cell-based il-12 immunotherapy in a transgenic murine ... · cell-based il-12 immunotherapy in a...

Cell-Based IL-12 Immunotherapy In A Transgenic Murine Breast Cancer Model

By

Michael Christopher Mielnik

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Medical Biophysics

University of Toronto

© Copyright by Michael Christopher Mielnik (2017)

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Cell-Based IL-12 Immunotherapy In A Transgenic Murine Breast Cancer Model

Michael Christopher Mielnik

Master of Science

Graduate Department of Medical Biophysics

University of Toronto

2017

Abstract

Interleukin-12 (IL-12) is a pro-inflammatory cytokine that plays a major role in

immune responses. IL-12 causes increases in T and NK cell proliferation, interferon-γ (IFN-

γ) production and leads to a TH1 differentiation bias and cell-mediated immunity. Due to the

pro-inflammatory effects, the use of IL-12 for cancer immunotherapy has been a constant

topic of debate. A promising approach has been studied by our lab where a small number of

transduced tumour cells expressing IL-12 are injected to initiate an immune response. These

IL-12 transduced host tumour cells cause sustained rejection, even to challenges with the

original parent tumour. The effectiveness of this model has been demonstrated in murine

leukaemias and solid tumour models.

In this thesis, I have developed a novel breast cancer cell line model derived from the

MMTV-PyMT transgenic breast cancer model to expand the cell-based IL-12 cancer

immunotherapy into a more clinically relevant solid tumour model.

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Acknowledgments

I would likely to sincerely thank my thesis supervisor, Dr. Christopher J. Paige for his

constant guidance and insight throughout my graduate degree.

I would like to thank all the past and present members of the Paige Lab. Specifically,

Dr. Alexandra Berger, Caren Furlonger, Joshua Moreau, Selena Cen, Dr. Charles Liu and Dr.

Megan Nelles for their constant support and guidance. A special thanks to Dr. Megan Nelles

and Joshua Moreau for their advice, assistance and being there to talk when working through

the tough aspects of this project.

I would like to sincerely thank my supervisory committee members, Dr. Tak Mak,

Dr. Jeffrey Medin and Dr. Mark Minden for their valuable insight in guiding this project.

I would like to thank Dr. Jeffrey Medin and his laboratory for providing us with the

murine IL-12 lentiviral vector.

Lastly, I would like to thank my friends and family for all their constant support

throughout this graduate degree. A special thanks to Ewa Hoffmann, Kasia Mielnik, Janusz

Mielnik and Jessica Mantha.

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Table of Contents

Abstract....................................................................................................................................................ii

Acknowledgments..............................................................................................................................iii

TableofContents.................................................................................................................................iv

ListofFigures&Tables...................................................................................................................vii

ListofAbbreviations.......................................................................................................................viii

CHAPTER1:INTRODUCTION............................................................................................................1

1.1 Breast Cancer ........................................................................................................................ 1

1.1.1 What is Breast Cancer? .................................................................................................. 1

1.1.2 Breast Cancer Staging and Classification ...................................................................... 2

1.1.3 Standard of Care Breast Cancer Treatments .................................................................. 4

1.2 Breast Cancer Immunology and Immunotherapy ................................................................. 8

1.2.1 Immunology of Breast Cancer ....................................................................................... 8

1.2.2 Breast Cancer Immunotherapies .................................................................................. 10

1.3 IL-12 Cancer Immunotherapy ............................................................................................. 14

1.3.1 What is IL-12? ............................................................................................................. 14

1.3.2 IL-12 Clinical Trials .................................................................................................... 15

1.3.3 Cell based IL-12 Cancer Immunotherapy .................................................................... 17

1.4 MMTV-PyMT Transgenic Breast Cancer Model ............................................................... 18

1.5 Current Experimental Framework ...................................................................................... 21

CHAPTER2:METHODS....................................................................................................................23

v

2.1 Animals ............................................................................................................................... 23

2.2 MMTV-PyMT Transgenic Mice Breeding ......................................................................... 23

2.3 Genotyping of MMTV-PyMT Transgenic Mice ................................................................ 23

2.4 Derivation and Standard Culturing of Breast Cancer Derived Epithelial Cell Lines ......... 24

2.5 IL-12 Lentiviral Transduction of MMTV-PyMT Derived Cells ........................................ 25

2.6 Assessing IL-12 Production by Intracellular Flow Cytometry and ELISA ........................ 25

2.7 Cloning of Epithelial Enriched Cells and IL-12 Transduced Cells .................................... 26

2.8 In Vivo Tumour Experiments with PyM1 Epithelial Tumour Initiating and IL-12

Transduced Clones .................................................................................................................... 27

2.9 Hematopoetic Cell Depletion .............................................................................................. 27

2.10 Cell Isolation and Flow Cytometry for Intracellular IL-12 and PyMT Phenotyping ....... 28

2.11 Development and Analysis of PyM1 and LV12 Conditioned Media ............................... 29

2.12 Statistical Analysis ............................................................................................................ 29

CHAPTER3:RESULTS.......................................................................................................................30

3.1 Development of the Breast Cancer Epithelial Tumour Initiating Cell Line PyM1 ............ 30

3.2 IL-12 Lentiviral Transduction of the Epithelial Tumour Initiating Cell Line PyM1 ......... 31

3.3 Cloning of MOI 10 Transduced PyM1 Cell Line ............................................................... 32

3.4 IL-12 Production of Transduced Subclones of PyM1 ........................................................ 32

3.5 Characterization of IL-12 Producing Cell Lines ................................................................. 33

3.6 IL-12 Producing Cell Lines Inhibit Tumour Development ................................................ 34

3.7 Small Percentages of IL-12 Producing Cells are Sufficient to Protect Mice from

Tumour Development ............................................................................................................... 34

3.8 IL-12 Producing Cells Mediate Delay in Tumour Development to PyM1 Challenge ....... 35

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3.9 Examining the Mechanism of IL-12 Producing Cell Line Rejection in the PyM1 Model . 36

3.10 Timing of IL-12 Producing Cell Treatment Alters Delay in Tumour Development ........ 37

3.11 Weekly Treatment of MMTV-PyMT Transgenic Mice with an IL-12 Producing Cell ... 38

CHAPTER4:FIGURES........................................................................................................................40

CHAPTER5:DISCUSSION.................................................................................................................52

5.1 General Discussion ............................................................................................................. 52

5.2 Future Directions ................................................................................................................ 69

5.2.1 Developing Improved Treatment Strategies for MMTV-PyMT Transgenic

Mice ...................................................................................................................................... 69

5.2.2 Effect of Cell-Based IL-12 Cancer Immunotherapy on Metastases ............................ 70

REFERENCES........................................................................................................................................72

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List of Figures & Tables Tables Table 1. MMTV-PyMT Genotyping PCR Reaction .............................................................. 41 Table 2. MMTV-PyMT Genotyping PCR Program .............................................................. 42 Figures Figure 1. Characterization of a MMTV-PyMT epithelial breast tumour initiating clone, PyM1 ........................................................................................................... 43 Figure 2. Transduction with an IL-12 lentiviral vector and cloning of PyM1 cells to isolate IL-12 producing subclones. ........................................................................ 44 Figure 3. Characterization of select IL-12 producing PyM1 subclones, LV12.1 and LV12.18 ................................................................................................................. 45 Figure 4. Injection of IL-12 producing subclones of PyM1 protects mice from tumour development ........................................................................................................... 46 Figure 5. A small number of IL-12 producing cells are required for protection of tumour development in mice .............................................................................................. 47 Figure 6. Injection of IL-12 producing subclones of PyM1 delay tumour development to PyM1 challenge ...................................................................................................... 48 Figure 7. Immune mechanism of tumour protection of IL-12 producing subclones ............. 49 Figure 8. Timing of IL-12 producing subclone treatment alters outcome to therapy ............ 50 Figure 9. Weekly treatment of MMTV-PyMT transgenic mice with an IL-12 producing subclone ................................................................................................................. 51

viii

List of Abbreviations

ACK Ammonium-Chloride-Potassium

ACT Adoptive T Cell Transfer

AJCC American Joint Committee on Cancer

ALL Acute Lymphocytic Leukaemia

AML Acute Myeloid Leukaemia

ANOVA Analysis of Variance

APC Allophycocyanin

BCS Breast-Conserving Surgery

BRCA1 Breast Cancer 1

BRCA2 Breast Cancer 2

CAR Chimeric Antigen Receptor

CCL C-C Motif Chemokine Ligand

CD Cluster of Differentiation

CEA Carcinoembryonic Antigen

CTL Cytotoxic T Lymphocyte

CXCL C-X-C Motif Chemokine

EDTA Ethylenediaminetetraacetic Acid

EF1- α Elongation Factor 1-α

ELISA Enzyme-Linked Immunosorbent Assay

ER Estrogen Receptor

FCS Fetal Calf Serum

FDA Food and Drug Administration

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G-CSF Granulocyte-Colony Stimulating Factor

GEM Genetically Engineered Mouse

GM-CSF Granulocyte/Macrophage-Colony Stimulating Factor

H&E Hematoxylin and Eosin

HER2 Human Epidermal Growth Factor Receptor 2

HNSCC Head and Neck Squamous Cell Carcinoma

HRT Hormone Replacement Therapy

HSA Heat Stable Antigen

IFN-γ Interferon-γ

IHC Immunohistochemistry

IL Interleukin

I.P. Intraperitoneal

IP-10 Interferon-γ-Induced Protein 10

I.V. Intravenous

JAK Janus Kinase

KC Keratinyte Chemoattractant

LIF Leukaemia Inhibitory Factor

LIX LPS-Induced CXC Chemokine

MAGE-A1 Melanoma-Associated Antigen 1

MART-1 Melanoma Antigen Recognized by T cells 1

MCP-1 Monocyte Chemoattractant Protein 1

M-CSF Macrophage-Colony Stimulating Factor

MDSC Myeloid Derived Suppressor Cell

x

MIG Monokine Induced by Interferon-γ

MIN Mammary Intraepithelial Neoplasia

MIP Macrophage Inflammatory Protein

MMTV Mouse Mammary Tumour Virus LTR Promoter

MOI Multiplicity of Infection

MUC-1 Mucin-1

NK Cell Natural Killer Cell

NOD/SCID Nonobese Diabetic/Severe Combined Immunodeficiency

PBL Peripheral Blood Lymphocytes

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

PDX Patient Derived Xenograph

PE Phycoerythrin

PE-Cy7 Phycoerythrin Cyanin 7

PR Progesterone Receptor

PSMA Prostate Specific Membrane Antigen

PyMT Polyoma Virus Middle T Antigen

RANTES Regulated on Activation, Normal T Cell Expressed and

Secreted

S.C. Subcutaneous

SPF Specific Pathogen-Free

STAT Signal Transducer and Activator of Transcription

TCR T Cell Receptor

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TGFβ Transforming Growth Factor β

TH1 T Helper Type 1 Cell

TH2 T Helper Type 2 Cell

TIL Tumour Infiltrating Lymphocyte

TNF-α Tumour Necrosis Factor α

TNM Tumour Status, Nodal Status, Metastasis Status

UICC Union for International Cancer Control

VEGF Vascular Endothelial Growth Factor

1

CHAPTER 1: INTRODUCTION

1.1 Breast Cancer

1.1.1 What is Breast Cancer?

Breast cancers are malignant tumours that originate from breast or mammary tissue,

which predominantly affect women. It is one of the most common forms of cancer afflicting

Canadian women today and is the second most common cause of cancer-related death in

Canadian women1. While breast cancer does predominantly affect women, there are cases of

it developing in males, but these are far less common with less than 1% new cases and

mortalities associated with it each year1. Based on figures from 2015, it is estimated that

there will be approximately 25,000 women diagnosed with breast cancer, which makes up

26% of all new cancers in women. The 5-year survival rate for breast cancer is

approximately 88% for women that are diagnosed1. The incidence rate for breast cancer in

Canada has fluctuated, both increased and decreased, through the 1990s and 2000s. The

increase in incidence rate seen has been associated with the improvement and standardization

of breast cancer screening1-3. Another reason for the increase has been due to the

implementation of hormone replacement therapies (HRTs) used amongst post-menopausal

women, which has been linked to increased rates of breast cancer. Following the discovery of

this link, the subsequent decrease in incidence rate seen in breast cancer is thought to be

associated with the decrease in HRT use1-3. Since approximately 2004, incidence rate of

breast cancer in Canada and the United States have remained stable1.

The breast is a complex organ that is comprised of mammary glands, which are

essentially a unique type of sweat gland4. Breasts are made up of a variety of different

substructures that are necessary for the regular function of milk production. The tissue types

2

that makes up the breast are either epithelial cells or stromal cells, comprising of

approximately 10-15% and 85-90% respectively4. The breast contains approximately 15 to

20 lobes surrounded by adipose fat tissue, with each lobe subdividing further into lobules

ranging from 20 to 40 in number4. The lobules are comprised of tubuloalveolar glands that

produce milk. These lobes drain into a major lactiferous duct that dilates into a lactiferous

sinus towards the areola and through the nipple4.

In most cases, the epithelial cells that line the duct are the cells of origin within

breast cancer5. The normal function of breast ducts is to carrying milk from the glands,

located in the lobes, to the nipple4. Breast cancers that arise from ductal epithelial cells are

broadly classified as ductal carcinomas. Another, less common, variation of breast cancer

arises from lobular epithelial cells, which are responsible for milk production4,5. Breast

cancers that arise from lobular epithelial cells are broadly termed lobular carcinoma. In

addition to these, there are other, less common histological, types of breast cancer such as

mucinous or tubular. However, these comprise a far smaller percentage of all breast cancer

instances5.

1.1.2 Breast Cancer Staging and Classification

Staging and classification are common processes by which cancers are diagnosed and

severity is assessed. Staging primarily focuses on specific parameters, which include tumour

burden and spread from the local site, to allow for an understanding of how much a patient’s

cancer has progressed. The purpose of staging is to coherently express the magnitude and

severity of a patient’s cancer, allow for accurate diagnosis, assessment of prognosis and, in

some cases, to help in treatment decision-making. A variety of staging criteria have been

developed for solid tumours, to effectively identify tumour burden and severity, with the

3

most common method being the tumour size, nodal status, and metastasis (TNM) staging

system6.

Another method of breast cancer classification is molecular characterization; the

identification of different breast cancer subtypes. Pioneering work was done by groups from

both Stanford and Norway, where they examined a cohort of breast cancer patients and

conducted gene expression analysis using microarrays followed by unsupervised gene

clustering7,8. From their analyses, the groups identified 5 clusters which they termed “tumour

subtypes”. These tumour subtypes have been identified as the following; the Basal-like, the

human epidermal growth factor receptor 2 positive (HER2+), the Normal Breast-like, the

Luminal A and the Luminal B8. In addition, the Stanford/Norway groups recapitulated the

gene clustering they initially observed in a variety of different breast cancer patient

cohorts8,9. Other groups expanded on the understanding of these subtypes by examining

estrogen receptor (ER), progesterone receptor (PR) and HER2 expression in breast

tumours10,11. Based on immunohistochemical (IHC) analysis, it was demonstrated that these

different breast cancer subtypes had differential expression of these three aforementioned

markers10,11. Upon analyses of these breast cancer subtypes, there are drastic differences in

the survival of patients8-11. It has also been demonstrated that these breast cancer subtypes

respond differently to therapy, which has been demonstrated in preoperative

chemotherapy12,13. Molecular subtyping is used in combination with TNM staging to provide

the information necessary to accurately diagnose, determine prognosis and guide treatment

decision-making.

4

1.1.3 Standard of Care Breast Cancer Treatments

Breast cancer is a diverse disease that can drastically differ from patient to patient,

which can mean that different therapies are necessary depending on a patient’s specific

disease state. Therefore, there are a variety of different possible therapies that are available

for breast cancer treatments, both broadly applied to breast cancer or specifically applied to

unique subtypes. A variety of different factors can be involved when dealing with therapy

decision-making for patients with breast cancer. Some factors that are involved include

tumour stage, molecular subtype, patient age, menopausal status and a family history of

inherited breast cancer associated genes such as BRCA1/2.

Surgery has been, and remains, one of the primary interventions for breast cancer. Dr.

William Halsted first proposed breast cancer surgeries and the approach by which they were

done in the late 1800s14. Surgery is focused on the removal of the tumour, which may or may

not involve the removal of surrounding healthy tissue. Over the years, surgical procedures

have evolved to include improved approaches that help to conserve the breast15. Two

possible surgeries can be done at the primary tumour sites; a lumpectomy or a mastectomy.

A lumpectomy, which is breast conserving, is a surgical procedure to remove the tumour

along with a small area of surrounding normal healthy tissue. This method leaves the

majority of the breast intact15. A mastectomy, on the other hand, which does not conserve the

breast, is a surgical procedure to remove the entirety of the tumour bearing breast, which

may include a large portion of healthy normal tissue. Variations may include a radical

mastectomy, which removes a large portion of lymph nodes in the underarm, or a double

mastectomy, which removes both breasts15. In some cases, where there is an increased risk

of breast cancer, a prophylactic mastectomy is considered16. In addition to these possible

5

breast-related surgeries, a patient can also receive lymph node removal of either the sentinel

or axillary lymph nodes for analysis. The specific type of surgery a patient will receive is

decided by the desires and judgment of the patient and doctor, which can be influenced by

the level of cancer risk15. With surgery, biopsies of the tumour mass, the surrounding tissue

and lymph nodes is commonly done to examine breast cancer staging and subtyping.

A second, broadly applied, breast cancer intervention is radiation therapy, which is

commonly utilized following surgical intervention. While surgery focuses on the removal of

any disease that has been detected, radiation therapy can be utilized to eliminate any

undetected disease that remains at local or distant sites17. Radiation therapy is commonly

given following some form of surgical intervention as a means of eliminating residual

disease, whether that be following breast-conserving surgery (BCS) or mastectomy. When a

patient receives BCS, such as a lumpectomy, or a mastectomy, a common site for possible

recurrence is within the remaining portion of the conserved breast or, in the axilla, if not

removed17. Post-BCS/mastectomy radiotherapy has been shown to decrease this recurrence

and decrease breast cancer related mortality based on meta-analyses of different studies17-19.

The most common type of radiation therapy is external-beam radiation therapy, utilizing a

machine that is exterior to the body. However, there also exist other variations that utilize a

probe, known as intra-operative radiation. Radiation therapy is given according to a specific

schedule, which consists of a number of treatments over a set period of time. A common

radiotherapy schedule in the treatment of breast cancer consists of 6 to 6.5 weeks of radiation

therapy with a patient receiving treatment 5 days a week17. Within this treatment schedule,

patients can receive whole breast irradiation, focused partial breast irradiation or a mixture of

the two17,20.

6

A third, broadly applied breast cancer intervention, is chemotherapy. Chemotherapy

is comprised of specialized pharmaceutical agents that target fast dividing cells, such as

cancer cells, and through various mechanisms, kills these rapidly dividing cells. With the

advent of molecular profiling, chemotherapy is generally only offered in specific cases15.

Patients with specific molecular profiles, such as large tumour size, lymph node

involvement, ER and PR negative tumours, and HER2 positive tumours, meet some of the

criteria for receiving chemotherapy15,21. Drugs such as doxorubicin, fluorouracil,

cyclophosphamide, methotrexate and docetaxel are just a handful of possible

chemotherapeutics that can be prescribed to a patient and are commonly given as a

combination. Possible chemotherapeutic cocktails include methotrexate, fluorouracil and

cyclophosphamide or doxorubicin, fluorouracil and cyclophosphamide15,22,23.

Chemotherapies can be used following an initial intervention such as surgical removal or

radiotherapy of the tumour to further eradicate residual undetected disease and prevent

relapse (post-operative adjuvant therapy). On the other hand, chemotherapies can be used as

a primary breast cancer therapy prior to any other intervention (neoadjuvant therapy)24,25.

Similar to radiation therapy, chemotherapy is administered following a specific schedule,

where patients they receive one or more agent. At present, various schedules have been and

are still being developed to determine the best method of chemotherapy administration26. A

variety of different chemotherapeutic pharmaceutical drugs are available and are used in a

diagnosis dependent manner, but some include capecitabine, carboplatin, cisplatin, docetaxel,

doxorubicin, 5-fluorouracil and various others15,23.

Other more specific and targeted therapies are generally only used for specific

molecular subtypes of breast cancer. One example of targeted therapy is hormone blockade

7

therapy, which is used in breast cancers that test positive for the expression of either the

estrogen (ER-positive) or progesterone (PR-positive) receptor27. In these types of tumours,

growth is driven by the expression of these endocrine hormones involved in normal breast

function. The blockade of these hormone pathways using specific agents, such as tamoxifen

(directly block the binding of estrogen to its receptor) and aromatase inhibitors (blocks

estrogen synthesis) causes endocrine deprivation to these estrogen responsive breast cancers

and has been shown to decrease death and cancer recurrences27-29. These therapies are

usually only suggested for use in postmenopausal women, as estrogen production within the

ovaries remains necessary prior to menopause15. Work analyzing the comparison of these

tamoxifen treatment to the use of aromatase inhibitors suggests that aromatase inhibitors are

superior to tamoxifen and have fewer adverse effects30. A second example of targeted

therapy is HER2 specific targeted therapy, primarily targeting the HER2+ molecular subtype

of breast cancer. The HER2+ subtype is characterized by the overexpression of the HER2

receptor on the cell membrane, which has been shown to promote cancer cell growth in these

subtypes of breast cancer. HER2 overexpression is seen in approximately 20-30% of all

breast cancers and is associated with a poor prognosis31,32. This family of receptors (which

includes the epidermal growth factor receptor) contains intracellular tyrosine kinase domains

for signaling and leads to a change in growth, differentiation, migration of breast cells that

promote cancer development32,33. HER2-specific monoclonal antibodies commonly target

extracellular domains that are responsible for ligand binding or dimerization, and therefore

prevent these normal functions of the receptor. On the other hand, HER2 receptor tyrosine

kinase inhibitors are small molecule inhibitors that block tyrosine kinase activity directly,

such as afatinib31,32,34. The most commonly used HER2 therapeutic agent is the monoclonal

8

antibody agent trastuzumab (known as Herceptin® from Genentech)31,32. The efficacy and

utility of trastuzumab has been tested in a variety of phase II and III clinical trials and used in

a variety of capacities, whether that be for primary for metastatic breast cancers32.

Trastuzumab has been used as an adjuvant therapy, in combination with chemotherapy, for

metastatic breast cancer or as a preoperative therapy in early stage breast cancer35-37.

While a variety of effective therapies are available for the treatment of primary and

metastatic breast cancer, success is not always possible. In many cases, the cancer may only

partially respond or may not respond at all and therefore recur or progress. These scenarios,

where a patient and doctor approach an impasse in treatment options, is where novel

therapies, such as cancer immunotherapies, can be utilized.

1.2 Breast Cancer Immunology and Immunotherapy

1.2.1 Immunology of Breast Cancer

In 2000, six “hallmarks” of cancer were proposed, which represented the unique and

complementary capabilities acquired by the cell that allow cancers to grow and metastasize38.

These hallmarks include such features as the evasion of growth suppressors, induction of

angiogenesis and others38. Since 2000, an increasing body of research has been assembled

that suggests that, in addition to the six classical hallmarks, there are two additional

characteristics that are acquired by cancerous cells; the deregulation of cellular energetics

and the avoidance of immune destruction, both of which are essential for the development of

cancer39.

Classically, the immune system is an organism’s biological network of structures and

processes that grant protection from a variety of harmful agents, along with keeping the body

in homeostasis. The immune system is responsible for the detection, containment and

9

elimination of harmful foreign pathogens such as bacteria or viruses while sparing normal

healthy self-tissue. To maintain homeostasis however, the body also monitors self-tissue in a

process called immune surveillance. Through immune editing, the immune system plays an

important role in the identification and elimination of transformed cancer cells.

The processes of immune editing and by which cancer escapes can be split into 3

distinct steps; elimination, equilibrium and escape. In the elimination stage, early cancerous

cells that have acquired initial alterations, such as mutations or antigen overexpression,

causing cells to acquire cancerous hallmarks, are recognized by the immune system40. In the

elimination stage, these early cancerous cells, that have expanded, are based on one or a

small number of alterations and are fairly homogeneous. When antigen-presenting cells take

up these altered antigens present on these early cancerous cells, they are presented to T

lymphocytes, activating an adaptive immune response leading to the complete elimination of

these cells40. In the majority of cases where cancerous cells arise, this method of immune

surveillance and immune editing is able to elicit effective elimination, preventing the

manifestation of cancers. While immune editing is extremely effective, it has long been

thought that during tumour formation, these immune responses actually help in the selection

of clones that are able to evade and survive immune editing, similar to the development of

resistant strains of viruses and bacteria40. This selection allows the tumour to enter the

equilibrium stage, where, through tumour heterogeneity, genomic instability (caused by the

acquisition of initial mutations) and immune selection, tumour cell clones with reduced

immunogenicity are able to evade the immune system40. This entire process of selection is

known as immune sculpting40. This equilibrium stage then leads to the final stage, the escape

stage. Through the process of genomic instability and immune selection, clones that are able

10

to survive within the intact immune system develop, allowing for the further acquisition of

mutations and alterations40. These selected clones then expand in an uncontrolled manner

possibly leading to metastases.

Immune cells present in the tumour microenvironment can have drastic impacts the

balance between tumour development and tumour clearance. Early acute inflammatory

responses to breast cancer are associated with a TH1 polarized adaptive immune response;

TH1 polarized CD4+ and CD8+ T cells mediate tumour cell cytotoxicity and regulate innate

immune cells towards tumour suppression such as M1 macrophages41. In contrast, chronic

immune responses, that are associated with tumour progression, are associated with a TH2

polarized adaptive immune response; TH2 polarized CD4+ T cells, myeloid derived

suppressor cells (MDSCs) and regulatory T cells (Tregs) lead to CD8+ T cell suppression and

lead to tumour progression through M2 macrophages and various pro-tumour cytokines (IL-

4, IL-6, IL-10 and TGFβ)41.

1.2.2 Breast Cancer Immunotherapies

While the standard of care therapies discussed previously, such as surgery, radiation

therapy and chemotherapy, have been used to treat primary breast cancer with some success,

no universally effective therapies exist in the treatment of invasive and metastatic breast

cancer. In recent years, a shift towards the use of immunotherapies has occurred for the

treatment of a range of cancers and in particular, focused on treating metastatic disease.

Animal models of breast cancer immunotherapies have demonstrated regression and an anti-

tumour immune response42. The lack of therapies for metastatic cancers and the apparent

efficacy of immunotherapies suggests that these therapies could be effective in the treatment

of patients with metastatic breast cancer. Immunotherapies that are currently being tested in

11

human breast cancer include antibody-based immunotherapies; cancer vaccine

immunotherapies, adoptive T cell transfer (ACT) immunotherapies and T cell receptor

(TCR) gene transfer immunotherapies.

Antibody-based therapies have already been briefly discussed, in the sense that they

can be used as a standard of care therapy for certain breast cancer subtypes, such as HER2

specific antibody treatment with trastuzumab (Herceptin) in HER2+ breast cancers. However,

other antibody based therapies do exist31,32. Other targets that have been explored in

antibody-based immunotherapies include antigens such as mucin-1 (MUC-1),

carcinoembryonic antigen (CEA) or NY-ESO-143-47. Antibody based therapies can either be

mediated by the in vivo production of antibodies through antigen specific stimulation or by

passive immunization via antibody transfer42. A variety of different mechanisms in the

antibody mediated elimination of cancer cells can occur following antibody binding but that

depends on the subclass of antibody used, including opsonization, cytotoxicity or activation

of complement42.

Cancer vaccine immunotherapies include a broad range of possible therapies, with the

underlying principle being to vaccinate a patient against specific cancer antigens. This can

include the use of tumour antigen-specific peptides, proteins, DNA, vectors, protein, and

tumour cell lysate-pulsed dendritic cells, or dendritic cell-tumour cell fusions that are all

focused on immunizing a cancer patient, in the attempt to produce an antigen driven anti-

cancer immune response42. These therapies aim to generate a cytotoxic T cell response to

lyse antigen expressing cancer cells. With these therapies, a primary prerequisite is a good

tumour specific antigen, which in some cases can limit the efficacy of these therapies42. A

number of different tumour antigens have been identified, with a large body of work focused

12

on melanoma specific antigens48,49. Examples of breast cancer antigens that have been

identified include HER2, MUC-1 and NY-ESO-142,50-52.

Adoptive T cell transfer and T cell receptor gene transfer immunotherapies go hand

in hand and are focused on the enrichment of T lymphocytes that can mediate an anti-cancer

immune response53. With adoptive T cell transfer immunotherapy, the enrichment of T

lymphocytes is commonly done from tumour infiltrating lymphocytes (TILs) that are

obtained from tumour biopsies or tumour surgery resections53. These cells are then isolated,

expanded and infused, at high numbers, into a patient, commonly co-administration with IL-

2, along with prior lymphocyte depleting chemotherapy53. Early developmental work for this

was done in melanoma patients, where they were able to establish and rapidly expand TIL

cultures from small fragments of tumours54. These studies were able to demonstrate tumour

specific TILs in up to 80% of melanoma patients54. Frozen sections of primary breast cancer

tissue from patients exhibited CD3, CD4 and CD8 staining by IHC, suggesting TILs were

already present within the tumours55. Subsequent work demonstrated that TILs could be

isolated from human breast cancer samples, but these studies tended to preferentially

generate CD4+ T cells over time rather than cytotoxic CD8+ T cells, leading to poorly lytic

cultures56,57. Further attempts were able to isolate TIL cultures that were tumour reactive42.

To present day, adoptive T cell therapy within breast cancer has shown limited efficacy in

clinical trials. In breast cancer patients with malignant pleural effusions, 12 of 81 patients

survived 5 or more years42. In a different trial, looking at malignant pleural effusions, 24

patients treated with TILs and IL-2 all saw decreases in CEA with some showing tumour

clearance42.

13

While adoptive T cell transfer does show promise in clinical trials, not all patients

have tumour lesions that can be used to obtain TILs. Even for those patients that do have

accessible tumour lesions, only 50% generate TIL cultures that can be used. However, in

melanoma treatment, this constitutes only 35% of patients42. For the other 65% of patients

that cannot be treated with adoptive T cell transfer, T cell receptor gene transfer is thought to

be a viable option. Similar to cancer vaccines, this therapy requires a good tumour specific

antigen for effective treatment58. Once a specific antigen is identified for an explicit cancer

type, a specific TCR or a chimeric antigen receptor (CAR) can be generated against the

antigen. The cDNA of the TCR or CAR can then be cloned into human peripheral blood

lymphocytes (PBLs), expanded and infused at high numbers into patients similar to adoptive

T cell transfer58. This approach has been used for a wide range of targets and cancers, which

include MART1 (Melanoma antigen recognized by T cells 1), MAGE-A1 (Melanoma-

associated antigen 1), CEA for colorectal cancer or PMSA (Prostate specific membrane

antigen)59-62. For breast cancer, HER2 has long been considered a potentially ideal target,

since it is highly expressed on the HER2+ subtype of breast cancer. Studies with HER2

specific CARs have demonstrated that they are functionally active (release cytokines in

response to HER2+ cell lines/primary tumours) and have high specificity (lyse HER2+ target

cells in vitro)47. Furthermore, other groups have also examined the efficacy and utility of

HER2-specific CARs for the treatment of HER2+ breast cancer63,64.

While there are many standard of care treatments, along with novel immunotherapies

available or in development for breast cancer, there still exists a subset of patients with

metastatic disease that see little to no response to the majority of available therapies, with the

same being true in a wide range of cancers. Due to this, our lab has worked on the

14

development of a cell based cancer vaccine immunotherapy that utilizes the cytokine IL-12.

While the work presented in this thesis focuses on the development in a mouse model of

metastatic breast cancer, it is more broadly aimed at gaining a better appreciation of the use

of the immunotherapy in the treatment of metastatic solid cancers as a whole.

1.3 IL-12 Cancer Immunotherapy

1.3.1 What is IL-12?

Interleukin-12 (IL-12) is an inflammatory cytokine and is one of the many molecules

involved in the normal functionality and execution of immune responses. Under normal

conditions, IL-12 is essential for the proper development of T lymphocyte based immune

responses. IL-12 is a heterodimeric cytokine composed of two different monomeric subunits,

p35 and p40, linked by disulfide bonds to create the active p70 molecule65. It is produced by

a variety of immune molecules such as monocytes, macrophages, dendritic cells and

neutrophils. While the p35 subunit is constitutively expressed, the p40 subunit has limited

expression in phagocytic cells that are able to produce the IL-12 heterodimer65. Upon

heterodimerization, IL-12 binds to its receptor (IL-12R), composed of the β1 and β2

subunits, with high affinity binding only occurring when both β1 and β2 are together. The

p40 portion of IL-12 interacts with the β1 subunit, while the p35 portion interacts with the β2

subunit65. Unlike other cytokine receptors, the IL-12R lacks intrinsic enzymatic activity and

relies upon the kinase activity of Janus kinases (JAKs). Upon ligand binding, JAKs are

activated by transphosphorylation, which leads to the phosphorylation of specific tyrosine

residues on the IL-12R, which then act as docking sites to signal transducers and activators

of transcription (STATs), primarily STAT465. The phosphorylation of STAT4 by JAKs leads

to their homo-/hetero-dimerization, translocation to the nucleus and subsequent activation of

15

IL-12 specific genes and a T cell phenotype skewing. One prominent gene activated through

this pathway is the interferon- γ (IFN-γ) gene, which is a primary effector molecule of IL-12

signaling65. Interestingly, there is a positive feedback loop with IL-12 production positively

regulating IFN-γ production, which is a product of IL-1265.

A primary function of IL-12 in a normal immune response is to facilitate the shift, or

polarization of CD4+ T cells to a “TH1” specific functionality65. Naïve CD4+ T cells, upon

antigen stimulation, can polarize to a variety of different possible phenotypes, with distinct

functions. The two most common polarizations are the TH1 and TH2 phenotypes, which yield

cellular and humoral immune responses respectively66. Naïve CD4+ T cells are polarized to

the TH2 phenotype by the cytokines IL-4 and IL-6, which leads to B cell activation, plasma

cell differentiation and antibody production66. In contrast, Naïve CD4+ T cells are polarized

to the TH1 phenotype by the cytokines IL-12 and IFN- γ, which leads to CD8+ cytotoxic T

lymphocyte (CTL) proliferation, activation and activity leading to increased cellular

mediated cell death65,66. In addition to a role in the mechanism of T lymphocyte regulation,

IL-12 also has an effect on natural killer (NK) cells, leading to their proliferation, IFN-γ

production and increased cytotoxicity65. Due to these many pro-immune effects, IL-12 has

long been considered a promising candidate cytokine to use in cancer immunotherapies, in

hopes of eliciting a potent immune response and overcoming the immunosuppression present

in breast cancer.

1.3.2 IL-12 Clinical Trials

Starting in the mid-90s, promising preclinical studies investigating the efficacy and

toxicity in animal models prompted the exploration of IL-12 as a cancer treatment. Early

experiments in animal models were able to show anti-tumour efficacy of recombinant IL-

16

1267, but also the associated hematological toxicities68. From this preclinical work, various

groups began clinical trials that initially focused on the use of recombinant IL-12 in a range

of cancers. These studies showed promising results with minimal toxicities in dose escalating

phase I trials69,70. This led to the initiation of a phase II clinical trial in renal cell carcinoma,

which was ultimately put on hold due to issues with toxicity in patients71. A number of side-

effects from the treatment developed in 12 of 17 patients, and led to two patient deaths71. The

trial was immediately suspended, along with all clinical trials involving IL-12. The toxic

effects seen with recombinant IL-12 injection were associated with systemic increases in

downstream products of IL-12, including IFN-γ and tumour necrosis factor α (TNF-α), an

effect seen in patients who were given an initial high dose of recombinant IL-1272. Further

examination and comparison of the phase I and II studies suggested that the increased

toxicities seen in the phase II trial were associated with the lack of an IL-12 priming dose

(used to determine pharmacokinetics of IL-12), which was administered in the phase I trials

and was crucial for the protective effects72. Upon consideration of these results the Food and

Drug Administration (FDA) reopened suspended clinical trials involving IL-1271.

Due to these initial complications with the use of recombinant IL-12, researchers

have developed other IL-12 based modalities in the hopes of taking advantage of the potent

pro-inflammatory effects of IL-12, while still avoiding the systemic rise in IFN-γ and

subsequent toxicities associated with recombinant IL-12. A variety of therapeutic techniques

have been developed, such as the introduction of IL-12 into viral vectors73, IL-12 vector

injection directly into tumours74, IL-12 engineered fibroblasts injected into tumours75, IL-12

engineered tumour antigen vaccines76, IL-12 engineered tumour cells77 or IL-12 engineered

T cells for ACT therapy78. Many of these therapies do show promise in preclinical murine

17

models, with minimal toxicities compared to the initial work done with recombinant IL-12.

Due to the many pro-immune and anti-cancer attributes of IL-12, our lab has worked on

refining and developing an improved understanding of a cell based cancer vaccine

immunotherapy that utilizes the genetic modification of cancer cells through the lentiviral

transduction with full length IL-12.

1.3.3 Cell based IL-12 Cancer Immunotherapy

To alleviate the toxicities associated with the direct injection of recombinant IL-12,

our lab utilizes a protocol whereby murine cancer cells are transduced with a full length IL-

12 lentiviral vector that is under the control of the EF1-a promoter that leads to its

constitutive expression. Previous studies have been done with the genetically modified

cancer cells leading to the expression of IL-12 and have shown promising results77,79. The

work being completed by our lab is focused on demonstrating the efficacy of IL-12

immunotherapy in a range of different cancers to better understand the mechanism by which

this therapy mediates rejection. Previous work from our lab, in various leukemia and solid

tumour cell line models demonstrated that while injection of the parental cancer cell lines

cause cancer development, mice injected with IL-12 transduced cancer cells are protected

from cancer development and these cells are rejected80,81.

Preliminary work from our lab with this experimental protocol was conducted in the

70Z/3 model of murine pre-B cell leukaemia, used as a model of human acute lymphocytic

leukaemia (ALL)82. To determine whether these findings could be expanded outside the

realm of leukaemias, testing in the head and neck squamous cell carcinoma model, SCCVII,

was conducted with consistent results81. These combined findings have prompted the

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development of a clinical trial for the treatment of acute myeloid leukaemia (AML) with IL-

12 transduced primary cancer cells.

While work is being done toward clinical trial development in leukaemias, a

knowledge gap still remains in our understanding of this therapeutic technique in the context

of solid tumours, particularly those with metastases, that represent the group that will see the

most benefit from immunotherapy treatment. To bridge this gap, our lab began testing this

therapy in transgenic mice that develop metastatic breast cancer, representing a more

clinically relevant model. Cell lines have been used extensively by our lab and others and

provide an excellent model for studying the molecular mechanisms of disease and the

mechanistic details of cancer immunotherapies84. While they generally have a faster time

frame for cancer development, based on their development, in some cases, they do not

accurately represent human cancers, particularly metastatic disease. To better mimic the

development of cancer in humans, transgenic mouse models are utilized. While they

commonly have a slower timeframe for cancer development, they better recapitulate the

development of cancers and metastases seen in humans and, therefore, allow for a better

understanding of how our IL-12 cancer immunotherapy might respond in humans with

metastatic disease. The model our lab has utilized is the MMTV-PyMT transgenic breast

cancer model.

1.4 MMTV-PyMT Transgenic Breast Cancer Model

The MMTV-PyMT breast cancer model was developed by Dr. William Muller’s Lab

during his tenure at McMaster University in Hamilton, Ontario84. In the MMTV-PyMT

breast cancer model, the oncoprotein polyoma middle T antigen (PyMT) is expressed under

the control of the mouse mammary tumour virus LTR promoter (MMTV), which leads to

19

mammary epithelium restricted expression of PyMT84. While PyMT is not a protein

expressed in human breast tumour cells, it acts as an extremely potent oncogene. The PyMT

protein is able to bind, and sabotage, several signaling pathways, such as the Src, ras and

PI3K pathways, which are all commonly altered in human breast cancers85. In addition,

MMTV-PyMT mice have increased c-myc levels in mammary tissue expressing PyMT,

which is a gene commonly amplified in human breast cancers85. Similar to that seen in some

forms of human breast cancer with poor prognoses, there is a loss of estrogen and

progesterone receptors (ERs and PRs) and an overexpression of HER2 and cyclin D1 in the

MMTV-PyMT model85. In addition, there is an increase in integrin-β expression as tumours

progress to malignancy. Work by Lin et al. demonstrated that based on histological and

molecular analysis, the MMTV-PyMT model recapitulates many biological processes found

in human breast cancer85. In the MMTV-PyMT model, 4 stages of progression have been

identified, which include hyperplasia, adenoma/mammary intraepithelial neoplasia (MIN),

early carcinoma and late carcinoma85.

The hyperplasia stage is a premalignant stage that occurs at approximately 4-6 weeks

and sees the initial development of tumours. Hyperplastic lesions generally consist of

clusters of densely packed lobules formed on the duct connecting to the main milk-collecting

duct. These cells are “bland”, with only minor increases in nuclear/cytoplasmic ratio

compared to normal ductal cells. In the hyperplasia stage, myoepithelial cells are rounded

rather than flat and are distributed unevenly around tumour acini. These tumour acini are still

contained within a defined basement membrane85.

The adenoma/MIN stage, is also a premalignant stage, that occurs at approximately 8-

9 weeks, with features of both adenoma and MIN lesions present. These lesions have more

20

florid epithelial proliferations and remain confined by a basement membrane with minimal

cytological changes, with no evidence of basement membrane invasion or metastasis. The

cellular proliferation that occurs in this stage causes a filling and expansion of the closely

packed acini and ducts, leading to an increase in primary tumour size. The acini are generally

completely filled with a solid sheet of cells, have increased vascularity and show an increase

in leukocytic infiltration relative to the hyperplastic stage85.

The early carcinoma stage is the initial malignant stage that occurs between 8-12

weeks. The defining feature of the transition from the adenoma/MIN stage to the early

carcinoma stage is the presence of greater cytological atypia and the identification of early

stromal invasion. This means, that there are usually lesions that possess features of both

adenoma/MIN and early carcinoma. The increase in cytological atypia is generally found in

the center of the primary tumour and as the cells transition to malignancy, the cells become

pleomorphic. There is a loss of the basement membrane, an increase in the number of

leukocytic infiltrates surrounding the tumour acini and further vascularization. This early

carcinoma stage is similar to human ductal carcinoma in situ with early stromal invasion85.

The last stage of tumour development in MMTV-PyMT mice is the late carcinoma

stage. By 10 weeks of age, 50% of MMTV-PyMT mice will have progressed to this

advanced carcinoma stage, where the tumour is almost entirely composed of solid epithelial

cell sheets with little or no remaining acinar structure remaining. There is a large amount of

variation in the cellular and nuclear size of the malignant tumour cells. At this stage, there is

no basement membrane and multiple tumour nodules develop throughout the mammary

glands with ductal hyperplasia present. In the late carcinoma stage, the tumours have many

21

characteristics that are similar to poorly differentiated human invasive ductal carcinoma, with

similar sheets of malignant cells85.

The MMTV-PyMT model serves as an effective model for recapitulating human

breast cancer. The different stages of tumour development in this model show a number of

similarities with human breast cancer, from its development of premalignant lesions to the

progression of highly invasive malignant carcinoma. In addition, MMTV-PyMT mice exhibit

a high frequency of pulmonary metastases, which is a common metastasis site seen in human

disease. These mice also serve as an elegant model due to their short tumour latency (late

carcinoma starting at 10 weeks) and pregnancy-independence. While oncogenesis is initiated

by the PyMT oncogene, which is not implicated in human disease, the model is able to

accurately depict the many distinct stages of human breast cancer providing validity for its

use. Importantly the consistent and aggressive lung metastases that occur in this model are an

attractive feature of these transgenic mice making them a valuable tool for gaining an

improved understanding of the efficacy of immunotherapies in patients with metastatic

disease84-86.

1.5 Current Experimental Framework

Using similar methodology to that demonstrated in previously tested leukaemia and

solid tumour models from our lab, the objective of this thesis is to develop our cell based IL-

12 cancer immunotherapy in the MMTV-PyMT model of metastatic breast cancer, as

evidenced by its similarity to human breast cancer development. The first aim is to isolate,

clone and transduce primary cells from MMTV-PyMT transgenic mice to develop a primary

breast cancer cell line model. The second aim is to demonstrate the ability of cell-based IL-

12 cancer immunotherapy to alter the course of cancer development, by conducting

22

experiments in a MMTV-PyMT derived breast cancer cell line to confirm results with

previous cell line work. Subsequently, expand these results by conducting experiments in the

transgenic MMTV-PyMT metastatic breast cancer model. It is hypothesized that a cell-

based IL-12 cancer immunotherapy will be able to delay the course of cancer

development and prolong survival in both MMTV-PyMT derived cell line and

transgenic mouse models.

23

CHAPTER 2: METHODS

2.1 Animals

Female C57BL/6 mice and Nonobese diabetic/severe combined immunodeficiency

(NOD/SCID) mice, 8-12 weeks old, were purchased from the Jackson Laboratories (Bar

Harbor, ME, USA). All mice were housed in sterile conditions in a specific pathogen-free

(SPF) animal facility at the Princess Margaret Cancer Centre, University Health Network,

Toronto, Ontario, Canada. Mice were fed standard irradiated diet and autoclaved water.

Animals were terminated by either CO2 asphyxiation or lethal doses of isoflurane followed

by cervical dislocation. The Animal Care Committee of the University Health Network

approved all experimental protocols employed.

2.2 MMTV-PyMT Transgenic Mice Breeding

Hemizygous male MMTV-PyMT transgenic mice on a C57BL/6 background were

obtained from Dr. Pamela Ohashi’s laboratory. Hemizygous males were bred to wild-type

C57BL/6 female mice purchased from The Jackson Laboratories (Bar Harbor, ME, USA).

Offspring were weaned at 21 days old and ear clipped for genotyping.

2.3 Genotyping of MMTV-PyMT Transgenic Mice

Ear clippings were collected and DNA was extracted using an adapted protocol of the

REDExtract-N-Amp Tissue PCR Kit from Sigma Aldrich (Oakville, Ontario, Canada). To

each ear clip, 25uL of Extraction Solution and 6.25uL of Tissue Preparation Solution were

added and the samples were vortexed. Samples were incubated for 20 min at room

temperature. The samples were then placed at 95°C for 10 min. To each ear clip, 25uL of

Neutralization Solution B were added and samples were vortexed. Samples were incubated at

24

room temperature O/N to increase DNA yield. Samples were then placed at 4°C for

prolonged storage.

Polymerase chain reaction (PCR) was set up for extracted DNA according to the

reaction in Table 1. Primers used were PyMT1 - GGA AGC AAG TAC TTC ACA AGG

and PyMT2 – GGA AAG TCA CTA GGA GCA GGG. Reaction samples were then run on a

Perkin Elmer GeneAmp PCR System 9600 (Waltham, Massachusetts, USA) according to the

PCR program in Table 2. Once PCRs were completed, samples were run on a 1.5% agarose

gel containing 0.4% ethidium bromide @ 100V for 45 – 90 min. Samples were visualized

using a Bio Rad UV Transilluminator (Mississauga, Ontario, Canada). Transgene positive

samples possess a 600 base pair band.

2.4 Derivation and Standard Culturing of Breast Cancer Derived Epithelial Cell Lines

A terminal MMTV-PyMT mouse with progressed mammary tumours (>16 weeks of

age) was sacrificed by CO2 asphyxiation and cerivally dislocated. Mammary tumours were

washed twice with phosphate buffered saline (PBS) (with CaCl2 and MgCl2) with

100units/mL penicillin, 100units/mL streptomycin and 0.25ug/mL Amphotericin B (Thermo

Fisher Scientific, Waltham, Massachusetts, USA). Washed tumours were minced and placed

in F12K digestion media (Thermo Fisher Scientific, Waltham, Massachusetts, USA)

containing 5ug/mL gentamicin (Thermo Fisher Scientific, Waltham, Massachusetts, USA)

and 1mg/mL collagenase A (Sigma Aldrich, Oakville, Ontario, Canada) for 1 hr. Digested

tumours were then passed through a 70um filter (Corning, Tewksbury, Massachusetts, USA)

and treated with ammonium-chloride-potassium (ACK) lysis buffer for 3 min to remove

residual red blood cells. Cells were then counted and plated @ 2x106 cells per well in a 6

well tissue culture plate in standard culture conditions of OptiMEM media (Thermo Fisher

25

Scientific, Waltham, Massachusetts, USA) containing 5-10% fetal calf serum (FCS) (Thermo

Fisher Scientific, Waltham, Massachusetts, USA), 100units/mL penicillin/100ug/mL

streptomycin (MultiCell, Woonsocket, Rhode Island, USA) or 100ug/mL kanamycin

(Thermo Fisher Scientific, Waltham, Massachusetts, USA), 2.4g/L NaHCO3 (BioShop,

Burlington, Ontario, Canada) and 55µM 2-mercaptoethanol (Thermo Fisher Scientific,

Waltham, Massachusetts, USA). Following prolonged culture of 1-2 months, epithelial

enrichment by trypsinization was conducted during routine culture of cells. Following a 5-

minute incubation with 0.05% trypsin + 0.53mM ethylenediaminetetraacetic acid (EDTA)

(MultiCell, Woonsocket, Rhode Island, USA), a gentle wash was conducted to remove the

epithelial cells, while a more thorough wash removed the fibroblast cells. Enriched epithelial

and fibroblastic cells were then plated in standard culture conditions.

2.5 IL-12 Lentiviral Transduction of MMTV-PyMT Derived Cells

MMTV-PyMT derived cells were cultured and plated at 1x105 cells in a 6 well plate

and placed at 37°C for 4 hrs. The cells are then transduced at a multiplicities of infection

(MOI) of 10 or 20 by adding 1x106 or 2x106 lentiviral vectors containing a full-length

recombinant IL-12, with expression driven by the elongation factor 1- α (EF1-α) promoter,

leading to constitutive expression (See Figure 2a for viral schematic). The cells are then

spun down at 800g for 1 hr and subsequently placed at 37°C overnight. The following day,

fresh media is placed on the cells to remove any residual viral particles.

2.6 Assessing IL-12 Production by Intracellular Flow Cytometry and ELISA

Up to 1x106 IL-12 transduced cells were plated in a 6 well plate and allowed to settle

overnight at 37°C. The following day the cells were incubated for 4 hrs at 37°C with

standard media conditions containing 1uL/mL of GolgiPlugTM (Brefeldin A) and

26

0.667uL/mL of GolgiStopTM (Monensin) from BD Biosciences (Mississauga, Ontario,

Canada). Following the 4 hr incubation, cells were incubated with 0.05% trypsin + 0.53mM

EDTA for 5-15 minutes to lift the adherent cells from the plate. Cells were washed with PBS

containing 2% FCS and stained for 20 minutes with the anti-mouse monoclonal antibody

phycoerythrin (PE) anti-IL-12 (C15.6; BD Biosciences). The cells were then analyzed using

the BD LSRII Fortessa from BD Biosciences (Mississauga, Ontario, Canada).

To quantify IL-12 production, 1x106 cells were plated in 1mL for 4 hrs at 37°C in

standard media conditions. Following incubation, supernatants were collected and IL-12

production was measured using a commercially available mIL-12 enzyme-linked

immunosorbent assay (ELISA) kit targeting the full-length p70 purchased from BD

Biosciences (Mississauga, Ontario, Canada). All ELISAs were done according to standard

protocol provided by BD Biosciences.

2.7 Cloning of Epithelial Enriched Cells and IL-12 Transduced Cells

Epithelial enriched cells were incubated with 0.05% trypsin + 0.53mM EDTA to

remove adherent cells from the flask. For limiting dilution cloning, cells were diluted to

concentrations of 2, 1, 0.5, 0.25, 0.125 cells/100µL/well in standard culture media. For each

cell concentration, a full 96 well plate was set up. The cells were then left for 7 days at 37°C.

Following 7 days, all plates were scored for wells with growing cells (positive wells). The

principles of limiting dilution dictate that cell dilutions with a positive well frequency of less

than 33% are statistically originated from single cells and therefore clonal87. Only positive

wells from cell dilutions with a positive well frequency of less than 33% were used.

For terasaki well cloning, cells were diluted to a concentration of 0.5 or 0.33

cells/10µL/well in standard culture media. 5-15 plates were plated. The cells were left for 30

27

to 60 minutes at 37°C to allow cells to settle to the bottom of the well. Following this

incubation, all plates were scored for wells with a single cell and therefore of clonal origin.

Following scoring, the plates were left for 2 to 7 days until clones had grown out. Only wells

that originated from a single cell based on scoring were expanded.

2.8 In Vivo Tumour Experiments with PyM1 Epithelial Tumour Initiating and IL-12

Transduced Clones

Cloned tumour cell lines were grown in standard culture conditions as described

above. Cells were incubated with 0.05% trypsin + 0.53mM EDTA, collected and spun down

by low-speed centrifugation, and washed with PBS prior to injection. Cells were injected

either subcutaneously into the flank or intraperitoneally. Cells were injected at a

concentration of 1x106 cells/100µL or 1x106 cells/200µL in PBS for subcutaneous and

intraperitoneal injections respectively. After injection, mice were monitored (from daily to

weekly monitoring) for tumour development and sacrificed at a humane endpoint of 1.5cm in

any dimension or progressive ulceration of the tumour. Euthanasia was performed by CO2

asphyxiation followed by cervical dislocation or a lethal dose of the inhaled anesthetic

isoflurane followed by cervical dislocation. At endpoint, blood serum, tumours, lungs,

draining lymph nodes, spleen and bone marrow were harvested for analysis. All tumour

volumes were calculated using the following formula: Volume = 4/3*length*width2*π.

2.9 Hematopoietic Cell Depletion

Specific antibodies were used to deplete for different hematopoietic cells, which

included CD4+ cells, CD8+ cells or NK+ cells. The hybridoma GK1.5 was used to deplete

CD4+ cells, YTS169 was used to deplete CD8+ cells, and HB9419 was used as an isotype

control. The hybridomas were obtained from the American Type Culture Collection (ATCC)

28

(Manassas, VA, USA). The protocol for hybridoma cell culture and antibody purification

were the same as previously described in Labbe et al. (2009). The mice were injected

intraperitoneally with 200µL of 2.5mg/mL antibody stock, producing a final dose of

0.5mg/injection/mouse. Mice were dosed on days -2, 3, 7 and weekly following until mice

reached humane endpoint (see above). The purchased monoclonal antibody anti-Asialo GM-

1, from Cedarlane Labs (Burlington, ON, Canada), was used to deplete NK cells. The mice

were injected intraperitoneally with 200µL of a 1:10 dilution of stock antibody. Mice were

dosed on days -2, 3, 7, 10, 14, 17, 21 and weekly following until mice reached human

endpoint (see above). The efficacy of depletion was tested via flow cytometry analysis of

peripheral blood prior and during experiments (data not shown).

2.10 Cell Isolation and Flow Cytometry for Intracellular IL-12 and PyMT Phenotyping



by low-speed centrifugation, and washed with PBS+2% FCS. For flow cytometry, cell

suspensions were stained with the following anti-mouse monoclonal antibodies: fluorescein

isothiocyanate (FITC) anti-Sca1 (Ly-6 A/E) (D7; Biolegend), PE anti-CD24 (heat stable

antigen (HSA)) (M1/69; BD Biosciences), allophycocyanin (APC) anti-CD49f (GoH3;

Biolegend), phycoerythrin cyanin 7 (PE-Cy7) anti-CD29 (HMβ1-1; Biolegend), PE anti-IL-

12 (C15.6; BD Biosciences) and Pacific Blue anti-lineage (Cat# - 133306; Biolegend). Flow

cytometry was conducted using a BD LSRIIFortessa 5 laser (325; 405; 488; 561; 632)

configuration (BD Biosciences).

29

2.11 Development and Analysis of PyM1 and LV12 Conditioned Media



by low-speed centrifugation, and washed with PBS. Cells were plated at 5x105 cells in

500µL per well in a 12 well plate and incubated for 24hrs at 37°C. Following the 24hr

incubation, conditioned media was collected and frozen and stored at -80°C. Samples were

sent to Eve Technologies (Calgary, AB, Canada) to conduct a mouse cytokine/chemokine

32-plex array which contained Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3,

IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-

10/CXCL10, KC/CXCL1, LIF, LIX/CXCL5, MCP-1, M-CSF, MIG/CXCL9, MIP-1α, MIP-

1β, MIP-2, RANTES/CCL5, TNFα, and VEGF.

2.12 Statistical Analysis

All statistical analysis of data was conducted using Prism 6 (GraphPad Software, La

Jolla, California, USA). For Figures 3c, 7a, 7b, and 8, two-way ANOVA was used. For

Figures 4b, 5b, 6b, and 9b, log-rank (Mantel-Cox) test was used. For Figure 9c, one-way

ANOVA was used. Figure 9d, unpaired Student’s T-test was used.

30

CHAPTER 3: RESULTS

3.1 Development of the Breast Cancer Epithelial Tumour Initiating Cell Line PyM1

The MMTV-PyMT breast cancer model possesses many characteristics that make it

an effective system comparable to human breast cancer85. To develop a simplistic model of

IL-12 immunotherapy in breast cancer, epithelial tumour initiating cells were derived from

the MMTV-PyMT model on a C57BL/6 background. Mammary tumours were initially

derived from MMTV-PyMT mice with progressive disease (tumours >1.5cm in diameter).

Tumour tissue was digested and plated at varying concentrations in vitro (0.5x106 cells/well

to 2x106 cells/well) to establish primary cell cultures with maintained growth. The cells that

were derived from the tumour tissue were a heterogeneous culture of epithelial and stromal

cells that all make up the tumour microenvironment.

Differential trypsinization was conducted on heterogeneous bulk primary cell cultures

to enrich for epithelial cells, which have been shown to be the tumour initiating cells in the

MMTV-PyMT model88. Enriched primary epithelial cell cultures and enriched primary

fibroblastic cell cultures were isolated initially based on morphology alone. To confirm these

morphological classifications, cellular phenotyping of cell cultures was conducted. Cells

were stained with markers that have been shown to be able to differentiate epithelial and

fibroblast cells (Figure 1a). Enriched epithelial cells have a predominant population that are

CD24+CD29-+CD49f+Sca1low, which have been characterized as epithelial cells and shown to

possess tumour initiating capacity. Enriched fibroblast cells have a predominant population

that are CD24-CD29+CD49f-Sca1high, which have been characterized as

fibroblast/mesenchymal cells and shown to possess no tumour initiating capacity. To fully

isolate pure epithelial and fibroblast cells, these enriched cell populations were cloned using

31

the limiting dilution cloning technique. From this, a variety of clonal cell lines were derived.

One such cell line derived from the epithelial enriched cells was PyM1. Similar cellular

phenotyping was conducted on PyM1 to ensure that this cloned cell line possessed only

epithelial tumour initiating cells. As seen in the representative plots in Figure 1a, the vast

majority (>95%) of the cells are CD24+CD29+CD49f+Sca1low, which is consistent with the

epithelial tumour initiating cell phenotype.

To further characterize the PyM1 epithelial cell line, we examined a limited protein

secretome of these cells. Conditioned media derived from PyM1 was sent to Eve

Technologies (Calgary, AB) to test for cytokine production using a 32-plex array. Figure 1b

shows the cytokine production profile of PyM1 cells. Based on cytokine array data, PyM1

produces an abundant amount of G-CSF and CXCL1 (>8000 pg/mL). PyM1 also produces

MCP-1 and LIF to a far lesser extent (>100 pg/mL). These results are consistent with

previous work that has examined cytokine production of MMTV-PyMT derived cell lines89.

To test tumour initiating capacity of the PyM1 epithelial cell line, C57BL/6 mice

were injected subcutaneously (S.C.) with 106 PyM1 cells and produced palpable tumours

within 7-14 days that grew until human endpoint (Figure 3c), yielding a median survival of

106 days (Figure 3d). These phenotypic, protein secretome and tumour initiating findings

are consistent with previous groups examining tumour initiating cells derived from MMTV-

PyMT mice (88; 89).

3.2 IL-12 Lentiviral Transduction of the Epithelial Tumour Initiating Cell Line PyM1

To generate tumour cells expressing IL-12, PyM1 was transduced with a recombinant

full-length IL-12 (p70) lentiviral vector construct developed by Dr. Jeffrey Medin’s

Laboratory (Figure 2a). Within the lentiviral vector, the recombinant full-length IL-12 is

32

under the control of the EF-1α promoter, leading to constitutive expression of IL-12. PyM1

cells were transduced with the IL-12 lentiviral vector at a multiplicity of infection (MOI) of

10 and subsequently tested for IL-12 production with intracellular flow cytometry analysis of

full-length IL-12. Cells were blocked for 4-6hrs with GolgiPlug and GolgiStop, which

inhibits golgi apparatus function and cytokine secretion, leading to accumulation of IL-12

within of the cell. Cells were fixed, permeabilized and stained with an anti-mouse IL-12

antibody and analyzed using the LSRIIFortessa. Figure 2b compares untransduced, MOI 10

transduced and MOI 20 transduced PyM1. Based on intracellular IL-12 analysis, there is an

increase in the number of cells containing intracellular IL-12 (IL-12 positive cells) in both

the MOI 10 and MOI 20 transduced PyM1 cells. The MOI 10 transduced PyM1 cells,

containing a large number of IL-12 positive cells, were used for subsequent subcloning to

generate IL-12 positive clonal cell populations.

3.3 Cloning of MOI 10 Transduced PyM1 Cell Line

PyM1 cells that had been transduced with a recombinant full-length IL-12 lentiviral

vector were used to generate IL-12 positive subclones of the parental line. To do this terasaki

plate cloning was conducted. Cells were plated at a concentration of 0.33-0.5 cells/well in

terasaki wells and scored 30-60min following plating for single cells in each well. Wells

containing single cells were scored positive and subsequently expanded if a population of

cells formed.

3.4 IL-12 Production of Transduced Subclones of PyM1

By conducting subcloning of the MOI 10 transduced PyM1 cell line, isolated clonal

cell lines (LV12 cell lines) were generated that produce a variety of different IL-12 amounts

based on genomic lentiviral integration. To measure IL-12 production of each of these

33

subclonal LV12 cell lines, cells were plated at 1x106 cells/mL/well for 4 hrs and supernatant

media was collected for IL-12 specific ELISA analysis. ELISAs were done with samples at

dilutions ranging from undiluted to 1:1000 diluted samples to accurately measure IL-12

concentration. Figure 2c presents the IL-12 production level of a large number of LV12 cell

lines (n=3 biological replicates with 3 technical replicates/biological replicate). Each LV12

cell line is expressed as ng/mL/1x106 cells/well/4hrs (shortened to ng/mL) to normalize

production between all lines. A large range of IL-12 production is seen between lines,

ranging from 3ng/mL for LV12.22 to 300ng/mL for LV12.23.

3.5 Characterization of IL-12 Producing Cell Lines

Newly developed LV12 cell lines were then analyzed for cellular phenotype (Figure

3a), cytokine production (Figure 3b) and proliferation (Figure 3c) to assess whether these

IL-12 positive subclones remained consistent with the parental cell line, PyM1.

Phenotypically, PyM1 and various LV12 cell lines had consistent expression of surface

markers. LV12.1 and LV12.18, two LV12 cell lines tested, all had a predominant population

of Lin-CD24+CD29+CD49f+Sca1low cells, as seen in the parental cell line PyM1 (Figure 3a).

Analysis of the same limited protein secretome used in Figure 1b confirmed that both IL-12

secreting subclones of PyM1 produce consistent cytokines at similar levels. Lastly,

proliferation of these cell lines, based on a time course of cell counts, is different between

different LV12 cell lines and PyM1. Interestingly, LV12.1, but not LV12.18, possesses a

greater proliferation rate compared to PyM1, which is statistically significant, adjusted

p<0.001, two-way ANOVA (Figure 3c).

34

3.6 IL-12 Producing Cell Lines Inhibit Tumour Development

The LV12 cell lines were injected S.C. into the left flank of C57BL/6 mice and were

monitored (from daily to weekly) for tumour development (Figure 4a) and survival (Figure

4b). As demonstrated in Figure 4a, mice injected with the parental cell line PyM1 developed

localized palpable tumours that continued to grow until they reached a humane endpoint of

1.5cm in any dimension. Mice that reached endpoint were euthanized according to

institutional guidelines. Conversely, all mice injected with a LV12 subclonal cell line were

protected from tumour development with the injected cells being rejected (n=5). Protection

was seen across a large range of IL-12 production. Cells producing as little as 3ng/mL and as

much as 300ng/mL all protected mice from tumour development with no evident signs of

toxicity. Similar results were seen in the survival, where all there all mice injected with

LV12 subclonal cell lines showed a drastic increase in survival compared to the parental line

PyM1 (p<0.007, n=15 for PyM1, n=5 for all LV12 clones, log-rank test) (Figure 4b).

3.7 Small Percentages of IL-12 Producing Cells Are Sufficient to Protect Mice from

Tumour Development

As previously described in murine ALL and murine HNSCC models utilized by our

lab, an important observation of this cell-based IL-12 cytokine cancer immunotherapy is that

the amount of IL-12 produced on a per cell basis is imperative80,81. It was demonstrated that

only a small number of high IL-12 producing cells was sufficient to protect from tumour

development and fewer high IL-12 producing cells were more effective than a greater

number of low IL-12 producing cells. To determine if this is also true in the PyM1 breast

cancer model, cell mixtures of two clones, LV12.1 and LV12.18 (185ng/mL and 32ng/mL

respectively), were made, with a constant number of the parental line, PyM1 and varying

35

numbers of either LV12 subclone. The following conditions were set up for both LV12

subclone: 106 PyM1, 106 LV12, 106 PyM1 + 106 LV12, 106 PyM1 + 105 LV12, and 106

PyM1 and 104 LV12 (n=5 per group). These mixtures were injected S.C. into the left flank of

mice and were monitored for tumour development and survival (Figure 5). Injection of 106

PyM1 cells, as previous demonstrated, produced palpable tumours in all mice, while

injection of 106 of either LV12 subclone produced no tumour in any mice with 100%

survival for the duration of the experiment. However, what is evident in the mixing

experiment is that any fractions of LV12 cells (both LV12.1 and LV12.18) were able to elicit

some delay of tumour growth and a survival advantage over the parental line PyM1 alone

(Figure 5).

3.8 IL-12 Producing Cells Mediate Delay in Tumour Development to PyM1

Challenge

To determine whether the anti-tumour immune response generated by an IL-12

producing cell treatment was long lasting, we intraperitoneally (I.P.) injected C57BL/6 mice

with 106 cells of LV12.18, LV12.22 or left mice untreated (naïve) (n=5 for naïve, n=9 for

LV12.18 treated, n=7 for LV12.22 treated). These two lines produce 32ng/mL and 3.5ng/mL

respectively. Mice were given 49 days to allow for IL-12 producing cell rejection. Following

this 49-day incubation, mice were challenged with 106 cells of the parental PyM1 cell line

S.C. into the left flank. Tumour growth (Figure 6a) and survival (Figure 6b) were

monitored. Naïve mice challenged with PyM1 developed tumours, whereas mice treated with

LV12.18 or LV12.22 showed a delay in tumour development (Figure 6a). While IL-12

producing cell treatment did not elicit complete memory, the delay in tumour development

suggests a partial memory immune response was elicited against the parental PyM1

36

challenge. Based on both tumour volume and survival, as demonstrated in Figure 6, the

effect of the treatment and the extent of the tumour progression delay was dose dependent,

with the clone LV12.18 (32ng/mL) having a greater effect relative to LV12.22 (3.5ng/mL).

Mice treated with LV12.18 and challenged with PyM1 demonstrated a significant increase in

survival relative to naïve mice challenged with PyM1 (p<0.025, log-rank test).

3.9 Examining the Mechanism of IL-12 Producing Cell Line Rejection in the PyM1

Model

Previous work from our lab in leukemia80 and solid tumour81 models have

demonstrated that the rejection and elimination of IL-12 producing cells was mediated in a T

cell-dependent manner leading to an anti-tumour immune response. To test whether this

mechanism is maintained in the PyM1 breast cancer model, we examined whether mice that

have been depleted for either CD4+ cells, CD8+ cells or both were still able to eliminate IL-

12 producing cells. For this experiment, LV12.18 (32ng/mL) was utilized as it produces

lower levels of IL-12 from the subclones generated and has been tested in vivo. The

following conditions were set up: 106 PyM1, 106 LV12.18 into PBS treated mice, 106

LV12.18 into Isotype treated mice, 106 LV12.18 into αCD4 treated mice, and 106 LV12.18

into αCD8 treated mice and 106 LV12.18 into αCD4/αCD8 treated mice (n=5 per group,

except n=3 for PBS and Isotype treated). The depleted mice and relevant control mice were

injected with LV12.18 and compared to the growth of the parental cell line, PyM1 (Figure

7a). The results demonstrate that LV12.18 does form a palpable tumour in the αCD4 and

αCD4αCD8 treated groups, but these tumours grow at a grossly reduced rate relative to the

parental line PyM1. Starting at 21 days, mice injected S.C. with PyM1 demonstrated

significantly larger tumours than all other groups (adjusted p<0.0001, two-way ANOVA).

37

To test whether this was an intrinsic trait of LV12.18 or related to the general

mechanism by which IL-12 producing cells are eliminated, we tested whether LV12.18 and

another IL-12 producing cell line, LV12.1, were able to form tumours in NK-depleted

NOD/SCID mice, which are devoid of any adaptive immune system. NK-depleted

NOD/SCID mice were injected with 106 of either PyM1, LV12.1 or LV12.18 cells and

monitored for tumour growth (Figure 7b). By day 35, a difference in tumour growth was

evident between these 3 cell lines tested. Mice injected with the parental cell line PyM1

developed tumours, which were also seen in mice injected with LV12.1. Similarly to what

was seen in Figure 7a with CD4+ and CD8+ cell depletion, injection of LV12.18 did not

form a tumour in depleted mice, suggesting that the lack of tumour formation is intrinsic to

LV12.18 rather than related to the mechanism of IL-12 producing cell elimination (Figure

7b). At 49 days post injection, both LV12.1 and LV12.18 injected mice had smaller tumours

than PyM1 injected mice (adjusted p<0.0001, two-way ANOVA). While LV12.1 injected

mice did have tumours smaller than PyM1 injected, in contrast to LV12.18 injected mice,

these tumours were expanding over the course of the study (At 49 days, adjusted p<0.05,

two-way ANOVA).

3.10 Timing of IL-12 Producing Cell Treatment Alters Delay in Tumour Development

To determine whether the timing of IL-12 producing cell therapy is important for an

immune response and the delay of tumour development, mice were injected on Day 0 with

106 PyM1 cells subcutaneously and then randomized to either no treatment, 106 LV12.1 cells

intraperitoneally on Day 3, Day 8, Day 14 or “weekly” (Day 3, Day 8, Day 14 and

subsequently weekly). As a control wild-type mice were given all injections that “weekly”

mice received to ensure no toxicities occurred with repeated LV12.1 administration (n=5 per

38

group). For all mice tumour growth kinetics were monitored weekly (Figure 8). At day 80, a

difference in tumour growth kinetics was evident between untreated mice and those receiving

any LV12.1 treatment (Day 3, Day 8, Day 14 or “weekly”). Mice treated with LV12.1 at Day

3, Day 8 or “weekly” all had a greatly significant delay in tumour development relative to

untreated mice (adjusted p<0.0001, two-way ANOVA). Mice treated with LV12.1 at Day 14

did also significantly delay tumour development but this was a far more modest delay

relative to any other treatment (adjusted p<0.001, two-way ANOVA). In addition, no

toxicities were observed in wild-type mice treated weekly with LV12.1.

3.11 Weekly Treatment of MMTV-PyMT Transgenic Mice with an IL-12 Producing

Cell

Weekly treatment of MMTV-PyMT transgenic mice was conducted to test the

efficacy of cell-based IL-12 cancer immunotherapy in a model that develops primary

tumours as well as metastatic disease. Immunotherapies, such as the therapy utilized in this

work, are less likely to be use as a first-line therapy for primary tumours, which in humans

are normally surgically resected and treated with chemo- or radiotherapy, and there is a

greater optimism that such therapies will take advantage of the systemic nature of immune

responses to reduce and eliminate metastatic cells. Hemizygous male MMTV-PyMT

transgenic mice were bred to wild-type female C57BL/6 mice to generate litter matched

wild-type and heterozygous female mice. Starting at the age of 6 weeks, age matched mice

were randomized to either weekly PBS or LV12.1 treatment (0.7-1x106 LV12.1 cells or

equivalent volume of PBS). Mice were monitored for both tumour growth kinetics (Figure

9a) and survival (Figure 9b). For wild-type mice, weekly treatment with LV12.1

demonstrated no impact on survival relative to PBS treated mice (n.s., log-rank test).

39

Regardless of whether mice were treated with PBS or LV12.1, MMTV-PyMT mice had a

significantly decreased survival rate relative to wild-type mice (adjusted p<0.0.0083, log-

rank test). When examining MMTV-PyMT mice, whether mice were treated with PBS or

LV12.1, no difference in survival was seen between therapies (n.s., log-rank test). In addition

to tumour volume and survival, the number of tumour sites present per mouse (Figure 9c)

and tumour development rate measured as time to reach 14cm3 (Figure 9d) were analyzed.

Examining number of tumour sites per mouse again demonstrated no difference between

PBS treated and LV12.1 treated MMTV-PyMT mice (n.s., one-way ANOVA). Similarly,

tumour development rate based on time to reach 14cm3 shows a similar lack of significant

difference between treatment for MMTV-PyMT mice (n.s., unpaired Student’s T-test). WT-

PBS, n=6. WT-LV12.1, n=7. PyMT-PBS, n=7. PyMT-LV12.1, n=8. For all mice, the left

lobes of the lungs were collected at time of animal euthanasia to determine the efficacy of

therapy on the development of metastatic disease. Lung tissues stained with hematoxylin and

eosin (H&E) were reviewed by a pathologist for the presence of lung metastases.

Unfortunately, a surprisingly low number of metastases (only 1/14 lungs between LV12.1

and PBS treated mice) were found, giving us little insight into the effect of treatment on lung

metastasis formation.

40

CHAPTER 4: FIGURES & TABLES

41

Table 1. MMTV-PyMT Genotyping PCR Reaction Reagent Volume MilliQ dH2O 1.7uL REDExtract-N-Amp PCR Reaction Mix 5uL PyMT-1 (Forward Primer – 20uM) 0.65uL PyMT-2 (Reverse Primer – 20uM) 0.65uL Tissue Extract 2uL Total Volume 10uL

42

Table 2. MMTV-PyMT Genotyping PCR Program Step Temperature Time Cycles Initial Denaturation 95°C 2 min 1 Denaturation 95°C 10 sec

35 Annealing 59°C 15 sec Extension 68°C 45 sec Final Extension 68°C 6 min 1 Hold 4°C Indefinitely

43

Eotaxin

G-CSF

GM-CSF

IFNg

IL-1

aIL

-1b

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-9

IL-1

0

IL-1

2 (p40

)

IL-1

2 (p70

)IL

-13IL

-15IL

-17IP

-10 KC LIF LIX

MCP-1

M-CSF

MIG

MIP-1a

MIP-1

bMIP

-2

RANTESTNFa

VEGF0

2000

4000

6000

8000

10000

pg/m

L

MediaPyM1

CD24

CD

49f

CD29

Sca

1

0 102 103 104 105

0

102

103

104

1050.582 98.3

0.9380.172

0 102 103 104 105

0

102

103

104

1057.83e-3 3.9

95.40.722

PyM1 Fibroblast Epithelial

0 102 103 104 105

0

102

103

104

1054.3 17

19.759

0 102 103 104 105

0

102

103

104

1055.18 70.5

18.16.22

0 102 103 104 105

0

102

103

104

1052.03 86.3

11.10.641

0 102 103 104 105

0

102

103

104

1051.03 27.7

67.63.66

CD

49f

CD

49f

Sca

1

Sca

1

A

B

0 20 40 60 80 100 120 1400

2

4

6

8

10

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our

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Tumour Volume

0 20 40 60 80 100 120 1400

20

40

60

80

100

TIme (Days)

% S

urvi

val

SurvivalC

Figure 1

D

Figure 1. Characterization of a MMTV-PyMT epithelial breast tumour initiating clone, PyM1. (A) Phenotypic flow cytometry analysis of bulk enriched cell cultures and cloned epithelial breast cancer cell line PyM1 based on expression of stem cell associated markers CD24, CD29, CD49f and Sca1. (B) Protein secretome analysis of PyM1 using a 32-plex cytokine array from Eve Technologies. For media, n=3. For PyM1, n=9. Tumour growth kinetics (C) and overall survival (D) of C57BL/6 mice injected subcutaneously with 1x106 PyM1 cells. For tumour growth kinetics and overall survival, n=5.

44

Figure 2 A ψ

IL-12 p70:

LTR ΔGag RRE cPPT EF1-α EGFP WPRE LTR/SIN

p35 p40

SD SA

B

IL-12

FSC

-A

FSC

-A

FSC

-A

Blocked for 4 hours with GolgiPlug and GolgiStop

0 102 103 104 1050

50K

100K

150K

200K

250K 0.853

0 102 103 104 1050

50K

100K

150K

200K

250K 24

0 102 103 104 1050

50K

100K

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200K

250K 29.1

PyM1 MOI 10 MOI 20

PyM1 LV12.1 LV12.4 LV12.8 LV12.9 LV12.12 LV12.13 LV12.15 LV12.16 LV12.17 LV12.18 LV12.21 LV12.22 LV12.23 LV12.240

40

80

120

160

200

240

280

320

360

IL-1

2 P

rodu

ctio

n (n

g/m

L/1x

106

cells

/4 h

rs)

C

Figure 2. Transduction with an IL-12 lentiviral vector and cloning of PyM1 cells to isolate IL-12 producing subclones. (A) Schematic representation of vector construct for IL-12 lentivirus utilized for transduction. (B) Intracellular flow cytometry of transduced PyM1 cells. Cells were incubated with GolgiPlug and GolgiStop for 4hrs prior to staining to halt cytokine secretion. (C) IL-12 secretion of PyM1 subclones plated at a concentration of 106 cells/mL/4hrs. For each subclone, 2-5 biological replicates were tested from samples set up on different days. For each biological replicate, 3 technical replicates were plated.

45

Eotaxin

G-CSF

GM-CSF

IFNg

IL-1

aIL

-1b

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-9

IL-1

0

IL-1

2 (p40

)

IL-1

2 (p70

)IL

-13IL

-15IL

-17IP

-10 KC LIF LIX

MCP-1

M-CSF

MIG

MIP-1a

MIP-1

bMIP

-2

RANTESTNFa

VEGF0

2000

4000

6000

8000

10000

12000

14000

16000

pg/m

L

MediaPyM1LV12.1LV12.18

Day0 Day 1 Day 2 Day 30

10

20

30

40

50

x104

cells

PyM1LV12.1LV12.18

0 102 103 104 105

0

102

103

104

1054.46e-3 1.63

97.90.452

0 102 103 104 105

0

102

103

104

1052.33 95.6

1.910.162

0 102 103 104 105

0

102

103

104

1050.0709 14.1

85.20.619

0 102 103 104 105

0

102

103

104

1054.25 95.3

0.3130.142

0 102 103 104 105

0

102

103

104

1050.582 98.3

0.9380.172

0 102 103 104 105

0

102

103

104

1057.83e-3 3.9

95.40.722

A Figure 3

LV12.18 PyM1 LV12.1

B

C *** ***

Figure 3. Characterization of select IL-12 producing PyM1 subclones, LV12.1 and LV12.18. (A) Phenotypic flow cytometry analysis of PyM1 and two two IL-12 producing cell lines, LV12.1 and LV12.18, which produce 185ng/mL and 32ng/mL respectively. Expression of stem cell associated markers CD24, CD29, CD49f and Sca1 were analyzed. (B) Protein secretome analysis of PyM1, LV12.1 and LV12.18 using a 32-plex cytokine array from Eve Technologies. For media, n=3. For PyM1, n=9. For LV12.1 and LV12.18, n=10. All samples, except media, are 4 biological replicates from different days, with each biological replicate having 1-3 technical replicate. (C) Proliferation of PyM1, LV12.1 and LV12.18 assessed by daily measurements of cell numbers over 3 days. Each bar represents 3 biological replicates set up separately. Each biological replicate contained 3 technical replicates. LV12.1 had significantly greater proliferation relative to PyM1 and LV12.18, adjusted p<0.001 (***), two-way ANOVA.

CD24

CD

49f

CD29

Sca

1

CD

49f

CD

49f

Sca

1

Sca

1

46

Figure 4. Injection of IL-12 producing subclones of PyM1 protects mice from tumour development. Tumour development (A) and survival (B) of PyM1 and seven IL-12 producing subclones (LV12.1, LV12.4, LV12.13, LV12.17, LV12.18, LV12.22, and LV12.23) with a range of IL-12 levels from 3.5ng/mL to 284ng/mL. C57BL/6 mice were injected with 106 cells subcutaneously. N=15 for PyM1 (pooled from 3 experiments), n=5 for all other groups. All IL-12 producing clones provided protection from tumour development, p<0.007, log-rank test.

0 20 40 60 80 100 120 14002468

1012141618

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(cm

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LV12.1

0 20 40 60 80 100 120 14002468

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Figure 4

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PyM1LV12.1LV12.4LV12.13LV12.17LV12.18LV12.22LV12.23

A

B p<0.007

47

0 20 40 60 80 100 120 140 160 180 200 2200

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Time (Days)

% S

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106 PyM1106 LV12.1106 LV12.18106 PyM1 + 106 LV12.1106 PyM1 + 106 LV12.18106 PyM1 + 105 LV12.1106 PyM1 + 105 LV12.18106 PyM1 + 104 LV12.1106 PyM1 + 104 LV12.18

0 40 80 120 160 20002468

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0 40 80 120 160 20002468

10121416182022

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(cm

3 )106 PyM1

Figure 5 A

B p<0.00625

p<0.00625

Figure 5. A small number of IL-12 producing cells are required for protection of tumour development in mice. Tumour growth kinetics (A) and survival (B) for C57BL/6 mice challenged with PyM1, an IL-12 producing clone, or mixed injections of both. Two IL-12 producing clones were used, LV12.1 (185ng/mL) and LV12.18 (32ng/mL). C57BL/6 mice were injected with 1-2x106 cells subcutaneously. Both LV12.1 and LV12.18 demonstrated a statistically significant increase in survival when cells were mixed 106 PyM1 with 105 IL-12 producing subclone (10:1). LV12.18 demonstrated superior protection relative to LV12.1 at a 10:1 mixture with 5 versus 1 survivor at 211 days post injection, p<0.00625. log-rank test. N=5 for each group.

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48

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 2000

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Figure 6

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Figure 6. Injection of IL-12 producing subclones of PyM1 delay tumour development to PyM1 challenge. C57BL/6 mice were treated with either LV12.18 (32ng/mL) or LV12.22 (3.5ng/mL) in the left flank. 49 days after treatment, mice were challenged with the parental PyM1 cell line subcutaneously in the left flank. Mice were monitored for tumour growth kinetics (A) and survival (B). PyM1, n=5. LV12.18, n=9, LV12.22, n=7. Mice treated with LV12.18 showed a statistically significant increase in survival relative to naïve mice, p<0.025, log-rank test.

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Figure 7. Immune mechanism of tumour protection of IL-12 producing subclones. (A) C57BL/6 mice were specifically depleted of CD4+ and/or CD8+ cells using antibodies. Depleted mice and appropriate controls were injected with 106 PyM1 or LV12.18 cells subcutaenously and monitored for tumour volume. From day 21 onward, tumours in PyM1 injected mice were significantly larger than any other group, adjusted p<0.0001, two-way ANOVA. (B) NK-depleted NOD/SCID mice were injected with 106 PyM1, LV12.1 or LV12.18 cells subcutaneously and monitored for tumour volume. On day 49, PyM1 injected mice have larger tumours than both LV12.1 and LV12.18 injected mice, adjusted p<0.0001, two-way ANOVA. Interestingly, LV12.1 injected mice have larger tumours than LV12.18 tumours, adjusted p<0.05, two-way ANOVA.

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Figure 8

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**

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Figure 8. Timing of IL-12 producing subclone treatment alters outcome of therapy. C57BL/6 mice were injected on Day 0 with 106 PyM1 cells subcutaenously to initiate tumours. These mice were then treated with LV12.1 (185ng/mL) on either post tumour initiation day 3, day 8, day 14 or weekly (days 3, 8, 14 and subsequently weekly) intraperitoneally with 0.5-1x106 LV12.1 cells. By day 80 post tumour initiation, a significantly lower tumour volume was seen in mice treated with LV12.1 at day 3, day 8 and weekly, adjusted p<0.0001, two-way ANOVA. While not as profound, by day 80 there is also a significantly lower tumour volume seen in mice treated with LV12.1 at day 14, adjusted p<0.001, two-way ANOVA.

51

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Figure 9. Weekly treatment of MMTV-PyMT transgenic mice with an IL-12 producing subclone. Starting at 6 week of age, heterozygous MMTV-PyMT transgenic mice and liter and age matched wild-type controls were randomized to either weekly PBS or LV12.1 (185ng/mL) treatment (0.7-1x106 LV12.1 cells or equilant volume of PBS). Mice were monitored weekly for tumour growth kinetics (A) and survival (B). For wild-type mice, treatment had no impact on survival, n.s., log-rank test. Regardless of therapy, MMTV-PyMT mice had a significantly decreased survival relative to wild-type mice, adjusted p<0.0083, log-rank test. Interestingly, for MMTV-PyMT mice, treatment with LV12.1 demonstrated no impact on survival relative to MMTV-PyMT mice treated with PBS, n.s., log-rank test. (C) When analyzing number of tumour sites per mouse, no difference was seen between MMTV-PyMT mice treated with weekly LV12.1 compared to PBS treated mice, n.s., one-way ANOVA. (D) Rate of tumour growth, as judged by time to reach 14cm3, no difference was seen between MMTV-PyMT mice treated with weekly LV12.1 compared to PBS treated mice, n.s., unpaired Student’s T-test. WT-PBS, n=6. WT-LV12.1, n=7. PyMT-PBS, n=7, PyMT-LV12.1, n=8.

ns

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52

CHAPTER 5: DISCUSSION

5.1 General Discussion

The standard of care for metastatic breast cancer that currently exist in the clinic are

inadequate for the treatment of afflicted patients, with some patients seeing little to no

benefit from approved treatments. Through the years, major leaps have been made at

improving breast cancer therapies and providing quality care to all patients. While improving

existing therapies such as chemotherapy or radiotherapy is a useful method at accomplishing

this goal, it has long been thought that the immune system is the single most potent weapon

for eliminating cancers. Under normal circumstances, the immune system is effective at

keeping our body in homeostasis. Through immune surveillance, the body is able to

eliminate the majority of mutated cells that arise, but due to various mechanisms of immune

evasion, these early cancerous cells are able to escape the initial elimination via mutations or

avoiding and suppressing the immune system and subsequently form cancers. Based on this

the answer seems simple, find a way to reactivate the immune system and recognize mutated

tumours. This has been the primary aim of IL-12 based cancer immunotherapies.

In early clinical trials, direct injections of IL-12 demonstrated promising results in

patients, but in a phase II clinical trial, toxicities were seen in 12 of 17 patients with two

patients dying71. These deaths were later associated with systemic increases in IFNγ, which

caused initial skepticism for the use of IL-12 as a cancer immunotherapy, the suspension of

clinical trials and thus the use of recombinant IL-12 was put on the backburner72. But the

potent pro-inflammatory effects of IL-12 remained too alluring for these toxicities to avert

research from its use in cancer immunotherapy and clinical trials were reopened71. To avoid

53

these toxicities associated with systemic IL-12, research in the development of alternative

methods for the use of IL-12 cancer immunotherapy began gaining steam.

Novel alternative IL-12 therapies that have been developed focus on avoiding

systemic increases in IL-12 and IFNγ, which have been shown to be the causes of toxicity72.

Rather than IL-12 administration that causes systemic rises in both cytokines, these

approaches aim to cause local increases in IL-12 in a variety of different ways. One

prominent approach is through the genetic modification of cells to express IL-12 as a method

of cancer immunotherapy, which is based on the use of plasmid-based transfection or

retroviral-based transduction to express IL-12 in a target cell population90. Work has shifted

to a focus on retroviral transduction due to a more stable genomic integration of the gene of

interest that is long lasting. Retroviral-based genetic modification using IL-12 has been

demonstrated with fibroblasts75, dendritic cells91, T lymphocytes78, and cancer cells77,79, all

which have shown promising results. In all of these genetic modification methods, specially

modified retroviruses are designed to insert a gene of interest into a target cell and in the

context of IL-12 cancer immunotherapy these vectors are specially designed to insert the p35

and p40 subunits of IL-12. While many gene modification modalities do exist, the genetic

modification of cancer cells is of particular interest as it serves as a target and source of

antigens for the immune cells, is the vehicle for IL-12 delivery and is eliminated by the

initiated immune response.

The majority of work using IL-12 transduced cancer cells has been done in murine

cancer cell line models. Early studies using murine cancer cell lines were performed with

sarcomas, melanomas and breast cancers73. In these studies, cells were transduced with a

retroviral vector containing IL-12. Bulk transduced samples were selected using G418, and

54

IL-12 concentration was measured from these enriched bulk cultures with all cultures

producing greater than 10ng/106 cells/48hrs. An antitumour immune response was observed

to primary challenge with IL-12 transduced sarcoma (MCA207 and MCA102), melanoma

(B16-F10) and breast cancer cells (TS/A) with 100% of mice being tumour free. These

tumour free mice were then rechallenged with the nontransduced parental line and were able

to reject these cells demonstrating long lasting memory in these cells73.

Based on the promising results of the experiments mentioned above and others using

the same framework of genetic modification of cancer cells to express IL-12, our lab

developed an interest in this method of cancer immunotherapy, with the primary aim of

developing a better understanding of the mechanism of IL-12 transduced cancer cells. Early

work in our lab began using the 70Z/3 pre-B cell leukaemia line. In this model, injection of

106 70Z/3 cells intraperitoneally into syngeneic BDF1 mice causes rapid tumour formation

and is lethal at approximately days 12-4080,92. 70Z/3 cells were then transduced with a

lentiviral vector (see Figure 2a) that causes constitutive expression of IL-12. Upon limiting

dilution cloning, single cell derived IL-12 producing clones were generated that had known

amounts of IL-12. Injection of these IL-12 producing cell lines with known stable IL-12

levels demonstrated that IL-12 production above a certain threshold, which is approximately

1ng/mL, allowed for protection from cancer development and prolonged survival relative to

the parental cell line. In addition, 1) it was demonstrated that a small number of high IL-12

producing cells is more effective than a large number of low IL-12 producing cells, 2) the

anti-cancer immune response is T cell mediated and 3) the immune response is specific and

long lasting80.

55

We then expanded our studies to other models. While additional leukaemia and solid

tumour models have been utilized, the most extensive research has been completed with the

SCCVII head and neck squamous cell carcinoma. In this model, we were able to recapitulate

the results seen in the 70Z/3 model demonstrating that IL-12 cytokine based cancer

immunotherapy show efficacy for a range of different tumour subtypes. This promising work

and the improved understanding of the mechanism gained prompted the initiation of a

clinical trial looking at the safety and efficacy of this therapy for use in patients diagnosed

with AML.

While major advancements have been made in AML toward getting this therapy from

the bench top to bedside, work still needs to be done to show its efficacy in solid tumours for

this same translation to clinical work. Solid tumours cover a range of different malignant

origins, with drastic differences in presentation, response to therapy and other factors seen

between them. For the majority of primary solid tumours, standard therapy such as surgery,

chemotherapy or radiotherapy are effective at treating patients, with the major challenge

being the treatment of recurrent and metastatic disease. Based on the range seen within solid

tumours, the focus of our lab is to better understand the mechanism of IL-12 cytokine based

cancer immunotherapy using a model that better represents tumour development seen in

patients with metastatic solid tumours. Cancer cell line models, which include 70Z/3 and

SCCVII, provide elegant models for understanding intricate mechanistic details such as the

importance of IL-12 production levels or establishment of memory. They also provide

homogeneity, as all cells and therefore all tumours originate from a single clonal cell, which

gives an understanding of the cell of origin, its phenotype and in some contexts, the prevalent

antigens. What cell line models can lack is an accurate modeling of cancer development, as

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human cancers are not based on the injection of a cancer cell line to initiate tumours but

rather develop over the lifetime of patients. To more accurately model human cancers, other

murine models have been developed.

Two murine models that can better reflect human cancer development are patient

derived xenograph (PDX) models and genetically engineered mouse (GEM) models. While

both PDXs and GEM models possess human cancer relevance, PDXs have an inherent flaw

for cancer immunotherapy research in that xenographs require the use of

immunocompromised mice that lack a fully functional adaptive immune system93. For IL-12

based immunotherapies, this is problematic as previous work from our lab has demonstrated

that the IL-12 cancer immunotherapy utilizing gene modification of cancer cells mediate a T

cell dependent response80,81. In contrast, GEM models do contain an intact adaptive immune

system and are therefore a better suited model for research in cancer immunotherapies93.

The MMTV-PyMT breast cancer model is a GEM model of metastatic breast cancer.

In this model, PyMT is expressed under the control of the MMTV promoter. This leads to the

mammary epithelium specific expression of the PyMT oncoprotein causing mammary

tumour formation in hetero/hemizygous mice expressing the transgene84. Importantly, while

the oncogene used represents an artificial cancer initiator, MMTV-PyMT transgenic mice

possess many features similar to that seen in human luminal subtype breast cancer and its

progression85. Both morphological and receptor/protein expression patterns as the mammary

tumours progress from initial hyperplasia to late carcinoma are similar to that seen in human

breast cancer84,85. In addition, the MMTV-PyMT transgenic mice consistently develop lung

metastases84,86. These features make it an excellent candidate for modeling human metastatic

57

breast cancer that and allows for a better understanding of the efficacy of genetically

modified IL-12 expressing cancer cells.

To establish an IL-12 cell-based cancer immunotherapy, it was important to

determine whether the MMTV-PyMT model responded in a similar capacity compared to

previous models such as 70Z/3 and SCCVII, and this was done by developing a MMTV-

PyMT derived cancer initiating cell line. From primary MMTV-PyMT tumour cultures, two

enriched cell culture populations could be identified based on cell morphology, epithelial and

fibroblast cells. Consistent with work by Ma et al.88, epithelial enriched cell cultures were

predominantly CD24+CD29+CD49f+Sca1low while fibroblast enriched cell cultures were

predominantly CD24−CD29+CD49f−Sca1high. PyM1, an epithelial cell line derived from

MMTV-PyMT nearing endpoint, was consistent with the phenotype of mouse mammary

epithelial stem cell/tumour initiating cell populations (>95% CD24+CD29+CD49f+Sca1low) as

seen in Figure 1a. Further characterization of the protein secretome of PyM1 demonstrates

that these cells produce large quantities of G-CSF and CXCL1 (KC) with small amounts of

CCL-2 (MCP-1) and other cytokines, which is consistent to what is seen from both freshly

derived primary MMTV-PyMT tumours and MMTV-PyMT derived cell lines as seen in

Figure 1b (89). Subcutaneous injection of PyM1 cells was able to initiate tumours in

syngeneic mice, which is consistent with the tumour initiating phenotype of the cells and

consistent with previous literature testing the tumour initiating capacity of a similar cell type.

Lacking in the reported literature is tumour growth kinetics and median survival of mice

injected with these cells, which has been recorded for tumours from PyM1 injected mice

(Figure 1c). In addition, phenotypically similar breast cancer initiating cells derived from

MMTV-PyMT mice have demonstrated plasticity. These MMTV-PyMT derived cell lines

58

have demonstrated changes in morphology and surface marker expression when incubated

with various factors such as BrdU, retinoic acid or TGFβ demonstrating a transition to a

more mesenchymal phenotype and morphology consistent with an epithelial to mesenchymal

transition (EMT)88,94.

Our rationale for the development of an epithelial cell line from MMTV-PyMT mice

was twofold. The first was to produce an in vivo breast cancer cell line model to recapitulate

the results previously tested in leukaemia and head and neck squamous cell carcinoma. The

second was that a derived breast cancer model would help predict possible validity of an IL-

12 cancer immunotherapy in MMTV-PyMT transgenic mice. Based on its characterization,

PyM1 represents a useful model of breast cancer initiating cells that can be used to test our

cell-based IL-12 cancer immunotherapy platform.

The next step in understanding the applicability of our IL-12 cancer immunotherapy

to breast cancer was to transduce cells with a lentivirus engineered to express full length

mouse IL-12 and subclone IL-12 producing cells (Figure 2a & 2b). From this process

fourteen IL-12 producing subclones of PyM1 were generated that secrete a range of IL-12

from as little as 3ng/mL up to 300ng/mL as seen in Figure 2c. Upon analysis of phenotype

(Figure 3a) and protein secretome (Figure 3b), these IL-12 producing subclones are

extremely similar to their parental counterpart PyM1. While these two metrics of phenotype

and protein secretome have not been tested previously, a similar consistency has been

demonstrated both in our work when cloning PyM1 from the epithelial cell culture for cell

surface marker expression88 as well as by others when examining the secreted products of

freshly derived MMTV-PyMT tumours compared to a derived cloned cell line89. Examining

the growth kinetics in vitro (Figure 3c) demonstrates that there are significant differences in

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proliferation evident between the cell lines, which is not an unexpected result. Previous work

from our lab has demonstrated that not all IL-12 producing subclones have consistent

proliferation with the parental cell line81.

For IL-12 cancer immunotherapy in the 70Z/3 ALL model, a range of IL-12

producing subclones were generated. Upon I.P. challenge with these IL-12 producing

subclones, a threshold of IL-12 production (approximately 1ng/mL/106 cells/2hrs) was

demonstrated that dictated whether mice would survive. Similarly, a range of IL-12

producing subclones of PyM1 were tested in wild-type synegeneic C57BL/6 mice. Based on

the results in Figure 4, mice injected S.C. with any tested IL-12 producing PyM1 subclone

did not generate tumours and showed 100% survival, from as low as 3.5ng/mL (LV12.22) to

as high as 284ng/mL (LV12.23), which is clearly evident based on tumour growth (Figure

4a) and survival analysis (Figure 4b). Interesting, even at the highest IL-12 production

(284ng/mL by LV12.23), no IL-12 associated toxicities were observed suggesting that these

doses produce local increases in IL-12 and avoid toxicities associated with systemic rises in

IL-12 and IFNγ. Both work previously done by other groups and work from our lab have

demonstrated that mice injected with IL-12 transduced cancer cells are able to reject these

cells, 100% of mice remain tumour free and have drastic increases in survival compared to

injection of the parental cancer cells. This has been demonstrated in models that utilized bulk

transduced and selected cells73 or cells cloned to have a known constant production level80,81.

The work from our lab has been important in cementing the fundamental understanding that

the amount of IL-12 produced by the transduced cells is essential.

We have demonstrated that when absolute amount of IL-12 is kept equal, a lesser

amount of a higher producing clone demonstrates an increase in survival and tumour free

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mice relative to a greater amount of a lower clone80,81. In Figure 5, a similar experiment was

conducted utilizing two IL-12 producing clones with varying production, LV12.1

(185ng/mL) and LV12.18 (32ng/mL) that had a 5.8x difference in IL-12 production. In the

PyM1 breast cancer model, the lower producing LV12.18 elicited a greater degree of

protection relative to the higher producing LV12.1, even at a lower number of cells. This is

especially evident when examining a mixing of 106 PyM1 cells with 105 IL-12 producing

cells. When this is done with LV12.18, mice remain tumour free past 200 days post injection,

while mice injected with LV12.1 developed tumours with a median survival of 204 days. A

potential explanation for these results may be due to a lack of dependency on IL-12 dose,

although this may also be caused by the difference in IL-12 levels between these two clones

not being sufficient to produce a dose dependent response. Another possible explanation

could be related to inherent differences between LV12.1 and LV12.18. Based on

phenotypic, protein secretion and proliferation analysis (Figure 3), there does not appear to

be any striking differences between these two clones and therefore may be related to

something else intrinsic to these clones. As described above (Figure 7) and discussed in

more detail below, work in NOD/SCID mice demonstrated that in comparison to PyM1 and

LV12.1, LV12.18 is non-tumourigenic and non-lethal. How this difference in tumour

forming capacity relates to an ability to prime an immune response remains to be determined.

Importantly, treatment with either clone generates a significant increase in survival relative

to injection of PyM1 alone.

An important aspect of IL-12 cancer immunotherapy is the generation of a memory

immune response. Mice that have been challenged and rejected an IL-12 producing clone

have demonstrated the ability to subsequently reject rechallenges with the parental line.

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These memory responses have been demonstrated in early work with limited memory seen

with bulk selected IL-12 transduced cells and complete long lasting memory by work in our

lab with ALL and HNSCC models73,80,81. The PyM1 breast cancer model however is not

consistent with this classical complete long lasting memory response seen in previous work

from our lab (Figure 6). Mice treated with LV12.18 (32ng/mL) and LV12.22 (3.5ng/mL) do

demonstrate a delay in tumour development, an increase in survival and some mice being

tumour free at 184 days post PyM1 challenge. This type of response is less consistent with

previous work from our lab where a complete memory response is seen upon rechallenge of

mice treated with IL-12 producing cells and in contrast far more consistent with early

literature that only demonstrated a portion of mice were tumour free at study completion73,77.

One possible parallel can be drawn from an IL-15 based cancer immunotherapy in prostate

and breast cancers95. IL-15 is a member of the IL-2 superfamily and primarily promotes

proliferation on T and NK cells and as an anti-apoptotic factor96. With this therapy, only a

delay in tumour development was demonstrated when mice were “vaccinated” with IL-

15/15Rα transduced prostate or breast cancer cells prior to a challenge with the parental line.

While the memory response seen in the PyM1 breast cancer model does show similarities to

earlier IL-12 gene modification therapies and those seen in the IL-15/15Rα transduced

cancer cell model, IL-12 and IL-15 do have distinct effects on the immune system. While

both demonstrate proliferative effects, IL-12 is essential for polarization of helper T cells to a

TH1 cell mediated immune response and therefore comparisons between cytokines must be

taken with heavy consideration. In addition, while not all mice are tumour free following IL-

12 based therapy in the PyM1 model (in contrast to work in other models from our lab), there

are a number of LV12.18 treated mice challenged with PyM1 that do show potent memory

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immune responses and are tumour free while the remaining mice that do develop tumours

still show a delay relative to naïve mice challenged with PyM1.

LV12.18, which produced the most profound response when mixed with the parental

cell line PyM1, demonstrating a memory immune response to PyM1 challenge, was used to

determine whether the anti-tumour immune response involved in eliminating IL-12

producing PyM1 subclones was T cell-dependent (Figure 7a). Interesting, mice injected S.C.

with LV12.18 did not develop tumours in CD4-depleted, CD8-depleted or CD4/8-depleted

mice, which is in stark contrast to what has previously been reported with IL-12 based cancer

immunotherapies. One interpretation of these results is that IL-12 producing PyM1 subclones

such as LV12.18 are eliminated in a T cell independent manner, which is uncharacteristic of

IL-12 based therapies, with IL-12 having such a prominent, but not exclusive, role in T cell

differentiation and activation. IL-12 has a direct effect on both CD4+ and CD8+ T cell

populations leading to T cell mediated responses. Work from our lab and others have

demonstrated that at least one T cell subset (CD4+ or CD8+) is essential for mediating tumour

elimination in mice injected with IL-12 transduced cells73,80,81. IL-12 however, also effects

NK cell activation and proliferation, envisaging a possible scenario for a T cell independent

mechanism for tumour rejection in mice injected with LV12.18. Alternatively, another

possible interpretation is that LV12.18 is non-tumourigenic and incapable of producing

tumours in vivo, irrespective of the immune system. To experimentally determine whether

one of these possible alternative mechanisms may be responsible, further testing was done in

NK-depleted NOD/SCID mice, which demonstrated that while mice injected with PyM1 or

LV12.1 were able to form tumours in immunocompromised mice, those injected with

LV12.18 were unable to (Figure 7b). Therefore, these results firmly support the notion that

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LV12.18 is a non-tumourigenic/non-lethal subclone of PyM1. The development of non-lethal

subclones has been observed in other models, as seen with 70Z/3-NL, which is a non-lethal

variant of 70Z/3 (termed 70Z/3-L)92. LV12.18 is unique relative to even 70Z/3-NL in that the

non-lethality of this ALL variant is the product of immune mediated clearance, while

LV12.18 is both non-lethal and non-tumourigenic. Work with 70Z/3-NL demonstrated that

treatment with a non-lethal variant is capable of producing a memory immune response when

mice are challenged with the lethal variant92. Experiments with LV12.18 demonstrate that

even a non-lethal/non-tumourigenic clone can evoke an immune response and generate

potent clearance of the parental cell line that is long lasting. These results put the efficacy of

LV12.18 over LV12.1 at delaying tumour development and prolonging survival at small

percentages with PyM1 into perspective. A possible implication of this could be that the

combination of the non-tumourigenic and IL-12 producing attributes of LV12.18 produce a

synergistic effect that leads to the improved efficacy over LV12.1 when combined with the

parental PyM1 cell line.

Optimizing treatment timing and frequency are necessary to gain a better

understanding of how to generate maximal immune responses for cell-based cell-based IL-12

cancer immunotherapies, which can have major implications for planning treatment

strategies in patients. While injection of equal numbers of IL-12 producing subclones and the

parental cell line demonstrate tumour cell rejection, as seen in Figure 5, delayed injection of

IL-12 producing cells at a distinct location (relative to injection of the parental cell line) has

not been adequately examined, but represents a more realistic scenario of treatment strategies

in patients who have existing cancer burden. With the PyM1 cell line model, it was

demonstrated that timing of an IL-12 producing subclone treatment alters the outcome of

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PyM1 injected mice (Figure 8). While all treatment regimens did demonstrate a delay in

tumour development relative to untreated mice (atleast adjusted p<0.001), treatment received

at day 14 elicits a far more modest delay in tumour development compared to treatment

received at day 3, day 8 or weekly (adjusted p<0.001 versus adjusted p<0.0001 respectively)

suggesting that treatment approaching 14 days in tumour bearing mice is not as effective at

delaying tumour development. The earliest work examining delayed treatment with IL-12

transduced cells was conducted using the MCA-207 sarcoma cell line73. Their work

demonstrated that a Day 3 treatment with IL-12 producing cells at a distinct location was

able to cause tumour rejection of the established tumours in some mice, while untreated and

control transduced mice showed no protection73. Our lab has demonstrated that in the 70Z/3

ALL model, both recombinant IL-12 and IL-12 producing cells were able to prolong survival

when injected into tumour bearing mice80. Interestingly, no literature exists examining the

efficacy of multiple repeated treatments with IL-12 producing cells on a primary breast

cancer tumour. One study examining the efficacy of repeated treatment with IL-12

transduced cells in a lung metastasis model was able to demonstrate that repeated treatment

with IL-12 producing cells (on days 3, 6, 9 and 12 post parental cell injection) increased

survival of mice relative to those repeatedly treated with IL-2 or untransduced cells77. While

at the current point in this experiment, little to no benefit is seen with weekly treatments over

a single treatment at day 8, the lack of toxicity from this treatment regime in wild-type

C57BL/6 mice suggests that repeated injections are well tolerated and are potentially useful

for inducing better outcomes. This lack of toxicity with repeated IL-12 producing cell

injections is an observation that was not examined by Rodolfo et al. and the first to

demonstrate that repeated weekly injections are well tolerated77.

65

Based on the overwhelming research demonstrating the success of cell-based IL-12

cancer immunotherapy in leukaemia, a Phase I dose escalating clinical trial has been initiated

to determine the potential for this therapy in patients. With work being done in HNSCC and

breast cancer models, our lab has aimed to extend this cell-based IL-12 cancer

immunotherapy to solid tumours, with models of metastatic solid tumour cancers allowing us

to gain further insights into the immune responses in such models and efficacy in both

primary and metastatic disease, mirroring the patients that will benefit most from this

immunotherapy. As previously discussed, the MMTV-PyMT transgenic mouse model is a

GEM model that has been demonstrated to show many characteristics and features of human

metastatic breast cancer such as expression of breast cancer associated markers and

development of metastases that are seen in human breast cancer progression84-86. Weekly

treatment of MMTV-PyMT transgenic mice was conducted to determine the efficacy of cell-

based IL-12 cancer immunotherapy in a model that develops primary and metastatic

tumours. MMTV-PyMT mice and wild-type litter matched controls were treated weekly

starting at 6 weeks of age with either PBS or the IL-12 producing clone of PyM1, LV12.1

(producing 185ng/mL). It was essential to examine the outcome in both primary and

metastatic tumour sites since immunotherapies, such as the therapy utilized in this work, are

less likely to be use as a first-line therapy for primary tumours, which in humans are

normally surgically resected and treated with chemo- or radiotherapy, and instead provide

greater utility in reducing or eliminating metastatic disease by taking advantage of the

systemic nature of the immune responses. To this end, tumour growth kinetics, survival,

tumour burden based on tumour sites per mouse, tumour development rate as measured by

time to reach 14cm3 and histological analysis of lungs for metastases were all conducted. The

66

results of the weekly treatment of MMTV-PyMT transgenic mice suggest that treatment with

the IL-12 producing subclone LV12.1 demonstrates no difference in outcome relative to PBS

treated mice when examining the effect on the primary tumour sites within the mammary fat

pads (Figure 9a-d). When examining the impact of therapy on development of lung

metastases via histology, a surprisingly low number of metastases were observed (1/14 lungs

between LV12.1 and PBS treated mice), which is uncharacteristic based on previous

literature regarding lung metastasis formation in MMTV-PyMT transgenic mice on a

C57BL/6 strain86,97. Due to a lack of metastasis formation irrespective of treatment, no

conclusions could be made from these results.

The lack of any change in outcome seen in LV12.1 treated MMTV-PyMT mice are

nonetheless interesting and important for guiding future work. A possible explanation for the

lack of response may be related to experimental design. The use of an IL-12 producing

subclone (LV12.1) of an epithelial derived clone (PyM1) as a treatment modality in MMTV-

PyMT mice may not be the ideal treatment regime as this represents the treatment of a

heterogeneous multi-clonal tumour with a mono-clonal cell line. While this may be true for

endpoint, well-established tumours (>16 weeks of age) that are generally heterogeneous and

of multi-clonal origin, weekly LV12.1 treatment onset begins at the early hyperplastic stage

(~6 weeks) where initial cancerous transformation occurs and these initially transformed

cells more closely represent the tumour initiating/tumour stem cells85. Similarly, LV12.1, the

IL-12 producing cell lines and the parental cell line PyM1 are all derived from MMTV-

PyMT mice and represent an epithelial tumour initiating cell phenotype based on expression

of stem cell associated markers (Figure 3a). Therefore, the tumour-initiating cell was

thought to be a good representation of the target cell at the early treatment time points when

67

mice are in the hyperplastic stage of cancer development (~6-8 weeks) as well as the cells

that give rise to early metastatic lesions in the lungs.

To improve outcome in MMTV-PyMT transgenic mice, a possible future direction

would be to alter and optimize the treatment regime. Weekly LV12.1 as a treatment already

is a fairly intense treatment regime and this suggests that the utility of IL-12 producing

subclones, representing a narrow cell type, as a treatment is not the ideal vehicle for therapy.

One possible method to improve outcome would be to utilize freshly derived and transduced

bulk cultures to better represent the heterogeneity seen in the primary or metastatic tumours.

It is important to note that the PyMT protein is extremely potent as an oncogene with

numerous transformation events occurring. As every mammary epithelial cell is forced to

express PyMT (via the MMTV promoter), each has the potential for transformation to a

cancerous cell. Therefore, the capacity for therapy may rely on the ability to clear each and

every mammary epithelial cell. While treatment of MMTV-PyMT transgenic mice showed

no difference in outcome for primary tumours, a greater emphasis should be placed on the

effect of treatment on the lung metastases, as patients with metastatic disease represent the

group with the fewest therapeutic options that will gain the greatest benefit. Although current

results do not provide insight into the effect of cell-based IL-12 cancer immunotherapy on

metastases, treatment of metastatic disease should be a focus of future work.

As discussed previously, the lack of metastases was a surprising observation,

considering previous literature supports the formation of lung metastases in the MMTV-

PyMT model86,97. Of particular note is the mouse strain utilized, which has long been known

to impact both primary and metastatic tumour development86,97. The vast majority of work on

the MMTV-PyMT has been conducted using the FVB/NJ strain, which has been

68

characterized as a “susceptible” strain, in terms of both primary and metastatic tumours86,97.

In the FVB/NJ model, by 16 weeks of age mice have tumours reaching endpoint and

extensive metastases in the lungs86. In contrast, the variant of the MMTV-PyMT model that

we have utilized in our lab is on the C57BL/6 strain, which has been characterized as a

“resistant” strain86,97. In the C57BL/6 model, tumour development is delayed for both the

primary and metastatic tumours, with a reported 6-week delay in mean latency between these

two strains86. Work by Davie et al. demonstrated that both strains develop metastases within

the lungs. While no statistical difference in metastatic burden (mm2/cm2) was reported

between these two strains, their data suggests that the C57BL/6 strain possesses a greater

variability of metastases relative to the FVB/NJ strain86. While this may give partial insight

into explaining the lack of metastases in our experiment in MMTV-PyMT mice on the

C57BL/6 strain, based on this literature it was expected that more than a single mouse would

have detectable lung metastases.

The work presented in this thesis has been focused on the expansion of a cell-based

IL-12 cancer immunotherapy into a model of invasive and metastatic breast cancer, used to

more broadly reflect treatment of solid tumours. Our work demonstrated that the MMTV-

PyMT transgenic metastatic breast cancer mice and the derived PyM1 breast cancer cell line

are useful solid tumour models that allow for assessment of cell-based IL-12 cancer

immunotherapy efficacy in vivo. Using the PyM1 cell line model, we were able to

recapitulate previous findings demonstrating rejection of IL-12 producing cells, local

responses with a small number of IL-12 producing cells and memory responses for mice

treated with IL-12 producing cells. In addition, the PyM1 model allowed us to expand upon

our understanding of a threshold for timing of treatment in mice with established tumours, an

69

observation that has not previously been examined in this immunotherapy. While

experiments testing the efficacy of the cell-based IL-12 cancer immunotherapy in the

transgenic MMTV-PyMT breast cancer model failed to show statistical differences from

normal cancer development of the primary mammary tumours and a lack of conclusive

results regarding the efficacy in metastases on the C57BL/6 strain, this model serves as a

useful tool for modeling metastatic solid tumour pathogenesis with many qualities and

characteristics of human breast cancer as demonstrated previously in various mouse

background strains85,86,97. The work presented demonstrates the efficacy of cell-based IL-12

cancer immunotherapy in a breast cancer cell line model and the opportunity in a transgenic

breast cancer GEM models. A primary focus was placed on expanding cell-based IL-12

cancer immunotherapies into models that better mimic metastatic solid cancers, centered

around the use of the MMTV-PyMT transgenic breast cancer model. While the current state

of experiments using the MMTV-PyMT model are in the preliminary stages, with further

experiments aimed at optimization of the treatment protocol and analyzing the potential

therapeutic effect on lung metastases, the expansion of cell-based IL-12 cancer treatment to

the treatment of metastatic disease remains promising for solid tumours.

5.2 Future Directions

5.2.1 Developing Improved Treatment Strategies for MMTV-PyMT Transgenic Mice

Future experiments in the MMTV-PyMT transgenic model are essential to bridge the

gap between cell line mouse work and clinical patient work. Finding ways to optimize the

treatment process with more realistic approaches are a key to understanding the efficacy of

this therapy when moving this work from the bench to bedside. While many questions still

remain in terms of gaining a better appreciation for the mechanistic details of this IL-12

70

based immunotherapy in the PyM1 breast cancer cell line, this model was used to

demonstrate that results in this model recapitulate those previously seen from our lab (such

as ALL and HNSCC models), which suggests a potential for therapeutic efficacy in the

MMTV-PyMT transgenic model. Our work demonstrates the efficacy of cell-based IL-12

cancer immunotherapy in a breast cancer derived cell line.

While transduction and subcloning of specific IL-12 producing cell populations is a

useful tool for understanding mechanistic details in a clinical setting, this does not provide an

accurate representation of how patients will be treated at the bedside. In a clinical setting for

AML, a patient with refractory disease will provide primary tumour cells via a blood sample

donation. These primary cells are transduced with a human grade IL-12 lentiviral vector,

with the optimal treatment strategy and dosing still to be determined. In the case of solid

tumours, patients with metastatic disease will likely benefit the most for this immunotherapy.

To more accurately model the method of cancer cell sampling in patients using the MMTV-

PyMT metastatic breast cancer model, a possible method would be to conduct transduction

of primary harvested tumours and treat syngeneic mice with these cells. This addresses the

lack of heterogeneity inherent in the treatment strategy conducted in the experiment

presented in Figure 9 where mice were treated with an IL-12 transduced clonal population of

cells, and also better represents the expected strategy for treatment of patients in clinical

trials.

5.2.2 Effect of Cell-Based IL-12 Cancer Immunotherapy on Metastases

The work thus far has focused upon demonstrating efficacy of treatment with IL-12

producing cancer cells for the primary tumours, with only early work examining the efficacy

in metastatic disease. While the primary tumour site is an important therapeutic target, a

71

combination of surgical resection, chemotherapy and radiotherapy are capable of eliminating

primary disease in many patients. In addition, many patients that enter clinical trials possess

cancers that have relapsed, are refractory, or possess pre-existing metastases. Based on work

from our lab and others, cell-based IL-12 cancer immunotherapy has demonstrated

tremendous success in treating primary tumours, but testing the efficacy in the treatment of

established metastases still requires further investigation. The MMTV-PyMT transgenic

model, in addition to its characteristic similarities to human breast cancer, develops

consistent lung metastases across a range of strains, adding greater utility to this model84,88,97.

In addition, while not fully characterized, the derived PyM1 breast cancer cell line has

demonstrated the potential to colonize the lung and form metastases, supported by studies

using MMTV-PyMT derived cells and cells from other models demonstrating intravenous

(I.V.) injection of breast cancer derived epithelial cells leads to the development of these

lung metastases77,98. Together these two models may serve useful to gain a better

understanding of not only the effect of cell-based IL-12 cancer immunotherapy on metastases

that commonly arise in patients with solid tumours.

72

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