ucalgary_2016_dastidar_himika.pdf

UNIVERSITY OF CALGARY
Enhancing Oncolytic Rhabdovirus Infection With Sunitinib In An IFN Responsive Tumour
Model
By
Himika Ghosh Dastidar
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTERS IN SCIENCE
GRADUATE PROGRAM IN MICROBIOLOGY AND INFECTIOUS DISEASES
CALGARY, ALBERTA
APRIL 2016
© Himika Ghosh Dastidar 2016

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Abstract
Oncolytic virus (OV) therapy for cancer is an emerging biotherapeutic strategy that
employs replicating viruses to selectively infect and kill cancer cells. While promising, host
innate immunity, namely type I IFN signaling, remains a barrier to OV therapy as it eliminates
the virus before it spreads efficiently through the tumour. Sunitinib (Su), a receptor tyrosine
kinase inhibitor, was recently shown to enhance OV infection by inhibiting IFN signaling in
tumour cells. We therefore hypothesized that Su might enhance oncolytic rhabdovirus (ORV)
infection in tumours. Indeed, Su treatment improved ORV productivity, tumour regression and
overall survival in an IFN responsive tumour model. Su reduced the number and function of IFN
producing myeloid cells such as cDCs and MΦ and thereby improved tumour infection with
ORV. Collectively, our findings provide further support for the clinical evaluation of Su/ORV
co-therapy in OV refractory tumors.

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Acknowledgements
I would like to express my sincere gratitude to my advisor Dr. Douglas Mahoney for the
continuous support of my Masters of Science study and related research, for his patience,
motivation, and immense knowledge. His guidance helped me in all the time of research and
writing of this thesis. I could not have imagined having a better advisor for my MSc study.
Besides my advisor, I would also like to thank the rest of my committee: Dr. Don Morris
and Dr. Donna Marie McCafferty, for their insightful comments and encouragement, but also for
the hard questions which pushed me to broaden my research horizons and perceptions. I would
also like to thank my external examiner Dr. Faisal Khan for taking the time to read and evaluate
my thesis. I would also like to extend a special thanks to Dr. Paul Beaudry for stimulating
discussion related to my particular research project and providing insightful comments, guidance
and encouragement.
I thank my fellow lab mates for the stimulating discussions, the sound advice and
company they provide during long days at the lab, and for all the fun we have had in the last
three years. I would especially like to thank Dr. Chunfen Zhang (lab manager) for the extensive
help with the in vivo studies. I am also grateful for Dr. Dale Balce (post-doctoral fellow) and
Rhiannon Campden for kindly providing me with bone marrow derived macrophages and
dendritic cells, Dr. Victor Naumenko and Dr. Dae Sun Kim for also providing assistance with
animal work. I would like to thank Dr. Franz Zemp for critically reading this thesis.
Last but not the least; I would like to thank the 2013 iGEM crew as well as my family
and friends for supporting me throughout the completion of my studies.

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Table of Contents
Abstract .......................................................................................................................................... ii
Acknowledgements ...................................................................................................................... iii
Table of Contents ......................................................................................................................... iv
List of tables.................................................................................................................................. vi
List of figures and illustrations .................................................................................................. vii
List of abbreviations .................................................................................................................. viii
CHAPTER 1: INTRODUCTION ................................................................................................ 2
1.1 Overview ........................................................................................................................... 2
1.2 Oncolytic viruses .............................................................................................................. 2
1.3 Oncolytic rhabdoviruses ................................................................................................ 10
1.3.1 Rhabdoviruses ........................................................................................................... 11
1.3.2 Lifecycle of rhabdoviruses ........................................................................................ 12
1.3.3 Development of clinically relevant ORVs ................................................................. 13
1.4 Mechanism of oncolytic rhabdovirus therapy ............................................................. 15
1.4.1 Oncolysis...................................................................................................................... 15
1.4.2 Immune Activation ...................................................................................................... 15
1.4.3 Vascular Collapse ........................................................................................................ 16
1.5 Barriers to oncolytic virus therapy ............................................................................... 17
1.5.1 Innate Immune responses that limit ORV ................................................................. 17
1.5.2 Tumour microenvironment as a barrier to ORV therapy ............................................. 21
1.6 Sunitinib as an adjuvant for OV therapy ....................................................................... 21
1.7 Research Objectives .......................................................................................................... 25
CHAPTER 2: EVALUATING SUNITINIB AS AN IMMUNO-ADJUVANT TO
ONCOLYTIC RHABDOVIRUS THERAPY IN AN IFN RESPONSIVE TUMOUR
MODEL ....................................................................................................................................... 26
2.1 Introduction: ..................................................................................................................... 26
2.2 Materials and Methods: ................................................................................................... 29
2.2.1 Reagents: ...................................................................................................................... 29
2.2.2 Dissolving Sunitinib Malate: ....................................................................................... 31
2.2.3 Purifying Oncolytic Rhabdoviruses: ............................................................................ 31
2.2.4 4T1 subcutaneous tumour model: ................................................................................ 32
2.2.5 In vivo survival experiments:....................................................................................... 33
2.2.6 Quantification of virus particles within the tumour: .................................................... 34
2.2.7 Bioluminescence Imaging:........................................................................................... 35
2.2.8 Bone marrow derived macrophage and DC cultures: .................................................. 36
2.2.9 IFN- assay (Eve Technologies): ................................................................................ 38
2.2.10 Measuring IFN- using ELISA: ................................................................................ 39
2.2.11 In vitro viability assay:............................................................................................... 40
2.2.12 Multistep viral growth curves: ................................................................................... 41
2.2.13 Western blot analysis: ................................................................................................ 42

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2.2.14 Flow cytometry: ......................................................................................................... 44
2.2.15 Statistical Methods: .................................................................................................... 46
2.3 Results ................................................................................................................................ 47
2.3.1 Sunitinib improves ORV mediated tumour control in the 4T1 model......................... 47
2.3.2 Sunitinib treatment improves ORV infection and reduces production of type I IFN in
the tumour and serum of ORV treated animals .................................................................... 51
2.3.3 Sunitinib does not inhibit IFN- production in 4T1 cells ............................................ 55
2.3.4 Sunitinib treatment reduced the number of IFN producing cells in the tumour and
spleen of 4T1 tumour bearing animals.................................................................................. 61
2.3.5 Sunitinib reduces type I IFN production from BMDM and BMDCs ex vivo ............. 67
2.3.6 Sunitinib treatment may make the tumour microenvironment more immune-
permissive ............................................................................................................................. 71
CHAPTER 3: DISCUSSION ..................................................................................................... 75
3.1 Summary of findings......................................................................................................... 75
3.2 Discussion of findings ....................................................................................................... 76
3.2.1 Sunitinib improves ORV infection in a tumour cell independent manner................... 76
3.2.2 Su treatment may improve immune contexture of 4T1 tumours ................................. 89
3.2.3 Su treatment may improve viral delivery to the tumours ............................................ 90
3.3 Limitations of this study ................................................................................................... 90
3.4 Future Directions .............................................................................................................. 92
3.5 Conclusion and Significance ............................................................................................ 96
REFERENCES: .......................................................................................................................... 97
APPENDIX:............................................................................................................................... 107
Appendix 1: Supplemental Figures ..................................................................................... 107