Tumor Angiogenesis Is All Tied up in Tie2- Expressing Macrophages

Tumor Angiogenesis is all Tied up in Tie2- Expressing Macrophages

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Mary A. Forget, B.S.

Graduate Program in Molecular Cellular and Developmental Biology

The Ohio State University

2012

Dissertation Committee:

Clay B. Marsh, Advisor

Mark Wewers

Susheela Tridandapani

David Bisaro

Mary A. Forget

2012

Abstract

The impact of the immune system on solid tumor formation and malignant disease has been the focus of much study over the last several decades. Mouse models of malignancy in which macrophages were suppressed resulted in a decrease in tumor angiogenesis, progression and malignancy. However, interventions that eliminate macrophages from tumors all together resulted in a rebound effect causing an increase in the production of progenitors from the bone marrow and homing to tumors resulting in a greater effect. Tie2-expressing monocytes (TEMs) are a distinct subset of pro-angiogenic monocytes selectively recruited to breast tumors in both mouse models of cancer and breast cancer patients. Because of the hypoxic nature of tumors, we investigated if oxygen regulated the trafficking of these cells into tumors or if this subset was differentiated once inside the tumor microenvironment. In part one of this study, (Chapter 2), we demonstrate that the Tie2 receptor is up regulated under low oxygen conditions and that a myeloid-specific knockout of the hypoxia-specific transcription factor, HIF-1α, inhibits Tie2 receptor expression at both the protein and mRNA levels. Further, we demonstrate that macrophage HIF-1α knockout blunts expression of a Tie2-luciferase reporter construct indicating direct transcriptional control. Given these results and that hypoxia commonly occurs during the development of solid tumors, we next investigated the effect of macrophage-specific deletion of HIF-

1α in a mouse model of PyMT breast cancer. Here genetic oblation of HIF-1α from macrophages decreased the rate of tumor growth, angiogenesis and tumor metastasis.

There was no difference in the percentage of TEMs in bone marrow (CD45+/Tie2+ cells) or peripheral blood (CD11b+/CD31-/Gr-1lo/Tie2+ cells) between the three mouse groups while HIF-1αfl/fl/LysMcre mice had a significantly smaller percentage of TEMs

(F4/80+/Tie2+) in tumors compared to wild type or HIF-2αfl/fl/LysMcre mice, even though the percentage of total F480+ macrophages was unchanged. Finally, the percent of tumor F4/80+/Tie2+ TEMs in the tumors significantly correlated with tumor cell intravasation while the percent of total tumor F4/80+ macrophages did not.

M-CSF is known to promote tumor vessel growth and progression through the production of angiogenic factors like VEGF. In the second part of this study (Chapter

3), we demonstrate that M-CSF can modulate Tie2 receptor expression on macrophages both in vitro and in vivo. Under ambient oxygen conditions, we found that M-CSF increased Tie2 receptor on macrophages. This up regulation synergistically increased migration of monocytes towards Angiopoietin-2 (Ang-2) and resulted in greater branch formation in an in vitro endothelial tube formation assay. A neutralizing antibody against the M-CSF receptor abrogated this activity as did knockdown of the Tie2 receptor through the use of a siRNA targeting the Tie2 receptor.

In a PyMT mouse model of breast cancer, recombinant M-CSF increased the TEM population while the total number of F4/80+ macrophages did not change between M-

CSF and vehicle control treatment groups. In this model, M-CSF administration enhanced tumor angiogenesis, while having no significant effect on tumor size. No metastatic effects were examined in any of the treatment groups as this study specifically examined early M-CSF and Tie2-related effects in the tumor. Given the

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observations that both HIF-1α and M-CSF could effect Tie2 receptor expression on macrophages, we investigated the mechanism underlying these events. We found that the PI3 kinase/Akt inhibitor, LY294002, suppressed the generation of TEMs in culture in response to M-CSF while MEK and NF-kB inhibitors did not abrogate Tie2 receptor expression. Further, we found that HIF-1α and M-CSF synergistically act to regulate the generation of TEMs as HIF-1α deletion from bone marrow-derived macrophages diminished the ability of M-CSF to enhance Tie2 expression. In the PyMT tumor model, M-CSF treatment of tumors failed to overcome the lack of tumor growth in HIF-

1αfl/fl/LysMcre mice.

These studies highlight the role of TEM modulation in tumor angiogenesis and progression. First, our results show that HIF-1α regulates TEM differentiation in the hypoxic environment of the solid tumor. Interestingly, HIF-1α myeloid knockout did not affect Tie2 receptor expression in the bone marrow or peripheral blood. M-CSF, to the contrary, expanded the TEM population not only in the tumor, but in the peripheral blood as well. This along with the increases in Ang-2 concentrations in M-CSF-treated tumors suggests a role for M-CSF in the recruitment of TEMs. These studies highlight role of TEMs specifically, and not total F4/80 macrophages, in tumor growth, angiogenesis and progression.

Dedication

This work is dedicated to my family. To Dad and Mom for your never-failing support and encouragement. To my nine brothers and the best men I know. To my sisters who are my best friends for life. Special thanks to my nephew John Paul who has gone before us in faith, “ero cras”. Thank you my grandfather, Thomas A. Doody.

Grandpa Tom immigrated to the U.S. in 1914, fought in WWI, built a home by hand and a good life for his family in Brooklyn N.Y. His example of perseverance and devotion lives on in his grandchildren and great-grandchildren.

Acknowledgements

I would like to thank my mentors Drs. Clay Marsh and Tim Eubank for their commitment, support and suggestions in mentoring me through this degree process.

Thank you for accepting me into the Marsh lab and Angiogenesis group and challenging me to do my best work.

I would like to thank my committee members Drs. Mark Wewers, Susheela

Tridanapani and Dave Bisaro for support of my dissertation process. Thank you for being available and for leading by your example of service.

Thank you to all the members of the Marsh Lab especially the Angiogenesis group. Thank you to Amy for help with completing final experiments this fall. Thank you to Randy for maintaining the mouse colonies and always having animals available for experiments. Thank you for all of your assistance with the processing of animal samples. Thank you to Jeff for help on those long sac days.

Thank you to Leni and Nic and to the entire Moldovan Group for discussions and input on endothelial cell functions and assays and for always being willing to help.

When most people hear the words, Biomedical Statistics, their first reaction is not a smile. Thank you to Xiaokui, who not made our work better with the stoical analysis but who did it with a wonderful smile.

Thank you to my fellow graduate students and trainees Cheryl, Kate, Yadira,

Aria, Devyn, Steve and Duaa for sharing this journey with me.

Special thanks to the staff of the core labs. In the flow cytometry core, thanks to Katrina, Bryan and Danny for teaching me the best ways to isolate our cell populations with flow, for staying late to run my samples and always treating my samples as if they were your own. Thank you also to the staff of the microscopy core,

Sara, Richard and Brain for answering all of my questions and helping me to take great pictures of the data. Thank you to Dr. Coppola, to Dan and to all the members of the genetically engineered mouse core lab for your work towards the Tie2 floxed mouse.

Most especially, thank you to my family for your unwavering support. Thank you to Dad, Mom, Mike, Andy, Steve, Rod, Ed, Tim, Joe, John, Anne, Margaret and

Jim. Thank you for your gracious friendship and for good examples your lives give to the world.

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Vita

May 1996 ...... B.S. Biology, Creighton University

2004-2009 ...... Graduate Student, MCDB program Department of

Veterinary Biosciences The Ohio State University.

2009-Present ...... Graduate Research Associate, MCDB program,

Department of Internal Medicine

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

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Table of Contents

Abstract ...... i

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

Fields of Study ...... vii

Table of Contents ...... viii

List of Tables ...... xii

List of Figures ...... xiii

Abbreviations ...... xv

Glossary ...... xviii

Chapter 1: Introduction

Normal Breast Tissue ...... 1

Breast Tissue is Susceptible to Transformation ...... 2

Mutations Associated with the Development of Breast Cancer ...... 3

Commonly Inherited Mutations ...... 3

BRCA1 and BRCA2 Mutations ...... 3

Li-Fraumeni cancer susceptibility syndrome:mutations inp53 and CHEK2 ...... 3

Ataxia-telangiectasia (ATM) ...... 4

Phosphatase and tensin homolog (PTEN) ...... 4

Spontaneous Mutations and Biology of Mammary Epithelial Cells ...... 4

Spontaneous Mutations and Estrogen in the Industrialized World ...... 6

Classification, Staging and Grading of Breast Tumors ...... 7

Biomarkers lead to personalized patient care ...... 14

Luminal A and Luminal B Subtypes ...... 14

HER2 Positive ...... 14

Triple Negative Breast Cancer ...... 15

The Oncotype DX test is a step towards personalized care ...... 15

An Overview of the Innate Immune System ...... 16

Overview of Monocytes and Macrophages ...... 18

The Macrophage Colony-Stimulating Factor (M-CSF) ...... 19

Tumor-Associated Macrophages ...... 21

Proangiogenic Tie2-Expressing Macrophages ...... 23

Overview of the Tie2 Receptor ...... 23

Tie2-Expressing Monocytes/Macrophages ...... 23

Tumor Metastasis ...... 27

The Hypoxia Inducible Factors ...... 29

Angiogenesis in Tumors ...... 30

Chapter 2: HIF-1α, but not HIF-2α, Regulates the Tie2 Receptor on Tie2- Expressing Monocytes in PyMT Breast Tumors and Augments Angiogenic Function and Metastasis

Absract ...... 33

Introduction ...... 34

Materials and Methods ...... 40

Results ...... 46

Hypoxia up-regulates the Tie2 receptor expression on human CD14+ monocytes ...... 46

HIF-1α regulates Tie2 receptor expression on BMDMs ...... 51

HIF-1α deletion suppresses Tie2 receptor promoter activity at hypoxia ...... 55

HIF-1α deficiency suppresses tumor growth and reduces the TEM population in PyMT tumors ...... 58

The hypoxic tumor microenvironment regulates the differentiation of TEMs ...... 62

Suppressed TEM population decreases tumor angiogenesis ...... 66

Loss of TEMs in tumors of HIF-1αfl/fl/LysMcre mice decrease tumor cell intravasation and pulmonary metastasis ...... 69

Discussion ...... 76

Acknowledgements ...... 81

Chapter 3: Macrophage Colony-Stimulating Factor Augments Tie2-Expressing Monocytes Angiogenic Function and Recruitment in a Mouse Model of Breast Cancer

Abstract ...... 82

Introduction ...... 84

Materials and Methods ...... 86

Results ...... 93

M-CSF regulates a transition from CD14+ monocytes to CD14+/TEMs ...... 93

M-CSF enhances CD14+ monocyte migration towards Ang-2 ...... 98

M-CSF augments the angiogenic potential of CD14+ monocytes on HUVECs via Tie2 receptor up-regulation ...... 102

M-CSF-treated CD14+ monocytes alters HUVEC phenotype ...... 107

M-CSF treatment increases the tumor F4/80+/Tie2+TEM population and augments angiogenesis while having no effect on tumor growth ...... 112

High serum levels of M-CSF expands a circulating TEM population ...... 118

M-CSF and HIF pathways can independently and synergistically regulate Tie2 receptor expression on TEMs in vitro ...... 122

M-CSF treatment cannot overcome HIF-1α-deficiency to augment tumor growth ...... 127

M-CSF recruits CD14+/Tie2+ TEMs into tumors by regulating Ang-2 and SDF- 1/CXCL12 ...... 130

Discussion ...... 134

Acknowledgements ...... 138

Chapter 4: Conclusions and Future Directions ...... 139

References ...... 144

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List of Tables

Table 1: Staging of Breast Cancer and Survival Rates ...... 13

Table 2: Characteristics of TEMs ...... 26

Table 3: Lineage of Tie2-expressing Monocytes/Macrophages (TEMs) ...... 39

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List of Figures

Figure 1: Hypoxia Up-Regulates the Tie2 Receptor on CD14+ Human Monocytes ...... 50

Figure 2: HIF-1α, but not HIF-2α, Regulates Tie2 Receptor Expression on F4/80 subset...... 54

Figure 3: HIF-1α Regulates Tie2 Promoter Activity ...... 57

Figure 4: HIF-1α Regulates Tumor Growth and F4/80+/Tie2+ TEMs ...... 61

Figure 5: HIF-1α Regulation of Tumor TEMs is Driven by Transdifferentiation and not Recruitment ...... 65

Figure 6: Reduced Population of Tumor TEMs Suppress Angiogenesis ...... 68

Figure 7: Tumor TEMs Regulate Tumor Cell Intravasation and Lung Metastasis ...... 73

Figure 8: Supplemental Data on TEM Isolation by Flow Cytometry of Bone Marrow, Blood and Tumors ...... 75

Figure 9: M-CSF Up-Regulates Tie2 Receptor on CD14+ Human Monocytes ...... 97

Figure 10: M-CSF Pre-treatment Augments the Migratory Response to Ang-2 by CD14+ Monocytes ...... 101

Figure 11: Conditioned Media from M-CSF Treated TEMs Augments HUVEC Branching ...... 106

Figure 12: Conditioned Media from Monocytes Pre-treated with M-CSF Induces a “tip” Characteristic on HUVECs ...... 111

Figure 13: M-CSF has no Effect on Tumor Growth but Increases TEM Numbers and Augments Angiogenesis ...... 116-117

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Figure 14: M-CSF Expands the TEM Population in Peripheral Blood ...... 121

Figure 15: M-CSF and HIF Pathways Independently and Synergistically Regulate the Tie2 Receptor Expression on Monocytes ...... 126

Figure 16: M-CSF Augments the Percent of Tumor TEMs Resulting in Increased Angiogenesis ...... 129

Figure 17: M-CSF Augments Ang-2 and SDF-1/CXCL12 Production in

PyMT Tumors ...... 133

ABBREVIATIONS

Tie2 ...... Tyrosine Kinase Receptor with IgG and EGF like

Domains

TEM ...... Tie2-Expressing Monocyte/Macrophage

EGF ...... Epithelial Growth Factor

HIF-1α ...... Hypoxia Inducible Factor 1 Alpha

HIF-2α/EPAS-1 ...... Hypoxia Inducible Factor 2 Alpha

HRE ...... Hypoxia Response Element

HIF-1β ...... Hypoxia Inducible Factor- 1 beta

Ang-1 ...... Angiopoietin-1

Ang-2 ...... Angiopoietin-2

LysMcre ...... CRE recombinase driven by the Lysozyme M promoter

sequence fl/fl ...... floxed/floxed

PyMT ...... polyoma middle T antigen

M-CSF ...... Macrophage Colony Stimulating Factor

MCSF-R ...... Macrophage Colony Stimulating Factor Receptor

MCSFR Nab ...... Macrophage Colony Stimulating Factor Neutralizing

Antibody

VEGF ...... Vascular Endothelial Growth Factor

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EPO ...... Erythropoietin

ECM ...... Extracellular Matrix

MCP-1/CCL2 ...... Monocyte Chemoattractant Protein 2

TNF-α ...... Tumor Necrosis Factor alpha siRNA ...... small interfering RNA siTie2 ...... small interfering RNA against Tie2 si scrmb ...... small interfering RNA with a non-specific sequence

Dll-4 ...... Delta like ligand 4

PBS ...... Phosphate Buffered Saline

Macs ...... Macrophages

FBS ...... Fetal Bovine Serum pB ...... Polymyxin B

HER2 ...... Human Epidermal Growth Factor Receptor 2

ELISA...... Enzyme-linked ImmunoSorbant Assay

TAF ...... Tumor Angiogenesis Factor

EMB ...... Endothelial Cell Basal Media

HUVEC ...... Human umbilical vein endothelial cells

MRC1 ...... mannose receptor

IL-10 ...... Interleukin 10

CSBT ...... Cathepsin B

TP ...... Thymidine phosphorylase

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Glossary

Hypoxia ...... An inadequate supply of oxygen or state of oxygen

deficiency

Tumor ...... growth of neoplastic cells

Angiogenesis ...... the process of new blood vessel growth from existing

vessels

Macrophage ...... a myeloid lineage cell produced from the differentiation

of a monocyte

Angiopoietins ...... family of growth factors that bind the TIE receptors and

function in blood vessel maintenance and angiogenesis.

Ang-1 binding maintains blood vessel integrity while

Ang-2 binding leads to vessel remodeling

M-CSF ...... macrophage colony- stimulating factor

VEGF ...... vascular endothelial cell growth factor

Endothelial Cells ...... line the surface of circulatory vessels

CD31 ...... cluster of differentiation 31

Mannose Receptor ...... a c-type lectin found on the surface of macrophages

M1 ...... Refers to classical inflammatory macrophages

M2 ...... refers to an immune regulatory macrophages phenotype

F4/80 ...... A 160 kDA glycoprotein specifically expressed on mouse

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macrophages

Malignancy ...... a general term typified by uncontrolled growth of

transformed cells

Metastasis ...... spread of a primary tumor to a secondary organ

Monocytes ...... peripheral blood mononuclear phagocytic cell that

survives about 3 days before either differentiating into a

macrophage or undergoing apoptosis

Oncotype DX ...... a diagnostic test used on early-on-set breast cancer to

evaluate the likelihood of reoccurrence

Extravasation ...... movement of cells out of blood and lymphatic vessels

into tissues

Intravasation ...... movement of cells out of tissues or tumors into blood and

lymphatic vessels

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Chapter 1: Introduction

“Human progress is neither automatic nor inevitable. Every step toward the goal of justice requires sacrifice, suffering and struggle the tireless exertions and passionate concern of dedicated individuals”

Martin Luther King Jr.

Normal Breast Tissue

Mammals are defined by their ability to nurse their young. Nursing reinforces the maternal-to-infant bond and provides the framework for the family system. Thus, the mammary gland is a fundamental tissue in mammalian life and reproduction.

Human mammary glands begin to develop prenatally in the sixth week of life in both female and males 1. During this period, breast tissue develops as positional information allows epidermal cells to form a “milk ridge”. The milk ridge extends from the upper inguinal area (the armpit) down to the groin on both sides of the body.

Following formation of the milk ridge, the rudimentary mammary gland regresses into the chest wall by the ninth week of fetal life leaving two breast buds. Following the development of these fundamental structures, the tissue will remain dormant until adolescence 2. At the onset of puberty hormones, specifically estrogen and progesterone signal the development of the mammalian breast. Positioned over the 1

pectoral muscle on the chest wall, normal breast tissue is composed of a series of lobules and ducts lined at the inner surface with epithelial cells. The epithelial cells are the functional cells of the breast producing and secrete milk into the lobules which connect to the ducts and empty into the nipple. The breast tissue is also composed of fibroblasts, adipocytes, immune cells and blood vessels. These accessory cells make up a matrix that surrounds and supports the epithelial cells of the ducts and lobules. At puberty as the human breast matures, a blood supply that will carry oxygen and nutrients to the breast tissue develops. The internal mammary artery supplies the inner portion of the breast and the axillary artery the outer portion of breast tissue 1-3.

Breast Tissue is Susceptible to Transformation

In developed countries, 1 in 8 women who live to the age 90 will develop breast cancer 3. The majority of breast cancers are epithelial cell carcinomas located in either the duct or the lobule 4. The work contained in this dissertation focuses on elucidating the mechanisms underlying angiogenesis and tumor progression in an epithelial carcinoma mouse model and therefore, I will focus background information on the development and progression of this type of breast tumors. The high incidence of breast cancer in our society leads one to ask the question, “why is this tissue so susceptible to carcinogenesis?” This vulnerability to tumor formation may be due to properties inherent in the epithelial cells of the breast or may be due to the influence of cells in the tissue surrounding the epithelial layer. To understand this, we will look at genetic mutations linked to breast cancer development and examine the biology of the

epithelial cells in the breast. Additionally, I will place these findings is the context of life in the developed world.

Mutations Associated with the Development of Breast Cancer

Commonly Inherited Mutations

Hypotheses regarding the development of tumors date back hundreds of years.

Payton Rous, for example, discovered that an infectious agent could induce tumors when injected into a healthy host. Over the next 70 years, further studies led to understanding that viruses can cause cancer through oncogenes. In 1979, the first human oncogene, RAS1, was cloned and found to be constitutively active 5, 6. This discovery in cancer research re-focused the field on the search for inherited mutations that underlie tumor formation.

BRAC1 and BRCA2 Mutations

By the mid-1990s, two mutations in the breast cancer susceptibility genes

(BRCA1 and 2) were discovered and described (6, 7). Mutations in either the BRAC1 or 2 gene lead to increased risk in an aggressive hereditary breast cancer. Women with mutations in the BRCA 1 or BRCA2 genes have a sixty percent higher incidence of breast cancer by age 90 than women with normal copies of these genes 4

Li-Fraumeni cancer susceptibility syndrome: mutations in p53 and CHEK2

Li-Fraumeni cancer susceptibility syndrome increases the risk of any individual for developing breast carcinomas 8. Mutations in both CHEK2 and in the tumor suppressor p53 are known to increase the risk for breast carcinomas. Normally, p53 functions to reduce the survival and proliferation of cells with DNA damage 9, 10.

Ataxia-telangiectasia (ATM)

Like p53, ATM normally functions to repair DNA. An autosomal recessive condition, Ataxia-telangiectasia is most common in Caucasians occurring at a rate of 1 in 300,000. Mutations in ATM are associated with a number of pathologies including an increased risk of developing leukemia during childhood and epithelial cancers in adulthood 11-14.

Phosphatase and tensin homolog (PTEN)

PTEN is a phosphatidylinositol-3,4,5-triphosphate 3-phosphatase. PTEN acts as a tumor suppressor negatively regulating cell cycle progression through regulation of Akt signal transduction 15, 16.

Spontaneous Mutations and Biology of Mammary Epithelial Cells

The steroid hormone estrogen is a major mediator of breast epithelial cell biology. To understand the susceptibility of breast epithelial cells to transformation, I will focus on explaining the mitogenic effects of estrogen. Estrogen, through binding to the estrogen receptor, stimulates epithelial cell proliferation and survival leading to localized tissue remodeling 17, 18. This occurs during pregnancy, lactation, and on a monthly basis due to the female reproductive cycle. Estrogen effects cell cycle regulatory proteins, the production of growth factors, expression of the progesterone receptor expression, increases cell survival and initiates tissue remodeling. First, the activated estrogen receptor binds to consensus sites in the promoter of Cyclin D1 19.

This binding increases transcription and subsequent protein production allowing for

Cyclin D1 to complex with the cyclin dependent kinases (CDKs). These complexes

phosphorylate proteins involved in cell cycle regulation such as the tumor suppressor,

Retinablastoma protein (Rb). Rb normally inhibits proliferative E2F-related transcription. However, when phosphorylated by the Cyclin D1/CDK complex, Rb dissociates from E2F proteins 20-24. Its repression of transcription is removed resulting in the proliferation of epithelial cells. Estrogen also increases production of growth factors, such as IGF and EGF, which bind to their respective tyrosine kinase receptors, increasing mitogenic cell signal transduction and proliferation of breast epithelial cells

25, 26. Estrogen intensifies production of the progesterone receptor 25, 27, 28. Therefore, if a breast carcinoma is estrogen receptor positive it will most likely also be positive for the progesterone receptor. This allows epithelial cells to be further stimulated by both hormones 29-31.

Estrogen promotes the survival of epithelial cells through the production of Bcl-

2 32-34. If DNA damage has occurred and Bcl-2 levels are high, the cell is more likely to undergo transformation initiating cancer formation. Estrogen increases production of cellular proteases which break down the extracellular matrix (ECM)35 . Normally, breakdown of the ECM leads to breast tissue remodeling. However, if transformation has occurred dissolving the ECM will allow for encapsulated in situ tumors to become invasive. Breakdown of the ECM also facilitates tumor cell intravasation in the blood and lymphatic vessels increasing tumor metastasis 36, 37.

Spontaneous Mutations and Estrogen Exposure in the Industrialized

World

Women in industrialized societies are often exposed to both physiological and exogenous estrogens. Exposure to estrogen increases the likelihood that a transformative mistake will be made initiating cancer. Proper nutrition has brought down the age menarche and an early onset of the menstrual cycle (at the age of 11 or before) along with the late onset of menopause (after the age of 55) is often seen in the modern world 38. This increases the number of year that women will be exposed to estrogen on a monthly basis and enhances the probability that a transformative mistake will take place. In addition to monthly hormonal changes, obesity and hormone replacement therapy, both common in the modern world, also increase the risk of developing breast cancer 4, 38-40. In addition to the physiological sources of estrogen, exogenous estrogens from dietary sources and synthetic estrogens produced by mankind also contribute to the development of breast carcinomas in the developed world. Plant foods are the main source of dietary estrogen 41-44. Soy and fiber containing foods are good examples of foods containing phytoestrogens and intake of these foods can raise serum estrogen levels 39. Food preservatives, insecticides and plasticizers all contain synthetic estrogens or molecules that contain aromatic rings that mimic the steroid hormones 42. These compounds are lipid soluble and can be stored in adipose tissue of the mammary gland. The hydrophobic structure also means that these estrogen mimics can be absorbed readily into cells where they can bind to estrogen receptors and exert changes in cell proliferation and survival.

The metabolism of synthetic agents to other biologically-active molecules remains a possibility and further study is needed to understand if and how the breakdown of

synthetic agents contributes to the formation of cancer. With these considerations in mind, the Environmental Protection Agency (EPA) in the United States and the World

Health Organization have begun to monitor and regulate the manufacture and use of synthetic estrogens and estrogen mimics. To this end, in 2009 the use of DDT has been outlawed in most countries 45-47.

Classification, Staging and Grading of Breast Tumors

Breast tumors are broadly divided into benign and malignant tumors. Benign tumors are the most common form of breast tumor occurring annually at a rate of 1 in

100 3, 4 . Benign lumps in breast tissue are commonly due to fibrosis or cyst formation and arise due to hormonal changes, infections, and necrosis of adipose tissue 48. A cyst is a fluid filled sac and althoug not directly harmful; cysts do increase the future risk of malignancy 3, 49.Fibrocystic disease increases the number of fibroblasts leading to scar like lesions in the breast tissue. In some cases such as fibroadenomas and intraductal papillomas, these scar like lesions can lead to increased risk of developing breast cancer. Fbrotic lesions which should be examined for cellular overexpression of the estrogen and progesterone receptors, monitored for overexpression of Her2 and for aneuploidy. Each of these changes makes it more likely that a malignant transformation will occur and progress to metastatic disease 50. The progression of a transformed cell to a primary tumor and then to metastatic disease occurs in stages.

Staging and classification systems are used to describe tumors and to assist in the selection of treatment for patients. Generally these classification schemes take into account histological characteristics, cellular attributes including nuclear morphology

and receptor status, genetic attributes and anatomical location including metastasis to distant sites sections.

Normal breast tissue is made up of epithelial cell-lined lobules that produce milk and a series of ducts that carry the milk to the nipple and the supporting tissue stroma

1. Cancers are named based on the cell type that is initially transformed. Sarcomas occur when the transformed cells were originally connective, adipose, muscle or vasculature tissue 49. These types of tumors are not common in breast cancer. A majority of breast cancers are carcinomas, arising from transformed epithelial cells in either a duct (ductal carcinoma) or lobule (lobular carcinomas) 3, 49. Adenocarcinomas occur in epithelial cells that secret a product as in breast tissue epithelial cells which produce milk. Early on-set tumors are characterized by clonal expansion of a transformed cell. Carcinoma in situ is confined to the duct (ductal carcinoma in situ or

DCIS) or lobule (lobular carcinomas in situ or LCIS) where it began 38, 49. The affected duct or lobule expands as it is filled with proliferating tumor cells. The extracellular matrix of the duct or lobule containing the tumor acts as a barrier enclosing the tumor and inhibiting spread to other parts of the mammary gland. The transformed cells have not invaded the surrounding healthy tissue. Progression corresponds to an invasive phenotype where further genetic and epigenetic events in the tumors cells allow for the breakdown of the extracellular matrix. Tumor cells are no longer encapsulated in a duct or lobule but spread throughout the local tissue and disperse through epithelial cell layers and the tissue stroma. DCIS has now progressed to IDC or invasive ductal carcinoma. Likewise, LCIS that has spread to surrounding tissue is referred to as invasive lobular carcinoma (ILC) 49, 51, 52. Finally, tumor cells escape through the blood

and lymphatic vessels moving into distant organs such as the bone and lung where secondary tumors can develop and these are called metastatic breast tumors.

The American Joint Committee on Cancer (AJCC) uses the TNM system to stage breast tumors 49. The TNM system uses tumor size (T), lymph node involvement

(N) and metastasis (M) to secondary organs to stage cancer progression. Tumor size

(T) and invasive spread to nearby tissue is given a score between 0 and 4 and the higher number indicates a larger tumor with spread nearby to local tissue. Briefly, the tumor size scores are as follows: “TX” is assigned when primary tumor size cannot be assessed. “T0” means there is no evidence of a primary tumor. “T” indicates ductal or lobular carcinoma in situ or Paget’s disease. Finally, scores of “T1”, “T2”, “T3”, and

“T4” refer to a primary tumor which is less than 2cm, between 2cm and 5cm, less than

5cm, and larger than 5cm, respectively4, 49. In addition to tumor size, the stage of a breast tumor is measured by the invasion to nearby lymph nodes (N) and is assigned a number between 0 and 3. “NX” indicates that nodal involvement cannot be assessed.

“N0” indicates that no lymph nodes contain transformed cells. When very small numbers of tumor cells (<200 cells) spread to the lymph nodes, these can be identified using cellular staining techniques “N0(i+)” or identified by RT-PCR “N0(mol+)” on transcripts specific to epithelial cells. “N1” indicates that between 1 and 3 axillary or mammary lymph nodes are tumor cell positive. “N2” specifies that 4 to 9 axillary lymph nodes or internal lymph nodes contain tumor cells. When 10 or more lymph nodes are involved a score of “N3” is assigned. Here the axillary nodes and lymph nodes under the clavicle bone are typically positive while the internal mammary lymph nodes are positive and greater than 2mm 4, 49. “M” or the metastatic index is used to indicate whether or not the tumor has spread to distant organs - most commonly the lungs or

the bone. “MX” means that metastasis cannot be assessed, while “M0” indicates no tumor cells have been identified by x-ray. A score of “cM0(i+)” means that a small number of tumor cell have been found in the blood, bone marrow, or distance lymph nodes. While a score of “M1” indicates a secondary tumor has formed 4, 49. Once the cancer has been scored in each category, the information is combined to give a stage of one (I) through 4 (IV); the details for each stage and the 5 year survival rate is presented in Table 1 49.

In addition to identifying the type of breast cancer and staging tumor progression, tumors will be graded upon surgical resection. Grading focuses on the appearance of transformed epithelial cells in relationship to normal breast epithelium.

Epithelial cells normally have a polarized morphology with a distinct apical and basolateral surfaces. The basolateral surface of the epithelial cells makes strong and direct contacts with the basement membrane. The nucleus of each epithelial cell is small and uniform in size. The tubules that comprise the ducts and lobules are ordered and the epithelial cells line up in a uniform fashion. As a breast carcinoma progresses, the transformed cells are dividing rapidly and lose their differentiated phenotype of an epithelial cell 49, 53, 54. The mitotic count in affected areas, increase 55. The nuclear phenotype of the transformed tissue becomes irregular in size and shape. The epithelial cells no longer line-up in a regular fashion and the integrity of the basement membrane in lost. In general, cells and tissue lose hallmarks of differentiation. Tumor grade increases as differentiation decreases and leukocyte infiltration increases. In the

United States, the Scarff-Bloom-Richardson (SBR) system was originally used to correlate histological grading with prognosis in breast cancer. Several other histological grading systems developed from this test including the Bloom-Richardson

grade, Elston-Ellis grade and Black system which stressed nuclear morphology as well as the Nottingham grading system, first used in Europe and then in the U.S, utilized more stringent criteria and gave reproducible data. Tumors are given a grade of 1 to 3 with higher numbers indicating a loss of epithelial cell phenotype, which divide rapidly causing a loss of normal tissue morphology of a breast duct or lobule 49, 56. Tumor grading systems are also based on the expression of specific cellular markers. These markers include cellular receptors and gene expression signatures. The use of these classification systems has led to enhanced clinical care.

Table 1: Staging of Breast Cancer and Survival Rates

Table 1 indicates the tumors staging system used for breast carcinomas. Stages are give as Stage 0 through Stage IV. A higher number indicates a higher level of progression and is based on the tumor size, “T”, spread to nearby lymphnodes, “N” and metastasis to distant organs, “M”. The total TMN score is then assigned based on the combination of characteristic s of the tumor. The indicated survival rates are based on stage assignment and provided in column three.

Table 1: Staging of Breast Cancer and Survival Rates

Biomarkers lead to personalized patient care

Determining clinical therapies based on histological classification and grading systems worked in part to reduce breast cancer progression and reoccurrence.

However, some tumors with favorable histological characteristics reoccur and others presenting a poor histological appearance would not. Given the variable nature of the clinical characteristics of tumors, genetic analysis was applied. So, in combination with histological grading, biological markers are used to grade the progression and invasiveness of breast cancers 57.

Luminal A and Luminal B Subtypes

These tumor subtypes are positive for expression of the estrogen receptor.

Luminal A tumors grow slowly and have a gene expression pattern that closely resembles the normal epithelial cells that line the ducts and lobules. Luminal B tumors grow faster and are associated with a poorer prognosis. Because these subtypes are estrogen receptor positive, chemotherapies are combined with hormonal therapies to treat luminal cancers 58-60.

HER2 positive

Approximately 25 to 30 percent of breast tumors overexpress the human epidermal growth factor receptor 2 (HER2). This is a tyrosine kinase receptor belonging to the four membered family of epidermal growth factors (EGFR). The HER2 receptor does not directly bind any epidermal receptor ligands rather it is the preferred dimerization partner for the other epidermal growth factors activating intracellular signaling. Cellular signaling processes affect cell migration, adhesion and cell proliferation 61. Patients with HER2 overexpressing breast cancer have tumors that are

more aggressive and an increased rate of metastasis 62 . Drugs targeting HER2 are used in combination with chemotherapy. The monoclonal antibody, trastuzumab, is effective on tumors that overexpress HER2 as is pertuzumab, which inhibits the dimerization of HER2 with HER3 63-66. Additionally, NeuVax is a peptide based vaccine that directs killer T cells to destroy HER2 positive cells 56, 56.

Triple Negative Breast Cancer

The first biomarkers used were receptors expressed on normal epithelia, the estrogen and progesterone receptors, and HER2. Expression of these three receptors decreases as the tumor stage and invasive phenotype increases. Tumors negative for all three of these receptors are referred to as “triple negative tumors” and will not respond to hormonal or HER2-based therapies. A majority, approximately seventy five percent, of triple negative breast cancers are of basal cell origin. Basal cells are found in the deeper epithelia layers of the duct and lobules. Triple negative tumors have the same metastatic potential as other breast cancers but relapse more often and the reoccurrence is more likely at metastatic sites. Chemotherapy is used against triple negative breast cancer; however, hormonal therapy and anti-HER2 therapies such as trastzumab and lapatinib are not effective 38, 49.

The Oncotype DX test a step towards personalized care

The hormone receptors and HER2 provide biomarkers for different subtypes of breast cancer and therapies can be directed toward these specific targets. In addition to therapies based on tumors subtypes, the Oncotype DX test assesses early-onset breast tumors. Following surgical resection of tumor tissue, the Oncotype DX test

determines the chances of tumor reoccurrence. Gene expression analysis is done on five control genes and measured against the expression levels of 16 genes involved in proliferation, tumor invasion, immune cell infiltration, as well as estrogen receptor and

HER2 expression. Based on this information, each individual is assigned a score from

0 to 100. Low scores are treated with radiation therapy because the likelihood of reoccurrence is low, while a high score indicates an aggressive tumor phenotype and is treated with a combination of radiation and chemotherapy 4, 38, 67 .

An Overview of the Innate Immune System

The immune system is the body’s defense against foreign invaders. Composed of the physical barriers, cells, cellular processes, and biological molecules, the immune system prevents a wide array of agents from overrunning the body. The immune system is composed of two main parts: the innate immune system - which provides immediate broad protection, and the adaptive system - a pathogen-specific type of immunity, takes several days to activate.

As a surface barrier, the skin protects the inner tissues and organs of the body from microorganisms as well as exposure to harmful chemicals and physical insult.

The integumentary system helps to regulate body temperature and maintains internal homeostasis. When microorganisms penetrate the skin, the innate immune system is activated. Within four hours of an insult, innate immune cells such as monocytes, macrophages, dendritic cells and neutrophils are triggered and begin to migrate into affected tissue. Acting with three main functions - cytokine production, phagocytosis, and antigen presentation - innate immunity is non-specific in nature. Pattern

recognition receptors that bind generalized microbial motifs are one example of the broad but effective way the innate immune system functions. Binding of pattern recognition receptors allows macrophages and dendritic cells to clear pathogens through phagocytosis. Following phagocytosis and digestion by internal lysozymes, microbial motifs are presented in conjunction with MHC molecules to stimulate antibody production. Activated monocytes produce pro-inflammatory cytokines and chemoattractant proteins such as TNF-α, MCP-1/CCL2, and RANTES/CCL5 to further recruit and activate an immune response68, 69.

In contrast to the immediate response of innate immunity, adaptive immunity is tailored to clear specific pathogens. In fact, it “remembers” former pathogen insult and intensifies its response during a second exposure. Cellular adaptive immunity is comprised primarily by T cells. Killer, or cytotoxic, T cells recognize antigens that are presented in complex with MHC receptors by antigen presenting cells. Once activated, T cell releases cytotoxins such as perforin which punch holes in the surface of the invading microbes which induces apoptosis due to changes in their intracellular molarity. Helper T cells do not directly kill pathogens but instead release cytokines that active macrophages and cytotoxic T cells, enhancing their ability to kill microorganisms. The humoral portion of the adaptive immune system is composed of antibody-producing B cells. Antibodies bind to and label microbes in a process called opsinization, which marks them for phagocytosis by macrophages 68, 69.

The same immune functions that clear the body of invading pathogens also work to remodel and repair tissue. In this process, dead and dying cells are cleared by phagocytosis. The immune system can also recognize transformed cells that express surface antigens not found on healthy tissue. It was long thought that the immune

system solely fought tumors in the same way it battled foreign invaders. However, beginning with the work of Judah Folkman, our understanding of the complexity with which the immune system functions in relationship to tumors has changed. In fact, immune system function – specifically, tumor-associated macrophages - can be usurped by tumors and used to support tumor progression and metastasis, which is the focus of this work.

Overview of Monocytes and Macrophages

In response to inflammation, and as part of the innate immune response, monocytes are released from the bone marrow into the peripheral blood. Physically small, 10-15 µm in diameter, monocytes have a distinct morphology including a bean- shaped nucleus and cytoplasmic granules containing lysosomes and phagocytic vesicles. In circulation, monocytes have a lifespan of about 3 days and will either apoptose due to caspase cleavage or migrate into tissue and differentiate into macrophages. As monocytes move into inflamed tissue, differentiation occurs. Known for their phagocytic ability, the word macrophage comes from two Greek words meaning “big eaters”. As differentiation occurs during migration into tissues, macrophages are named for their location. For example, alveolar macrophages are found in the lung, kupffer cells in the kidney, microglia in the brain, and osteoclasts in the bone 69, 70.

Macrophages function in a variety of ways. Through the production of reactive oxygen species, nitric oxide and through phagocytic lysozymes, macrophages function directly to kill microbes. Through antigen presentation, macrophages work to activate

and assist in the adaptive immune response. Macrophages also act in tissue remodeling and repair through the production of angiogenic factors, growth factors, and proteases 68, 70. As the various functions of macrophages were characterized, macrophages were divided into pro-inflammatory and immune-modulatory phenotypes.

The differentiation of macrophages in response to microbes elicits a “classical” or M1 macrophage phenotype. Microbial exposure of lipopolysaccharide (LPS) induces interferon-γ, the Granulocyte Macrophage Colony-Stimulating Factor, and TNF-α which skew macrophages towards a pro-inflammatory phenotype. Classically-activated macrophages produce high amounts of inducible nitric oxide synthase, IL-12 and TNF-

α. In contrast, macrophages are pushed toward an “alternatively-activated” or M2 phenotype by M-CSF, IL-4 and IL-13. These immune modulatory cells produce high amounts of IL-10, VEGF and MMPs 71-74.

The Macrophage Colony-Stimulating Factor (M-CSF)

The colony stimulating factors (CSFs) were discovered by Pluznik and Metcalf in the mid-1960s. These scientists observed that hematopoietic cells would grow in agar when treated with conditioned media from feeder cells 75, 76. Building on these initial experiments, three colony stimulating factors were isolated and characterized.

The first discovered was CSF-1 or Macrophage Colony-Stimulating Factor (M-CSF).

M-CSF is a pleiotropic growth factor best known for promoting the survival, proliferation and differentiation of macrophages from monocytes and bone marrow precursor cells

77-80.

M-CSF is expressed from a single gene and alternatively-spliced into several different isoforms; a secreted, soluble, and membrane-bound form. Post-translational modifications of soluble M-CSF produce both a glycoprotein and proteoglycan form of this growth factor 81. M-CSF is produced by a variety of cells including endothelial cells, osteoblast, and by epithelial cells in the oviduct and uterus during pregnancy 82.

Upon binding to its cognate receptor, the M-CSF receptor (M-CSF-R), M-CSF causes receptor dimerization and phosphorylation of intracellular tyrosine kinases 83-85 .

Phosphorylation of the M-CSF-R results in activation of intracellular signal transduction including signaling through PI3 Kinase/Akt, Ras/Mapk, Stat1 and 3, Jak1,

Phospholipase A2 and 1C 86-91. Stimulation of intracellular signals affects numerous cellular events that enable an innate immune response, tissue remodeling and angiogenesis 92-94. Because of its actions in macrophage maturation, M-CSF is important in resistance to numerous pathogens, such as listeria infection 82.

Deregulation of M-CSF has been associated with a number of diseases including the development of arthritis and the formation of atherosclerotic plaques. M-CSF also plays a role in the pathology of Lupus and glomerulonephritis as well as kidney allograft rejection. Reduced levels of M-CSF are known to contribute to leukemia.

However, the most widely examined relationship between M-CSF and disease is in the progression and metastasis of solid tumors. In fact, M-CSF can be expressed by some tumor cells (Kacinski 1990/Scholl 1994) and high serum concentrations of M-CSF correlate with poor prognosis in many tumor types including ovarian, endometrial, and breast cancers 95-98.

Tumor-Associated Macrophages

The correlation between increased M-CSF serum levels in cancer patients led to studies examining the role of macrophages in tumor progression. A meta-analysis by Claire Lewis’s group examined published work correlating patient outcomes with macrophage numbers in different tumor types. This analysis revealed that macrophage numbers correspond to prognosis in approximately 80% of solid tumors 95.

The strongest correlation between increased macrophage numbers and poor patient prognosis was found in human breast cancer where all four studies revealed a direct connection. In 1993, Jeffery Pollard’s group conducted a seminal study in which macrophages were omitted through transgenic knockout of M-CSF 98, 99. This report was the first to show that macrophages were a causative factor in breast cancer progression. Since then, tumor- tumor-associated macrophages or TAMs have been shown to migrate into tumors and localize in the stroma, near blood vessels and areas of low oxygen tension (hypoxia).97, 100, 101. TAMs takeover the normal reparative function of macrophages and support tumor progression.

Virchow first hypothesized that inflammation plays a role in cancer development. He observed that irritants which caused inflammation could also lead to tumor formation. Chronic inflammation is known to be directly associated with certain types of cancers including inflammatory bowel disease and colon cancer as well as hepatitis C which contributes to liver cancer102-105. Macrophages can participate in tumor initiation through the production of reactive oxygen species and nitric oxide.

These factors, in addition to eliminating microbes, increase the likelihood of DNA mutations that transform cells. Additionally, ROS contribute to the survival of transformed cells by oxidizing and inhibiting oncosuppressor proteins that increase the

survival of newly transformed cells 82, 106, 107 . Macrophages facilitate tumor progression and metastasis in a number of ways. TAMs produce VEGF which activate endothelial cells and initiate angiogenesis by supplying the growing tumor with oxygen and nutrients and providing an avenue for waste removal. Proteases produced by macrophages, such as MMP-9 and cathepsins, dissolve the ECM allowing tumor cells to migrate through the tissue and intravasate the blood and lymphatics 108. Dissolving the ECM frees bound growth factors such as VEGF and M-CSF which further enhances tumor progression through action on endothelial cells and macrophages 102.

TAMs also participate in a paracrine feedback loop with tumor cells which increases intravasation and seeding at distance site. Here, macrophages produce epidermal growth factor (EGF) which stimulates tumor cell motility. At the same time, tumor cells produce M-CSF which increases macrophage survival and differentiation towards an

M2 phenotype 109, 110. Alternatively activated macrophages produce more proteases and angiogenic factors leading to further tumor progression. This paracrine effect facilitates a tumor cells exit through the vasculature. In fact, live imaging within PyMT mouse breast tumors using multi-photonmuti photon imaging demonstrated that tumor cells and macrophages actually alternate exiting the tumor by entering blood vessels

111. Further, TAMs trigger the epithelial-to-mesenchymal transition (EMT). As detailed in the section on tumor progression, EMT augments the exit of cells from the primary tumor, cell survival in circulation, and adherence and proliferation at secondary organs.

TAMs secrete TGF-β, FGF-2 and PDGF, as well as produce MMPs which facilitate

EMT.transition 108, 112-114.

Proangiogenic Tie2-Expressing Macrophages

Overview of the Tie2 Receptor

The Tie2 receptor is a tyrosine kinase receptor with IgG- and EGF-like domains

(Tie or TEK). First discovered on endothelial cells, there are two members of the TEK family, Tie1 and Tie2. Tie2 was originally described in 1992 and contain structural similarities to other growth factor receptors. Its roles in vascular formation and remodeling were first described when a Tie2 receptor knockout mouse was developed.

In this mouse, large vessels such as the aorta developed normally; however, formation of vessel networks did not occur. Tissue analysis revealed few endothelial cells lining blood vessels. Further, the endothelial cells had lost intracellular adhesion and contact with supporting pericytes. In normal tissues, the Tie2 receptor is highly-expressed in areas of remodeling and angiogenesis.

Discovery of the ligands for the Tie2 receptor, the angiopoietins (Ang-1 and -

2), led to further characterization of roles of this receptor in both blood vessel maintenance and reorganization 115-117. When Ang-1 is bound to the Tie2 receptor, endothelial connections to the basal lamina, to supporting cells, and to other endothelial cells are strong 118, 119. The endothelial cell remains quiescent and vessels are largely impermeable. However, when Ang-2 binds the Tie2 receptor, endothelial cells disengage from each other and from surrounding support structures. Vessel remodeling or regression follows depending on availability of VEGF and MMPs 120, 121

Tie2-expressing monocytes/macrophages (TEMs)

TEMs were identified in Tie2 receptor-GFP mice where green fluorescent protein is expressed from the Tie2 promoter 122, 123. In these mice, it was noted that in 23

addition to endothelial cells, a number of myeloid lineage cells expressed the Tie2 receptor. Since these initial studies, Tie2-expressing monocytes/macrophages have been identified in mouse and human peripheral blood, normal tissue, and tumors. In vitro work demonstrated that TEMs migrate toward Ang-2 in a dose dependent manner and activate endothelial cells 124. Conditioned media from TEMs increases HUVEC branching and tube formation significantly more than Tie2 receptor-negative monocytes 125. TEMs resemble an M2 phenotype and are thought to function in paracrine manner. Co-injection of TEMs with glioma cells enhances angiogenesis while TEM depletion impairs it 124, 126. Localizing to blood vessels in tumors, TEMs do not incorporate into the endothelial layer as was once hypothesized 127. Instead, TEMs produce high levels of proangiogenic cytokines such as VEGF and TNF-α. TEMs also produce proteases including MMP-9, CTSB, and TP as well as anti-inflammatory mediators like IL-10. The immune regulatory properties of consist primarily of its ability to decrease T cell proliferation and increase the regulatory T cell phenotype 129.

Interestingly, TEMs produce lower amounts of tumor-activating EGF and a consequence of this in tumor progression has yet to be examined 125. The work presented in this dissertation illustrates that expression of the Tie2 receptor on TEMs is dynamic. Expression can be modulated by both HIF-1α and M-CSF. These factors function indepentantly and synergistically to up-regulate Tie2 expression on macrophages. Further, we show that M-CSF pre-treatment of monocytes increase the

TEM population and augments the proangiogenic properties of the cell.

Table 2: Characteristics of TEMs

Tie2-Expressing Monocytes/Macrophages are a tumor infiltrating myeloid cell. First identified in Tie2 GFP-expressing mice the myeloid cells have been shown to facilitate tumor progression. TEMs are known to express F4/80+, CD11b+, MRC-1+ and Tie2+.

Interestingly TEMs lack expression of CD31- and GR-1low and do not incorporate into the endothelial cell layer. Genetic profiling of TEMs has revealed that these cells express high amounts of SDF-1/CXCL12 and IGF-1.

Table 2: Characteristics of TEMs

Tumor Metastasis

Metastasis is the process through which transformed cells leave the primary tumor and spread to other locations in the body. Mortality is associated with metastasis

90 percent of the time 130. In fact, most therapies that treat primary tumors succeed; the reoccurrence of early-stage in situ breast cancer is relatively small. However, this number drops significantly with the spread of transformed cells to surrounding tissue and to secondary organs. As with primary tumor progression, metastasis occurs in stages. The “Seed and Soil” hypothesis describes the stages of metastasis and was proposed by James Ewing in 1928 131. The following discussion presents an overview of the metastatic process highlighting some important facilitators of these events but is by no means and exhaustive discussion of the molecular events leading to the formation of secondary tumors. During metastatic spread, the tumor cells first penetrate the surrounding tissue and then exit the tumor moving into the blood or lymphatic vessels (intravasation)132, 133. The tumor cells are then transported through the vasculature and move into a new tissue (extravasation) 133-135. The tumor cells must now survive and proliferate to form a secondary tumor 130. During invasion into surrounding tissue, the extracellular matrix (ECM) is broken down by proteases such as matrix metalloproteinases (MMPs) and the serine protease urokinase (uPA) which degrades the structural proteins, glycosaminoglycans and proteoglycans, in the ECM

134, 135. Breakdown of the structural proteins increase the interstitial space facilitating cell migration. Additionally, peptides formed by the breakdown of collagen and elastin can stimulate tumor cell movement and this, along with local chemokines, draw tumor cells towards vessel exit sites. Dissolution of the ECM intensifies intravasation as fibrin peptides from the degradation of fibronectin increase vascular permeability 136, 137.

In order to invade surrounding tissue and leave through the vessel, tumor cells must detach from the primary tumor. The process of epithelial-to-mesenchymal transition (EMT) facilities this through changes in the differentiation state of transformed epithelial cells. EMT was first described during embryonic development in chickens138. Later, it was discovered that EMT facilitated trophectoderm development in mammalian embryos 139. In cancer, EMT is reversible and thought to be essential in metastasis of breast cancer. In a primary tumor, EMT enables migration and intravasation while the reverse mesenchymal-to-epithelial transition (MET) allows for attachment and growth of a secondary tumor in ancillary organs 138, 140. Healthy epithelial cells are polarized having an apical and basal surface - the latter of which is affixed to the basement membrane. Epithelial cells adhere to the ECM and to other cells via tight junctions, cadherins, and other intercellular connections that maintain immobility 141, 142. Mesenchymal cells, on the other hand, lack cellular polarization and are defined by their ability to migrate. Migration is facilitated by the lack the cellular adhesions and cytoskeletal elements can rearrange easily fostering movement 138, 141,

142. In addition to acquiring invasive characteristics, mesenchymal cells arrest in G1 of the cell cycle making them resistant to apoptosis 143. Cell cycle arrest and inhibition of p53 activity by mesenchymal Twist and Snail proteins makes it more likely that these cells will survive travel through the vasculature and extravasation into a new organ 144-

146. Soluble factors that induce EMT include TGF-β, FGF-2 which are produced by macrophages, as well as MMPs, αvβ3-integrins, and PDGF which are produced by a subset of macrophages that express the Tie2 receptor, TEMs - the focus of this work.

The Hypoxia Inducible Factors

Hypoxia is defined as low oxygen tension in tissue and cells. It occurs when the demand for oxygen is greater than the supply. During periods of low oxygen tension, the Hypoxia Inducible Transcription Factors (HIFs) are stabilized and function to bring the cell and the tissue environment back to homeostasis. The HIF system was first identified as a regulator of transcription for the erythropoietin (EPO) gene. EPO, produced by renal fibroblasts in the kidney, drives differentiation of red blood cells from bone marrow progenitors. Production of EPO increases the oxygen carrying potential of red blood cells 147.

At the molecular level, the HIFs function as heterodimers with α- and β- subunits. The α-subunit of the heterodimer contains one of HIF-1α, HIF-2α (EPAS-1) or

HIF-3α (IPAs or inhibitor of PAS) proteins. At normoxia, the α-subunits are quickly marked for degradation by the E3 ubiquitin ligase through proline hydroxylation at the

Pro403 and/or Pro564 amino acid residues. The exterior pocket of the von Hippel-

Lindua (VHL) protein recognizes the hydroxyproline and expedites polyubiquitination and degradation by the proteasome. However, low oxygen tension inhibits hydroxylation of the HIF-α subunits, increasing their half-life and enabling them to be translocated to the nucleus 148-150. Once inside the nucleus, the HIF-α subunit binds with a HIF-β subunit to form a heterodimer. Binding of the heterodimer to a hypoxia response element (HRE) activates transcription inducing changes in cellular metabolism, blood vessel dilation, angiogenesis and hematopoiesis. HRE core sequence contain a 5’-[A/G]CGTG-3’ and reside within the promoters of HIF responsive genes allowing for binding and transactivation by these HIF transcription factors151.

Hypoxia is a hallmark of solid tumor development where rapidly dividing tumor cells create a need for oxygen. Both clinically and in animal models of solid tumors, the presence of hypoxic and necrotic areas are characteristics of aggressive tumors.

During hypoxia, global RNA transcription is blunted as the cell attempts to limit energy use. However, HIF-related transcription intensifies leading to expression of genes regulating angiogenesis and metabolism 152. HIF-1α and HIF-2α contain 48% amino acid homology and have similar tertiary structure. In terms of restoring oxygen, the

HIFs have both overlapping and unique roles. HIF-1α was the first described hypoxia transcription factor and is known as the “master regulator” of cellular response to oxygen stress. Active during short periods of hypoxia (2-24 hours), HIF-1α drives the initial cellular repsonse to hypoxia and promotes cellular survival at very low oxygen concentrations (< 0.1%) 153-155. HIF-2α was initially identified as an endothelial specific protein and is also known as the endothelial PAS domain protein (EPAS-1) 156. Since its discovery in endothelial cells, HIF-2α has been shown to be transcriptionally-active in a variety of tissue including lung, brain, liver, heart, intestine and pancreas. More recently, HIF-2α has been shown to activate gene transcription during continuous

(greater than 48 hours) and mild hypoxia (less than 5% oxygen) 148, 156, 157.

Understanding these roles, we examined which HIF might be responsible for regulating the Tie2 receptor on TEMs under low oxygen tension. We found that HIF-1α knockouts in myeloid lineage cells diminished the effect of hypoxia on the Tie2 receptor.

Angiogenesis in Tumors

As transformed cells rapidly divide, small tumors begin to form. However, the diffusion constant of oxygen allows the malignant mass to grow to a maximal size of

approximately 2 mm3. These small tumors pose little risk to the individual and are thought to comprise a majority of tumors. However, proliferating tumor cells stimulate remodeling and growth of new blood vessels from pre-existing vessels, or angiogenesis, to supply the oxygen and nutrients required to survive. In 1971, Judah

Folkman demonstrated that tumor cells secreted a tumor angiogenic factor (TAF) that stimulated new vessels to sprout from nearby systemic blood vessels. Folkman postulated that without production of TAF the tumor would remain a few millimeters in size, avascular and dormant 158. However, production of TAF allowed new blood vessels to form and provided oxygen and nutrients to the tumor, thereby stimulating further angiogenesis, tumor growth, progression and metastasis. The vascular endothelial growth factor (VEGF) is produced by hypoxic cells, diffuse to nearby vascular endothelial cells binding to VEGF receptors and initiates angiogenesis.

Through binding to VEGF-R1, VEGF induces endothelial cells to exit their quiescent state and re-enter the cell cycle 159, 160. As endothelial cells are proliferating, the hypoxic tumor also produces SDF-1/CXCL12, a chemoattractant for monocytes/macrophages 113, 161, 162. Macrophages produce proteases which degrade the basal lamina covering endothelial cells. This allows pericytes to retract back from blood vessels permitting the proliferating endothelial cells to migrate. Migration occurs toward increasing concentrations of VEGF - in other words, towards the hypoxic tumor.

The proliferating and migrating endothelial cells first form a solid vessel sprout, then the lumen of the vessel is hollowed out. Finally, connections are made between a network of vessel sprouts and blood flows into the area. With a supply of oxygen and nutrients, the proliferating tumor grows and differentiates163-165.

Normal tissue blood vessels are characterized by strong connections between the endothelial cells, the pericytes, and the surrounding ECM. Strong cellular adhesions make the vessels impermeable and provide a regular shape and even distribution of blood though the tissue. Cellular connections, not VEGF, provide the survival signals and keep endothelial cells in a quiescent state. In contrast to this, tumor blood vessels have weak and leaky intracellular adhesions. Poor connections give tumor vessels a disorganized, twisted and irregular shape. Endothelial cells in tumor blood vessels rely on VEGF for survival and expression of various integrins stimulates further angiogenesis 166-168

Chapter 2: HIF-1α, but not HIF-2α, Regulates the Tie2 Receptor on Tie2-

Expressing Monocytes in PyMT Breast Tumors and Augments

Angiogenic Function and Metastases

“Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time”

Thomas Edison

Abstract

Tie2-expressing monocytes (TEMs) are a distinct subset of pro-angiogenic monocytes selectively recruited to tumors in both mouse models and breast cancer patients. Because of the hypoxic nature of tumors, we investigated if oxygen regulated the trafficking of these cells into tumors or if this subset was transdifferentiated once inside the tumor microenvironment. Real-time PCR and flow cytometry confirmed the up-regulation of Tie2 receptor mRNA and protein on CD14+ monocytes in response to low oxygen. To delineate which hypoxia inducible factor-α (HIF-α) subunit was responsible, we derived macrophages from bone marrow of wild type LysMcre, HIF-

1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and found the population of F4/80+/Tie2+ 33

TEMs from wild type and HIF-2α-deficient macrophages increased when exposed to hypoxia with no such increase in HIF-1α-deficient cells. To understand if HIF-1α regulates F4/80+/Tie2+ chemotaxis or the tumor environment drives the differentiation of F4/80+/Tie2- cells to Tie2-positivity, we implanted PyMT breast tumor cells into wild type, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and evaluated TEM infiltration.

There was no difference in the percentage of TEMs in bone marrow (CD45+/Tie2+) or peripheral blood (CD11b+/CD31-/Gr-1lo/Tie2+) compartments between the mouse groups. But, the HIF-1αfl/fl/LysMcre mice had a significantly smaller percentage of

TEMs in tumors compared to wild type or HIF-2αfl/fl/LysMcre mice resulting in reduced angiogenesis, even though the percentage of total F480+ macrophages was unchanged. Finally, the percent of TEMs in the tumors significantly correlated with tumor cell intravasation and lung metastases while the percent of total tumor F4/80+ macrophages did not.

Introduction

Hypoxia occurs when the physiological demand for oxygen outweighs supply.

Observed in solid tumors, rapidly dividing cells create a need for oxygen. Hypoxia is a hallmark of tumor progression in both human tumors and animal models 153, 169, 170. In this setting, changes in cellular metabolism, vascular tone, angiogenesis and hematopoiesis occur as the system attempts to recapture oxygen homeostasis.

Globally, hypoxia induces an overall reduction in RNA transcription in an attempt to maintain cellular processes required for cell survival 152. A family of transcription factors, the Hypoxia Inducible Factors (HIFs), increases transcription of survival genes in an attempt to regain oxygen delivery to dying cells. Regulated by the presence of oxygen, the prolyl hydroxylases (PHD-1, -2, and -3) tag the HIF-α subunits for

ubiquitination by E3 ubiquitin ligase and proteasome degradation 148-150. The absence of oxygen inhibits the ability of the PHDs to hydroxylate the HIF-α’s. Their stabilization leads to heterodimerization with the HIF-β subunits and subsequent translocation to the nucleus. Once in the nucleus, binding of the heterodimer to gene promoters containing a hypoxia response element (HRE) activates transcription. The result is changes in glycolysis, glucose transport, erythropoiesis, blood vessel dilation, reorganization and angiogenesis 151.

HIF-1α regulates changes in glycolysis, up-regulating glucose absorption through Glut-1 and glucose metabolism through genes such as, phosphofructokinase

153, 170. As the master regulator of oxygen homeostasis, HIF-1α facilitates angiogenesis by up-regulating the potent angiogenic protein, vascular endothelial growth factor

(VEGF) 171. Studies in mouse models of breast cancer illustrate that deletion of HIF-1α from tumor cells inhibits production of SDF-1/CXCL12, a major chemoattractant for lymphocytes and endothelial progenitor cells (EPCs). While HIF-1α drives expression of survival genes, the predominant role of its HRE-binding family member, HIF-2α, may be to regulate more global homeostatic mechanisms. For example, Johnson et al suggests that HIF-2α governs macrophage polarization towards M2 157. In recent studies, we showed an alternate profile of HIF-2α in driving the expression of the antiangiogenic and soluble form of VEGF receptor-1 173-176, 176. Further, we have shown disparate roles of HIF-1α and HIF-2α in tumor angiogenesis. HIF-1α drives VEGF production and new blood vessel infiltration, while HIF-2α regulates sVEGFR-1 expression hindering VEGF bioactivity.

Classic CD14+ blood monocytes arise from bone marrow precursors and their differentiation is regulated by macrophage colony-stimulating factor (M-CSF).

Monocytes are recruited by chemokines, such as CCL2, released at sites of 35

inflammation where tissue macrophages are required 36, 161, 177-179. Through integrin expression, monocytes roll and stick at inflammatory sites then diapedes the endothelial barrier. Chemokines gradients, produced by tumor and stromal cells stimulate this migration. Extravasation into a tissue environment is accompanied by and coordinated with differentiation of monocytes into macrophages 94. Once inside the tumor proper, EGF-like module-containing mucin-like hormone receptor-like 1 positive

(EMR-1, human homologue to F4/80 in mice) tumor-associated macrophages (TAMs) accumulate at regions separating hypoxic patches and normal tissue 180, 181. These

TAMs have been shown to promote angiogenesis and drive for tumor progression and metastasis 178, 182-184. In human breast, ovarian, cervical, prostate, and bladder cancers, increased numbers of macrophages correlate with tumor progression and poor prognosis 178.

Recently, a subpopulation of mononuclear phagocytes that express the endothelial cell tyrosine kinase receptor, Tie2, has been identified 124, 185. Tie2- expressing monocytes/macrophages (TEMs) have been shown to facilitate tumor angiogenesis by both expressing angiogenic factors that regulate blood vessel development, like VEGF, and physically associating with new vessel sprouts 125, 186.

Hypoxia has been shown to increase Tie2 receptor expression on human monocytes in vitro 185. Because we have observed disparate roles for the hypoxia inducible transcription factors in regulating tumor angiogenesis, we sought to determine the role of HIF-α in regulating TEM differentiation and function in a PyMT mouse model of breast cancer. We hypothesize that HIF-1α and HIF-2α will regulate

Tie2 receptor expression differently on incoming, less mature macrophage populations and that the hypoxic tumor microenvironment will expand a population of macrophages expressing the Tie2 receptor regulated by HIFs facilitating tumor angiogenesis and 36

metastasis. Importantly, this work utilizes the definition of TEMs put forth by the seminal works of the Naldini and Lewis. Both of these groups have characterized the

Tie2-expressing monocyte/macrophage subset in bone marrow, peripheral blood and breast tumors (Table 3).

In the current study, transgenic mice deficient in macrophage HIF-1α or HIF-2α were used to determine the role of the HIF pathway in breast tumor TEM recruitment.

Our results show that HIF-1α regulates TEM differentiation in the hypoxic tumor microenvironment. In our studies the accumulation of TEMs within the tumors are from monocytes recruitment from the bone marrow or peripheral blood. The loss of HIF-1α resulted in a decreased in the population of total tumor F4/80+ cells gaining Tie2- positivity, leading to reduced tumor growth rates and less angiogenesis.

Table 3: Characterization of Tie2-Expressing Monocytes/Macrophages. As seen in table 3 surface marker expression profiling has been accomplished to identify the markers on the monocyte/macrophage subset, TEMs. This marker profile for mice includes Tie2+, CD45+, CD11b+, F4/80+, MRC-1+ with CD31- and GR-1low. The human marker profile for TEMs is Tie2+, CD45+, CD14+, CD16+, and CD11b+ with CD31-.

Mouse References Bone marrow: CD45+/Tie2+ [69]; [67] Peripheral blood: CD11b+/CD31-/GR-1lo/Tie2+ [67]; [430]; [338] Breast tumors: CD11b+/CD31-/Gr-1lo/Tie2+ [67]; [430]; [62] F4/80+/Tie2+ [338]; [48]; [47]; [1] F4/80+/MRC-1+/Tie2+ [261]

Human Bone marrow: Not yet characterized Peripheral blood: CD14+/Tie2+ [278] CD14+/Tie2++ [48]; [47] CD14+/CD16+/Tie2+ [430] Breast tumors: CD14+/CD45+/CD16+/CD11b+/CD31-/Tie2+ [430]

Table 3. Lineage of Tie2-expressing monocytes/macrophages (TEMs):

Materials and Methods Monocyte Isolation

Human monocytes were isolated from the peripheral blood of healthy volunteers following Institution Review Board protocol 2011H007IRB. Peripheral blood mononuclear cells were isolated by a Ficoll gradient and CD14+ monocytes were collected by positive bead separation as per manufacturer’s protocol (Miltenyi Biotech).

Isolated CD14+ monocytes (2x106) were incubated in RPMI media (BioWhittaker) with

1% FBS (certified as containing <0.06 EU/ml endotoxin levels from HyClone, Logan,

UT), penicillin (100 U/ml), streptomycin (100 U/ml), amphotericin B (0.25 µg/ml) (Gibco) and 10 µg/ml polymyxin B (Calbiochem).

Flow Cytometry

CD14+ human monocytes were incubated at 0.5% (hypoxia) or 21% O2 (normoxia) for

6, 18, 48 or 72 hours. The cells were then blocked with anti-FcR antibody (R&D

System) for 15 minutes at 4ºC to prevent non-specific binding. Cells were incubated with 10 µl PE-conjugated anti-human Tie2 (clone 83715, R&D Systems) per 1x106 cells at 4ºC for 30 minutes per manufactures instructions or 10 µl of the isotype control PE- conjugated anti-human IgG (R&D Systems). Tie2-expressing monocytes were subsequently analyzed using the Aria III flow cytometer at The Ohio State University

Flow Cytometry Core Facility.

Cobalt Chloride Treatment

Human CD14+ monocytes were isolated as described above and cultured for 24 hours at 21% oxygen with or without 100 µM cobalt chloride (Sigma). Cells were immunostained as described above and subjected to flow cytometry analysis for Tie2 receptor expression.

Isolation and Differentiation of Bone Marrow-Derived Macrophages

Bone marrow from 6-8 week wild type LysMcre (control), HIF-1αfl/fl/LysMcre, and HIF-

2αfl/fl/LysMcre mice was obtained by flushing the femurs into a 50 ml conical tube containing RPMI media on ice. Bone marrow cells were washed twice with sterile PBS by centrifugation at 1500 rpm for 15 minutes. The cells were resuspended and added to 6-well culture plates at 5x106 per well in 1 ml of low endotoxin RPMI media containing 10% FBS and 5 µg/ml polymyxin B. Macrophages were differentiated with

20 ng/ml recombinant murine (rm)M-CSF (R&D Systems) and the media was refreshed every other day. Macrophage differentiation was verified using F4/80 antibody (clone

Cl:A3-1, AbDSerotech) labeling and flow cytometry analysis. To examine the effect of hypoxia on Tie2 expression following seven days of differentiation, macrophages were incubated at 0.5% O2 or 21% O2 for 0, 1, 3, or 7 days. At each time point, macrophages were harvested and antibody labeled using anti-Tie2 PE (clone TEK4,

BioLegend) and F4/80 APC (clone Cl:A3-1). Flow cytometric analysis was analyzed using the Aria III flow cytometer at The Ohio State University Flow Cytometry Core

Facility.

Luciferase Promoter Activity Assay

A Tie2 luciferase reporter construct (1 μg) encompassing the 2.1kb promoter and the

1.7kb 5’ intronic enhancer regions, or the transfection control Renilla TK (1 μg), was transfected into mouse bone marrow-derived macrophages (BMDMs) using the Amaxa

Nucleofector (Lonza) as previously reported. Briefly, BMDMs (5x106) were trypsinized and washed twice with sterile PBS using centrifugation at 1500 rpm. BMDMs were resuspended in Mouse Macrophage Transfection Solution containing 1 µg of the Tie2

Luciferase Reporter construct and the p-RL-TK-luc construct (Promega). Cells were transfected using the Amaxa Y-001 program, washed in RPMI media and plated into 6- well plates. BMDMs were incubated at 21% O2 and 5% CO2 and three independent replicates for each condition were harvested at 24 hours post-transfection with the addition of passive lysis buffer. Reporter activity and transfection efficiency was measured using the Dual-Luciferase Reporter System (Promega). 20 µl cell lysates were added to a 96-well plate. Luciferase assay substrate, LAR II, at 100 µl per well was added and measures were recorded for 2 seconds per sample on the Victor 3 illuminometer (Perkin Elmer). For the control measurements, STOP and GLO (100 µl) was added to each sample and Renilla luciferase readings measured for 2 seconds.

PyMT Tumor Model

PyMT tumor cells from C57Bl/6 mice were cultured in Dulbecco’s Modified Eagles

Medium (DMEM) supplemented with 1% PSA, 10% FBS, 10 µg/ml human insulin, and

5 µg/ml rmEGF. Tumor cells (1x106 cells in 100 µl) were injected orthotopically into the number four mammary fat pad of C57Bl/6 LysMCre, HIF-1α(fl/fl)LysMcre, or HIF-

2α(fl/fl)LysMcre mice. Tumor growth was measured 3 times per week using calipers and

tumor volume was calculated using the equation: Tumor volume = 0.5 x [(large diameter) x (small diameter)2].

Tumor Cell Isolation

Age-matched female C57Bl/6 mice previously injected orthotopically in the mammary fat pad with PyMT tumor cells were sacrificed by asphyxiation followed by cervical dislocation. Peripheral blood was removed by cardiac puncture. Bone marrow was isolated from the femurs as described, and the tumors were harvested. Tumors were cut into small sections using a razor blade and then digested to homogenization in 1 ml of PBS containing collagenase (0.55 mg/ml) and DNase (0.1 mg/ml). The tumors were incubated at 37ºC and vortexed every 5 minutes for 30 minutes. Each sample was passed over a 100 μm mesh cell strainer (Fisher Scientific) and washed twice with 10 mls PBS using centrifugation at 1500 rpm for 10 minutes. The lymphocytic layer from peripheral blood and tumor homogenate was separated by a Ficoll gradient. The lymphocytic layer was removed and washed twice with PBS. The red blood cells were lysed using Red Cell Lysis Buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA; pH=7.4) for 5 minutes at room temperature. Bone marrow cells were centrifuged, resuspended in PBS and red blood cells lysed. Remaining cells were washed twice with PBS and resuspended in MACS buffer (Miltenyi Biotech). For flow cytometric analysis and cell sorting, cells isolated from tumors, peripheral blood and bone marrow were blocked with whole IgG for 15 minutes at 4ºC. Samples were immunostained with antibodies specific for Tie2 (PE-conjugate clone TEK4, eBiosciences), CD11b (Percy

5.5 and PE-conjugate clone M1/70), CD31 (PE-Cy7 and APC-conjugate clone 390),

Gr-1 (Percy 5.5 and PE-conjugate clone RB6-8C5) (R&D Systems) and F4/80 (PE-Cy7 or APC-conjugate clone Cl:A3-1, Serotec). Isotype control samples were 43

immunostained with rat anti-mouse IgG2a and IgG2b with the corresponding fluorescent conjugates (R&D Systems). TEMs in this study are described as:

CD45+/Tie2+ (bone marrow), CD11b+/CD31-/Gr-1low/Tie2+ (peripheral blood and tumor for comparing different compartments), and F4/80+/Tie2+ (for comparative studies in tumor tissue by flow cytometry and immunohistochemistry). All flow cytometry was measured using the Aria III flow cytometer at The Ohio State University

Flow Cytometry Core Facility.

Confocal Microscopy

Upon harvesting tumors as described above, each sample was dissected into 15 mm sections and fixed in 4% paraformaldehyde for 48 hours. Samples were then washed in PBS for 24 hours and cryoprotected by equilibrating in 20% sucrose in PBS until sections floated on top, approximately 48 hours. Tumor sections were flash frozen in

Tissue-TEK OCT compound (VRW International) with liquid nitrogen and stored at -

80ºC. Tissue sections were cut using a cryostat and mounted onto SuperFrost Plus slides from (Fisher Scientific). Samples were then post-fixed for 30 minutes with 4% paraformaldehyde and washed 3 times with PBS. Sections were permeabilized with

0.3% Triton in PBS for 30 minutes and blocked with PBS containing 5% normal goat serum and BSA fraction V (1 mg/ml). Primary antibody incubations were done overnight at 4ºC and samples washed 7 times for 5 minutes each with PBS containing

BSA. Samples were then incubated with secondary antibodies for 1 hour at room temperature and then again washed 7 times for 5 minutes each with PBS containing

BSA. In the last wash, DAPI (100 ng/ml) was added as a nuclear counterstain. Three drops of Immuno Mount (Electron Microscopy Sciences) was added to each sample prior to cover slip. Samples were visualized using the Olympus Flow View FV 1000 44

laser scanning confocal microscopy at The Ohio State University Microscopy Core

Facility.

Intravasation Assay

After four weeks post-PyMT tumor cell implantation, whole blood was collected by cardiac puncture from LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre PyMT-tumor bearing mice. The blood was cultured in DMEM media containing 10% FBS, 5 µg/ml

EGF, 10 µg/ml insulin, and 1% PSA for 24 hours. After, any non-adherent cells were removed by washing and adherent cells were allowed to grow for two more weeks and the numbers of colonies formed were blindly counted as described.

Tumor Angiogenesis and TEM determination

Tumors from PyMT tumor-bearing wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-

2αfl/fl/LysMcre mice were collected at time of sacrifice as described above. Tumor sections were immunostained with CD31 antibody and imaged by fluorescent microscopy using a ×20 objective lens. Five random images per tumor per group were captured in a blinded manner, analyzed for CD31-positivity (red pixels) and quantified using Adobe Photoshop CS2 (Adobe Systems) histogram analysis

Real-time PCR

Lungs from PyMT tumor-bearing LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice were collected at time of sacrifice and snap frozen in liquid nitrogen. The lungs were pulverized in liquid nitrogen and dissolved in Trizol reagent (Invitrogen) followed by further homogenation using a syringe. RNA was extracted in chloroform and then purified using the RNeasy Minikit (Qiagen). cDNA was generated from 1 µg of RNA 45

using the Superscript First Strand Synthesis System (Invitrogen) and was used for real- time PCR with the primers: PyMTf: 5’-GGAAGCAAGTACTTCACAAGGG-3’ and

PyMTr: 5’-GGAAAGTCACTAGGAGCAGGG-3’ and SYBR Green PCR Master Mix

(Applied Biosciences) according to the manufacturer’s instructions. Data were analyzed according to the comparative threshold method and normalized against the

GAPDH internal control transcript.

Statistical Analyses

In the experiments of human donor cells across different conditions (Human

Monocytes Normoxia vs Hypoxia; Tie2 Normoxia Hypoxia Real-time PCR; Cobalt

Chloride Treatment; and F480 Macs Normoxia vs Hypoxia), we used mixed effect models considering observational dependencies to analyze the data. In other experiments (Luciferase Data, Percent TEMs in Tumors, Bone Marrow and Blood, and

PyMT Intravasation, Tumor Angiogenesis, PyMT Lung Metastases), one way ANOVA was used. P-values were adjusted using the Holm’s procedure to conserve the type I error at 0.05 due to multiple comparisons. For tumor growth data, changes in tumor volume over time were assessed via a longitudinal model. Statistical outliers were determined by Grubb’s Outlier Test. For all analyses, p ≤ 0.05 was considered statistically significant.

Results

Hypoxia up-regulates Tie2 receptor expression on human CD14+ monocytes

In endothelial cells, when the Tie2 receptor (TEK receptor tyrosine kinase) binds its ligand, Angiopoetin-2, endothelial cell connections are loosened allowing for 46

endothelial cell and blood vessel reorganization 187, 188. Because it has been reported that hypoxia induces HIF transcription factors to increase expression of genes required for blood vessel reorganization in endothelium 189, we assayed the ability of hypoxia to up-regulate Tie2 on a population of freshly-isolated CD14+ human monocytes. To elucidate this effect, monocytes were isolated from whole blood using CD14+ microbeads and incubated in non-adherent culture tubes in RPMI media containing fetal bovine serum and placed at normoxia (21% O2) or hypoxia (0.5% O2) for 24 hours.

After, monocytes were immunostained with an antibody specific for the human Tie2 receptor or isotype antibody then analyzed by flow cytometry (Figure 1A). The data is represented as CD14+/Tie2+ cells as a percent of total CD14+ cells. Our data suggests that CD14+ cells incubated at normoxia express the Tie2 receptor similar to freshly-isolated CD14+ cells (fresh: 9.1±0.9%, normoxia: 6.8±0.8%; p=NS and N=12 per group). In contrast, CD14+ monocytes exposed to hypoxia showed increased Tie2 receptor expression compared to cells cultured at normoxia (hypoxia: 18.6±0.3%; p<0.0001 and N=12) (Figure 1B).

To determine whether hypoxia-enhanced Tie2 receptor expression was regulated transcriptionally, we assayed these monocytes for Tie2 receptor mRNA by

RT-PCR. Tie2 receptor mRNA expressed by CD14+ monocytes significantly increased with exposure to hypoxia starting at 6 hours (6 hrs: 5.0±1.2-fold; p=0.003,

N=6 per group) (Figure 1C). Maximal mRNA expression was achieved after 18 hours of incubation, showing a 10.6-fold increase compared to those cells maintained at normoxia for the same duration (p<0.0001) and an increase in Tie2 receptor mRNA expression was maintained during all time points tested up to 72 hours compared to freshly-isolated CD14+ cells (18 hrs: 10.6±1.3-fold, 48 hrs: 11.3±1.2-fold, 72 hrs:

13.0±1.0-fold, p<0.0001, n=6 for each condition and time point). This data was 47

confirmed by culturing CD14+ monocytes with cobalt chloride. CoCl2 functions as a hypoxia mimetic by occupying the VHL-binding domain of the HIF-α subunits and

190 preventing proteasome degradation . Treatment of monocytes with CoCl2 resulted in increased in Tie2 receptor expression after 24 hours compared to cells cultured without

CoCl2 (isotype: 1.0±0.2%; fresh: 9.3±0.7%; -CoCl2: 8.4±0.9%; +CoCl2: 19.2±1.0%; p<0.0001 and N=10 per condition) (Figure 1D).

Figure 1. Hypoxia up-regulates Tie2 receptor on CD14+ human monocytes. (A)

CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or the isotype control antibody immediately following isolation (Fresh) or after subjected to 21% oxygen (Normoxia) or 0.5% oxygen (Hypoxia) for 24 hours. (B) Hypoxia significantly up-regulates Tie2 receptor expression after 24 hours. Results represent the mean ±

SEM of Tie2-positivity. (C) Total mRNA was collected from freshly-isolated

CD14+/Tie2+ monocytes or after exposure to hypoxia for 6, 18, 48, or 72 hours using

Trizol. Tie2 receptor mRNA is increased at each time point compared to cells cultured at normoxia at the same time point as determined by real-time PCR. Results represent the mean ± SEM for Tie2 mRNA levels and N=6 per condition. (D) CD14+ monocytes were cultured without (-CoCl2) or with 100 µM cobalt chloride (+CoCl2) for 24 hours.

Results represent the mean ± SEM of Tie2-positivity by flow cytometry.

A Isotype control Freshly isolated

CD14+ cell isolation from PE-Cy7-Tie2 PE-Cy7-Tie2 whole blood using CD14+ Normoxia Hypoxia beads

PE-Cy7-Tie2 PE-Cy7-Tie2

p<0.0001 B 20

Isotype control 16 p=NS Freshly isolated

Normoxia cells + cells Hypoxia 12 + /Tie2 + /Tie2

+ 8 CD14 % CD14 4

0 Isotype Freshly Normoxia Hypoxia control isolated Log Fluorescence Intensity 24 hours

p<0.0001

C 14 D 20 p=NS 12 * * * 15 10 cells + 8

/Tie2 10 6 + * 4 5 % CD14 2 to normoxic cells at the same time Fold change in Tie2 mRNA relative mRNA Fold change in Tie2 0 0

6 18 48 72 Isotype Freshly - CoCl2 + CoCl2 control isolated Hours 24 hours

*p<0.0001 vs normoxia at the same time point

Figure 1. Hypoxia up-regulates Tie2 receptor on CD14+ human monocytes. 50

HIF-1α regulates Tie2 receptor expression on BMDMs

Tie2-expressing monocytes/macrophages are reported to resemble the genetic profile of the M2 pro-inflammatory F4/80+ tumor-associated macrophages (TAMs), yet they express distinct characteristics 127, 191. Interestingly, HIF-1α and HIF-2α have recently been described as having differential function in macrophage nitric oxide homeostasis and M1/M2 polarity 157. The Tie2 promoter contains HIF core binding sequences (hypoxia regulatory elements, HREs), 5′-RCGTG-3′ 151, upstream of the start codon, and another HRE located in the first intron. Because we observed an increase in Tie2 receptor mRNA and protein in CD14+ monocytes in response to hypoxia, we hypothesized that the HIF-1α and HIF-2α transcription factors may disparately affect Tie2 receptor expression on these cells. We utilized our colony of

HIF-1αfl/fl/LysMcre and HIF-2αfl/fl/LysMcre mice in which HIF-1α or HIF-2α are deleted in the mononuclear phagocyte population 192. We harvested bone marrow from these mice and differentiated macrophages (BMDMs) over 7 days using endotoxin-free RPMI with serum supplemented with rmM-CSF. Differentiation was confirmed by immunostaining with the mouse macrophage-specific marker F4/80 antibody and flow cytometry (data not shown) and expression of LysMCre as well as absence of HIF-1α or HIF-2α was confirmed by genotyping (data not shown). BMDMs were then exposed to normoxia or hypoxia for 1, 3, and 7 days. At each time point, BMDMs were co- immunolabeled with F4/80 and Tie2 receptor antibodies and quantified by flow cytometry (Figure 2A). We observed no difference in the number of F4/80+/Tie2+ cells at any time point between the wild type, HIF-1α- or HIF-2α-deficient cells when exposed to normoxia (wild type: day 1: 11.7±2.0%, day 3: 12.8±1.5%, day 7:

12.4±1.5%; HIF-1α KO: day 1: 14.0±2.1%, day 3: 12.0±2.3%, day 7: 14.1±2.6%; HIF-

2α KO: day 1: 14.5±1.2%, day 3: 12.0±1.4%, day 7: 11.0±0.9%; p=NS, N=5 for all 51

samples). In contrast, hypoxia exposure significantly augmented the number of

F4/80+/Tie2+ cells at each time point from both the wild type BMDMs (day 1:

31.4±1.4%, day 3: 27.8±1.8%, day 7: 29.5±3.0%) and the HIF-2α KO BMDMs (day 1:

29.5±2.6%, day 3: 28.2±3.6%, day 7: 30.9±4.2%) compared to normoxic exposure

(p<0.0001 for wild type BMDMs and p<0.001 for HIF-2α KO BMDMs, N=5 for all samples) while the number of F4/80+/Tie2+ cells from the HIF-1α KO BMDMs exposed to hypoxia were not significantly different compared to normoxia exposure (day 1:

13.8±2.0%, day 3: 13.4±1.3%, day 7: 13.7±1.8%; p=NS, N=5 for all samples) and significantly reduced compared to both wild type and HIF-2α BMDMs at hypoxia

(p<0.0001 versus wild type and HIF-2α BMDMs at 0.5% O2, N=5 for all samples)

(Figure 2B).

Figure 2. HIF-1α, but not HIF-2α, regulates Tie2 receptor expression on F4/80+ subset. (A) Bone marrow was collected from femurs of wild type LysMcre, HIF-

1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and differentiated over 7 days with 20 ng/ml rmM-CSF to a monolayer of macrophages (BMDMs). BMDMs were then cultured on tissue culture plates at 21% oxygen or 0.5% oxygen for 1, 3, or 7 days. The cells were fixed and co-immunostained using F4/80 and Tie2 receptor antibodies and subjected to flow cytometry. (B) After each 1, 3 and 7 days, there was no difference between mouse groups in the percent of F4/80+/Tie2+ cells exposed to 21% oxygen. The percent of

F4/80+/Tie2+ cells from the HIF-1αfl/fl/LysMcre mice were significantly reduced at each day assayed compared to the wild type LysMcre and HIF-2αfl/fl/LysMcre mice when exposed to 0.5% oxygen. Results represent the mean percent ± SEM F4/80+/Tie2+ cells.

wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre 88.0% 1.3% 86.4% 1.5% 83.9% 1.3%

21% O2 APC-F4/80 APC-F4/80 APC-F4/80

10.7% 0% 12.1% 0% 14.8% 0%

PE-Cy7-Tie2 PE-Cy7-Tie2 PE-Cy7-Tie2

wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre 66.2% 27.3% 91.7% 1.9% 62.4% 28.7%

0.5% O2 APC-F4/80 APC-F4/80 APC-F4/80

6.5% 0% 6.4% 0% 8.9% 0%

PE-Cy7-Tie2 PE-Cy7-Tie2 PE-Cy7-Tie2

p<0.0001 p<0.0001

50 p<0.0001 p<0.0001 Day 1 Day 3 Day 7 40

p=NS cells

+ 30 /Tie2 + 20 % F4/80 10

0 21% O2 0.5% O2 21% O2 0.5% O2 21% O2 0.5% O2

wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre

Figure 2. HIF-1α, but not HIF-2α, regulates Tie2 receptor expression on F4/80+ subset. 54

HIF-1α deletion suppresses Tie2 receptor promoter activity at hypoxia

Because we observed that hypoxia increased Tie2 receptor expression on

CD14+ monocytes, increased the population of F4/80+/Tie2+ BMDMs, and that HIF-

1α- but not HIF-2α-deficiency abrogated this outcome, we wanted to observe the effect of HIF-1α-deficiency on Tie2 promoter activity at hypoxia. We transfected a Tie2- luciferase reporter plasmid encompassing the 2.1kb murine Tie2 promoter and the

1.7kb 5’-intronic enhancer, along with the transfection control plasmid, p-RL-TK- luciferase, into BMDMs derived from wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice to measure luciferase production as a function of Tie2 promoter activity in response to 0.5% O2. Measurements of luciferase production were recorded

24 hours subsequent to transfection using a luminometer. After standardization, our data shows that luciferase expression in wild type BMDMs is significantly higher than those non-transfected wild type BMDMs (WT: 3741±353 relative units, non-transfected:

334±52 relative units; p<0.0001 and N=5 for all samples). Not surprisingly, the Tie2 promoter from the HIF-2α KO BMDMs expressed similar levels of luciferase to that of the Tie2 promoter from the wild type BMDMs in response to hypoxia (HIF-2α KO:

4069±479 relative units; p=NS and N=5 for all samples) while Tie2 promoter activity was significantly suppressed in BMDMs with HIF-1α-deficiency (HIF-1α KO: 735±141 relative units; p<0.0001 vs. both WT and HIF-1α KO BMDMs for luciferase expression;

N=5 for all samples) (Figure 3).

Figure 3. HIF-1α regulates Tie2 promoter activity.

Bone marrow was collected from the femurs of wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice and differentiated over 7 days with 20 ng/ml rmM-CSF to a monolayer of macrophages. A Tie2-luciferase reporter plasmid and control plasmid p-

RL-TK-luciferase was transfected into these BMDMs and exposed to 0.5% oxygen for

24 hours. After standardization and luminometer analysis, there was no statistical difference between luciferase expression in the wild type LysMcre and HIF-

2αfl/fl/LysMcre BMDMs while those BMDMs from the HIF-1αfl/fl/LysMcre mice were significantly reduced. Results represent the mean ± SEM relative units of luciferase expression.

p=NS

p<0.0001 p<0.0001 20

non-transfected cells 4 Fold change luciferase over

0 Wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre

Figure 3. HIF-1α regulates Tie2 promoter activity.

HIF-1α deficiency suppresses tumor growth and reduces the TEM population in PyMT tumors

In solid tumors, the demand for oxygen from rapidly dividing cells exceeds oxygen diffusion distance resulting in regions of hypoxia. Given that TEMs have been implicated in tumor angiogenesis 193, 194 and because our data illustrates the importance of HIF-1α in Tie2 receptor expression on these TEMs, we used a monocytic lineage depletion of HIFs to examine the role of Tie2 expression in response to tumor hypoxia. We orthotopically injected 1x106 C57Bl/6 PyMT tumor cells into the number four mammary fat pad of wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice. Once palpable, about two weeks post-implantation, we recorded tumor volumes and again after 14 days and calculated tumor growth rates of each group. Not surprisingly, the tumors from the HIF-1αfl/fl/LysMcre mice grew at a significantly slower rate than the tumors from both the wild type LysMcre mice (WT:

0.31±0.11 cm3/day vs. HIF-1α KO: 0.11±0.03 cm3/day, p<0.0001 and N=6 mice for WT and N=9 mice for HIF-1α KO groups) and HIF-2αfl/fl/LysMcre mice (HIF-2α KO:

0.70±0.23 cm3/day, p=0.0004 and N=9 mice for HIF-2α KO group) (Figure 4A). These data emphasize the impact of HIF-1α from a host immune cell, and not tumor cells themselves, as enough to significantly hinder tumor growth. In comparison, tumors from the HIF-2α-deficient mice grew at a markedly, but not quite statistically significant

(p=0.260), faster rate compared to mice with wild type levels of HIF-1α/HIF-2α.

Because we observed differences in tumor growth rates in these mice, and because we found that HIF-1α up-regulates Tie2 expression on F4/80+ macrophages in culture, we asked if the HIF-1α-deficiency would have the same effect in vivo and reduce the number of tumor TEMs. So, the PyMT tumors from the tumor growth study above were resected, homogenized and co-immunolabeled with antibodies specific for 58

F4/80 and Tie2 receptor and the data represented as F4/80+/Tie2+ as a percent of total tumor F4/80+ cells. Our findings suggest that while the percent of total F4/80+ cells in the tumors were unchanged across the wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre groups (WT: 35.6±1.7%, HIF-1α KO: 32.4±0.9%, HIF-2α KO:

33.5±1.6%; p=NS, N=5 for all mouse groups), the percent of F4/80+/Tie2+ cells in the tumors of the HIF-1αfl/fl/LysMcre mice were statistically reduced compared to tumors from both the wild type tumors and the tumors from the HIF-2αfl/fl/LysMcre mice (WT:

21.8±1.5%, HIF-1α KO: 5.1±1.0%, HIF-2α KO: 23.4±1.2%; p<0.0001) (Figure 4B).

These data suggest that HIF-1α regulates the migration of only those F4/80+ cells that express the Tie2 receptor in the PyMT tumors.

Because we observed differences in tumor growth rates and differences in the percent of F4/80+ macrophages that are also Tie2+ (TEMs), we correlated each mouse group tumor growth rates as a function of percent TEMs (Figure 4C, top) or as a function of total F4/80+ macrophages (Figure 4C, bottom). Tumor growth rates from the wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice correlate better with the percent of TEMs (F4/80+/Tie2+) (r2=0.5641) than total tumor F4/80+ cells

(r2=0.0095).

Figure 4. HIF-1α regulates tumor growth and F4/80+/Tie2+ TEMs.

(A) 1x106 C57Bl/6 PyMT tumor cells were injected into the number 4 mammary fat pad of 6-8 week old C57Bl/6 female wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice. Once palpable (two weeks post-implantation), tumor measures are recorded and again after two more weeks. Tumors from the HIF-1αfl/fl/LysMcre mice grew at a significantly slower rate than tumors from the wild type LysMcre and HIF-

2αfl/fl/LysMcre mice. Circles (○) represent each mouse and bar represents the mean tumor volume in each group. (B) From the tumor growth studies in (A) above, the tumors were removed, subjected to collagenase and DNase digestion, fixed, immunostained with F4/80 and Tie2 receptor antibodies and analyzed by flow cytometry for the percent F4/80+/Tie2+ cells in the tumors. There was no statistical difference in the percent of total F4/80+ cells (black box) in the tumors between the wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice. But, there was a significant reduction in the percent of F4/80+/Tie2+ cells (shaded box) in the tumors from the HIF-1αfl/fl/LysMcre mice. Results represent the mean ± SEM percent total

F4/80+ cells and percent F4/80+/Tie2+ cells in the tumors. (C) Tumor growth rates from wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice were correlated as a function of percent F4/80+/Tie2+ cells of total F4/80+ (black circle) or as a function of total tumor F4/80+ cells (shaded circle). Tumor growth rates better correlated with percent F4/80+/Tie2+ cells (r2=0.6757) than the percent total tumor

F4/80+ cells (r2=0.0258).

All Macs (F4/80+) TEMs (F4/80+/Tie2+)

A B p=NS p=NS

p=0.260 p=NS p=NS

2.5 p<0.0001 p<0.0001 p=0.0004 45

40 -1 2.0 35 day 3 p<0.0001 30 1.5 25

20 1.0 15 % Cell population

0.5 10 Tumor growth rate, cm Tumor 5

0 0 Wild type HIF-1αfl/fl HIF-2αfl/fl wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre LysMcre LysMcre LysMcre

C All Macs (F4/80+) 1.6 TEMs (F4/80+/Tie2+) -1 1.4

day HIF-2α KO 3 1.2

1.0

0.8 r2= 0.5641 0.6 r2= 0.0095 0.4 WT 0.2

Tumor growth rate, cm Tumor HIF-1α KO 0 0 5 10 15 20 25 30 35 40 45 % Cell population

Figure 4. HIF-1α regulates tumor growth and F4/80+/Tie2+ TEMs.

The hypoxic tumor microenvironment regulates the differentiation of

TEMs

Because we observed a reduction in the percent of TEMs infiltrating the tumors in the HIF-1αfl/fl/LysMcre mice, we next sought to understand if the HIFs regulated TEM recruitment from peripheral blood or if the hypoxic nature of the tumor microenvironment regulated differentiation of these cells. We orthotopically injected

1x106 C57Bl/6 PyMT tumor cells into wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice and allowed the tumors to grow for four weeks. After, the femurs were removed for bone marrow collection, peripheral blood was collected by cardiac puncture, and tumors resected and homogenized using collagenase. The bone marrow was co-immunostained with antibodies specific for CD45 and Tie2 and analyzed by flow cytometry (Supplemental Figure 8A). Blood and tumor homogenates were subjected to a Ficoll gradient for the isolation of the leukocyte layer followed by co- immunostaining with antibodies specific for CD11b, CD31, Gr-1, and Tie2 and the percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells relative to total CD11b+/CD31-/Gr-1lo cells in each compartment was determined by flow cytometry (Supplemental Figure 8B).

Importantly, because Pucci et al. classified mammary tumor CD11b+/CD31-/Gr-

1lo/Tie2+ cells as F4/80+/Tie2+ TEMs 195, we wanted to track these cells from the blood into the tumors using the same clusters of differentiation to minimize variability that may exist between the CD11b, CD31, Gr-1, and F4/80 antibodies. We observed no difference in the percent CD45+/Tie2+ cells of total CD45+ cells between the wild type

LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice in the bone marrow (WT:

2.4±0.5%, HIF-1α KO: 3.7±0.5, HIF-2α KO: 3.0±0.7%; p=NS and N=6 for WT, 5 for each HIF-1α KO and HIF-2α KO groups) (Figure 5). Further, we observed no difference in the percent CD11b+/CD31-/Gr-1lo/Tie2+ cells of total CD11b+/CD31-/Gr-1lo cells in 62

the peripheral blood compartment of these mice (WT: 9.2±0.8%, HIF-1α KO: 8.5±0.7%,

HIF-2α KO: 9.8±1.3%; p=NS and N=6 for WT, 5 for each HIF-1α KO and HIF-2α KO groups) (Figure 5). In the tumors, however, the HIF-1αfl/fl/LysMcre mice had a significantly smaller percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells than both the tumors from the wild type LysMcre mice (WT: 34.4±1.8%, HIF-1α KO: 8.4 ± 2.0%; p<0.0001 and N=11 for WT, N=10 for HIF-1α KO groups) and from the tumors of the HIF-

2αfl/fl/LysMcre mice (HIF-1α KO: 8.4 ± 2.0%, HIF-2α KO: 36.0 ± 3.0%; p<0.0001 and

N=10 HIF-2α KO group) (Figure 5). These data suggest disparate roles for HIF-1α and

HIF-2α in the transdifferentiation of TEMs within the tumor microenvironment.

Figure 5. HIF-1α regulation of tumor TEMs is driven by transdifferentiation and not recruitment.

1x106 C57Bl/6 PyMT tumor cells were injected into the number 4 mammary fat pad of 6-8 week old C57Bl/6 female wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice.

The tumors were allowed to grow for four weeks. Bone marrow cells were fixed and co- immunostained with FITC-conjugated CD45 and PE-conjugated Tie2 antibodies and analyzed by flow cytometry for CD45+/Tie2+ cells. Blood and tumor homogenates were separately fixed and co-immunostained with PerCP-Cy5-conjugated Gr-1 antibody, an

APC-conjugated CD31 antibody, a PE-Cy7-conjugated CD11b antibody, and a PE- conjugated Tie2 antibody and analyzed by flow cytometry. The percent of CD11b+/CD31-

/Gr-1lo/Tie2+ cells were determined for both the blood and tumor compartments from each mouse group. There was no difference in the percent of CD45+/Tie2+ cells in the bone marrow or the percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells in the blood from wild type

LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice. There was a significant reduction in the percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells in the tumors from the HIF-

1αfl/fl/LysMcre mice. Results represent the mean ± SEM for each compartment TEM population.

p<0.0001

50 50 Wild type LysMcre p<0.0001 HIF-1αfl/fl LysMcre p<0.0001 total CD11b fl fl cells HIF-2α / LysMcre + 40 40 % TEM population of

30 30 + /CD31 - 20 20 /Gr-1

p=NS lo cells

10 10 p=NS % TEM population of total CD45 0 0 Bone marrow Blood Tumors (CD45+/Tie2+) (CD11b+/CD31-/Gr-1lo/Tie2+)

Figure 5. HIF-1α regulation of tumor TEMs is driven by transdifferentiation and not recruitment.

Suppressed TEM population decreases tumor angiogenesis

It is well known that stabilization of HIF-1α during tissue hypoxia induces VEGF and other factors that stimulate angiogenesis 154, 155, 196, 197, 197. But we recently reported an anti-angiogenic role for macrophage HIF-2α, demonstrating disparate roles of the

HIF-α subunits in regulating angiogenesis 173-175. TEMs within the tumor microenvironment have been shown to localize to the tumor vasculature in vessel regions undergoing angiogenesis 186 and function in a paracrine manner to stimulate vessel growth 129, 198. Because we observed both reduced tumor growth rates and a smaller percentage of TEMs in the tumors of the HIF-1αfl/fl/LysMcre mice, we asked if there was a detrimental effect on angiogenesis as a result of fewer TEMs. Once again, we implanted PyMT tumor cells into the wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice and allowed them to grow for four weeks. After, the tumors were resected and prepared for fluorescence immunohistochemistry. We observed a significant reduction (62% of wild type) in the area of CD31-stained blood vessels in the tumors from the HIF-1αfl/fl/LysMcre mice compared to tumors from the wild type

LysMcre mice (WT: 2.1±0.2% CD31+ pixels, HIF-1α KO: 0.8±0.1% CD31+ pixels; p=0.003, N=5 fields per tumor and 5 mice per group) and HIF-2αfl/fl/LysMcre mice (82% of HIF-2αfl/fl/LysMcre mice) (HIF-1α KO: 0.8±0.1% CD31+ pixels, HIF-2α KO: 4.5±0.7%

CD31+ pixels; p<0.0001, N=5 fields per tumor and 5 mice per group) while we observed no difference in CD31+ pixels in the tumors between the wild type and HIF-

2αfl/fl/LysMcre mice (Figure 6). These data together support the role of both HIF-1α and

TEMs in the regulation of tumor angiogenesis.

Figure 6. Reduced population of tumor TEMs suppress angiogenesis

1x106 C57Bl/6 PyMT tumor cells were injected into the number 4 mammary fat pad of

6-8 week old C57Bl/6 female wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice. After four weeks, the tumors were removed, fixed, sectioned and immunostained with an APC-conjugated CD31 antibody. Tumors from the HIF-

1αfl/fl/LysMcre mice had less angiogenesis (CD31+ pixels) than tumors from wild type or HIF-2αfl/fl/LysMcre mice. Results represent the mean ± SEM percent of CD31+ pixels.

Wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre CD31 CD31 CD31

20x 40x 20x 40x 20x 40x

p=0.0324

p=0.003 p<0.0001 8.0

7.0

6.0

5.0

4.0

+ pixels per HPF 3.0

2.0 CD31

1.0

0.0 Wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre

Figure 6. Reduced population of tumor TEMs suppress angiogenesis

Loss of TEMs in the tumors of HIF-1αfl/fl/LysMcre mice decrease tumor cell intravasation and pulmonary metastasis

Clinically, increased numbers of macrophages have been reported to correlate to poor prognosis in human breast cancer patients 96. In a PyMT mouse model of breast cancer, a transgenic deficiency in the macrophage differentiation factor, M-CSF

(osteopetrotic (op/op) mice), decreased tumor vascular area and metastasis to the lung

98. In the same PyMT model, mammary tumor cell intravasation has been shown to occur in association with perivascular macrophages in the absence of local angiogenesis 199. Because of our observed diminished angiogenesis, we asked if the

HIF-1α deficiency would hinder tumor cell intravasation and metastatic potential in the

PyMT tumors. We implanted PyMT tumors into wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-2αfl/fl/LysMcre mice. After four weeks, we collected the whole blood by cardiac puncture and lungs. The blood was plated with FBS-containing media for 24 hours.

After, any non-adherent cells were removed by washing and adherent cells were allowed to grow in DMEM media containing serum, EGF and insulin. Tumor cell colonies were allowed to grow for 2 weeks post-plating and the numbers of colonies formed were blindly counted as described 199. The tumor cells were harvested and the presence of the PyMT oncogene confirmed by RT-PCR (data not shown). Blood from

HIF-1αfl/fl/LysMcre mice formed less PyMT tumor cell colonies than blood from the wild type LysMcre (HIF-1α KO: 11±0.4 colonies, WT: 18±0.5 colonies; p=0.044 and N=5 per group) and HIF-2αfl/fl/LysMcre mice (HIF-2α KO: 21±1 colonies; p=0.015 and N=5 per group), while there was no difference between the wild type and HIF-2αfl/fl/LysMcre mice (p=0.274) (Figure 7A). These data indicate that the loss of HIF-1α in the TEMs

and subsequent reduction in the percent of TEMs in the PyMT tumors result in decreased tumor cell intravasation into peripheral blood.

To understand if the TEM population alone is regulating tumor cell intravasation, we correlated the number of PyMT colonies formed from the whole blood of the wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice (intravasation assay) as a function of the percent of tumor F4/80+/Tie2+ cells in each group compared to the percent of total F4/80+/(Tie2+ and Tie2-) cells in these tumors. Our data indicates that the percent of tumor F4/80+/Tie2+ cells correlates highly with PyMT tumor cell intravasation (r2=0.9328) while the percent of total tumor F4/80+ cells does not (r2=0.2621) (Figure 7B). These data suggest that the percent of TEMs are more predictive regarding tumor cell infiltration into blood vessels than overall F4/80+ cells.

Because we observed reduced PyMT tumor cell colony formation from the blood of the

HIF-1αfl/fl/LysMcre mice, we hypothesized that metastasis may be suppressed in these animals as well. We performed RT-PCR for micrometastases of PyMT mRNA in the lungs of the tumor-bearing wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-

2αfl/fl/LysMcre mice. We found that the lungs of the HIF-1αfl/fl/LysMcre mice contained significantly less PyMT mRNA than the lungs from both the wild type mice (4.6-fold less) and HIF-2αfl/fl/LysMcre mice (4.0-fold less) (WT: 0.00071±0.00018 relative units,

HIF-1α KO: 0.00015±0.00002 relative units, HIF-2α KO: 0.00062±0.00014 relative units; p=0.08 for WT vs HIF-1α KO, p=0.12 for HIF-1α KO vs HIF-2α KO, and p=0.69 for WT vs HIF-2α KO and N=4 per group) (Figure 7C). The pattern of lung metastases compares with the tumor cell intravasation assay and percent of TEMs in the tumors of these mice. To understand if the TEM population alone is regulating PyMT lung metastases, we correlated the PyMT lung metastases mRNA from the lungs of the wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre mice as a function of the 70

percent of tumor F4/80+/Tie2+ cells in each group compared to the percent of total

F4/80+/(Tie2+ and Tie2-) cells in these tumors. Our data indicates that the percent of tumor F4/80+/Tie2+ cells correlates better with PyMT lung metastases (r2=0.9184) than the percent of total tumor F4/80+ cells (r2=0.7556) (Figure 7D). These data suggest that the percent of TEMs are more predictive regarding tumor metastases than overall

F4/80+ cells.

Figure 7. Tumor TEMs regulate tumor cell intravasation and lung metastases

(A) 1x106 C57Bl/6 PyMT tumor cells were injected into the number 4 mammary fat pad of 6-8 week old C57Bl/6 female wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice. After four weeks, whole blood was collected and plated with FBS- containing media for 24 hours in DMEM media containing serum, 5 µg/ml EGF and 10

µg/ml insulin. Tumor cell colonies were allowed to grow for 2 weeks post-plating and the numbers of colonies formed were blindly counted. Blood from wild type LysMcre mice and the HIF-2αfl/fl/LysMcre mice formed significantly more PyMT tumor cell colonies on the plate that the blood from the HIF-1αfl/fl/LysMcre mice. Results represent the mean ± SEM percent of PyMT tumor cell colonies formed. (B) PyMT tumor cell colonies formed from each wild type LysMcre (WT), HIF-1αfl/fl/LysMcre (HIF-1α KO), or

HIF-2αfl/fl/LysMcre (HIF-1α KO) mice as a function of percent F4/80+/Tie2+ cells

(shaded circles) or percent of total F4/80+ cells (black circles). PyMT tumor cell colonies from the blood correlates higher with the percent F4/80+/Tie2+ cells

(r2=0.9328) compared to the percent total tumor F4/80+ cells in the PyMT tumors

(r2=0.2621). (C) The lungs from the PyMT tumor model described in (A) above were homogenized in Trizol, total RNA was isolated and cDNA synthesized for RT-PCR analysis for the presence of PyMT mRNA. The lungs from the HIF-1αfl/fl/LysMcre mice had significantly less PyMT mRNA than the wild type LysMcre or HIF-2αfl/fl/LysMcre mice. Results represent the mean ± SEM fold change in PyMT mRNA. (D) PyMT mRNA from the lungs of wild type LysMcre (WT), HIF-1αfl/fl/LysMcre (HIF-1α KO), and

HIF-2αfl/fl/LysMcre mice (HIF-2α KO) as a function of percent F4/80+/Tie2+ cells

(shaded circles) or percent of total F4/80+ cells (black circles). PyMT mRNA from the lungs correlates higher with the percent F4/80+/Tie2+ cells (r2=0.9184) compared to the percent total tumor F4/80+ cells (r2=0.7556). 72

A p=0.275 B p=0.044 p=0.015 24 All Macs (F4/80+) 25 TEMs (F4/80+/Tie2+) 20 HIF-2α KO

20 16 2 r = 0.9328 WT

15 12 r2= 0.2621 HIF-1α KO 10 8 PyMT colonies formed 4 5 PyMT colonies formed

0 0 0 5 10 15 20 25 30 35 40 45 Wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre % Cell population

C D p=0.69

12 p=0.08 p=0.12

) 12

-4 All Macs (F4/80+) 10 ) -4 10 TEMs (F4/80+/Tie2+) 8 8 6 WT 6 r2= 0.9184 HIF-2α KO

PyMT mRNA 4 2

PyMT mRNA 4 r = 0.7556 HIF-1α KO 2

(relative expression, x10 2 (relative expression, x10

0 0 0 5 10 15 20 25 30 35 40 45 Wild type HIF-1αfl/fl HIF-2αfl/fl LysMcre LysMcre LysMcre % Cell population

Figure 7. Tumor TEMs regulate tumor cell intravasation and lung metastases

Figure 8. Supplemental Data on TEM isolation by flow cytometry of bone marrow, blood, and tumors.

(A) 1x106 C57Bl/6 PyMT tumor cells were injected into the number 4 mammary fat pad of 6-8 week old C57Bl/6 female wild type LysMcre, HIF-1αfl/fl/LysMcre, or HIF-

2αfl/fl/LysMcre mice. The tumors were allowed to grow for four weeks. Bone marrow cells were fixed and co-immunostained with FITC-conjugated CD45 and PE- conjugated Tie2 antibodies and analyzed by flow cytometry for CD45+/Tie2+ cells. (B)

Blood and tumor homogenates were separately fixed and co-immunostained with

PerCP-Cy5-conjugated Gr-1 antibody, an APC-conjugated CD31 antibody, a PE-Cy7- conjugated CD11b antibody, and a PE-conjugated Tie2 antibody and analyzed by flow cytometry. Those cells identified as Gr-1lo/CD31- were included in the gate to exclude both myeloid-derived suppressor cells (MDSCs) and endothelial progenitor cells. The percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells were determined for both the blood and tumor compartments from each mouse group.

A Bone marrow cells

FITC-CD45 PE-Tie2 CD45+/Tie2+ cells from bone marrow: Wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre 0.1% 2.3% 0.1% 2.4% 0.1% 3.0% PE-Tie2 PE-Tie2 PE-Tie2

17.2% 80.4% 16.3% 81.2% 10.6% 86.3%

FITC-CD45 FITC-CD45 FITC-CD45

Blood/Tumors cells B MDSCs excluded

PerCP-Cy5.5-Gr-1 PerCP-Cy5.5-Gr-1

APC-CD31

Endothelial progenitors excluded

APC-CD31 PE-Cy7-CD11b PE-Tie2

CD11b+/CD31-/Gr-1lo/Tie2+ cells from blood: Wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre 1.1% 7.9% 0.3% 11.3% 0.4% 8.8% PE-Tie2 PE-Tie2 PE-Tie2

25.6% 65.4% 26.0% 62.7% 20.8% 70.0%

PE-Cy7-CD11b PE-Cy7-CD11b PE-Cy7-CD11b

CD11b+/CD31-/Gr-1lo/Tie2+ cells from tumors: Wild type LysMcre HIF-1αfl/fl LysMcre HIF-2αfl/fl LysMcre 0.0% 29.9% 0.0% 2.5% 0.0% 26.6% PE-Tie2 PE-Tie2 PE-Tie2

15.2% 54.8% 9.3% 88.3% 14.5% 58.9%

PE-Cy7-CD11b PE-Cy7-CD11b PE-Cy7-CD11b

Figure 8. Supplemental Data on TEM isolation by flow cytometry of bone marrow, blood, and tumors. 75

Discussion

This study establishes a novel role for the hypoxia inducible protein, HIF-1α, in regulating the expression of the Tie2 receptor on a subpopulation of monocytes with characteristics reported to resemble tumor-associated macrophages (TAMs) of an M2 phenotype 127, 129, 195. The impetus of this study originated from reports that hypoxia results in increased numbers of these Tie2-expressing monocytes (TEMs) in human breast cancer. Further, mononuclear phagocyte-specific Tie2 receptor knockout studies in mice are shown to decrease angiogenic area and metastasis in breast cancer models 122, 129, 186.

Tissue hypoxia is a result of cell proliferation boundaries overcoming the oxygen diffusion distance (~100 µm) required to deliver oxygen in areas devoid of blood vessels. Once this threshold is reached, the expanding cells switch into “survival mode” and activate the hypoxia pathway, inducing the expression of genes able to increase transcription, neovascularization, anaerobic metabolism and cell survival 148,

159, 200. Before the seminal studies by Naldini 124and Lewis 185 characterizing Tie2 expression on subsets of the monocyte/macrophage lineage, Tie2 receptor expression was almost exclusively known to endothelial cell biology. The importance of the HIF pathway on Tie2 expression in endothelial cells have been reported as the expression of a dominant-negative HIF mutant inhibited transcriptional activation by HIF-1 and

HIF-2 in endothelial cells 201. We report in our current study that hypoxia results in a

HIF-1α-, but not HIF-2α-, -dependent up-regulation of the Tie2 receptor on both human and mouse monocytes. Interestingly, this paradigm is reversed in endothelial cells as it has been shown that overexpression of HIF-2α activates transcription of a Tie2-driven reporter gene 202. In our LysMcre model, TEMs (F4/80+/Tie2+ BMDMs) with the HIF-

2α-deficiency still expressed luciferase at wild type BMDM levels while those cells 76

lacking HIF-1α lost this ability. It is possible that HIF-1α and HIF-2α have redundant roles in regulating Tie2 on endothelial cells as hypoxia is essential for embryonic heart and blood vessel development, there does exist a high homology in amino acid sequence between the two 201. HIF-1α was the first described hypoxia transcription factor and is known as the “master regulator” of cellular response to hypoxia 203. HIF-

1α drives the initial cellular changes to hypoxia as it is stabilized during shorter (2-24 hours), more acute episodes of hypoxia, typically under 0.1% oxygen 204-206. HIF-2α on the other hand was initially identified as an endothelial specific protein as it was first named endothelial PAS domain protein (EPAS-1). Since its discovery in endothelial cells, HIF-2α has been shown to direct responses to hypoxia in a number of tissues including lung, brain, liver, heart, intestine and pancreas. More recently, HIF-2α has been shown to activate gene transcription during continuous (greater than 48 hours) and mild hypoxia, less than 5% oxygen 207, 207, 208 In recent reports, we showed that

HIF-2α drove a more anti-angiogenic phenotype from tumor macrophages directing their expression of sVEGFR-1, which limits VEGF activity 175. Of further interest to tumor biology, it seems that intermittent hypoxia, which occurs in the volatile tumor microenvironment from erratic and unstable vessel function, favors the stability of HIF-

1α. In fact, in vitro studies have shown that 60 cycles of normoxia/hypoxia (intermittent hypoxia) is sufficient to degrade HIF-2α while HIF-1α is unaffected, and that return to stable hypoxia rescues HIF-2α stability 209.

Interestingly, hypoxia induced-expression of Tie2 on endothelial cells has been attributed to both HIF-1α and HIF-2α. In these demonstrations, overexpression of HIF-

2α increases Tie2 expression on human endothelial cells while incubation of endothelial cells under hypoxia has also been shown to increase Tie2 receptor expression through increased translation facilitated by HIF-1α 210. Given these 77

seemingly incongruent roles for the HIFs, we set out to determine the role for the HIF-α transcription factor subunits in Tie2 receptor expression on monocytes.

Given our observation that HIF-1α-deficiency abrogates the ability of hypoxia to increase Tie2 receptor expression on monocytes in vitro, we examined this effect in a mouse model of breast cancer. It has been shown that HIF-1α knockout in tumor cells themselves results in a decrease in SDF-1/CXCL12 leading to a decrease in the recruitment of all CXCR4 positive cells, including TEMS which express the cognate receptor 162. We found that the percentage of TEMs did not change in tumors from the wild type mice or mice with macrophages deficient in HIF-2α but the TEMs from the

HIF-1αfl/fl/LysMcre mice did, showing reduced percentages relative to total F4/80+ cells.

It seems that hypoxia not only up-regulates receptors responsible for the migratory potential of TEMs, but that they also drive ligand expression from other cells in the tumor microenvironment 211, 212.

HIF-1α knockout has also been shown to affect the differentiation of myeloid suppressor (CD11+ and GR-1+) cells into macrophages, in vitro, as only about half of the CD11b+/Gr-1+ cells acquired the macrophage F4/80 marker 213. In our model, we did not find this to be the case as in vitro differentiation of BMDMs from wild type

LysMcre, HIF-1αfl/fl/LysMcre, and HIF-2αfl/fl/LysMcre did not result in a loss of F4/80 expression. At the same time, when these macrophages were exposed to hypoxia, only the HIF-1α knockout macrophages failed to up-regulate the Tie2 receptor. Further, the percentage of total F4/80 cells in the PyMT tumors from all these three mouse groups was not different while the percent of those F4/80 macrophages that expressed the Tie2 receptor changed significantly with the presence of HIF-1α in those cells.

These data raise the possibility that the number of F4/80+ cells that correlate with poor prognosis in patients with breast cancer are derived from a smaller, more specific 78

subpopulation of F4/80+ cells, namely the F4/80+/Tie2+ TEMs. It has been shown that these TEMs express high levels of MMP-9, VEGF, MRC-1 (mannose receptor), SDF-

1/CXCL12 for cell recruitment, and anti-inflammatory IL-10 127, 129, 195.

TEM function in the PyMT tumor model was highlighted as our knockout of HIF-

1α, but not HIF-2α, resulted in a decrease in the overall angiogenic area. This supports recent data on TEMs in tumors where co-injection of Tie2+ monocytes with glioblastoma cells increased tumor angiogenesis whereas co-injection of monocytes lacking the Tie2 receptor had the opposite effect 124. Further, microRNA-directed knockdown of the Tie2 receptor on monocytes also decreased the angiogenic area and branching of tumor vasculature 186. We found decreases in tumor cell intravasation paralleled the percentage of TEMs found in tumors. This is interesting considering that it has been shown that macrophages and tumor cells alternate leaving the tumors by moving out into tumor vasculature in the initial stages of metastases 199, 214. Given that

TEMs have been shown to produce high levels of MMP-9 and thymidine phosphorylase 125, 195, it may be that this angiogenic subset of macrophages which degrades tumor extracellular matrix facilitating tumor cell invasion and exist through the blood and lymph vessels. Genetic manipulation that allows for specific macrophage

Tie2 receptor labeling will assist in further examination of the role of TEMs in tumor cell intravasation. MicroRNA directed knockdown of Tie2 on monocytes has been shown to reduce tumor metastasis to the lungs 186. In would be interesting to delineate the role TEMs compared to total F4/80+ macrophages play in metastases.

The fact that we observed no differences in the number of TEMs in the bone marrow or peripheral blood from wild type LysMcre, HIF-1αfl/fl/LysMcre, and HIF-

2αfl/fl/LysMcre mice, but saw significant changes in the percent of F4/80+ macrophages that express Tie2, suggests the possibility that that the process of TEM differentiation 79

takes place within the tumor microenvironment. Because we saw differences between

HIF-1α and HIF-2α in TEM population regulation illustrates a specific role for HIF-1α.

We are currently investigating the possibility that an “intermediate” monocyte precursor resides in the peripheral blood of macrophage-specific HIF-α-deficient mice. In humans, a population of intermediate monocytes between classical CD14+/CD16- and the mature pro-inflammatory CD14++/CD16++ exists (the mouse CD11b+/CD31-/GR-

1lo/Tie2+ TEMs homologue) that may help identify cells in transition from classical monocytes to TEMs.

In summary, we illustrate that hypoxia increases Tie2 receptor expression at both the protein and mRNA levels on a pro-angiogenic subset of monocytes/macrophages called TEMs. We further distinguished these hypoxic effects by delineating differences between HIF-1α and HIF-2α in regulating this activity as monocytes/macrophages-deficient in HIF-1α but not HIF-2α demonstrates reduced expression of Tie2 in response to low oxygen. To test our hypothesis in vivo, we used

PyMT tumor-bearing mice deficient in mononuclear phagocyte HIF-1α and observed a decrease in the number of tumor TEMs compared to wild type and HIF-2α-deficient mice. This decrease in TEMs resulted in reduced angiogenesis and tumor cell intravasation as well as fewer tumor lung metastases.

To our knowledge, this is the first study demonstrating not only the disparate roles of HIF-1α and HIF-2α on the recruitment and function of TEMs in breast tumors, but also the regulatory role that HIF-1α plays in the differentiation of classical circulating monocytes (CD14+/Tie2- in humans and CD11b+/CD31-/Gr-1lo/Tie2- in mice) to the pro-angiogenic and M2-like TEMs.

Acknowledgements

This work was supported by National Cancer Institute Grant R00 CA131552 (to

T.D.E.), start-up funds (to T.D.E.), and R01 HL067167 (to C.B.M.).

Chapter 3: Macrophage Colony-Stimulating Factor Augments Tie2- Expressing on Monocytes and Augments their Angiogenic Function and Recruitment in a Mouse Model of Breast Cancer

Abstract

In previous work, we reported that Macrophage Colony-Stimulating Factor (M-

CSF) induced human monocytes to produce the potent pro-angiogenic factor, VEGF.

Other studies reported the role of M-CSF in breast tumor progression, angiogenesis, and metastases in mouse models, as well as the prognostic value of macrophage numbers in breast cancer patients. Recently, a relatively uncommon subset of CD14+ monocyte has been characterized as expressing the Tie2 receptor, once thought to be exclusively expressed on endothelial cells. The percentage of Tie2-expressing monocytes/macrophages (TEMs) is increased in breast tumors compared to their relative levels in circulation. To understand if increased levels of M-CSF in breast tumors can play a role in the transdifferentiation of Tie2- monocytes to a Tie2+ phenotype, we subjected freshly-isolated CD14+ monocytes to M-CSF and found a significant increase in CD14+/Tie2+ positivity. To see if M-CSF-induced Tie2 expression on these cells improved their migratory ability, we used Boyden chemotaxis chambers containing angiopoietin-2 (Ang-2), a chemotactic ligand for the Tie2 receptor, to observe if M-CSF pre-treatment conditioned these CD14+ monocytes to

increase their response to Ang-2. We found that pre-treatment with M-CSF significantly augmented chemotaxis and that Tie2 up-regulation was responsible as transfection with a siRNA targeting the Tie2 receptor abrogated this effect. Further, to show that M-

CSF-derived TEMs maintained their pro-angiogenic potential, we used conditioned media from M-CSF-pre-treated monocytes on human umbilical vein endothelial cells

(HUVECs) in a tube formation assay. We found that while M-CSF-pre-treated TEMs induced branching, a neutralizing antibody against the M-CSF receptor abrogated this activity. To illustrate it was the Tie2 receptor responsible for HUVEC branching, siRNA against Tie2 reduced the ability of TEM conditioned media to induce branching while a scrambled siRNA was ineffective. In the PyMT mouse model of breast cancer, recombinant M-CSF increased both the number of total F4/80+ cells and Tie2- positivity, as well as tumor angiogenesis, while having no effect on tumor size. By investigating the mechanism of Tie2 up-regulation on F4/80+ cells by M-CSF, we found that the PI3 kinase/Akt inhibitor, LY294002, suppressed the generation of TEMs in culture while the MEK inhibitor and NF-kB inhibitors did not. Further, we found that

HIF-1α contributes to Tie2 up-regulation on these cells as HIF-1α deletion from bone marrow-derived macrophages derived from HIF-1αfl/fl/LysMcre mice diminished the ability of M-CSF to augment Tie2 expression while M-CSF augmented tumor growth in

PyMT-tumor bearing HIF-1αfl/fl/LysMcre mice, suggesting that M-CSF overcomes the lack of TEMs in the macrophage HIF-1α-deficient tumors. Finally, we found that M-CSF treatment augments the chemokine SDF-1/CXCL12 and Ang-2 production in the tumors summoning TEMs as we saw no difference in response to M-CSF compared to

PBS-treated tumors.

Introduction Macrophage colony-stimulating factor (M-CSF) drives the survival and differentiation of monocytes recruited from the bone marrow to macrophages. Upon binding to the M-CSF receptor (M-CSF-R/c-fms), M-CSF causes receptor dimerization and phosphorylation of tyrosine kinase sites on the intracellular215-218 domain of the receptor. Phosphorylation of the intracellular domain of M-CSF-R results in activation of intracellular signal transduction pathways involving PI3 Kinase-Akt, Ras/MAPK,

Stats-1 and -3, Jak1, Phospholipase A2 and 1C and this activation regulates numerous cellular events that facilitate an innate immune response 88-91, 217, 218.

Tumor-associated macrophages (TAMs) arise from peripheral blood monocytes that diapedis into tissue, through the endothelial barrier, is response to chemokines released during an inflammatory response or by neoplastic and tumor stromal cells 82 and this extravasation into tumor tissue is accompanied by and coordinated with the differentiation of monocytes into macrophages 71, 219, 220. TAMs can have both a positive and negative effect on tumors. Macrophages exert anti-tumor effects through cytotoxic killing of tumor cells and through increasing presentation of tumor antigens. At the cellular level, TAMs facilitate tumor growth and progression through the production of proteases such as MMP-9, which break down extracellular matrix that facilitate tumor vessel sprouting 125, 165, 199, 221. In patient studies, increased numbers of macrophages correlate with poor prognosis in human breast carcinomas 95.

In recent years, subpopulations of macrophages have been shown to exert these contrasting positive or negative effects on tumors. Differentiation of classical or

M1 macrophages is induced by inflammatory stimuli 222, 223. These anti-tumor macrophages express high levels of factors that facilitate cytotoxic ability and antigen presentation facilitating immune cell influx and tumor cell cytotoxicity 224, 225. For

example, high levels of reactive oxygen species produced by M1 increase tumor cell

DNA damage leading to apoptosis 101, 105, 107, 226. Similarly, IL-1β and IL-6 production leads to the activation and differentiation of cytotoxic T cells 82, 227, 228. To the contrary,

M2 macrophages are anti-inflammatory and represent a tissue repair phenotype involving the expression of IL-10, arginase-1, TGF-β, and mannose receptor for increased ability to recognize cellular pathogens 71. In addition, M2 macrophages produce factors that facilitate angiogenesis 96. For example, treatment of macrophages with M-CSF up-regulates the potent pro-angiogenic molecule vascular endothelial growth factor (VEGF) which increases endothelial cell proliferation and migration to drive new blood vessel growth 229. The production of these factors has been demonstrated in solid tumors, such as breast cancer, where M-CSF increases angiogenic branching and blood vessel density. In mouse models, mice deficient in M-

CSF not only have less vessel density but also fewer tumor metastases than mice producing normal levels of M-CSF 182. Moreover, the re-introduction of M-CSF locally in the tumor resulted in restoration of the tumor macrophage population, tumor vessel density, and metastasis 230.

Recently, a subpopulation of macrophages resembling the genetic expression profile of macrophages with M2 polarity and expressing the endothelial cell tyrosine kinase receptor, Tie2, has been identified 124, 185. These Tie2-expressing monocytes/macrophages (TEMs) have been shown to be major contributors to tumor angiogenesis, progression and metastasis 126, 186, 231. For example, co-injection of isolated TEMs with glioma cells in mice increased tumor angiogenic area 124. Myeloid- specific knockdown of Tie2 via miRNA techniques decreased tumor volume and tumor cell metastasis to the lung 122, 186. In tube formation assays, the conditioned media from

TEMs produced more branch points than Tie2 receptor negative monocytes 125, 195. The 85

IL-10 produced by monocytes following Ang-2 production results in suppression of tumor immunity through decreasing T cell proliferation and cytotoxicity 129. TEMs also produce high amounts of remodeling molecules, such as MMP-9 facilitating macrophage migration to hypoxic and necrotic areas 125.

In our recent study, we demonstrated an oxygen-dependent mechanism of Tie2 receptor regulation on tumor macrophages and differentiation of a subpopulation on tumor-supporting macrophages independent of other F/480+/Tie2- cells once within the tumor. In that study we did not observe differences in TEM populations in either the bone marrow or peripheral blood, only in the tumor proper. Here, we observe an expansion of the Tie2-expressing monocyte population in the peripheral blood with addition of M-CSF.

Materials and Methods

Monocyte Isolation

Isolated CD14+ monocytes (2x106) were incubated in RPMI media (BioWhittaker) with

1% FBS (certified as containing <0.06 EU/ml endotoxin levels from HyClone, Logan,

UT), penicillin (100 U/ml), streptomycin (100 U/ml), amphotericin B (0.25 µg/ml) (Gibco) and 10 µg/ml polymyxin B (Calbiochem).

Flow Cytometry

Freshly-isolated CD14+ human monocytes were incubated for specified times. The cells were incubated with rhAng-1 (100 ng/ml), rhAng-2 (100 ng/ml), rhM-CSF (0.1, 1,

10, or 100 ng/ml), or pre-treated with a neutralizing antibody targeting the M-CSF receptor (R&D Systems clone 61701). The cells were fixed then blocked with anti-FcR antibody (R&D System) for 15 minutes at 4ºC to prevent non-specific binding. Cells were incubated with 10 µl PE-conjugated anti-human Tie2 (clone 83715, R&D

Systems) per 1x106 cells at 4ºC for 30 minutes per manufactures instructions or 10 µl of the isotype control PE-conjugated anti-human IgG (R&D Systems). Tie2-expressing monocytes were subsequently analyzed using the Aria III flow cytometer at The Ohio

State University Flow Cytometry Core Facility.

Migration Assay

CD14+ human monocytes were isolated as described above and cultured in RPMI media containing 10% FBS without or with 10 ng/ml rhM-CSF in the bottom well of

Boyden chemotaxis chambers for 18 hours. The cells were washed 3x with PBS and the media replaced with FBS-containing RPMI without or with a dose escalation of rhAng-2 (0.1, 1, 10, 100, 300 ng/ml) in the top chamber. After 24 hours, the filters were inspected for the migration of monocytes to the top chamber (chemotaxis) and cells blindly counted by microscopy. In another experiment, the CD14+ monocytes were treated as above except some were transfected with a scrambled siRNA-GFP conjugate or siRNA-GFP targeting the human Tie2 receptor (Sant Cruz). The cells were washed and sorted using flow assisted cell sorting for GFP expression to confirm successful transfection followed by Boyden chamber chemotaxis assay as described above. 87

HUVEC Branching Assay

5x103 HUVEC (ATCC) per well in a 24-well plate were cultured on top of 120 µls growth factor-reduced Matrigel (BD Discovery Labware) in conditioned media derived from CD14+ human monocytes cultured in RPMI media containing 10% FBS alone, with rhVEGF (10 ng/ml), rhM-CSF (10 ng/ml), rhAng-2 (10 ng/ml), or with 10 ng/ml rhM-CSF for 18 hours followed by 3 PBS washes and subsequent incubation in Ang-2

(10 ng/ml)-containing media for 24 hours. The HUVECs were cultured for 6 hours in the conditioned media and branch points counted in a blinded manner. In another condition, the CD14+ monocytes were first treated with a neutralizing antibody against

M-CSF receptor (50 ng/ml) for 30 minutes before 18 hours incubation with rhM-CSF

(10 ng/ml), 3 PBS washes, and rhAng-2 treatment (10 ng/ml) for 24 hours. In another experiment, the CD14+ monocytes were treated as above except some were transfected with a scrambled siRNA-GFP conjugate or siRNA-GFP targeting the human Tie2 receptor. The cells were washed and sorted using flow assisted cell sorting for GFP expression to confirm successful transfection followed plating and collection of conditioned media as described above.

Real-time PCR

HUVECs from the branching assay were collected from the Matrigel using dispase and dissolved in Trizol reagent (Invitrogen). RNA was extracted in chloroform and then purified using the RNeasy Minikit (Qiagen). cDNA was generated from 1 µg of RNA using the Superscript First Strand Synthesis System (Invitrogen) and was used for real- time PCR for Dll-4 mRNA with the primers: hDll-4f: 5’-ACCGGGTCATCTGCAGTGA-3’ and hDll-4r: 5’-GTGGCGAAGTGGTCATTG-3’ and hJagged-1f: 5’-

TGTGGAGGAGGCGTGGGATTC-3’ and hJagged-1r: 5’- 88

TGATCATGCCGAGTGAGAA-3’ and SYBR Green PCR Master Mix (Applied

Biosciences) according to the manufacturer’s instructions. Tumors from female

C57Bl/6 wild type PyMT tumor-bearing mice were collected at time of sacrifice and snap frozen in liquid nitrogen. Tumors were pulverized in liquid nitrogen and dissolved in Trizol followed by further homogenation using a syringe. RNA was extracted in chloroform and then purified using the RNeasy Minikit. cDNA was generated from 1 µg of RNA and used for real-time PCR for tumor angiopoietin-2 mRNA with the primers: mVEGFf: 5’-TTACACGGTGCCGATTTCAG-3’ and mVEGFr: 5’-

TGTTAGCATGAGAGCGCATTTG-3’, mAng-2f: 5’-GGCTGGGCAATGAGTTTGTC-3’ and mAng-2r: 5’-CCCAGTCCTTCAGCTGGATCT-3’, and mVEGFf: 5’-

TTACTGCTGTACCTCCACC-3’ and mVEGFr: 5’-ACAGGACGGCTTGAA GATG-3’.

SYBR Green PCR Master Mix according to the manufacturer’s instructions. Data were analyzed according to the comparative threshold method and normalized against the

GAPDH internal control transcript.

PyMT Tumor Model

PyMT tumor cells from C57Bl/6 mice were cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) supplemented with 1% PSA, 10% FBS, 10 µg/ml human insulin, and

5 µg/ml rmEGF. Tumor cells (1x106 cells in 100 µl) were injected orthotopically into the number four mammary fat pad of naïve 6-8 week old female C57Bl/6 wild type mice or

C57Bl/6 wild type LysMcre or HIF-1αfl/fl/LysMcre mice. Beginning with palpation (about

2 weeks post-injection), tumor growth was measured 3 times per week using calipers and tumor volume calculated using the equation: Tumor volume = 0.5 x [(large diameter) x (small diameter)2]. In one study, the tumors were treated when palpable with 50 μls of PBS, M-CSF-R NAb (50 mg/kg), or IgG Ab (50 mg/kg). Four hours later, 89

the tumors were injected with 50 μls PBS or rmM-CSF (100 ng/ml). This treatment paradigm was delivered every other day for two weeks.

Tumor Cell Isolation

Age-matched female C57Bl/6 mice injected orthotopically in the mammary fat pad with

PyMT tumor cells were sacrificed by asphyxiation followed by cervical dislocation.

Peripheral blood was removed by cardiac puncture. Bone marrow was isolated from the femurs and the tumors were harvested. Tumors were cut into small sections using a razor blade and then digested in 1 ml of PBS containing collagenase (0.55 mg/ml) and DNase (0.1 mg/ml). The tumors were incubated at 37ºC and vortexed every 5 minutes for 30 minutes. Each sample was passed over a 100 μm mesh cell strainer

(Fisher Scientific) and washed twice with 10 mls PBS using centrifugation at 1500 rpm for 10 minutes. The lymphocytic layer from peripheral blood and tumor homogenate was separated by a Ficoll gradient removed and washed twice with PBS. The red blood cells were lysed using Red Cell Lysis Buffer (155 mM NH4Cl, 10 mM NaHCO3,

0.1 mM EDTA; pH=7.4) for 5 minutes at room temperature. Remaining cells were washed twice with PBS and resuspended in MACS buffer (Miltenyi Biotech). For flow cytometric analysis and cell sorting, cells isolated from tumors, peripheral blood and bone marrow were blocked with whole IgG for 15 minutes at 4ºC. Samples were immunostained with antibodies specific for Tie2 (PE-conjugate clone TEK4, eBiosciences), CD11b (Percy 5.5 and PE-conjugate clone M1/70), CD31 (PE-Cy7 and

APC-conjugate clone 390), Gr-1 (Percy 5.5 and PE-conjugate clone RB6-8C5) (R&D

Systems) and F4/80 (PE-Cy7 or APC-conjugate clone Cl:A3-1, Serotec). Isotype control samples were immunostained with rat anti-mouse IgG2a and IgG2b with the corresponding fluorescent conjugates (R&D Systems). TEMs in this study are 90

described as: CD45+/Tie2+ (bone marrow), CD11b+/CD31-/Gr-1low/Tie2+ (peripheral blood and tumor for comparing different compartments), and F4/80+/Tie2+ (for comparative studies in tumor tissue by flow cytometry and immunohistochemistry). All flow cytometry was measured using the Aria III flow cytometer at The Ohio State

University Flow Cytometry Core Facility.

Tumor Angiogenesis

Tumors from PyMT tumor-bearing 6-8 week old female C57Bl/6 mice were collected at time of sacrifice as described above. Tumor sections were immunostained with CD31 antibody and imaged by fluorescent microscopy using a ×20 objective lens. Five random images per tumor per group were captured in a blinded manner, analyzed for

CD31-positivity (red pixels) and quantified using Adobe Photoshop CS2 (Adobe

Systems) histogram analysis.

Serum M-CSF by ELISA

6-8 week old female C57Bl/6 mice were injected intravenously with 50 µls of PBS or 50

µls of recombinant murine M-CSF (2.5 ng) (#416-ML-010, R&D Systems) for three consecutive days prior to mouse sacrifice. After sacrifice, cardiac blood was captured and placed into serum separator tubes, centrifuged, and serum collected and frozen for

ELISA. Serum M-CSF was detected by the murine M-CSF Duoset ELISA Kit (#DY416,

R&D Systems).

Inhibitor studies

Macrophages were differentiated from the bone marrow of 6-8 week old female

C57Bl/6 wild type mice LysMcre or C57Bl/6 HIF-1αfl/fl/LysMcre mice over five days 91

using 20 ng/ml rmM-CSF in endotoxin-free RPMI, 10% FBS, and 10 µg/ml polymyxin

B. After, the cells were pre-treated with the appropriate vehicle or the PI3 kinase/Akt inhibitor LY294002 (50 μM), the MEK inhibitor U0126 (10 μM), or the NF-B inhibitor

PDTC (100 μM) for 30 minutes followed by stimulation with 100 ng/ml rhM-CSF or not for 24 hours. The cells were collected, fixed, and immunostained using F4/80 and Tie2 receptor as described above and subjected to flow cytometry for percent F4/80+/Tie2+ cells.

Statistical Analyses

For the assays determining the effects of Tie2 expression by M-CSF, the migration and

HUVEC sprouting assays, and the mRNA expression of Dll-4 and Jagged-1, the data was analyzed by linear mixed effect model which considers observational dependencies across treatments groups. For the assays determining percent TEMs or total F4/80+ cells in bone marrow, blood, or tumors, tumor volume determinations, small molecule inhibitor studies, and tumor angiogenesis, the data was analyzed by

ANOVA. Holm’s method was used to adjust multiplicity for primary comparisons when necessary. SAS 9.3 software was used for analysis (SAS, Inc. Cary, NC).

Results

M-CSF regulates a transition from CD14+ monocytes to CD14+/Tie2+

TEMs

M-CSF drives the differentiation of peripheral blood monocytes to macrophages resulting in cells with a pro-angiogenic and immune regulatory phenotype 229. In breast tumors, M-CSF expression has been found to increase the number of macrophages which enhances tumor vessel density and metastasis 178, 182, 183. Further, loss of M-CSF

(op/op mice) hinders the development of tumor blood vessels and hinders tumor progression 182. Recently, Tie2-expressing monocytes/macrophages (TEMs) have been reported to be a subset of myeloid cells that function similar to M2, tumor- associated macrophages (TAMs) as they increase tumor blood vessel area and intensify metastasis in mouse models of breast cancer 186. As stated above, M-CSF plays a role in normal mammary gland physiology and ductal development 98. But, in breast cancer patients, M-CSF levels are augmented and predictive of poor outcome

232. In 2003, we reported M-CSF stimulation of monocytes resulted in VEGF production

229. But, in that study we did not consider the differentiation of alternate subpopulations of monocytes that M-CSF may derive. So, in our current study we examined whether a direct link exists between M-CSF and the expression of the Tie2 receptor on CD14+ human monocytes to become TEMs. To elucidate this effect, monocytes were isolated from whole blood using CD14+ microbeads and incubated in non-adherent culture tubes in RPMI media containing fetal bovine serum and cultured for 24 hours with or without 100 ng/ml recombinant human (rh)M-CSF. We used 100 ng/ml M-CSF in our initial trials as it has been reported that cancer patients can have serum concentrations which reach these levels 233. After, monocytes were immunostained with an antibody specific for the human Tie2 receptor or isotype antibody and analyzed by flow 93

cytometry (Figure 9A). Our data suggests that CD14+ cells incubated in the absence of rhM-CSF for 24 hours express the Tie2 receptor similar to freshly-isolated CD14+ cells

(fresh: 9.8±0.9%, -M-CSF: 10.2±0.7%; p=NS and N=10 per group). In contrast, CD14+ monocytes cultured in the presence of rhM-CSF for 24 hours significantly increased

Tie2 receptor expression (+M-CSF: 45.1±2.4%; p<0.0001 and N=10) (Figure 9A). Cell viability of all monocytes was confirmed by trypan blue staining (data not shown).

Next, we assayed the ability of low to moderate serum levels of M-CSF (0.1 to

10 ng/ml) to induce the differentiation of CD14+ monocytes to CD14+/Tie2+ TEMs. We cultured freshly-isolated CD14+ monocytes as described above in increasing doses of rhM-CSF. A trend analysis for dose-dependency suggests that dose escalation of rhM-

CSF significantly increases Tie2 receptor expression on these cells (0: 10.6±0.9%, 0.1 ng/ml: 22.4±1.7%, 1.0 ng/ml: 31.0±4.4%, 10 ng/ml: 38.9±2.3%, 100 ng/ml: 43.7±3.0%;

1 ng/ml dose increase of rhM-CSF results in a Tie2 receptor expression increase of

0.2%, p=0.0003 and N=10 for each group) (Figure 9B).

To compare the ability of M-CSF to induce Tie2 expression on CD14+ monocytes to known inducers of Tie2 expression on both endothelial cells and TEMs, we cultured these cells in 100 ng/ml angiopoietin-1 (Ang-1) or angiopoietin-2 (Ang-2).

While Ang-1 had no effect on Tie2 expression compared to untreated cells (Ang-1:

10.4±0.7% and -M-CSF: 10.7±1.0%; p=NS and N=10), Ang-2 induced a significant increase in Tie2 expression (Ang-2: 29.5±1.2%; p<0.0001 and N=10) but less than 100 ng/ml M-CSF (43.7±3.0%) (Figure 9B). This data indicates that M-CSF concentration found in either the serum or within tumors can induce a CD14+/Tie2- to CD14+/Tie2+ phenotype transition. To ensure that it is M-CSF responsible for this Tie2+ transition, we pre-treated freshly-isolated CD14+ monocytes with a neutralizing antibody to the M-

CSF receptor for 30 minutes then treated with 10 ng/ml rhM-CSF and analyzed these 94

cells for Tie2 expression. We found that pre-incubation with the M-CSF-R antibody significantly inhibited Tie2 expression on CD14+ cells compared to M-CSF-treated cells after 24 hours (+M-CSF: 30.1±5.6%, M-CSF-R NAb+M-CSF: 16.7±0.9%; p<0.0001 and N=8) and similar to untreated cells (untreated: 7.7±0.6%; p=NS and N=8 per group). To illustrate the effect of the M-CSF-R NAb on M-CSF signaling pathway activation, CD14+ monocytes were pre-treated for 30 minutes with the M-CSF-R NAb followed by stimulation with 10 ng/ml rhM-CSF or left untreated for 10 minutes.

Western blot analysis reveals a reduction in PI3 kinase/Akt activation as the phosphorylation of Akt1 is suppressed in the presence of the antibody (Figure 9D).

Figure 9. M-CSF up-regulates Tie2 receptor on CD14+ human monocytes.

(A) CD14+ monocytes were isolated from whole blood using CD14+ microbeads. Cells were fixed and immunostained using anti-human Tie2 receptor antibody or the isotype control antibody immediately following isolation (Freshly isolated) or after treated without (-M-CSF) or with rhM-CSF (100 ng/ml) (+M-CSF) for 24 hours. Results represent the mean ± SEM of Tie2-positivity. (B) CD14+ monocytes treated with rhAng-1 (100 ng/ml), rhAng-2 (100 ng/ml) or a dose-response of rhM-CSF (0, 0.1, 1,

10, 100 ng/ml). Ang-2 up-regulated Tie2 expression compared to Ang-1 and M-CSF induces a dose-escalation of Tie2 on CD14+ monocytes. Results represent the mean ±

SEM of Tie2-positivity. (C) CD14+ monocytes were left untreated (Utx) or treated with rhAng-2 (100 ng/ml) (Ang-2), rhM-CSF (100 ng/ml) (M-CSF), M-CSF-R neutralizing antibody alone, or pre-treated with the M-CSF-R NAb for 30 minutes prior to stimulation with rhM-CSF (100 ng/ml) (M-CSF-R NAb+M-CSF) for 24 hours. Ang-2- and M-CSF-treatment significantly increased Tie2 expression while the M-CSF-R NAb abrogated this effect. Results represent the mean ± SEM of Tie2-positivity by flow cytometry. (D) CD14+ monocytes were left untreated (Untreated), pre-treated with M-

CSF-R NAb (40 g or 80 g) for 30 minutes then treated with rhM-CSF (100 ng/ml) (M-

CSF-R NAb+M-CSF), or with rhM-CSF (100 ng/ml) alone (M-CSF) for 10 minutes.

Western blot analysis indicates that the M-CSF-R NAb was effective at reducing Akt1 phosphorylation.

p<0.0001 A 50 Isotype control Freshly isolated - M-CSF 40 +M-CSF cells + cells

+ 30

/Tie2 p=NS + /Tie2

+ 20 CD14 % CD14 10

0 Isotype Freshly - M-CSF + M-CSF control isolated Log Fluorescence Intensity 24 hours

B 100 ng/ml Ang-1 100 ng/ml Ang-2

40 p<0.0001 PE-Tie2 PE-Tie2

0.1 ng/ml M-CSF 1 ng/ml M-CSF cells + 30 Trend Analysis for dose-dependency: p=0.0003 /Tie2 + 20

% CD14 10 PE-Tie2 PE-Tie2 10 ng/ml M-CSF 100 ng/ml M-CSF 0 Isotype Ang-1 Ang-2 0 0.1 1 10 100 control 100 ng/ml M-CSF, ng/ml

PE-Tie2 PE-Tie2

C Untreated Ang-2 40 D p<0.0001 p<0.0001 10’ 35 p<0.0001

30 cells + 25 Untreated M-CSF M-CSF-R NAb (40 µ g) + M-CSF M-CSF-R NAb (80 µ g) + M-CSF

PE-Tie2 PE-Tie2 /Tie2 p-Akt + 20 M-CSF M-CSF + M-CSF Nab total Akt 10 % CD14 5

0 Utx Ang-2 M-CSF-R M-CSF M-CSF + PE-Tie2 PE-Tie2 NAb M-CSF-R NAb Figure 9. M-CSF up-regulates Tie2 receptor on CD14+ human monocytes. 97

M-CSF enhances CD14+ monocyte migration towards Ang-2

Ang-2 is reported to bind the Tie2 receptor on endothelial cells allowing for the detachment of cellular adhesions and facilitate the migration of these cells during angiogenesis 120, 121. We next sought to determine if M-CSF up-regulation of Tie2 receptor on CD14+ monocytes modulated migration towards Ang-2. We pre-treated

CD14+ monocytes with 10 ng/ml rhM-CSF or media in the bottom well of Boyden chemotaxis chambers for 18 hours then washed the cells and replaced the media with

FBS-containing media without or with a dose escalation of rhAng-2 in the top chamber.

After 24 hours, the filters were inspected for the migration of monocytes to the top chamber (chemotaxis) and cells blindly counted by microscopy. We observed that Ang-

2 stimulation alone induced significant migration at 100 ng/ml compared to Ang-2-free media (+Ang-2: 300 ng/ml: 44.6±4.8 cells, 100 ng/ml: 31.1±2.9 cells, 10 ng/ml:

12.6±3.7 cells, 1 ng/ml: 13.5±1.5 cells, 0.1 ng/ml: 11.5±1.7 cells; -Ang-2 media:

11.3±2.3 cells; p<0.0001 and N=8 per group) (Figure 10A). Interestingly, pre-treatment with 10 ng/ml M-CSF of the CD14+ monocytes significantly increased the number of migrating cells responsive to Ang-2 compared to monocytes not pre-treated with M-

CSF but also subjected to Ang-2 and reduced the significant migration response to

Ang-2 from 100 ng/ml to 1 ng/ml (+M-CSF/+Ang-2: 300 ng/ml: 64.5±3.5 cells, 100 ng/ml: 92.0±6.7 cells, 10 ng/ml: 73.6±3.1 cells, 1 ng/ml: 37.1±3.0 cells, 0.1 ng/ml:

10.1±1.6 cells, -Ang-2 media: 11.3±2.3 cells; p<0.0001 and N=8 per group) (Figure

10A) suggesting that M-CSF pre-treatment increased the number of CD14+/Tie2+ cells during the 18 hour pre-incubation and resulted in a larger population of CD14+/Tie2+ cells able to respond to Ang-2.

CD14+ monocytes express c-fms, the receptor for M-CSF, and are reported to have migratory potential in response to M-CSF 234. Because of this, we asked if 98

monocyte migration was a result of M-CSF or Ang-2, the ligand for the Tie2 receptor.

We again plated freshly-isolated CD14+ monocytes into the bottom wells of Boyden chambers and pre-treated or not with 10 ng/ml rhM-CSF. After 18 hours and washing of the cells, fresh media was replaced and media containing 10 ng/ml rhM-CSF or 10 ng/ml rhAng-2 was placed in the top chamber. We found no difference in monocyte migration to the top chamber between the M-CSF- and Ang-2-containing media (Ang-2:

13.6±3.4 and M-CSF: 13.1±2.1 cells/well; p=NS and N=8 per group) (Figure 10B). This data suggests that CD14+ monocytes that transition to Tie2+ in response to pre- treatment with M-CSF no longer migrate in response to M-CSF. To confirm that it is the

Tie2 receptor regulating the migratory ability of these cells, we transfected CD14+ monocytes with either a scrambled siRNA (scrambled siRNA-GFP) or a siRNA targeting the Tie2 receptor (Tie2 siRNA-GFP) for 18 hours. Subsequent to transfection, we separated CD14+ cells into positive and negative fractions based on GFP expression using flow-assisted cell sorting followed by pre-treatment for 18 hours with rhM-CSF. After, we replaced the media and added 10 ng/ml rhAng-2 in the top chamber for 4 hours. We found significantly more migration from the cells transfected with the scrambled siRNA compared to those cells transfected with the Tie2 siRNA

(scrambled: 61.1±3.0 and siTie2: 21.3±1.5 cells/well; p<0.0001 and N=8 per group) suggesting that it is the Tie2 receptor regulating the migration of these CD14+ monocytes (Figure 10B).

Figure 10. M-CSF pre-treatment augments the migratory response to Ang-2 by CD14+ monocytes.

(A) CD14+ monocytes were isolated and cultured in Boyden chemotaxis chambers in minimal media alone (Media) in 0.1, 1, 10, 100 or 300 ng/ml rhAng-2, or with the same

Ang-2 doses but first pre-treated for 24 hours with 10 ng/ml rhM-CSF and analyzed for their migratory ability. A significant synergistic effect of M-CSF pre-treatment was first observed at 1 ng/ml rhAng-2 and peaked at 100 ng/ml rhAng-2. Results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber. (B) CD14+ monocytes were treated with rhAng-2 (10 ng/ml) (Ang-2) or rhM-CSF (10 ng/ml) (M-

CSF) alone, or pre-treated with rhM-CSF (10 ng/ml) for 24 hours, washed, then treated with rhAng-2 (10 ng/ml) for another 24 hours and transfected with a scrambled siRNA or an siRNA targeting the human Tie2 receptor. While Ang-2 and M-CSF did not induce significant migration, the M-CSF-pre-treated cells transfected with the scrambled siRNA migrated significantly more than those cells transfected with Tie2- targeting siRNA Tie2. Results represent the mean ± SEM of CD14+ monocyte migration through the Boyden chamber.

100

A p<0.0001

Ang-2 100 M-CSF pre-treatment p<0.0001 + Ang-2 p<0.0001 80

60 p=0.0003

p=NS

Migrating cells per well 20

0 Media 300 100 10 1 0.1 Ang-2, ng/ml

B p<0.0001 80 p<0.0001 p<0.0001

20 Migrating cells per well

0 Ang-2 M-CSF scrambled Tie2 10 ng/ml 10 ng/ml siRNA siRNA

Ang-2

M-CSF (10 ng/ml) pre-treament

Figure 10. M-CSF pre-treatment augments the migratory response to Ang-2 by CD14+ monocytes.

101

M-CSF augments the angiogenic potential of CD14+ monocytes on

HUVECs via Tie2 receptor up-regulation

Tumor angiogenesis requires endothelial cell proliferation, mobilization, and sprouting blood vessels. The human umbilical vein endothelial cell (HUVEC) tube branching assay is a method to analyze the angiogenic potential of endothelial cells in vitro. It has been shown that conditioned media from M-CSF-treated human monocytes can contribute to endothelial cell tube formation 229. More specifically, conditioned media from TEMs has been shown to increase endothelial tube branching over that observed by conditioned media from a TEM-depleted monocyte population 125. Given our observation that M-CSF increases Tie2 receptor expression on CD14+ monocytes, we hypothesized that pre-treatment of monocytes with M-CSF would increase HUVEC tube branching and that blocking Tie2 receptor expression with an siRNA targeting

Tie2 would abrogate this effect. To determine the angiogenic effects of M-CSF up- regulation of the Tie2 receptor on monocytes, we isolated and transfected CD14+ monocytes with a scrambled siRNA or siRNA targeting Tie2 as described above. Fresh media was added to each sample and conditioned for 24 hours. The cell-free media was collected and used to culture HUVEC cells on growth factor-reduced Matrigel for 6 hours followed by tube branching analysis. Our data suggests that conditioned media from monocytes treated with Ang-2, rhM-CSF, or rhVEGF resulted in augmented branch formation in each case compared to HUVECs cultured in minimal media

(Media: 12.0±1.2, Ang-2: 93.4±9.3, M-CSF: 73.4±3.4, and VEGF: 80.6±6.2 branch points; p=0.0001 and N=5 per group) (Figure 11A). More importantly, conditioned media from those CD14+ cells pre-treated with M-CSF then Ang-2 induced significantly more HUVEC branch points than conditioned media from Ang-2-treated cells alone (M-

CSF/Ang-2: 301.6±7.0 branch points; p<0.0001 and N=5) conditioned media from 102

monocytes pre-treated with the M-CSF-R NAb abrogated the ability of M-CSF/Ang-2 to induce branch points on HUVECs (M-CSF-R NAb/M-CSF/Ang-2: 89.2±7.0 branch points; p<0.0001 vs M-CSF/Ang-2, N=5 and p=NS vs VEGF, Ang-2, or M-CSF and

N=5) (Figure 11A). These data suggest that M-CSF up-regulation of Tie2 induces a more pro-angiogenic CD14+ phenotype. Finally, the role of the Tie2 receptor on

HUVEC branching was confirmed as conditioned media from the CD14+ monocytes transfected with the Tie2 siRNA induced significantly less HUVEC branching compared to conditioned media from the M-CSF/Ang-2 treated cells (siRNA Tie2/M-CSF/Ang-2:

23.2±1.9 branch points; p<0.0001 and N=5) and conditioned media from the scrambled control siRNA (scrambled siRNA: 174.6±10.0 branch points; p<0.0001 and N=5)

(Figure 11B). This data confirms the importance of Tie2 on the angiogenic function of

CD14+/Tie2+ cells.

103

Figure 11. Conditioned media from M-CSF-treated TEMs augments HUVEC branching.

(A) Human monocytes were isolated from whole blood and differentiated to macrophages over five days using rhM-CSF (20 ng/ml). The cells were serum-starved in endotoxin-free RPMI containing polymyxin B (10 µg/ml) for 24 hours then treated with either a neutralizing antibody for M-CSF receptor or isotype antibody (50 ng/ml) for

1 hour prior to stimulation with M-CSF (10 ng/ml) for 18 hours. The cells were then washed three times and treated with Ang-2 (10 ng/ml) for 24 more hours. The conditioned media from these cells (M-CSF/Ang-2; M-CSF-R NAb/M-CSF/Ang-2) or minimal media alone, (minimal), Ang-2 alone (10 ng/ml) (Ang-2), M-CSF alone (10 ng/ml) (M-CSF), or VEGF (10ng/ml) (VEGF) were used to culture 1.5x104 human umbilical vein endothelial cells (HUVEC) on growth factor-reduced Matrigel for 8 hours.

Digital images were taken to determine HUVEC branching. The number of branches was quantified in a blinded manner per field. M-CSF, VEGF, and Ang-2 each stimulated significantly more branch points from the HUVECs than minimal media. The conditioned media from the macrophages pre-treated with M-CSF induced significantly more branch points than M-CSF, VEGF, and Ang-2 treatment alone and pre-treatment of the macrophages with the M-CSF receptor neutralizing antibody significantly reduced branch points comparable to VEGF, M-CSF, and Ang-2 alone levels. Results represent the mean ± SEM of HUVEC branch points. (B) In the same manner as above, human monocytes were differentiated to macrophages over five days and serum-starved for 24 hours. The cells were transfected with a siRNA targeting human

Tie2 receptor (40 nM) or a scrambled siRNA (40 nM) for 24 hours. The macrophages were washed three times with PBS and then treated with M-CSF (10 ng/ml) for 18 more hours. These conditioned media were collected and used to culture HUVEC cells 104

grown on growth factor-reduced Matrigel for eight hours to detect branch points. Digital images were taken to determine HUVEC branching. The macrophages pre-treated with

M-CSF then Ang-2 (M-CSF/Ang-2) induced a significant number of branch points compared to HUVEC cultured in media alone (Media). The conditioned media from the macrophages transfected with siTie2 then treated with M-CSF then Ang-2 (Tie2 siRNA/M-CSF/Ang-2) had significantly less branch points than the M-CSF-conditioned media and similar to the minimal conditions. The macrophages transfected with the scrambled siRNA and pre-treated with M-CSF then Ang-2 (scrmb siRNA/M-CSF/Ang-

2) induced significantly more branch points than the conditioned media from the siTie2 samples but still significantly less than the M-CSF conditioned media. Results represent the mean ± SEM of HUVEC branch points.

105

A p<0.0001 p<0.0001 350

300

250

200

Minimal media VEGF M-CSF 150

Branch points 100 * * * 50

0 Minimal VEGF M-CSF Ang-2 Ang-2 Ang-2 media Ang-2 Ang-2 Ang-2 M-CSF M-CSF M-CSF M-CSF *p<0.0001 vs Minimal media M-CSF-R NAb M-CSF-R NAb

B p<0.0001 p<0.0001 p<0.0001 350

300 p<0.0001

250

200

Minimal media Ang-2 150 M-CSF Branch points 100

0 Media Ang-2 Ang-2 Ang-2

M-CSF M-CSF M-CSF Ang-2 Ang-2 Tie2 scrmb siRNA siRNA M-CSF M-CSF

Tie2 scrmb siRNA siRNA

Figure 11. Conditioned media from M-CSF-treated TEMs augments HUVEC branching.

106

M-CSF-treated CD14+ monocytes alters HUVEC phenotype

Because M-CSF treatment induced CD14+ monocytes to increase Tie2 receptor expression and because conditioned media from these cells augmented the number of branches from HUVECs, we harvested RNA from the HUVECs used in the branching assay to assess changes in mRNA expression levels of transcripts known to be involved in endothelial cell tube formation. Notch signaling is critical for vascular development 235-237. As endothelial cells proliferate to form new vessels, these cells can remain in the vessel stalk or differentiate into tip endothelial cells which reside at the leading edge of new blood vessels. Endothelial tip cells determine new vessel branch points on emerging vasculature. Recently, it has been shown that the Notch signaling molecules Jagged-1 and Delta-like ligand 4 (Dll-4) are important in selecting the proliferating endothelial stalk cells that will take the position of a leading edge tip cell

238, 239. Specifically, high levels of Dll-4 indicate an endothelial cell will remain a stalk cells while high levels of Jagged-1 will mobilize endothelial cells toward tip cell formation. We found that while the expression levels of Dll-4 remained statistically unchanged in all conditions assayed, HUVECs cultured in CD14+ conditioned media pre-treated with M-CSF significantly increased the Jagged-1:Dll-4 mRNA expression ratio toward favoring tip growth over stalk growth. Further, we found that conditioned media from CD14+ cells pre-treated with the M-CSF-R NAb or those cells transfected with siRNA targeting Tie2 receptor suppressed Jagged-1 mRNA up-regulation from

HUVECs. For Dll-4 mRNA expression, Ang-2 or M-CSF increased mRNA expression by 1.4 and 6.1-fold, respectively, when compared with media alone. The combination of the two increased expression by 4.8-fold, which was not significantly lower than each of them alone (p=0.27) (Figure 12). For Jagged-1 mRNA expression, Ang-2 or M-

CSF increased mRNA expression by 4.9 and 10.2-fold, respectively, when compared 107

with media alone. The combination of Ang-2 and MCSF increased Jagged-1 mRNA by

194-fold which is significantly lower than the additive effect of each of the two treatments alone (p=0.0012). Also, gene expression of Dll-4 and Jagged-1 mRNA from

HUVECs treated with Ang-2+M-CSF in combination indicated that Jagged-1 mRNA expression is significantly higher than Dll-4 mRNA (p<0.0001) after adjusting for the baseline media levels. These data suggests that M-CSF treatment of CD14+ monocytes induces the expression of genes that represents a more sprouting phenotype from endothelial cells than Ang-2-treated monocytes alone.

108

Figure 12. Conditioned media from monocytes pre-treated with M-CSF induces a “tip” characteristic on HUVEC.

HUVEC that were cultured in the conditioned media from the assay above were collected from the Matrigel by dispase digestion for 1.5 hours and total RNA collected and cDNA synthesized for RT-PCR analysis of Dll-4 and Jagged-1 mRNA. VEGF (10 ng/ml) (VEGF) induced more Dll-4 mRNA than media alone while Ang-2 (10 ng/ml)

(Ang-2) alone and M-CSF (10 ng/ml) (M-CSF) alone in minimal media had no significant difference between Dll-4 and Jagged-1 mRNA expression, but significantly more than media alone (data not shown) of each gene. Conditioned media from macrophages pre-treated with M-CSF (10 ng/ml) then Ang-2 (10 ng/ml) (M-CSF/Ang-2) induced significantly more Jagged-1 mRNA than Dll-4 mRNA while the neutralizing antibody for M-CSF receptor then M-CSF then Ang-2 conditioned media abrogated this effect on Jagged-1 mRNA expression (M-CSF-R NAb/M-CSF/Ang-2). Conditioned media from macrophages transfected with siTie2 then M-CSF pre-treatment then Ang-

2 (Tie2 siRNA/M-CSF/Ang-2) removed the induction of Jagged-1 mRNA to levels similar to Dll-4 mRNA while the phenotype of the HUVEC cultured in conditioned media from macrophages transfected with scrambled siRNA then M-CSF pre-treatment then

Ang-2 (scrmb siRNA/M-CSF/Ang-2) had Jagged-1 mRNA levels no different than the

M-CSF/Ang-2 samples. Conditioned media from macrophages transfected with either

Tie2 siRNA or scrambled siRNA then pre-treated with M-CSF-R NAb followed by M-

CSF then Ang-2 (Tie2 siRNA/M-CSF-R NAb/M-CSF/Ang-2 or scrmb siRNA/M-CSF-R

NAb/M-CSF/Ang-2) each induced less expression of Jagged-1 mRNA than their non

M-CSF-R NAb-containing counterparts (scrmb siRNA/M-CSF/Ang-2 and Tie2 siRNA/M-CSF/Ang-2). Results represent the mean ± SEM of fold change Jagged-1 or

109

Dll-4 mRNA relative to media alone conditions and standardized to GAPDH control mRNA as determined by RT-PCR.

110

DII-4 Jagged-1 21 p=0.0012 p<0.0001 p<0.0001 20 19 p=0.270 p=0.480 p=0.480 18

17 p<0.0001 16 15 14

dCt over GAPDH 13 12 11 10 VEGF M-CSF Ang-2 Ang-2 Ang-2 Ang-2 Ang-2 Ang-2 Ang-2

M-CSF M-CSF M-CSF M-CSF M-CSF M-CSF

M-CSF-R scrmb M-CSF-R Tie2 M-CSF-R NAb siRNA NAb siRNA NAb

scrmb Tie2 siRNA siRNA

Jagged-1: sprout growth

DII-4: stalk growth

Figure 12. Conditioned media from monocytes pre-treated with M-CSF induces a “tip” characteristic on HUVEC.

111

M-CSF treatment increases the tumor F4/80+/Tie2+ TEM population and augments angiogenesis while having no effect on tumor growth

High serum levels of M-CSF correlate with poor prognosis and increased metastasis in patients with breast cancer 240. Further, increased levels of M-CSF in the tumor microenvironment correlate with increased vessel density and metastasis.

Because TEMs are reported as being one of the most potent cell types in supporting vascularization, and because we found that M-CSF up-regulates Tie2 receptor expression on CD14+ monocytes, we asked if M-CSF treatment would expand the number of tumor TEMs and if increased numbers would alter angiogenesis. PyMT tumor cells were injected subcutaneously into the number 4 mammary fat pad of wild type C57/Bl6 mice and the tumors were grown until palpable (0.3 mm x 0.3 mm). After, intratumoral injections with PBS, M-CSF, an M-CSF-R neutralizing antibody, M-CSF-R

NAb pre-treatment in combination with M-CSF, isotype IgG antibody, or isotype IgG antibody pre-treatment in combination with M-CSF were performed every other day for two weeks. Tumor growth data illustrates no significant increase in tumor growth rate from tumors treated with M-CSF compared to PBS. Interestingly, there was a significant increase in tumor growth rate from the M-CSF-R NAb treatment as well as a significant marked decrease in tumors treated with isotype IgG Ab alone (Trend differences: PBS vs M-CSF: 0.38 mm3/day, p=0.2581; M-CSF vs M-CSF+M-CSF-R

NAb: 0.7194 mm3/day, p=0.0336; M-CSF+M-CSF-R NAb+PBS: 1.2845 mm3/day, p=0.0004; N=7 mice per group) (Figure 13A).

Upon sacrifice, these tumor were collected, homogenized using collagenase, immunostained with F4/80 and Tie2 receptor antibodies and subjected to flow cytometry for the percent total F4/80+ cells and percent F4/80+/Tie2+ TEMs relative to tumor cells. M-CSF treatment induced a significant increase in both overall tumor 112

F4/80+ cells (PBS: 29.6±1.8%, M-CSF: 38.6±1.4%, M-CSF-R NAb: 14.9±3.8%, M-

CSF-R NAb/M-CSF: 27.0±2.8%, M-CSF/isotype IgG: 28.5±3.6%, and isotype IgG:

24.5±5.8% total F4/80+ cells; p=NS for PBS vs M-CSF, p<0.0001 for M-CSF vs M-

CSF-R NAb/M-CSF, and p<0.0001 for M-CSF-R NAb/M-CSF vs IgG/M-CSF and N=at least 5 mice per treatment group) and the population of tumor F4/80+/Tie2+ TEMs

(PBS: 60.8±1.5%, M-CSF: 70.7±1.8%, M-CSF-R NAb: 44.4±10.6%, M-CSF-R NAb/M-

CSF: 52.1±2.1%, isotype IgG/M-CSF: 67.0±3.4%, and isotype IgG: 41.5±8.4%

F4/80+/Tie2+ TEMs; p=0.0003 for PBS vs M-CSF, p<0.0001 for M-CSF vs M-CSF-R

NAb/M-CSF, and p<0.0001 for M-CSF-R NAb/M-CSF vs IgG/M-CSF and N=at least 5 mice per treatment group) (Figure 13B). These data suggest that M-CSF contributes more to the expansion of the population of tumor TEMs than overall F4/80+ cells.

Because macrophages are reported to drive tumor progression and angiogenesis, we analyzed sections of these tumors for an increase in blood vessel formation in response to M-CSF. We observed significantly more CD31+ blood vessels in the tumors treated with M-CSF compared to those tumors treated with PBS or those tumors pre-treated with the neutralizing antibody toward the M-CSF receptor plus M-

CSF treatment (PBS: 1.7±0.1%, M-CSF: 4.4±0.2%, M-CSF-R NAb+M-CSF: 1.4±0.2%,

M-CSF-R NAb: 1.2±0.3%, M-CSF+IgG: 3.0±0.5%, IgG: 2.0±0.5% CD31+ pixels per

HPF; p<0.0001 for PBS vs M-CSF-treatment, p<0.0001 for M-CSF vs M-CSF-R

NAb+M-CSF, and p=0.0085 for M-CSF vs M-CSF+IgG and N=5 tumor sections from 5 mice per group) (Figure 13C). Further, the M-CSF-R antibody reduced blood vessel formation to levels similar to PBS (p=NS). These data highlight the role of M-CSF in tumor angiogenesis.

113

Figure 13. M-CSF has no effect on tumor growth but increases TEM numbers and augments angiogenesis.

(A) 1x106 PyMT tumor cells were implanted into the number 4 mammary fat pad of wild type C57Bl/6 female mice. After becoming palpable (about 2 weeks), the tumors were intratumorally injected with PBS, M-CSF (100 ng in 100 µls) (M-CSF), M-CSF receptor neutralizing antibody (50 ng/ml) (M-CSF-R NAb), M-CSF (100 ng in 100 µls)+M-CSF-R

NAb (50 ng/ml) (M-CSF-R NAb+M-CSF), M-CSF (100 ng in 100 µls)+isotype antibody

(50 ng/ml) (M-CSF+IgG), or isotype antibody alone (50 ng/ml) (IgG) for 2 additional weeks. M-CSF treatment had no significant effect on tumor growth compared to PBS- treated tumors. Results represent the mean ± SEM of tumor volume. (B) After two weeks of treatment, these tumors were removed, homogenized and immunostained with antibodies specific for F4/80 and Tie2 to identify total F4/80+ cells and

F4/80+/Tie2+ cells (Tie2-expressing macrophages, TEMs). While there was a marked increase in total F4/80+ macrophages with M-CSF treatment, the percent of

F4/80+/Tie2+ TEMs was significantly increased in response to M-CSF suggesting a regulatory role for M-CSF in expanding the TEM population. Results represent the mean ± SEM of total F4/80+ and F4/80+/Tie2+ TEMs within the tumors. (C, top)

Orthotopically implanted PyMT mammary tumors in wild type C57Bl/6 female mice were allowed to become palpable then intratumoral treated with PBS (PBS), M-CSF

(100 ng in 100 µls) (M-CSF), a neutralizing antibody for the M-CSF receptor 4 hours prior to M-CSF treatment (100 ng in 100 µls) (M-CSF-R NAb+M-CSF), the M-CSF receptor antibody alone (M-CSF-R NAb), an isotype antibody 4 hours prior to M-CSF

(100 ng in 100 µls) treatment (M-CSF+IgG), or the isotype antibody alone (IgG) three times per week for two additional weeks. The tumors were homogenized and immunostained with a CD31-Alexa Flour 546 antibody to recognize endothelial cells 114

that comprise blood vessels. Qualitatively, M-CSF treatment increased the percent of

CD31-postitive pixels per high powered field compared to PBS treated tumors, while the neutralizing antibody to M-CSF receptor suppressed the M-CSF effect on angiogenesis. (C, bottom) Quantitatively, the percent of CD31+ pixels per high powered field were quantified as blood vessels (angiogenesis) using Adobe Photoshop histogram analysis. M-CSF treatment significantly increased CD31-positive pixels

(angiogenesis) compared to PBS. The neutralizing antibody for M-CSF receptor significantly reduced the ability of M-CSF to up-regulate angiogenesis. Results represent the mean ± SEM of percent CD31-positive pixels per high powered field

(HPF).

115

A 0.9 PBS 0.8 M-CSF M-CSF-R NAb 0.7 M-CSF+M-CSF-R NAb M-CSF+IgG IgG 3 0.6

0.5 p=0.0004

0.4 p=0.2581

0.3 Tumor volume, cm Tumor

0.2

0.1

0 1 2 3 4 5 6 7 Measure day

B All Macs (F4/80+) p<0.0001 TEMs (F4/80+/Tie2+) p<0.0001 p=0.0003 80 80 % F4/80+/Tie2+ TEMs of total F4/80+ cells (gray)

70 70

60 60

50 50

40 40

30 30

20 p=NS 20

10 10 p<0.0001 p<0.0001 0 0 % Total F4/80+ cells relative to tumor (black) % Total PBS M-CSF M-CSF M-R IgG IgG + M-R NAb +M-CSF NAb

Figure 13. M-CSF has no effect on tumor growth but increases TEM numbers and augments angiogenesis.

116 Continued Figure 13 Continued

C PBS M-CSF M-CSF-R NAb + M-CSF CD31 CD31 CD31

20x 40x 20x 40x 20x 40x

M-CSF-R NAb M-CSF + IgG IgG CD31 CD31 CD31

20x 40x 20x 40x 20x 40x

8.0 p=0.0085

7.0 p<0.0001 p<0.0001

6.0

5.0

4.0

3.0

2.0 CD31 + pixels per HPF

1.0

0.0 PBS M-CSF M-CSF-R M-CSF-R M-CSF IgG NAb NAb + IgG + M-CSF

117

High serum levels of M-CSF expands a circulating TEM population

Because we observed an increase in the number of TEMs within the tumors of the M-CSF-treated mice, we hypothesized that M-CSF may be inducing a more global effect on TEM expansion. Because breast cancer patients can have significant increases in serum M-CSF levels 233, we emulated these levels in non-tumor bearing wild type mice to see if in the absence of a tumor that M-CSF could expand the circulating levels of TEMs. We intravenously injected PBS or M-CSF at a concentration which emulates human breast cancer patients (20 ng/ml). We found no difference between treatment groups in CD45+/Tie2+ cells in the bone marrow (described as

TEM bone marrow precursors 122, 123) (PBS: 1.7±0.2%, M-CSF: 1.7±0.2% TEMs; p=NS and N=5 mice per group). Interestingly, we observed a significant reduction in the percent of CD11b+/CD31-/Gr-1lo/Tie2- monocytes in (PBS: 85.5±0.5%, M-CSF:

76.1±0.7% TEMs; p<0.0001 and N=5 mice per group) while at the same time observing a significant increase in the percent of CD11b+/CD31-/Gr-1lo/Tie2+ cells

(TEMs) with M-CSF treatment (PBS: 16.9±1.4%, M-CSF: 30.5±1.6% TEMs; p<0.0001 and N=5 mice per group) (Figure 14A). This data suggests that breast tumors producing M-CSF can regulate TEM expansion while in circulation and not solely once monocytes enter the tumor proper.

To understand if PyMT tumor-bearing mice produce and release a significant amount of M-CSF into circulation compared to non-tumor-bearing wild type mice, we collected atrial blood from mice treated with PBS or M-CSF as described above, or from wild type mice which did not have tumors and subjected the serum to M-CSF

ELISA. Not surprisingly, compared to non-tumor bearing wild type mice, the tumor- bearing mice receiving exogenous intratumoral M-CSF had significantly higher levels of

M-CSF while the tumor-bearing mice receiving PBS had higher levels but not 118

statistically different than the wild type mice (Control, non-tumor-bearing: 6.5±0.9, PBS- treated tumor-bearing: 8.8±1.2, and M-CSF-treated tumor bearing: 10.9±0.6 pg/ml; p=0.05 for Control vs M-CSF-treated, p=NS for control vs PBS-treated and PBS- treated vs M-CSF-treated and n=4 per group) (Figure 14B). These data indicate that tumors produce M-CSF and increase these levels in the serum.

119

Figure 14. M-CSF expands the TEM population in peripheral blood.

Atrial blood was collected at sacrifice from the PyMT tumor-bearing mice subjected to

M-CSF or PBS treatment. Also, atrial blood was collected from wild type, non-tumor bearing mice as a control. Serum was isolated and subjected to M-CSF ELISA. M-CSF was significantly higher in the serum of M-CSF-treated tumors compared to control, non-tumor bearing mice and markedly increased but not statistically different than those PyMT tumor-bearing mice given PBS. Results represent the mean ± SEM of serum M-CSF in pg/ml. To reach serum levels of M-CSF similar to breast cancer patients and determine that effect on TEM expansion, PBS or M-CSF (20 ng/ml) was intravenously injected into non-tumor bearing wild type C57Bl/6 female mice every other day for a total of three treatments. Bone marrow and atrial blood was collected and immunostained with CD45 and Tie2 antibodies (for bone marrow) or CD11b,

CD31, Gr-1, and Tie2 antibodies (for blood). There was no difference in the percentage of TEMs in the bone marrow of the PBS- and M-CSF-treated mice. In peripheral blood,

M-CSF treatment significantly reduced the percent of CD11b+/CD31+/Gr-1lo/Tie2- cells while significantly increasing CD11b+/CD31+/Gr-1lo/Tie2+ TEMs. Results represent the mean ± SEM of percent total CD45+ and CD45+/Tie2+ cells in the bone marrow, and the percent of CD11b+/CD31-/Gr-1lo/Tie2- and CD11b+/CD31-/Gr-

1lo/Tie2+ TEMs in the blood.

120

A 100 p<0.0001 100

90 PBS 90 M-CSF 80 80

p<0.0001 % Cell population 70 70

60 60

50 50

40 40

30 30

20 20

10 p=NS 10 % TEM population of total CD45+ cells 0 0 Bone marrow Tie2- Tie2+ (CD45+/Tie2+) Blood (CD11b+/CD31-/Gr-1lo)

B p=0.05 p=NS 12 p=NS

4 Serum M-CSF, pg/ml Serum M-CSF, 2

0 Control PBS M-CSF Tumor-bearing mice

Figure 14. M-CSF expands the TEM population in peripheral blood.

121

M-CSF and HIF pathways can independently and synergistically regulate

Tie2 receptor expression on TEMs in vitro

In a previous study, we found that Tie2 receptor expression on monocytes is regulated by oxygen and the HIF pathway, specifically HIF-1α, by showing that bone marrow-derived macrophages from HIF-1αfl/fl/LysMcre mice was unable to increase

Tie2 expression on F4/80+ macrophages during hypoxic episodes while macrophages from wild type LysMcre and HIF-2αfl/fl/LysMcre mice augment Tie2 expression on these cells. Because we show in our current study, we show that M-CSF can also regulate

Tie2 expression on monocytes, we asked if the M-CSF and HIF pathways merge to regulate Tie2 receptor expression on monocytes to generate TEMs. So, we differentiated bone marrow-derived macrophages from HIF-1αfl/fl/LysMcre and wild type

LysMcre mice and stimulated these cells under normal oxygen levels with PBS or 100 ng/ml M-CSF. After 24 hours, we analyzed these F4/80+ cells for expression of Tie2 by flow cytometry. We found no difference in the percent of F4/80+ macrophages expressing Tie2 in the vehicle-treated wild type compared to HIF-1α-deficient cells

(WT: 8.8±0.9% and HIF-1α KO: 9.0±0.9% TEMs; p=NS and N=5 per group) (Figure

15A). In the M-CSF-treated cells, we found that the loss of HIF-1α significantly inhibited the ability of these F4/80+ macrophages from expressing Tie2 (WT: 37.2±1.1% and

HIF-1α: 28.4±2.4% TEMs; p<0.0001 and N=5 per group) (Figure 15A). These data suggest a role for HIF-1α in M-CSF augmentation of TEMs.

Because an appreciable percentage of F4/80+ macrophages still expressed

Tie2-positivity in the HIF-1α KO macrophages treated with M-CSF compared to vehicle treatment, we asked which signaling pathway downstream of the M-CSF receptor regulated Tie2 expression on TEMs. We pre-treated with the appropriate vehicle or the

PI3 kinase/Akt inhibitor LY294002 (50 μM), the MEK inhibitor U0126 (10 μM), or the 122

NF-B inhibitor PDTC (100 μM) for 30 minutes then stimulated with 100 ng/ml M-CSF or not for 24 hours. M-CSF has been reported to activate all three of these signaling pathways 13, 90, 217, 218. Of the three inhibitors assayed, only the inhibitor abrogating PI3 kinase/Akt activity (LY294002) significantly suppressed the percentage of TEMs generated by M-CSF stimulation (Vehicle: 8.8±0.9%, M-CSF: 37.2±1.1%, LY294002:

7.9±1.0% LY294002+M-CSF: 11.3±0.9%, U0126: 8.4±1.5% UO126+M-CSF:

36.2±1.1%, PDTC: 7.1±0.6% PDTC+M-SCF: 36.4±0.8% TEMs; p<0.0001 for vehicle vs M-CSF and p=0.0804 for LY294002 vs LY294002+M-CSF and N=5 per group.

UO126 and PDTC vs their M-CSF-treated counterparts p<0.0001) (Figure 15B).

Further, when we assayed wild type or HIF-1α KO macrophages in the absence or presence of LY294002, we found that the ability of M-CSF to augment Tie2 receptor expression was completely abolished to vehicle-treated levels (WT Vehicle: 7.8±0.9% vs HIF-1α KO Vehicle: 9.0±0.9%; p=NS and WT LY294002+M-CSF: 11.3±0.9% vs

HIF-1α KO LY294002+M-CSF: 7.8±1.1%; p=0.5 and N=5 per group) (Figure 15C).

Taken together, these data suggest that independently, M-CSF and HIF-1α pathways can regulate Tie2 expression on TEMs, but also that there is some level of synergy between to the pathways regulating this activity.

123

Figure 15. M-CSF and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes.

(A) Bone marrow-derived macrophages were differentiated from age-matched wild type LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were treated with PBS or M-CSF (100 ng/ml) for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. M-CSF induced an increase in F4/80+/Tie2+ cells over PBS-treated cells from the macrophages derived from the bone marrow of wild type LysMcre mice. The macrophages derived from the HIF-1αfl/fl/LysMcre bone marrow and treated with M-CSF had a significantly smaller percentage of F4/80+ Tie2+ cells than those from the wild type mice. Results represent the mean ± SEM of percent

F4/80+/Tie2+ cells of total F4/80+ cells. (B) Macrophages were derived from the bone marrow of wild type female mice over five days in non-adherent tubes. The cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were pre-treated with

DMSO (vehicle), the PI3 kinase/Akt inhibitor LY294002 (50 μM) (LY294002), the MEK inhibitor U0126 (10 μM) (U0126), or the NF-Kb inhibitor PDTC (100 μM) (PDTC) for 30 minutes. After, the cells were treated with M-CSF (100 ng/ml) (Vehicle+M-CSF),

LY294002+M-CSF (100 ng/ml) (LY294002+M-CSF), U0126+M-CSF (100 ng/ml)

(U0126+M-CSF), or PDTC+M-CSF (100 ng/ml) (PDTC+M-CSF) or left untreated for 24 hours followed by immunstaining with antibodies specific for F4/80 and Tie2. M-CSF induced an increase in the percent of F4/80+/Tie2+ cells compared to vehicle alone.

The inhibitors LY294002, U0126, and PDTC alone had no effect on TEM levels.

LY294002 pre-treatment significantly reduced the percent of TEMs regulated by M-

CSF while U0126 and PDTC had no effect on M-CSF expansion of TEMs. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells. (C) 124

Bone marrow-derived macrophages were differentiated from age-matched wild type

LysMcre and HIF-1αfl/fl/LysMcre mice over five days in non-adherent tubes. After, the cells were serum-starved in endotoxin-free RMPI for 24 hours. The cells were then pre- treated with DMSO or LY294002 (50 μM) for 30 minutes followed by M-CSF (100 ng/ml) (LY294002+M-CSF) or not (vehicle) for 24 hours and then immunostained with antibodies specific for F4/80 and Tie2. The macrophages derived from HIF-

1αfl/fl/LysMcre mice in combination with the PI3 kinase inhibitor LY294002 significantly reduced the percent TEMs to that similar to untreated levels. Results represent the mean ± SEM of percent F4/80+/Tie2+ cells of total F4/80+ cells.

125

A p<0.0001 p<0.0001

p<0.0001 40 wild type LysMcre 35 HIF-1αfl/fl LysMcre

15 p=NS 10 % F4/80+/Tie2+ cells % F4/80+/Tie2+

0 Vehicle M-CSF (100 ng/ml)

p<0.0001 p<0.0001 p<0.0001 p<0.0001 B 40

25 p=0.0804 20

10 % F4/80+/Tie2+ cells % F4/80+/Tie2+

0 Vehicle Vehicle LY294002 LY294002 U0126 U0126 PDTC PDTC + + + + M-CSF M-CSF M-CSF M-CSF

14 C p=0.05 wild type LysMcre HIF-1αfl/fl LysMcre 12

10 p=NS

4 % F4/80+/Tie2+ cells % F4/80+/Tie2+ 2

0 Vehicle LY294002 + M-CSF Figure 15. M-CSF and HIF pathways independently and synergistically regulate Tie2 receptor expression on monocytes. 126

M-CSF treatment cannot overcome HIF-1α-deficiency to augment tumor growth

In our previous work, we demonstrated a direct correlation between tumor growth rate and the percent of TEMs within PyMT breast tumors. Because we showed in our current study that M-CSF can not only expand the percent of TEMs within tumors, we next examined the effect of M-CSF treatment and resulting expansion of

TEM population on tumor volume in HIF-1αfl/fl/LysMcre mice which lack the ability to up-regulate Tie2 once monocytes enter the tumor proper and as a result grow at a significantly slower rate. We orthotopically injected PyMT tumor cells into the number four mammary fat pad of wild type LysMcre or HIF-1αfl/fl/LysMcre mice. Once palpable, we treated these tumors with M-CSF or PBS every other day for 8 days. In the

LysMcre control mice, M-CSF induced a slight but not significant increase in tumor volume compared to PBS treatment. Not surprisingly, the tumors from the HIF-

1αfl/fl/LysMcre mice grew at a significantly slower rate than the PBS-treated tumors from the wild type LysMcre mice (p<0.0001 and N=5 mice) (Figure 16). The tumors from both the wild type LysMcre and HIF-1αfl/fl/LysMcre mice treated with M-CSF grew slightly, but not significantly, faster (p=NS for PBS- vs M-CSF-treated for both wild type and HIF-1α KO mice and N=5 mice per group). These data suggest that while macrophage HIF-1α is essential for tumor growth, M-CSF receptor activation is not.

127

Figure 16. M-CSF cannot overcome the HIF-1α-deficiency to augment tumor growth

1x106 PyMT tumor cells were orthotopically injected into the number 4 mammary fat pad of age-matched female wild type LysMcre or HIF-1αfl/fl/LysMcre mice. After becoming palpable, the tumors were treated intratumorally with PBS or M-CSF (100 ng in 100 uls) every other day for eight additional days. Tumor measures were recorded on treatment days and volume calculated. There was a significant decrease in tumor growth between the PBS-treated wild type mice (wild type LysMcre+PBS) and the HIF-

1α-deficient mice treated with PBS (HIF-1αfl/fl/LysMcre+PBS) indicating the role of HIF-

1a in tumor growth. M-CSF treatment of both groups (wild type LysMcre+M-CSF and

HIF-1αfl/fl/LysMcre+M-CSF) slightly increased tumor growth, but not significantly.

Results represent the mean ± SEM of tumor volume in cm3 on each measure day subtracting the mean volume from the initial tumor volumes to standardize all groups.

128

6.0

wild type LysMcre + PBS Tx 5.0 wild type LysMcre + M-CSF Tx HIF-1αfl/fl LysMcre + PBS Tx HIF-1αfl/fl LysMcre + M-CSF Tx

3 4.0

p=NS

3.0

Tumor volume, cm Tumor 2.0 p<0.0001

p=NS 1.0

0 1 2 3 4 Measure day

Figure 16. M-CSF cannot overcome the HIF-1α-deficiency to augment tumor growth

129

M-CSF recruits CD14+/Tie2+ TEMs into tumors by regulating Ang-2 and

SDF-1/CXCL12

Angiopoietin-1 and -2 (Ang-1 and Ang-2) are ligands for the Tie2 receptor. Both

Ang-1 and Ang-2 have been shown to be required for the formation of mature blood vessels 115, 118-120. While Ang-1 activates intracellular signaling cascades leading to secure endothelial intracellular adhesions, Ang-2 binding to the Tie2 receptor on endothelial cells loosens cellular adhesion allowing factors such as β-FGF and VEGF to facilitate and maintain endothelial cell proliferation and blood vessel sprouting.

Within the tumor microenvironment, Ang-2 concentrations have been shown to increase and correlate to infiltration of transformed cells to lymph nodes and with metastatic disease in breast cancer 241-243. In addition to Ang-2, SDF-1/CXCL12 is expressed in many tumor types and recruits TEMs, not by the Tie2 receptor, but by the ligand for SDF-1/CXCL12, CXCR4. TEMs express CXCR4 and have been shown to react to SDF-1/CXCL12 161, 162. Because TEMs express CXCR$ 124, 185, 244, and with our observations that M-CSF increases Tie2 receptor expression on monocytes we examined if exogenously-administered M-CSF would regulate Ang-1, Ang-2, SDF-

1/CXCL12 mRNA levels in the PyMT breast tumors. Again, we injected PyMT tumor cells were injected subcutaneously into the number 4 mammary fat pad of wild type

C57/Bl6 mice and the tumors were grown until palpable. After, intratumoral injections with PBS or M-CSF (100 ng in 100 µl) were performed every other day for two weeks.

The tumors were evaluated for two chemokines known to recruit TEMs, Ang-2 and

SDF-1/CXCL12, by RT-PCR. We found a significant upregulation of both Ang-2 mRNA

(2.2-fold) and SDF-1/CXCL12 mRNA (73.5-fold) in the tumors treated with M-CSF compared to PBS-treated tumors (p=0.013 and p<0.0001 for Ang-2 mRNA from M-

CSF-treated tumors vs PBS treatment and SDF-1/CXCL12 mRNA, respectively, and 130

N=4 tumor per group) (Figure 17). Next, because we observed an increase in tumor angiogenesis (CD31+ blood vessels) in response to M-CSF-treatment, we asked if we could detect increases in VEGF mRNA as well. We found that M-CSF induces an 18.0- fold increase in VEGF mRNA compared to the PBS-treated tumors (p<0.0001 and N=4 tumors per group). Taken together, these data suggest that both Ang-2 and SDF-

1/CXCL12 regulate TEM migration into the PyMT tumors and that once there, they express higher levels of VEGF mRNA inducing angiogenesis.

131

Figure 17. M-CSF augments Ang-2 and SDF-1/CXCL12 production in PyMT tumors.

PyMT tumors were collected from wild type LysMcre female mice that received PBS or

M-CSF (100 ng in 100 µls) intratumorally every other day for two weeks. The tumors were homogenized in Trizol and total RNA isolated, cDNA synthesized and subjected to RT-PCR for Ang-2, SDF-1/CXCL12, and VEGF mRNA using GAPDH mRNA as the housekeeping control gene. M-CSF treatment induced a significant increase in each

Ang-2, SDF-1/CXCL12 and VEGF mRNA compared to PBS-treated tumors. Results represent the mean ± SEM of mRNA expression from M-CSF-treated tumors standardized to GAPDH mRNA relative to PBS-treated tumors.

132

75 **

30 **

4 *

realtive to PBS-treated tumors 2 Fold change in mRNA expression Fold change in mRNA 0 Ang-2 SDF-1/ VEGF CXCL12

*p=0.013 vs PBS-treated tumors **p<0.0001 vs PBS-treated tumors

Figure 17. M-CSF augments Ang-2 and SDF-1/CXCL12 production in PyMT tumors.

133

Discussion

This paper establishes a novel role for M-CSF in regulating the expression of the Tie2 receptor on macrophages both in vitro and in vivo in a model of murine breast cancer. Impedes for this study stemmed from independent reports that M-CSF and

TEMs increased tumor vascular density and pulmonary metastasis 98, 122, 126, 182, 186. We hypothesized that because M-CSF has been shown to differentiate macrophages with a pro-tumor, M2 phenotype that M-CSF might also be differentiating a subpopulation of monocytes and macrophages with characteristics of tumor-associated macrophages known as Tie2-expressing monocytes/macrophages (TEMs), and recruited into breast tumors 125, 186.

Although not classified as M2 macrophages, TEMs are reported to maintain a pro-angiogenic, M2-like profile 125, 195. For example, co-injection of isolated TEMs with glioma tumor cells increased vascular area over those glioma cells injected with Tie2 receptor negative monocytes 124. On endothelial cells, the expression of the Tie2 receptor is dynamic, increasing during hypoxia through binding of its ligand, Ang-2, and resulting stimulation of transcription factors Gata3 and Ets-1245, 246. In our current study, we demonstrate that M-CSF treatment induces increased pro-angiogenic activity on both human and mouse monocytes when the Tie2 receptor is expressed. Further we demonstrate that the Tie2 receptor is responsible for this activity. We examined the functional consequences of an expanded TEM population as it pertains to monocyte migration and endotheial cell tube formation. M-CSF augmented Tie2 receptor-driven migration of monocytes toward Ang-2. Previous studies have shown that the Tie2 receptor is required for TEM migration towards Ang-2 and we found that pre-treatment with M-CSF decreases the threshold of Ang-2 required for this response and increases the number of migrating cells, in vitro. This study is of importance because Ang-2 134

levels are known to be expressed in a variety of solid tumors, including breast cancer

241-243, 247. In fact, although Ang-2 overexpression has been shown to increase the number of TEMs in breast cancer 129, monoclonal antibodies blocking Ang-2 inhibits

TEM interaction with blood vessel endothelial cells in a PyMT mouse model of breast cancer, limiting their supportive nature and pro-angiogenic function on endothelium 186.

We found that pre-treatment of human CD14+ monocytes with M-CSF resulted in Tie2 overexpression and a significant increase in endothelial cell branch points from

HUVEC cells when cultured with conditioned media from these TEMs. In both of our

TEM functional studies, antibody neutralization of the M-CSF receptor and knockdown of the Tie2 receptor on monocytes using siRNA targeting Tie2 abrogated the synergistic effect of M-CSF and Ang-2 treatment.

Tie2-expressing monocytes have been shown to express higher levels of both

IL-6 and TNF-α than their Tie2-negative monocyte counterparts to prime endothelial cells to vacate their quiescent state and mobilize to facilitate angiogenesis 125, 195. This

TNF-α effect was shown in a Matrigel plug assay where short-term TNF-α exposure increased CD31+ cell invasion248, 249. TNF-α induces a number of responses from endothelial cells including expression of matrix metalloproteinases, as well as integrin and adhesion molecules 250, 251. One way in which TNF-α facilitates angiogenesis is its instruction on endothelial cells to become “tip cells” during the vessel sprouting process regulated by Notch signaling. Notch has been shown as essential in the formation and restructuring of functional blood vessels 238, 239 in a process in which rapidly proliferating endothelial cells push forward as a sprout at the end of the “stalk” portion of a newly forming vessel. At the leading edge of the vessel, proliferation slows as the endothelial tip cells respond to chemokines and form branch points in accordance with local cytokine signals. In a similar manner, we found that conditioned media from M-CSF 135

and Ang-2 pre-treated monocytes skewed endothelial cells towards a tip cell phenotype by increasing the ratio of tip cell (Jagged-1) to stalk cell (Dll-4) expression.

Because of our observation of expanding TEM population and increase in endothelial cell function with M-CSF pre-treatment, we next asked what effects exogenous M-CSF treatment would have in a PyMT tumor model of breast cancer. We showed that intratumoral administration of M-CSF had a slight but not significant effect on the overall percent of F4/80 positive macrophages in these tumors, while the percentage of F4/80+/Tie2+ TEMs were significantly increased. As stated previously,

TEMs are cells of a pro-angiogenic phenotype which express high levels of VEGF,

MMPs, cathepsin B, and thymidine phosphorylase which break down extracellular matrix facilitating angiogenesis 125. Likewise, M-CSF is known to increase expression of these same factors from macrophages in vitro as well as in models of breast cancer

88, 98, 99, 182, 229, 252. We observed changes in vascular density assayed by CD31-positivity in the tumors treated with M-CSF which corresponded to augmentation of the TEM population.

Tumor cells are reported to express high levels of M-CSF as shown in the serum of patients with various cancer types 178, 233 Serum M-CSF mobilizes bone marrow cells to release and migrate to the tumor site, then activate these cells, differentiate into macrophages and skew their phenotype supporting a Th2 response

253, 254. In turn, these macrophages produce epidermal growth factor (EGF) which enhances tumor cell proliferation 111. This EGF/M-CSF paracrine loop has been shown to increase tumor cell intravasation and, in fact, tumor cells and macrophages alternate leaving the tumor via intravasation into circulation 199. Given the functional similarities between M-CSF and TEMs and our data showing a connection between M-CSF and

TEM expansion, further work to explore a link between TEMs and mechanisms 136

regulating tumor angiogenesis and progression are warranted. Our data support a role for Ang-2 in this process as we found that M-CSF increased Ang-2 levels in our PyMT tumor model. Further, Ang-2-stimulated monocytes have been shown to produce immunoregulatory IL-10 which suppresses T cell proliferation in vitro 129. In tumors, T cells produce IL-4 which primes macrophages to produce EGF to augment tumor cell proliferation 255.

The fact that we observed an expansion of the F4/80+/Tie2+ TEM population yet not an overall expansion of F4/80+/Tie2- macrophages in circulation when delivering M-CSF similar to that of breast cancer patients is relevant. This data suggests that, unlike our previous study of the role of the HIF pathway in the generation of TEMs within the tumors and not expanded in the peripheral blood, that

M-CSF can actuate TEM differentiation from monocytes already in circulation which have potential to migrate to the tumor and induce Tie2 expression and recruit them in yet another manner besides MCP-1/CCL2, by Ang-2.

In summary, we hypothesized that M-CSF, which induces an M2 macrophage phenotype, would expand the population of macrophages that also express the Tie2 receptor, normally expressed on endothelial cells. We found that M-CSF can differentiate TEMs and that these cells are more responsive to Ang-2 stimulation – resulting in increased cell migration and pro-angiogenic potential on HUVECs. Further, we found that M-CSF treatment in a mouse model of breast cancer increases tumor angiogenesis but had limited effect on tumor growth. This activity was determined to be regulated by each the PI3 kinase pathway and HIF pathway independently, or in a synergistic manner. Finally, we found that M-CSF can expand the TEM population while in circulation and that M-SCF treatment augment SDF-1/CXCL12 levels in the

137

tumor to recruit these TEMs. This is relevant because breast cancer patients have elevated levels of serum M-CSF.

Acknowledgements

This work was supported by National Cancer Institute Grant R00 CA131552 (to

T.D.E.), start-up funds (to T.D.E.), and R01 HL067167 (to C.B.M.).

138

Chapter 4: Conclusions and Future Directions

These studies demonstrate the effect of the tumor microenvironment on the immune system, specifically on a subset of macrophages identified as

TEMs. The phenotype of a macrophage is known to be affected by the local tissue environment. Factors produced during microbial infections lead the development of pro-inflammatory or M1 macrophages whereas the tumor microenvironment skews myeloid cell towards a repair and immune modulatory phenotype an M2. This work establishes a novel role for HIF-1α and M-CSF in the regulation of the proangiogenic receptor Tie2 on monocytes and macrophages. Before the seminal studies by Naldini 124, 185, 195 and Lewis 185 characterizing Tie2 expression on myeloid lineage cells, Tie2 receptor expression was exclusively known to endothelial biology. Since this identification, TEMs have been characterized as a fixed population. However, data from the literature did not support this view. For example, the percent of

TEMs isolated from human peripheral blood varies from between 2 to 20 percent of the isolated CD14+ cells 124, 185. Further, on endothelial cells, expression of the Tie2 receptor is known to fluctuate, as it increases during embryology and periods of growth and is down regulated on mature vessels 256- 139

258. In our initial study, we demonstrate that expression of the Tie2 receptor can be modulated on myeloid lineage cells. Further, we demonstrate that HIF-1α transcriptional activity affects the differentiation of TEMs found in tumors.

Interestingly, the number of TEMs and not total macrophages corresponds to increases in tumor volume and the intravasation of tumor cells to the vasculature. In these studies, the tumors that contained more TEMs showed a greater metastatic potential resulting in the formation of more pulmonary micrometastases. This is important because macrophages are known to produce angiogenic factors such as VEGF and cellular proteases which facilitates these processes. TEMs have recently been shown to produce these factors in significantly higher amounts than their Tie2 receptor negative counterparts 125, 195. Finding that TEMs, and not total macrophages, correlate to tumor cell intravasation and metastasis illustrates that further study is needed to define the role of TEMs in driving these processes.

Previous work from our group established that M-CSF induced monocytes and macrophages to produce VEGF, but we did not extend the study to characterize the phenotype of these cells after M-CSF activation 229.

Our current study establishes a new role for M-CSF in tumor angiogenesis and progression through regulation of the Tie2 receptor on monocytes. We demonstrate that M-CSF augmentation of the receptor increases monocyte motility as conditioned media from M-CSF-treated TEMs augments HUVEC vessel branching. Further, we demonstrate that M-CSF increases the percentage of TEMs in the peripheral blood and boosts Ang-2 levels in tumors 140

facilitating TEM tumor infiltration resulting in greater tumor angiogenic area.

Further, we observed that M-CSF does not significantly increase the total number of F4/80+ macrophages, but instead significantly expands the TEM subpopulation. It is important to note that our work examines early tumor events as M-CSF has been shown to enhance the overall number of macrophages in tumors late in development. Further study is needed to define how an expanded TEM population may lead to an increase in total macrophages later in tumor progression. Seminal studies from Pollard’s group illustrate a direct correlation between macrophage number and tumor progression 98, 99, 259. As his group illustrated, knockout of M-CSF resulted in a significant reduction in tumor macrophages; however, this report fails to examine the effect of macrophage phenotype on this process. Initially, depletion of macrophages from tumors seemed a possible therapeutic route to inhibit tumor progression. However, clinical therapies that target tumor macrophages result in a rebound effect in which macrophage progenitors are quickly released from the bone marrow migrate to the tumors and result in an overall increase in progression. Given our results that the percent of tumor

TEMs correspond to up regulation of angiogenesis, intravasation and metastasis, more specific and targeted therapies aimed at the Tie2 receptor on macrophages may prove more advantageous in limiting progression.

Macrophages have been shown to participate in metastatic spread through the

M-CSF and EGF paracrine loop. It is hypothesized that TEMs function in a paracrine manner and these cells have been shown to have a specific cytokine 141

profile that enable such activity. Examination into the direct effects of TEMs on tumor cell motility and EMT transition is warranted. We have shown that TEMs migrate toward Ang-2 which requires activation and reorganization of the cytoskeleton; further work is needed to examine what possible effects the

Tie2/Ang-2 axis may have on tumor cell mobility. The EMT allows tumor cells to lose epithelial cellular adhesions which increase motility and metastasis. It would be interesting to examine if TEMs play a role in the differentiation of tumor cells.

These studies highlight the importance of Tie2 receptor expression on mononuclear phagocytes and define two cellular pathways, HIF-1α and M-CSF, which modulate expression of the Tie2 receptor on myeloid cells. However, direct genetic oblation of the Tie2 receptor from macrophages is needed. To this end, a floxed Tie2 mouse is being developed in which loxP restriction sites have been incorporated around the Tie2 promoter gene for removal upon Cre recombinase overexpression. Several TEM knockout strategies have been presented in the literature. The first developed was a total Tie2 receptor knockout and was embryonic lethal. In the study of TEMs, miRNA and HSV-TK strategies have also been developed: miRNA strategies including knockdown of the Tie2 receptor through the expression of miRNA142 and miRNA126 by lentiviral infection 122, 186, 195. However, with these lentiviral systems, the integration site is critical to expression for the targeted knockdown. Further, miRNAs are known to be promiscuous effecting the expression of a number of functionally-linked transcripts. For example miRNA126 was chosen because it 142

is expresses late in hematopoiesis and would knockdown expression of the

Tie2 receptor without affecting hematopoietic progenitor cells. However, endothelial progenitor cells which also express the Tie2 receptor arise from both the bone marrow stroma and through the CD45 lineage cells. EPCs function in tumor angiogenesis and this strategy may have effected this critical sub-population of cells and alter the phenotype attributed to Tie2 knockdown on myeloid cells 260-262. Knockdown of the Tie2 receptor using HSV TK strategies also pose problems. For example, timing the administration of ganciclovir can result in early versus late tumor effects. The best strategy to eliminate the Tie2 receptor from myeloid lineage cells is through the use of tissue-specific promoters. The Tie2-floxed mouse will allow knockout of the Tie2 receptor in a lineage specific manner. The LysMcre transgenic mouse places the Cre recombinase under the control to the macrophages specific lysosome M promoter. Genetic crossing the Tie2-folxed mouse with LysMcre mice will allow investigation into the effect of Tie2 knockout on macrophages, specifically, and how it effects tumor growth, angiogenesis and metastasis.

143

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