficacy. A

demand for novel therapeutic alternatives therefore exists, and our lab is currently evaluating

such an approach. Previous studies conducted by Le Boeuf et al. (2012) have implicated that

human papillomavirus (HPV) infection sensitizes tumors to oncolytic viral therapy by rendering

type-1 interferon mediated cellular immunity obsolete. Since 40% of the pathogenesis of

HNSCCs are the result of HPV infections, we hypothesized that HPV infections would sensitize

HNSCCs to oncolytic viral therapy. Specifically vesicular stomatitis virus (VSV), which is

particularly sensitive to interferon signaling, was evaluated as an oncolytic virus that represents

an alternative to chemo radiation therapy for HPV driven HNSCCs. VSV is anticipated to be

selective towards targeting HPV cancer cells without targeting healthy normal cells, as only HPV

infected cells would have impaired interferon activity. Our results show that the efficacy of

oncolytic viral therapy is significantly increased in HPV positive HNSCCs particularly in

samples HN 14-13 and HN 14-09 compared to HPV non-infected cells including melanoma,

lung, and HNSCC HPV negative samples.

i

Acknowledgements

The works presented in this study were a collaborative effort with members of the Dimitroulakos

lab under the guidance of Dr. Jim Dimitroulakos. I would like to thank him and his lab for

welcoming me into their workplace and showing me the fundamentals required to carry out this

project. I would also like to extend my gratitude to members of the department of thoracic

surgery at the Ottawa Hospital for providing the tumor cores that were assessed in this study.

Declaration

The study was conducted during the fall and winter semesters of the year 2014-2015 (September

2014 – April 2015).

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Table of ContentsABSTRACT...................................................................................................................................................I

ACKNOWLEDGEMENTS........................................................................................................................II

DECLARATION.........................................................................................................................................II

LIST OF FIGURES...................................................................................................................................IV

STATEMENT OF CONTRIBUTION.......................................................................................................V

SECTION 1: INTRODUCTION.................................................................................................................11.1 HUMAN PAPILLOMAVIRUS AND HEAD & NECK SQUAMOUS CELL CARCINOMA..................................11.2 ONCOLYTIC VIRAL THERAPY................................................................................................................21.3 DIMITROULAKOS LABS PREVIOUS ASSESSMENT OF THE EFFICACY OF VSV.......................................41.4 RATIONALE AND HYPOTHESIS...............................................................................................................6

SECTION 2: MATERIALS AND METHODS..........................................................................................8

SECTION 3: RESULTS.............................................................................................................................11

SECTION 4: DISCUSSION.......................................................................................................................154.1 ANALYZING THE FINDINGS OF THE STUDY.........................................................................................154.2 FUTURE DIRECTIONS...........................................................................................................................194.3 TROUBLESHOOTING AND TECHNICAL DIFFICULTIES...........................................................................194.4 DESIGN FLAWS AND ALTERNATIVE APPROACH..................................................................................20

REFERENCES...........................................................................................................................................22

APPENDICES.............................................................................................................................................24

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List of Figures

1. Examining the sensitivity of CCs (A) and HNSCCs (B) to VSV via MTT.………………….…………………....5

2. Comparing the sensitivity of HPV negative SCC25 cell line before and after E6 transfection to VSV…………...6

3. Visual Representation of Viral Titers for Head and Neck Squamous cell carcinoma for HPV positive (A)

and HPV negative (B) samples……………………………………………………………………...……….…..11

4. Graphical Analysis of Viral Titers for Head and Neck Squamous cell carcinoma for HPV positive (A) and

HPV negative (B) samples……………………………………………………………………………………….12

5. Graphical Analysis of Viral Titers for Lung (A) and Melanoma (B) samples ………………………………….....14

iv

Statement of Contribution

My supervisor, Dr. Jim Dimitroulakos, designed the project and objectives of this study, and the experimental procedure for viral tittering was obtained from Dr. Fabrice LeBoeuf.

Members of the Department of Thoracic Surgery at the Ottawa Hospital performed the surgical resections to retrieve the tumor samples, and Jennifer Hanson completed the initial viral infections using VSV.

Special recognition also goes out to Tabassom Baghai, who conducted the plaque assays for the lung samples found in the results section of this study.

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SECTION 1: INTRODUCTION

1.1 Human Papillomavirus and Head & Neck Squamous Cell Carcinoma

The human papillomavirus (HPV) is a double-stranded DNA pathogenic virus that has

been identified with a tumorigenic capacity in certain subtypes (Hausen, 1996). There are a

several different genotypes of the virus that are documented, of which some are higher risk types

for the development of cancerous tissue: type 16, 18, 31, and 45 (Ramanakumar, 2010).

Particularly, these subtypes with higher risk denote active expression of E6 and E7 oncogenes in

the virus that regulate the malignant progression of tumors. High expression of E6 and E7 is

retained in most cancers that are HPV positive and are thereby considered important components

for the malignant conversion of healthy cells from the viral infection (McLaughlin-Drubin &

Munger, 2010).

HPV infects keratinocytes of the skin and mucous membranes, to induce the formation of

warts and papillomas (Sterling et al., 1993). They have been discovered to be an emerging risk

factor for the pathogenesis of cancers such as cervical carcinoma, anogenital carcinoma, and head

and neck squamous cell carcinoma (HNSCC). The unique carcinogenic ability comes from E6

and E7 viral proteins that promote genetic instability and hyperproliferative cellular tendencies.

The mechanism by which each oncoprotein promotes these qualities varies by the particular

mechanisms they are involved in. The E6 constituent of HPV specifically associates with the p53

protein using an adaptor protein (E6-associated protein) found in primary human keratinocytes.

With its adaptor protein the complex degrades p53 by an ubiquitin dependent proteolysis

mechanism, which ultimately compromises cellular functions that require p53 (Howley, 1996).

These include mechanisms such as nucleotide excision repair, apoptosis, and cell growth control

1

that are important for maintaining cellular response to genotoxic stresses and preventing

hyperproliferation (Smith et al. 1995). The E7 constituent of HPV imposes similar implications

to E6 by interacting with retinoblastoma proteins to enhance cell cycle progression; furthermore,

promoting immortalization of human keratinocytes (Tommasino & Crawford, 1995).

The synergistic interactions of the two constituents of HPV give the virus its oncogenic

potential and of the many different types of cancers it can cause, this study focuses on HNSCCs.

Head and neck squamous cell carcinomas are a type of head and neck cancer found in the

aerodigestive tract including the nasopharynx, oropharynx, larynx, oral cavity, lip, and nose

(Syrjanen, 2005). HNSCCs account for 90% of all head and neck cancer and are characterized by

their metastatic and heterogenic property (Marur & Forastiere, 2008) . HPV is associated with

the aetiology of HNSCC and is responsible for approximately 60% of all HNSCC cases (Kreimer

et al., 2005). The residual cause of HNSCC remains attributed to environmental and behavioral

factors that include smoking and alcohol consumption. The prevalence of HNSCC cases make it

the 6th most common form of cancer and constitute a worldwide public health issue because of its

high morbidity and high mortality rates (Mellin et al., 2000). The current treatment options for

the advanced form of this cancer is limited to types of radiotherapy combined with chemotherapy

(Bonner et al., 2006). There is a very poor prognosis associated with these limiting treatment

options as well as side effects that limit their use; therefore, novel therapeutic approaches are

required to rectify the concurrent issue. In this study, the proposed therapeutic alternative to

chemoradiotherapy is the use of oncolytic viruses.

1.2 Oncolytic Viral Therapy

Oncolytic viruses (OV) are genetically engineered microorganisms that have been

synthesized to selectively grow inside tumor cells. They have been programmed to manipulate

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cellular defects that are unique to, and are typically responsible for, the malignant transformation.

These can be tumor-specific mutations or components to tumor dependent signaling pathways

that are exclusive to cancer cells, which allow these cells to be preferentially targeted. In

principle, the natural propensities of OVs to target, replicate in, and kill cells with a high degree

of specificity will be exploited to selectively target cancerous cells. OVs therefore offer a mode

of oncolytic therapy that carries advantages over conventional anticancer drugs. Unlike drugs,

OVs are extremely adaptable as they can be tailored using recombinant DNA technology, not just

at a molecular level, but also at the level of genes. They can be engineered with the ability to

discriminate tumors from normal cells, which confers a huge therapeutic advantage. They hold

great promise to improve cancer patient outcomes, as the selective nature of viruses would

theoretically limit the possibility of side effects (Parato et al., 2005).

Pertaining to this study the vesicular stomatitis virus (VSV) was examined as a hopeful

therapeutic alternative for its unique viral qualities. VSV is a member of the Rhabdoviridae

family, and is a nonpathogenic negative-stranded RNA virus that has been engineered into an OV

(Lazzarini et al., 1981). The virus is a good candidate for oncolytic viral therapy based on its

simplicity with respect to its genetic composition, and its ability to grow to high titers in most

tissue culture cell lines. VSV is also well documented to be particularly sensitive to interferon

(IFN) mediated cellular immunity, which is the underlying reason as to why this virus was

selected over other possible alternatives (Balachandran & Barber, 2000).

VSVs sensitivity to IFN mediated cellular immunity is an important characteristic of the

virus because of the ability for HPV infections to disrupt the activity of IFN signaling and the

production of associated cytokines. The E6 oncoprotein that is expressed in high-risk types of

HPV has an added ability to inhibit cellular anti-viral IFN response through interacting with

tyrosine kinase 2 (Tyk2). Tyk2 is an important component to IFN signaling that regulates the

3

activity of the type 1 interferon receptor (IFNAR-1). The activation of IFNAR-1 is imperative to

IFN anti-viral responses and is only active upon binding Tyk2. In the presence of E6, the JH6 and

JH7 binding domains Tyk2 that facilitate the interaction between itself and IFNAR1 remains

occupied by the oncoprotein and thus, IFN mediated immunity is impaired by HPV (Li et al.,

1999). With defective innate immunity then, these cells will be extremely sensitive to VSV

infections. This is significant because only cells that are actively expressing the E6 protein will

have defective IFN mediated immunity, and only HPV positive cells will be capable of

expressing this protein. Furthermore, since HPV infections will be specific to tumor cells, this

property will allow VSV to discriminate tumor cells from normal healthy cells, which will be

resistant to the OV infection.

1.3 Dimitroulakos Labs Previous Assessment of the Efficacy of VSV

A study conducted by Le Boeuf et al. (2012) investigated the efficacy of VSV treatments

to HPV positive cervical cancers (CC) and HPV negative HNSCCs using MTT viability assays to

assess the response of certain cancer cell lines to VSV OV therapy. A total of four CC cell lines

were examined, ME180, SiHa, HeLa, and Caski, all notably HPV positive, in comparison with

four HPV negative HNSCC cell lines SCC25, Cal27, SCC9, and FADU. Each cell line was given

varying doses of VSV in vitro, and the viability of the cells was graphically analyzed (Fig. 1).

There was a clear difference between the sensitivity of CC and HNSCC to VSV concentrations,

whereby the viability of cells were significantly lower in CC compared to HNSCC especially at

higher viral concentrations. The study essentially concluded that the HPV positive CCs were

more susceptible to VSV cytotoxicity compared to the HPV negative HNSCCs.

4

Figure 1. Examining the sensitivity of CCs (A) and HNSCCs (B) to VSV via MTT. Four HPV-positive CCs (ME180, SiHA, HeLa, CaSki) and four HPV-negative HNSCCs (SCC25, Cal27, SCC9, FADU) were infected with VSV at different MOIs. The percentage of cell survival was determined by a MTT cell viability assay. (LeBoeuf et al., 2012).

Another finding in this publication was the confirmation of the role of E6 protein in

inducing tumor susceptibility to VSV cytotoxicity. The Dimitroulakos lab tried transfecting the

E6 oncogene into the SCC25 cell line that had been notably unresponsive to VSV treatments.

The SCC25 cell line, upon expressing E6, then experienced an increase in responsiveness to VSV

concentrations with significantly lower cellular viability at high viral loads compared to the wild

type SCC25 cell line. They essentially proved that E6 expression plays an important role at

increasing the efficacy of VSV treatments as expected based upon the association with the

oncoprotein and IFN mediated cellular immunity.

5

Figure 2. Comparing the sensitivity of HPV negative SCC25 cell line before and after E6 transfection to VSV. A MTT cell viability assay was performed to determine the sensitivity of SCC25 cell containing either an empty vector or expressing the HPV E6 protein to VSV (LeBoeuf et al., 2012).

1.4 Rationale and Hypothesis

The previous study had indicated that HPV infections in cancer cells could be used as a

biomarker or an indication that a particular cancer type will be susceptible to VSV treatments.

HPV positive CC cell lines were proven to be more sensitive to OV therapy compared to the

HPV negative HNSCC cell lines. The study also proposes that the difference in sensitivity

towards VSV treatments between the two different types of cancers is the result of the presence

or absence of E6 expression. There are, however, limitations to the study in its inability to

confirm that the same increase in efficacy of OV therapy will be seen in HPV positive HNSCCs.

Even though they were able to prove that induced expression of E6 in an HPV negative HNSCC

cell line sensitized the cancer to VSV cytotoxicity, whether or not innately HPV positive HNSCC

cancers will have the same result is unknown. None of the HPV positive cell lines assessed in the

previous study were HNSCC and therefore it cannot be concluded based on their findings alone

6

that VSV cytotoxicity will be seen HNSCC predisposed to HPV infections.

It is therefore the objective of this study to investigate the sensitivity of HPV positive

HNSCCs to VSV cytotoxicity using surgically resected tumor cores retrieved from patients at the

Ottawa Hospital. The investigation is performed ex-vivo to quantify the sensitivity of HNSCCs in

response to VSV treatments. We hypothesize, based on previous findings, that HPV infections

will sensitive HNSCCs to VSV infections. In order to measure the relative sensitivity of HPV

positive HNSCCs, melanoma, lung carcinoma, and HPV negative HNSCCs will be assayed using

the same quantification method for comparative purposes. Melanoma and lung cancer samples in

particular are notably HPV negative since the pathogenesis of either form of cancer is unrelated

to HPV, and represent a form of negative control alongside HPV negative HNSCCs. If in fact,

HPV infections do sensitize HNSCCs to VSV treatments we expect to see increased viral activity

in HPV positive HNSCCs compared to the negative control samples detectable through plaque

based assays. Viral tittering, or plaque assays, will be conducted to quantify the viral activity in

each tumor type. Higher viral activity measured in plaque forming units (PFU) will be indicative

of higher sensitivity of the examined sample to VSV treatments.

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SECTION 2: MATERIALS AND METHODS

Cell Maintenance - Vero cell lines obtained from Dr. J.Bell (Ottawa Hospital Research Institute,

Ottawa) were cultured with complete media consisting of Dulbecco’s Modification of Eagle’s

Medium (DMEM, Media services, Ottawa Regional Cancer Centre) in the presence of 10%

HyClone Fetal Bovine Serum (FBS, Fischer Scientific Co., Toronto, ON), 100U/mL penicillin

(Sigma, St. Louis, MI), and 100ug/mL streptomycin (Sigma, St. Louis, MI). The cells were

maintained as monolayers on cell-culture plates with growing conditions set at 37oC and 5% CO2

in a HERA_cell incubator (Kendro Laboratory Products, Newtown, CT). Optimal cellular

growing conditions were maintained by frequent replacement of complete media (every 48 hours)

in addition to frequent monitoring of cellular confluency. Cells were permitted to grow until a

confluency of approximately 80-90% was achieved, whereby confluent plates were passaged

using Phosphate Buffered Saline (PBS, Cellgro, Manassas, VA) and Trypsin EDTA 1X (Cellgro,

Manassas, VA). PBS, warmed to 37oC, was used to wash cell cultures in confluent plates, which

were treated with Trypsin, warmed to 37oC, to cleave the cells off the plates. The released cells

were then resuspended in DMEM media and stored at 37oC and 5% CO2.

Tissue Explantation and Infection - Primary cancer cell tissue specimens were surgically

removed from consenting patients to be analyzed. Fresh specimens, within 48 hours post-surgery,

were processed in complete media consisting of DMEM supplemented with 10% (v/v) FBS, and

1% (v/v) penicillin/streptomycin. Metal forceps and disposable blades, sterilized with ethanol,

were used to dissect tissue samples into 0.5mm3 pieces. Each analyte was incubated in DMEM

containing either 1 x 107 pfu or 5 x 107 pfu of GFP labeled vesicular stomatitis virus (VSV) at

8

37°C for 48 hours. Viral infectivity was confirmed by microscopy 24 hours post infection, and

infected tissue samples were stored at -80°C until used.

Viral Quantification – Plaque Assay - Confluent Vero cell cultures were passaged and

resuspended in complete media to determine the concentration of viable cells using the trypan

blue dye exclusion method as part of the Vi-Cell® XR Cell Viability Analyzer (Beckman Coulter,

Mississauga, ON). Vero cells were plated on six-well culture plates (Corning Inc., Corning, NY)

at 6 x 105cells/well and incubated for 24 hours at 37°C and 5% CO2 to develop a monolayer

covering the entire surface area of each well on the plate. VSV infected patient samples and an

unaffected patient sample were homogenized individually in 0.5 mL of PBS on ice and

centrifuged at 4000 rpm and 4°C for 10 minutes. The supernatant was serially diluted into

fractions with DMEM with respective dilution factors of: 1 in 10, 1 in 200, 1 in 4000, 1 in 40000,

and 1 in 4000000 per homogenized tissue sample. Media contained in prepared multi-well plates,

incubated with Vero cells, were aspirated and each well was allotted 0.2 mL of each dilution

along with one negative control. Treated plates were incubated at 37°C and 5% CO2 for one hour.

Post-incubation, analyte was aspirated from each well and 2 mL of a semi-solid overlay (1:1 ratio

of 2X DMEM and 0.75% agarose (amresco, Solon, Ohio)), heated to 42.5°C, was added. The

agarose solution was allowed to solidify, and samples were subsequently incubated for 24 hours

at 37°C and 5% CO2. Conroy Fixative (methanol:acetic acid in a 3:1 ratio) (Fischer Scientific

Co., Toronto, ON) was then administered on top of the overlay for 2 hours and the resultant gel

was gently removed with water . The fixed monolayers of cells were then stained with a

coomassie blue mixture (70% (v/v) dH2O, 20% (v/v) methanol, 10% (v/v) acetic acid, and 0.1%

(w/v) coomassie blue R) for 45 minutes. Plaques were counted and analyzed.

9

Graphical Analysis - Graphpad Prism 6 software was used to obtain histograms pertaining to

data retrieved from viral quantification assays.

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SECTION 3: RESULTS

The objective of the study was to quantify the sensitivity of HNSCC samples to VSV

treatments. HPV infected HNSCC samples were hypothesized to experience increased sensitivity

to VSV cytotoxicity. To validate the assertion, plaque assays were performed on HNSCC

samples that were predisposed to HPV infections and compare the results to plaque assays

performed on HNSCC samples that were not infected with the virus. Whether or not a tumor core

was exposed to HPV infections was pre-determined by identification of P16, an HPV protein

marker, through polymerase chain reactions (PCR). Plaques were quantified and graphically

represented using PRISM 6 software to generate histograms based on plaque forming units

presented through plaque assays (PFU). A total of 5 HPV positive HNSCC and 15 HPV negative

HNSCC samples were analyzed. We wanted to see an amplified response in HPV infected

samples in comparison with HPV negative samples.

Figure 3. Visual Representation of Viral Titers for Head and Neck Squamous cell carcinoma for HPV positive (A) and HPV negative (B) samples. Viral titers were performed on 2 different head and neck tumors received from different consenting patients that underwent tumor resections. Samples were processed into 0.5mm 3 pieces and infected with GFP labeled VSV at 1 x 107pfu within 48 hours post-surgery. Infection was permitted for 48 hours at 37°C and infected samples were later homogenized in PBS to treat Vero cells that were cultured on six-well cell culture plates. Tissue samples infected with virus were also accompanied with an uninfected sample from the same patient to serve as a negative control (top right well in both figures). All infected tissues were serially diluted in DMEM where the most concentrated analyte was allotted on the bottom left wells on both plates. Concentration of

A B

11

virus was progressively diluted going from left to right and bottom to top as shown. Plaques formed 24 hours post-treatment were stained with coomassie blue staining reagent and subsequently counted.

Figure 4. Graphical Analysis of Viral Titers for Head and Neck Squamous cell carcinoma for HPV positive (A) and HPV negative (B) samples. Viral titers were performed on 15 HPV negative and 5 HPV positive head and neck tumors received from different consenting patients that underwent tumor resections. Samples were processed into 0.5mm3 pieces and infected with GFP labeled VSV at 1 x 107, 2 x 107, or 5 x 107 pfu as listed, within 48 hours post-surgery. Infected tissues were then used to treat Vero cells that have been plated on multi-well plates. Plaques were formed 24 hours post-treatment and stained with coomassie blue staining reagent to be counted. Each viral concentration per sample was averaged between two replicates and their corresponding pfu’s (plaque forming units) were determined via accounting for dilution factors and the amount of virus administered. HN: Head and Neck Cancer.

PFU reflect the number of viral particles that were capable of infecting VERO cells

cultured on multi-well plates. The ability of the virus to infect VERO cells is measured by the

forming of plaques or unstained spots shown in figure 3 that is counted and quantified as PFU.

PFU therefore, measures the functional capabilities of the viral particles in each tumor samples

and not the absolute quantity of virus. It is elemental to measure sensitivity of the tissue samples

to VSV treatments by PFU and not the quantity of virus since we are specifically looking at how

A BB

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the vulnerability of HPV infected tumors compare with those that are not infected with the virus.

Figure 4 depicts high PFU’s, particularly in HN14-09 and HN14-13, for HPV positive

samples compared to HPV negative samples. Some head and neck samples that were HPV

positive did not however show sensitivity towards oncolytic viral therapy contrary to the

hypothesis: HN14-20 and HN14-21. Figure 4B illustrates mildly sensitive tumor populations

compared to HN14-09 and HN14-13, where only a few samples responded to the viral

treatments.

In order to get a better idea, for comparison, of how HPV negative samples respond to

this mode of oncolytic treatment, melanoma and lung cancer samples were also analyzed by the

same method: plaque assay. HPV is not involved in the pathogenesis of either melanoma or lung

cancer; therefore, these samples are negative controls that are used to assess how VSV infectivity

is presented in other HPV negative tumor populations outside of HNSCCs. A total of 12 lung

cancer samples were analyzed as well as 4 melanoma tumors. Each lung cancer sample was

analyzed in addition to a biopsy of healthy lung tissue from the same patient.

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Figure 5. Graphical Analysis of Viral Titers for Lung (A) and Melanoma (B) samples . Viral titers were performed on 12 Lung and 4 Melanoma tumors received from different consenting patients that underwent tumor resections. All lung samples were analyzed alongside a sample of healthy lung tissue from the same patient. Within 48 hours of surgery, samples were processed into 0.5mm3 pieces and infected with GFP labeled VSV at 1 x 107 pfu or as listed on each figure. Infected tissues were used to treat Vero cells that have been plated on multi-well plates and plaques were grown 24 hours post-treatment. Each viral concentration per sample was averaged between two replicates, and their corresponding pfu’s were determined via accounting for dilution factors and the amount of virus administered. N: Healthy Lung Tissue LU: Lung Tumor MEL: Melanoma Tumor. Data for lung tissue samples were collected by Tabassom Baghai, and analyzed by Nicholas Kim.

In the context of sensitivity, measured in PFU, the melanoma and lung samples in figure 5

were all insensitive to VSV treatment relative to HPV positive HNSCC. All lung and melanoma

resected tumors had PFU/slice lower than 105, which is substantially lower than the PFU/slice

seen in HPV positive HNSCC samples.

A B

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SECTION 4: DISCUSSION

4.1 Analyzing the Findings of the Study

One of the emerging causes of head and neck squamous cell carcinoma (HNSCC) are

notably predisposed infections to the human papillomavirus (HPV) (LeBoeuf et al., 2012). In

terms of epidemiological evidence, recent studies have shown that 40% of HNSCC pathogenesis

is directly related to HPV infection among other types of cancers the virus can cause (Fakhry,

2008). The high morbidity, and high mortality rates associated with HNSCC; however, is the

reason why this study was aimed at investigating this particular form of cancer. Currently, there

are no effective methods of treatment for patients suffering from HNSCC outside of

chemoradiotherapy, which negatively impacts patients’ quality of life. In hopes of determining a

therapeutic alternative, the efficacies of oncolytic viral treatments were therefore investigated.

Oncolytic viruses offer therapeutic properties that are promising in theory at selectively

targeting cancer cells without compromising the health of patients receiving treatment. They have

the ability to kill cells through viral replication and consequently cell lysis (Parato et al. 2005).

The specificity of the virus that allow it to preferentially kill cancer cells is presented by the

unique properties of the tumor type relative to surrounding healthy tissues. HNSCCs that have

been generated by HPV infection is thought to have this unique attribute based on HPVs ability

to disrupt Type-1 Interferon (IFN) mediated cellular immunity. HPV expresses E6 protein that

grants its tumorigenic capacity and furthermore inhibits IFN response in infected cells by

compromising the integrity of the JAK/Stat pathway (Li et al.,1999). With a defective innate

immune response specific to cells that are infected with HPV, these cells are thought to be

exclusively sensitive to oncolytic viral treatments. A study conducted by LeBoeuf (2012)

15

supports this assertion as they investigated cervical cancers, which are 100% HPV positive and

determined that they are more susceptible to oncolysis from vesicular stomatitis virus (VSV)

compared to HPV negative HNSCC cell lines. Whether or not HPV positive HNSCC samples

will display the same degree of amplified sensitivity to VSV treatments compared to HPV

negative HNSCC samples is then examined to expand on the findings of this previous study. It is

hypothesized that HNSCC are sensitized by HPV infection to VSV oncolysis.

Based on the findings of this study, it can be concluded that HPV infections are increasing

tumor vulnerability to VSV infections. HPV positive HNSCC samples, particularly HN14-09 and

HN14-13, displayed significantly higher PFU from plaque assays compared to all other HPV

negative samples including HPV uninfected HNSCC, melanoma, and lung tissues. The high

PFUs in HPV positive HNSCC is indicative of high sensitivity to VSV administration, as it is a

direct quantification of the amount of functional viruses in each tumor sample. VSV is

exclusively capable of replicating in susceptible cells within the tumor and therefore tumors that

have a higher sensitivity to the oncolytic virus are expected to uptake more replicating viruses

and conclusively produce higher PFUs in plaque assays. Since the majority of the 5 HPV positive

HNSCC samples had higher PFUs than the uninfected samples, these samples were concluded to

be more sensitive to VSV.

The increased sensitivity in these particular samples was anticipated, however, there were

still some discrepancies in the data that have not been accounted for. Even though the majority of

HPV positive cells experienced an increase in sensitivity to VSV treatments there were select

samples (HN 14-20 and HN 14-21) that did not respond to VSV presence. These were thought to

be the result of several possible factors including the potential for necrotic activity in the

collected tumor samples that would compromise the viability of its comprised cells. The tumor

samples were homogenized after being stored at -80°C, but never tested in terms of viability. If

16

some of the tissue samples were not viable hosts to facilitate viral replication because of necrosis,

these samples would have appeared as though they were not sensitive to VSV treatments. The

viability of the cells could therefore influence the way the tumors appeared to respond to VSV.

Furthermore, a difference in the subtype of HPV that caused the head and neck tumor in the

unresponsive HPV positive samples compared to the responsive samples may also account for the

discrepancy. The method used in this study, diagnosed tumors with HPV through PCR screening

for P16 gene expression. P16 is a recognized marker for high risk tumorigenic HPVs that

particularly have elevated expression levels of E6 and E7 oncoproteins compared to other HPV

subtypes. The P16 gene works as a surrogate marker for E6 expression since it is involved in a

complementary pathway that regulates cell cycle degradation (Rampias et al., 2013). The marker

however, does not always accurately depict HPV E6 expression as shown in a study by Roncaglia

et al. (2013). Though P16 expression may have been sustained in all HPV positive samples in

this study, it is possible that E6 expression varied between samples. If E6 expression were not as

prevalent in select samples, these tumors would in theory experience less sensitivity towards the

oncolytic virus.

Variability in E6 expression may also have been sustained between tumor samples based

upon the progression of the tumors themselves. Tumors at different stages of development often

have different phenotypes and composition. Some will be more malignant than others and have

different qualities that would elicit varying responses to varying modes of treatment. Yamakawa-

Kakuta et al. (2009) have found that HPV-initiated tumors lose their original characteristics

during tumor progression, which could involve a decrease in E6 expression. As the heterogeneity

of a tumor increases with its development, the expressions of certain genes become obsolete as a

result of selective pressures in the system. E6 may have been an important component in

tumorigenesis, but irrelevant in the later stages of development. The stage of the tumor could

17

therefore be a contributing factor to the unexpected insensitivity of samples HN14-20 and HN14-

21.

Another interesting finding in this investigation was the appearance of mildly sensitive

HPV negative tumor samples particularly in the HPV negative HNSCCs. Some tumor samples

that were uninfected with HPV, particularly HN14-12 (VSV 1E7) and HN14-16 (VSV 5E7) that

had PFUs over 105, showed slight vulnerability to viral treatments.

In ideal circumstances, VSV cytotoxicity would be limited to cells with impaired IFN

signaling capabilities, but the apparent sensitivity in HPV negative HNSCC suggests that the

virus can be cytotoxic even to HPV negative cells. The result can be explained by the expression

of viral matrix protein (M) associated with VSV that has the potential to inhibit interferon gene

expression (Ahmed et al., 2003). The M protein allows VSV to suppress the expression of host

IFN genes by blocking nuclear-cytoplasmic exchange of cellular mRNAs relevant to IFN

expression. Our results therefore reinforce a potential need to use attenuated strains of the virus to

mitigate the chance of potential side effects from VSV exposure. Though HPV negative cells are

only mildly sensitive to VSV, utilizing attenuated strains with truncated/mutated M protein may

enhance its therapeutic index (LeBoeuf et al., 2012).

In summation to the results discussed in this investigation, HPV infections are seen to

sensitize HNSCCs to VSV treatments as hypothesized from comparing PFUs between HPV

infected HNSCCs and uninfected tumors. The results however are inadequate to define the

relationship between HPV and HNSCC sensitivity to VSV with a high degree of statistical

significance due to the small sample size of HPV positive tumors. More HPV positive HNSCC

samples are needed to compute a T-test analysis between the different tumor types to define the

statistical significance of the results.

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4.2 Future Directions

The results obtained in this study hold promise in the use of VSV as a hopeful therapeutic

alternative to chemoradiotherapy in treating HPV positive HNSCCs. HNSCCs that are

predisposed to HPV infections are displaying an increased sensitivity to OV treatments with

VSV, but the results in this study alone are not sufficient to draw the conclusion with absolute

certainty. The sample size that represents the HPV positive HNSCCs is currently inadequate to

illustrate a positive relationship between HPV and HNSCCs in terms of therapeutic response with

a high degree of statistical significance. As such, more samples are needed to further increase the

validity of our findings in order to move on to in vivo models. Once we have analyzed more ex-

vivo samples, we are hopeful that murine models will be examined in the near future. The

ultimate objective of our research is to have VSVs used in a clinical setting on human patients to

treat advanced forms of HNSCCs.

4.3 Troubleshooting and Technical Difficulties

Though results were obtained successfully towards the end of the work term, there had

been a lot of technical difficulties at the beginning of the semester. The Dimitroulakos lab is

fairly new to plaque assays, and therefore the first couple of months were devoted to mastering

the technique in order to properly assess the viral activity of VSV in tumor samples. The main

problem encountered in this investigation was the finding of gaps that formed in wells of the

multi-well plates after they were stained with coomassie blue staining reagent. There were

localized regions that were not being properly stained most likely due to an absence of cells in the

unstained areas. This was thought to be the outcome of one of two possibilities: (i) the cells were

dying as a result of being burned from the overlay (agarose) mixture that was added on top of the

cells or (ii) the cells were being removed from the plates along with the agarose when the overlay

19

was removed. To remedy the situation, we have had several unsuccessful attempts of lowering

the temperature of agarose and increasing the time the fixative remained onto the multi-well

plates. The problem was only resolved, later, when the procedure was modified to have the

fixative on top of the overlay for a duration of 2 hours as opposed to 30 minutes.

4.4 Design Flaws and Improvements

Although the experiment did not have many design flaws, there were still areas that could

have been improved to increase the accuracy of the findings. For starters, the PCR method

employed to decipher the HPV status of tumor cores should be adjusted to specifically address E6

activity, potentially through immunohistochemical staining. Though P16, in literature, is strongly

associated with E6 expression the unpredictable nature of tumors based on their heterogenic

characteristic demands a need to specifically monitor E6 expression on its own. This is especially

important because E6 expression is thought to be the underlying reason as to why HPV positive

HNSCCs are more sensitized to OV therapy. For the same reason, the stage of the cancer should

also be incorporated as an experimental parameter as the progression of the tumor is capable of

influencing the disease phenotype. It may also be worth measuring the viability of the cells via

RNA analysis in future experiments since compromised cellular viability can give the illusion

that the tumor cores are not responsive to VSV treatments as it impairs the ability of the tumor

cells to facilitate viral replication.

I would also like to acknowledge the fact that the testing of HPV status should follow the

viral quantification assays, whether it is through viral tittering or MTT, as the pre-identification

of the HPV condition of the HNSCC tumor core could implement an element of bias when the

samples are being analyzed.

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4.4 Conclusion

The potential for the pathogenesis of HNSCC is a well-documented consequence of HPV

infections. It was the objective of our lab to therefore investigate whether the predisposed

infection to HPV in HNSCCs increased their sensitivity towards oncolytic viral treatments:

specifically VSV. We hypothesized, based on the HPVs ability to inhibit interferon mediated

cellular immunity, that HPV infected HNSCC tumors will display an increased sensitivity

towards VSV cytotoxicity compared to tumor samples that have not been exposed to the

papillomavirus. The results obtained in this study ultimately support the hypothesis, as HNSCCs

that were HPV positive were vulnerable to VSV treatments to a higher degree compared to the

HPV negative tumor cores that were assessed. The use of VSV as an OV and therapeutic

alternative to chemoradiotherapy looks promising, however more samples need to be analyzed

prior to the advancement from this preclinical experiment to an actual clinical trial that will

assess an effective and safe viral dose in human patients.

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Appendices

Appendix A

Equation used to define PFU:

PFU=Number of Plaques∗Dilution FactorVolume of dilution added ¿

well ¿

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