CANCER IMMUNOTHERAPY: TARGETEDCELLULAR VEHICLE-MEDIATEDIMMUNOGENE THERAPY AND DENDRITIC
CELL-BASED VACCINEYOVITA IDA PURWANTINATIONAL UNIVERSITY OF SINGAPORE

2013

CANCER IMMUNOTHERAPY: TARGETEDCELLULAR VEHICLE-MEDIATEDIMMUNOGENE THERAPY AND DENDRITIC
CELL-BASED VACCINEYOVITA IDA PURWANTI(B.Sc.Hons., National University of Singapore)

A THESIS SUBMITTEDFOR THE DEGREE OF DOCTOR OF
PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE&
INSTITUTE OF BIOENGINEERING AND
NANOTECHNOLOGY (A*STAR)
2013

DECLARATIONI hereby declare that the thesis is my original work and it has been written by
me in its entirety. I have duly acknowledged all the sources of information
which have been used in the thesis.
This thesis has also not been submitted for any degree in any university
previously.

Yovita Ida Purwanti
20 Aug 2013

Acknowledgements
I would like to express my gratitude to my supervisor A/P Wang Shu forproviding me the opportunity to work on this project. Thank you for yoursupport and guidance which have allowed me to learn and make tremendous
progress in my research and thinking abilities throughout my candidature.
I would like to thank my past and present lab mates in IBN and DBS, NUS. I
deeply appreciate all the help and advices I have received for my project.
Special thanks to Tim and Lam for the fun, laughter and friendships that havemade my lab life fruitful and memorable.
I am grateful for my loving parents and sisters. Thank you for supporting mydecision to embark on this journey and for the care and reliance that I canalways turn to.
I would also like to thank Alvin, Meirita, Elis, Budi, Sin Man, Yunika and allother good friends of mine whom I cannot possibly name one by one. I am
grateful for all the encouragements which have motivated me a great deal
throughout this PhD journey.
Lastly, I would like to acknowledge the National University of Singapore andthe Institute of Bioengineering and Nanotechnology for the opportunity andsupport granted to me to do a PhD.

“Bless the Lord, O my soul, and do not forget all His benefits”– Psalm 103:2

iTable of Contents

ACKNOWLEDGEMENTS ....................................................................................................... ITABLE OF CONTENTS .......................................................................................................... II
SUMMARY .............................................................................................................................. V
LIST OF TABLES ................................................................................................................. VII
LIST OF FIGURES ............................................................................................................... VIII
LIST OF ABBREVIATIONS ................................................................................................... X
LIST OF PUBLICATIONS ................................................................................................... XIII
CHAPTER I: INTRODUCTION ............................................................................................... 1
1.1
CANCER IMMUNOLOGY.............................................................................................. 2
1.1.1 Tumor antigen recognition and presentation by dendritic cells ........................... 2
1.1.1.1 Dendritic cells as professional antigen presenting cells ...................................... 2
1.1.1.2 Tumor antigen presentation.................................................................................. 3
1.1.1.3 Dendritic cells bridge the innate and adaptive immunities .................................. 5
1.1.2 Cytotoxic T Lymphocytes: professional killers of immune system ........................ 6
1.1.2.1 Activation of cytotoxic T lymphocytes .................................................................. 6
1.1.2.2 Antitumor effects of cytotoxic T lymphocytes ....................................................... 7
1.1.3 Tumor evasions of dendritic cells surveillance and cytotoxic T lymphocytes
killing mechanisms ............................................................................................................ 8
1.2
CANCER IMMUNOTHERAPY ...................................................................................... 10
1.2.1 Stem cells as cellular delivery vehicle for cancer gene immunotherapy ............ 10
1.2.1.1 Stem cell candidates for immunotherapy ............................................................ 10
1.2.1.2 Stem cell delivery of cytokine for cancer immunotherapy .................................. 12
1.2.1.3 Immunotherapy via in situ antibodies delivery by stem cells ............................. 13
1.2.2 Dendritic cell-based vaccinations ...................................................................... 15
1.2.2.1 Dendritic cells as an excellent candidate for developing therapeutic vaccines
against cancer ................................................................................................................. 15
1.2.2.2 Loading dendritic cells with tumor-specific antigens ......................................... 16
1.2.3 Other approaches ............................................................................................... 18
1.2.3.1 Adoptive T cells for cancer therapy .................................................................... 18
1.2.3.2 Genetic engineering of T cells ............................................................................ 19
1.2.4 Challenges in cancer immunotherapy ................................................................ 20
1.3
PURPOSES AND MOTIVATIONS.................................................................................. 22
CHAPTER II: ANTITUMOR EFFECTS OF CD40 LIGAND-EXPRESSING
ENDOTHELIAL PROGENITOR CELLS DERIVED FROM HUMAN IPS CELLS IN AMETASTATIC BREAST CANCER MODEL ........................................................................ 24
2.1
INTRODUCTION ........................................................................................................ 25
2.1.1 EPCs ................................................................................................................... 25
2.1.1.1 Definition, Sources and characterization ........................................................... 25
2.1.1.2 EPCs gene therapy strategies ............................................................................. 26
2.1.1.2.1
Suicide gene therapy .................................................................................. 26
2.1.1.2.2
Antiangiogenic therapy .............................................................................. 27
2.1.1.2.3
Immunotherapy .......................................................................................... 28
2.1.2 CD40 ligand ....................................................................................................... 29
2.1.3 Induced pluripotent stem cells ............................................................................ 30

ii

2.1.4 Objective and Aim of Study ................................................................................ 31
2.2
MATERIAL AND METHODS....................................................................................... 33
2.2.1 Cell culture ......................................................................................................... 33
2.2.2 Stromal-based EPC derivation method .............................................................. 35
2.2.2.1 OP9 co-culture ................................................................................................... 35
2.2.2.2 M2-10B4 co-culture............................................................................................ 36
2.2.3 Non-stromal-based EPC derivation method ....................................................... 36
2.2.3.1 2-D culture ......................................................................................................... 36
2.2.3.2 Embryoid bodies method .................................................................................... 37
2.2.4 Characterization of EPCs ................................................................................... 38
2.2.4.1 Flow cytometry ................................................................................................... 38
2.2.4.2 Immunostaining .................................................................................................. 38
2.2.4.3 Tubulogenesis assay ........................................................................................... 38
2.2.4.4 DiI-Ac-LDL assay ............................................................................................... 39
2.2.5 Baculoviral vector preparation .......................................................................... 39
2.2.6 Animal studies .................................................................................................... 41
2.2.6.1 Animals ............................................................................................................... 41
2.2.6.2 Dual in vivo imaging system ............................................................................... 41
2.2.6.3 Biodistribution of EPCs in intracranial 2M1 tumor model ................................ 42
2.2.6.4 Therapeutic studies of EPCs ............................................................................... 42
2.2.7 Histology ............................................................................................................ 43
2.2.8 Statistical analyses ............................................................................................. 43
2.3
RESULTS .................................................................................................................. 44
2.3.1 Generation of EPCs from Human Pluripotent Stem Cells .................................. 44
2.3.1.1 OP9 co-culture method ....................................................................................... 44
2.3.1.2 M2-10B4 co-culture method ............................................................................... 47
2.3.1.3 Non-stromal 2-D differentiation method ............................................................ 51
2.3.1.4 Human iPS cell-derived EPCs via embryoid bodies formation .......................... 53
2.3.2 Tumor tropism of iPS-EPCs ............................................................................... 58
2.3.2.1 Homing of hPSC-EPCs to 4T1-luc orthotopic breast cancer model .................. 58
2.3.2.2 Homing of iPS-EPCs to breast cancer lung metastasis model ........................... 63
2.3.2.3 Tumor tropism of iPS-EPCs to 2M1 invasive glioma model .............................. 65
2.3.3 Effects of iPS-EPCs on tumor development and metastasis ............................... 67
2.3.4 Genetic modification of EPCs ............................................................................ 72
2.3.5 EPCs therapeutic effects ..................................................................................... 74
2.3.5.1 iPS-EPC expressing CD40L impede tumor development in a breast cancer lungmetastasis model .............................................................................................................. 74
2.3.5.2 iPS-EPCs expressing HSV-tk.............................................................................. 76
2.3.5.3 iPS-EPCs expressing Isthmin ............................................................................. 77
2.4
DISCUSSION ............................................................................................................. 80
2.4.1 Derivation of EPCs............................................................................................. 80
2.4.2 Tumor tropism of iPS-EPCs ............................................................................... 85
2.4.3 Effect of iPS-EPCs in cancer growth and metastasis ......................................... 86
2.4.4 Immunotherapy of EPCs using CD40L .............................................................. 87
2.4.5 Challenges and future direction ......................................................................... 90
CHAPTER III: TARGETED CANCER THERAPY USING CYTOTOXIC TLYMPHOCYTES ACTIVATED BY DENDRITIC CELLS PULSED WITH CANCERSTEM CELL-LIKE CELLS ..................................................................................................... 94
3.1
INTRODUCTION ........................................................................................................ 95
3.1.1 Cancer stem cells ................................................................................................ 95
3.1.2 Objective ............................................................................................................. 96

iii

3.2
MATERIAL AND METHODS ....................................................................................... 99
3.2.1 DCs and naïve T cells derivation from PBMC ................................................... 99
3.2.2 Tumor lysate preparation ................................................................................... 99
3.2.3 DCs pulsing with tumor lysate and maturation ................................................ 100
3.2.4 CTL stimulation and expansion ........................................................................ 100
3.2.5 Flow cytometry ................................................................................................. 100
3.2.6 ELISPOT .......................................................................................................... 101
3.2.7 Statistical analyses ........................................................................................... 102
3.3
RESULTS ................................................................................................................ 102
3.3.1 DCs derivation and characterization ............................................................... 102
3.3.2 Naïve T cells selection and characterization .................................................... 107
3.3.3 IFNγ production of CTL activated by CSC-like-CRC-pulsed DC..................... 109
3.3.4 IFNγ production of CTL activated by CSC-like-glioma-pulsed DC ................. 110
3.4
DISCUSSION ........................................................................................................... 112
3.4.1 DC differentiation and characterization .......................................................... 112
3.4.2 Activated CTLs display appropriate co-stimulatory molecules and antigenspecific targeting ........................................................................................................... 114
3.5
FUTURE DIRECTION ............................................................................................... 116
CHAPTER IV: CONCLUSION ............................................................................................. 119
CHAPTER V: BIBLIOGRAPHY .......................................................................................... 124
APPENDICES ........................................................................................................................ 138iv

Summary

Cancer immunotherapies have treated many cancer patients and improved
their quality of life. In spite of their clinical effects, the available treatments
using cytokines and antibodies are still hindered by their toxic effects, half-lifeand efficacies. In this project, we are interested in the developments ofimmunotherapies using the stem cell vehicles to deliver immunogene products
and the dendritic cell (DC)-based vaccination approach.
Targeted immuno-gene therapy approach using the stem cell delivery
vehicle is based on the inherent tumor tropism of stem cells. Endothelial
progenitor cells (EPCs) is particularly attractive, not only due to their intrinsic
tumor tropism but also their involvement in cancer angiogenesis. However,collecting a sufficient amount of EPCs is one of the challenging issues criticalto achieving effective clinical translation of this new approach. In this study,
we sought to explore whether human induced pluripotent stem (iPS) cells
could be used as a reliable and accessible cell source to generate uniform
human EPCs with cancer gene therapy potential. We showed that by using anembryoid body formation method, CD133+CD34+ EPCs could be efficiently
derived from human iPS cells. The generated EPCs expressed endothelial
markers such as CD31, Flk1 and VE-cadherin but not the CD45 hematopoietic
marker. Subsequently, we showed that intravenously injected iPS cell-derived
EPCs migrated towards orthotopic and lung metastatic tumors in the mouse
4T1 breast cancer model, and that injection of the EPCs alone did not escalate
tumor growth and metastatic progression. Most importantly, the systemic
injection of EPCs transduced with baculovirus encoding the potent DC cov

stimulatory molecule CD40 ligand could impede tumor growth, leading toprolonged survival of the tumor-bearing mice. Therefore, our findings suggestthat human iPS cell-derived EPCs could potentially serve as tumor-targeted
cellular vehicles for anticancer gene immunotherapy.
Despite their proven effectiveness in reducing the tumor burden, mostof the available cancer treatments, including chemotherapy and radiation
therapy, fail in eradicating cancer stem cells (CSCs). With their capability for
self-renewal and differentiation, CSCs are capable of re-establishing the tumor
mass, resulting in the relapse of tumors in patients. By utilizing baculoviruszinc-finger technology, we have reprogrammed human glioma and colorectal
cancer cell lines into CSC-like cells. We generated whole tumor lysates fromthese enriched CSCs using freeze-thaw-cycles and used them to pulse PBMCderived DCs. We showed that we could obtain sufficient functional DCs thatwere capable of stimulating naïve T cells into cytotoxic T lymphocytes(CTLs). The stimulated CTLs were capable of producing IFNγ cytokine in a
CSC-like antigen-specific manner. Our findings suggest that DC-based
immunotherapy approach can be used to target CSC-like cell population.

vi

List of Tables

Table 3.1. Grouping of ELISPOT for T cells after activation by DCs pulsedwith CRC stem cell-like cells. ....................................................................... 110
Table 3.2. Grouping of ELISPOT for T cells after activation by DCs pulsedwith U87 glioma stem cell like cells. ............................................................. 111

vii

List of Figures

Figure 2.1. Derivation of hESC-derived EPCs via mouse stromal OP9 coculture .............................................................................................................. 46
Figure 2.2. iPS-derived EPCs produced via mouse stromal OP9 co-culture ... 47
Figure 2.3. Derivation of hESC-derived EPCs via mouse stromal M2-10B4 coculture .............................................................................................................. 48
Figure 2.4. H9 hESC line-derived EPCs via mouse stromal M2-10B4 coculture .............................................................................................................. 49
Figure 2.5. iPS cells-derived EPCs via mouse stromal M2-10B4 co-culture .. 50
Figure 2.6. H1 hESC-derived EPCs via 2-D differentiation method............... 52
Figure 2.7. iPS cells-derived EPCs via 2-D differentiation method ................ 53
Figure 2.8. Generation of EPCs from human iPS cells via EB method........... 56
Figure 2.9. Characterization of iPS cells-derived EPCs .................................. 57
Figure 2.10. Dual in vivo imaging system ....................................................... 59
Figure 2.11. Tumor tropism of iPS cells-derived EPCs in the 4T1 orthotopic
mouse model of breast cancer .......................................................................... 60
Figure 2.12. In vivo migration of hESC-EPCs toward 4T1 breast cancer cells
in 4T1 orthotopic immunocompromised NSG mice ........................................ 62
Figure 2.13. Tumor tropism of iPS cells-derived EPCs in the 4T1 breast cancer
lung metastasis model ...................................................................................... 64
Figure 2.14. Biodistribution of EPCs in intracranial 2M1 glioma model atprimary tumor mass ......................................................................................... 66
Figure 2.15. Biodistribution of EPCs in intracranial 2M1 glioma model atsecondary tumor foci........................................................................................ 67
Figure 2.16. Effects of iPS-EPCs on 4T1 tumor development and metastasis in
the 4T1 orthotopic mouse model of breast cancer ........................................... 69
Figure 2.17. Effect of iPS-EPCs on tumor development and metastasis in 4T1
mammary pad model........................................................................................ 70

viii

Figure 2.18. Effect of iPS-EPCs on 4T1luc tumor development in the 4T1
breast cancer lung metastasis model ................................................................ 71
Figure 2.19. Genetic modifications of EPCs ................................................... 72
Figure 2.20. Therapeutic effects of CD40L-expressing iPS-EPCs in the 4T1
breast cancer lung metastasis model ................................................................ 76
Figure 2.21. Therapeutic effect of iPS-EPCs expressing HSV-tk in 4T1luclung metastatic Balbc/nude mice ..................................................................... 77
Figure 2.22. Therapeutic effects of EPCs encoding mIsthmin ........................ 79
Figure 3.1. Antigen presentation and CTL activation...................................... 98
Figure 3.2. Dendritic Cells and T cells derivation from PBMC .................... 103
Figure 3.3. Characterization of PBMCs by flow cytometry .......................... 105
Figure 3.4. Characterization of DCs by flow cytometry before and after
pulsing and maturation................................................................................... 106
Figure 3.5. Characterization of CTL by flow cytometry before and after
priming with DCs ........................................................................................... 108
Figure 3.6. Production of IFNγ by CTL after activation by DCs pulsed withreprogrammed CRC cell lysate ...................................................................... 110
Figure 3.7. Production of IFNγ by CTL after activation by DCs pulsed withreprogrammed glioma cell lysate ................................................................... 112

ix

List of Abbreviations
2-D

2-Dimension

4F-BV-ZFN
5-FU
AAVS1
Ac-LDLADCC
AdCD40L
AIDS
AML
ANOVA
anti-HER
APCs
bFGF
BHQ1
BM
BSA
BV
CAF
CAR
CCD camera
CCL
CCR
CD
CD/5-FC
CLL
CML
CMV
Cre-RMCE
CSCs
CTL
CTLA-4
CXCL
CXCR
DCs
DMEM
DPBS
EBs
ECs
EDTA
eGFP
EGM-2
ELCs
EpCAM
EPCs

4 Factors – Baculovirus – Zinc Finger Nuclease5-fluorouracil
Adeno-Associated Virus Integration Site 1
Acetylated-Low Density Lipoprotein
Antibody-Dependent Cell Mediated Cytotoxicity
Adenoviral-CD40Ligand
Acquired Immunodeficiency Syndrome
Acute Myeloid Leukemia
Analysis Of Varianceanti-herceptin
Antigen Presenting Cells
basic Fibroblast Growth Factor
Black Hole Quencher
Bone Marrow
Bovine Serum Albumin
Baculovirus
Carcinoma-Associated Fibroblasts
Chimeric Antigen Receptor
Charge-Coupled Devices camera
C-C Chemokine Ligand
C-C Chemokine Receptor
Cluster of Differentiation
Cytosine Deaminase/5-Fluoro Cytosine
Chronic Lymphocytic Leukemia
Chronic Myelogenous Leukemia
Cytomegalovirus
Cre-Recombinase-Mediated Cassette Exchange
Cancer Stem Cells
Cytotoxic T Lymphocytes
Cytotoxic T Lymphocyte Antigen-4
Chemokine (C-X-C motif) Ligand
Chemokine (C-X-C motif) Receptor
Dendritic Cells
Dulbecco's Modified Eagle Medium
Dulbecco’s Phosphate-Buffered Saline
Embryoid Bodies
Endothelial Cells
Ethylenediaminetetraacetic Acidenhanced Green Fluorescent Protein
Endothelial cell Growth Medium-2
Endothelial Lineage Cells
Epithelial Cell Adhesion Molecule
Endothelial Progenitor Cells

x

FBS
FDA
GCV
GFP
GITR
GM-CSF
GVHD
HCC

Fetal Bovine Serum
Food and Drug Administration
Ganciclovir
Green Fluorescent Protein
Glucocorticoid-induced TNF-related Receptor
Granulocyte-macrophage colony-stimulating factor
Graft Versus Host Disease
Hepatocellular Carcinoma

HER2

Human Epidermal growth factor Receptor 2

hESC
HLA
HPSCs
HPV
HSCs
HSV-tk
HSV-ttk
HUVEC
i.p
i.v
IFN
IL
IMDM
iPS cells
iPS-EPCs
iPS-NSCs
ISM
IVIS
LPS
Luc
mAbs
MACS
MART-1
M-CSF
MHC
MMPs
MOI
mRNA
MSCs
MSCs
MTG
NK
NKT
NSCs
NSG
P/S
P-/UP-DCs
PBMCs

Human Embryonic Stem cells
Human Leukocyte Antigen
Human Pluripotent Stem Cells

Human Papillomavirus
Hematopoietic Stem Cells
Herpes Simplex Virus-thymidine kinase
Herpes Simplex Virus-truncated thymidine kinase
Human Umbilical Vein Endothelial Cells
intraperitoneal
intravenous
Interferon
Interleukin
Iscove's Modified Dulbecco's Media
induced Pluripotent Stem cells
induced Pluripotent Stem cell – derived Endothelial Progenitor Cells
induced Pluripotent Stem cell – derived Neural Stem Cells
Isthmin
In Vivo Imaging System
Lipopolysaccharide
Luciferase
monoclonal Antibodies
Magnetic-activated cell sorting
Melanoma Antigen Recognized by T cells-1
Macrophage-Colony Stimulating Factor (M-CSF)
Major Histocompatibility Complex
Matrix Metalloproteinases
Multiplicity Of Infection
messenger Ribonucleic Acid
Mesenchymal Stem Cells
Mesenchymal Stem Cells
Monothioglycerol
Natural Killer
Natural Killer T
Neural Stem Cells
NOD scid gamma
Penicillin/Streptomycin
Pulsed-/Unpulsed-Dendritic Cells
Peripheral Blood Mononuclear Cells

xi

PD
PDL
PRRs
qPCR
RCC
RPMI-1640
s.d/ SD
SCID

Programmed cell Death
Programmed cell Death Ligand
Patter Recognition Receptors
quantitative Polymerase Chain Reaction
Renal Cell Carcinoma
Roswell Park Memorial Institute-1640
Standard Deviation
Severe Combined Immunodeficiency

SDF-1

Stromal-derived Factor-1

Sf-9
SFM
TAP
TCR
TGF
TH1
TH2
TILs
TRAIL
Treg
VE-cadherin
VEGF
VEGFR
VSVG
vWF
WPRE
WT
ZFN

Spodoptera frugiperda-9
Serum-Free Media
Transporter associated with antigen processing
T cell receptor
Transforming Growth Factor
T Helper 1
T Helper 2
Tumor Infiltrating Lymphocytes
TNF-related apoptosis-inducing ligand
T regulatory
Vascular Endothelial-cadherin
Vascular Endothelial Growth Factor
Vascular Endothelial Growth Factor Receptor
Vesicular Stomatitis Virus-G
von Willebrand Factor
Woodchuck hepatitis virus Post-transcriptional Regulatory Elements
Wild TypeZinc Finger Nucleasesxii

List of Publications

1. Purwanti YI, Chen C, Lam DH, Wu CX, Wang S. Antitumor Effects ofCD40 Ligand-expressing Endothelial Progenitor Cells Derived from
Human iPS Cells in a Metastatic Breast Cancer Model. (Submitted).
2. Zhu D, Lam DH, Purwanti YI, Goh SL, Wu C, Zeng J, Fan W, Wang S.
Systemic Delivery of Fusogenic Membrane Glycoprotein-expressing
Neural Stem Cells to Selectively Kill Tumor Cells. Mol Ther. (2013);
doi:10.1038/mt.2013.123.
3. Chen C, Wang Y, Goh SS, Yang J, Lam DH, Choudhury Y, Tay FC, Du S,
Tan WK, Purwanti YI, Fan W, Wang S. Inhibition of neuronal nitric oxide
synthase activity promotes migration of human-induced pluripotent stem
cell-derived neural stem cells toward cancer cells. J Neurochem. 2013
Aug;126(3):318-30.
4. Shahbazi M, Kwang TW, Purwanti YI, Fan W, Wang S. Inhibitory effects
of neural stem cells derived from human embryonic stem cells on
differentiation and function of monocyte-derived dendritic cells. J Neurol
Sci. 2013 Jul 15;330(1-2):85-93

xiii

CHAPTER I: INTRODUCTION

1

1.1 Cancer immunology
Cancer is a pathological condition affecting people regardless of their age andgender, and is one of the leading causes of death worldwide1. While vast
amounts of research have been conducted to treat and improve the health andlife expectancy of cancer patients, an ideal cure has not yet been found.
Though cancer seems formidable, our own body’s immune system is builtwith the capability to recognize and destroy malignantly transformed
autologous cells. Dendritic cells (DCs), the body’s designated professional
antigen presenting cells (APCs), play a critical role in recognizing tumor cells
and activating arrays of immune effectors to eliminate them. One such effector
that has a central role in eradicating tumor cells is cytotoxic T lymphocytes(CTLs). The intricate mechanisms which control how our immune system
recognizes and kills the cancerous cells, as well as the evolving mechanismsof the tumor to evade this system, will be discussed briefly below.
1.1.1

Tumor antigen recognition and presentation by dendritic cells

1.1.1.1 Dendritic cells as professional antigen presenting cells
Macrophages and dendritic cells in the innate immune system possess
phagocytic capability and antigen recognition ability. These cells use innate
non-clonal receptors, such as Toll-like receptors, lectins, scavenger receptors,
FC receptors and other pattern recognition receptors (PRRs) to perceive andrecognize different types of antigens2, 3. However, unlike macrophages whichhave antimicrobial and scavenging functions, DCs are inefficient in destroying
the antigen-expressing cells2, 3. The phagocytic activities of DCs are designed
to facilitate antigen processing and presentation to the T cells for the initiation2

of antigen-specific adaptive immune response. As professional APCs, DCs
exist in two functional states: the immature and mature states4. Immature DCs
are adept at acquiring the antigens whereas the mature DCs are responsible forstimulating T cells by presenting the acquired antigens.
Tumor cells express proteins or peptides which are considered foreign
by the immune system of the body. Tumor development is associated with the
chaotic proliferation of viable tumor cells as well as disordered tumor cell
deaths in the form of apoptosis and necrosis. Moreover, tumor cells are known
to secrete soluble proteins and exosomes which carry antigens5. The immature
DCs in the periphery are actively engulfing antigens (both self and foreign) via
endocytic processes including phagocytosis, pinocytosis and clathrin-mediatedendocytosis4. Thus, tumor antigens may be transferred into the dendritic cells
via phagocytosis of apoptotic or necrotic tumor cells, pinocytosis of soluble
antigens or capture of exosomes.
Upon tumor antigen encounter, DCs undergo maturation, upregulate
chemokine receptors to facilitate migration into the lymph nodes via the bloodor lymph and increase their expression of co-stimulatory molecules forengaging T cells2, 3, 6. In the lymph nodes, their job is to properly present thesetumor antigens to the adaptive immune effectors in the context of MHC(Major Histocompatibility Complex) to their cell surface.
1.1.1.2 Tumor antigen presentation
Antigen presentation is the process by which protein fragments are complexedwith MHC products and posted to the surface of dendritic cells, resulting in
the orchestration of immune responses repertoire. MHC molecules are3encoded by a large gene family that is located on chromosome 6 in humans7.
MHC class I and II regions encode for genes which are involved in antigen
presentation to T cells and hence control immune reactions against antigens
such as, grafts acceptance/rejection and anti-tumor responses7, 8.
In MHC I antigen presentation pathways, tumor antigens are acquired
and presented in a process known as cross-presentation3, 5. The acquired tumor
antigens will be targeted for destruction and proteolysis processes resulting insmaller peptides in the cytosol. Subsequently, these processed peptides aredelivered and bound to the MHC class I molecules in the endoplasmic
reticulum with the help of TAP (Transporter associated with Antigen
Processing) proteins9, 10. Subsequently, these MHC-peptide complexes aretransported to the cell membrane of the DCs and presented to the CD8 + T
cells.
MHC II molecules (HLA-DR, -DQ and -DP), unlike MHC I moleculesthat are expressed by all nucleated cells, are only found on APCs. Tumor
antigen presentation by MHC II molecules expressed on the DCs triggers theactivation of T helper cells (TH) via interaction with the CD4 receptor. The
stimulation of TH cells results in the secretion of cytokines to boost the CTL
responses and the production of antibodies by B cells8, 11. Upon tumor antigen
recognition, which can be in the form of foreign nucleic acid, glycolipid,
peptide or carbohydrate, the antigen-specific B cell will undergo clonal
expansion, producing high affinity antibodies12. Thus, the presentation oftumor antigen-MHC complexes by APCs directs the activation of adaptive
immune arsenals, both T and B cells, to kill the tumor cells.

41.1.1.3 Dendritic cells bridge the innate and adaptive immunities
The components of innate immunity, which include APCs, natural killer (NK)cells and complement system, are quick in responding to danger signals givenby the tumors. Although fast in its response, innate immunity is not powerfulenough to eradicate the tumor cells. Activation, production and clonal
expansion of tumor specific T cells and B cell antibodies are required to kill
the tumor cells more effectively. The DCs play a central role in the cross-talks
between adaptive and innate immunities.
As the DCs uptake, process and present tumor antigens to activate theadaptive immune effectors, NK cells act as the first line of defense against thetumors13. The NK cells recognize neoplastic cells due to the down-regulationof MHC I expression (‘missing-self’ signal) and expression of NKG2d(‘induced-self’ signal) on these target cells13-15. Once activated, NK cells exert
cytolytic effect on the target cells via perforin and granzymes production14.
NK cells also secrete various cytokines such as IFNγ to help DC maturation16.
Moreover, the killing of target cells by NK cells can enhance presentation of
antigens from the apoptotic cells by DCs17. Consequently, the mature DCs will
activate T cells via antigen presentation. In turn, the activated T cells produce
cytokines such as IL2, which can enhance and stabilize the NK cell activities
further18.
Aside from presenting antigens, DCs also produce various cytokinesthat are important in regulating both innate and adaptive immune responses
against tumors. One such cytokine is IL12, which has been shown to be an
essential regulator for skewing toward TH1 responses. IL12 is also important5

in further enhancing cytolytic activities of NK cells14, 17, 19. Thus, through thepresentation of antigens, cell-cell interaction and cytokine production, DCs
communicate with both arms of the immune system to ensure proper
responses against cancer.
1.1.2

Cytotoxic T Lymphocytes: professional killers of immune system

1.1.2.1 Activation of cytotoxic T lymphocytes
The adaptive immune cells, both B and T cells, are produced in the bonemarrow. However, as the name implies, T cells undergo maturation in thethymus. T cells are segregated into different subtypes including CD8+ naïve T
cells which can differentiate into CTLs and memory cells as well as CD4+ TH
cells20, 21. These different T cell populations play different roles in the immune
system. Memory T cells provide long-term immunity against previously
encountered antigens. TH cells, mainly categorized into TH1 and TH2, provide
signals in the form of direct cellular contact and cytokines to enhance both B
and T cell responses22. TH1 cells produce cytokines such as IFNs and IL2
cytokines to promote the CTL-mediated immune response11, 22. TH2 cells
produce cytokines such as IL4 to enhance antibody production11, 22. However,
the mechanism of tumor cell elimination relies largely on CD8+ CTLs.
CTL activation is initiated when the CD8+ T cell receptor (TCR)
recognizes the antigen peptide-MHC I complex on an APC which leads to T
cell differentiation and extensive proliferation into CTLs22, 23. However,antigen recognition alone is insufficient to fully activate CTL. Interaction of
co-stimulatory signals between APCs and T cells is needed to promote T cell
survival, proliferation and migration towards the tumor microenvironment 24.
6

For example, CD28 interaction with CD80 and CD86 on APCs is needed tostimulate T cell activation via amplification of the signals from TCR11, 25. The
development of optimal CTL activation also requires survival signals provided
by cytokines that are produced by mature DCs (such as IL6, IL12 and IFNs)
and TH1 cells25.
1.1.2.2 Antitumor effects of cytotoxic T lymphocytes
If DCs are the professional APCs, CTLs are the professional killers which can
specifically target and eliminate malignant cells. CTLs inspect MHC I
molecules that are found in all nucleated cells through the binding of CD8
receptor11. MHC I gene products, which are designated as HLA (Human
Leukocyte Antigen) -A, -B and -C, are encoded by different loci whereby eachlocus possesses extensive allelic variations7. The recognition of tumor antigens
by CTLs is HLA restricted, in that CTLs and tumor cells must have the same
HLA type for appropriate recognition11.
When the cells expressing the antigen that has been reported by theDCs have been found, they will be targeted by CTLs for destruction. CTLs kill
the target cells in a contact-dependent manner22. Upon target cell recognition,
CTL granules are mobilized towards the target cells, followed by fusion of the
granule membranes with the plasma membrane of the target cells. Exocytosisof the granule contents, which are the granzymes and perforin, into the target
cells triggers cell lysis22. Apoptotic pathways can also be activated through theexpression of the Fas ligand on the CTLs which engages the Fas (CD95)
receptor on the target cell membrane22. CTLs also exert their antitumor effects
via the production of cytokines such as IFNγ and TNFα that can arrest
7

malignant cell proliferation24.

Effective antigen-specific cytolysis whichspares normal cells is the inherent characteristic of CTLs and has become theultimate goal of cancer immunotherapies. Unfortunately, the current available
treatments still evoke some degree of off-target toxic effects.
Tumor tissues are often infiltrated with activated adaptive immune
cells, indicating the presence of vigorous responses against cancer26, 27.
Although adaptive immune effectors provide powerful antitumor effects, theiractivation requires time. Eventually, the interplay of innate and adaptive
immune responses is important for fully-mounted immunity against cancer.
When all the parts are serving their purposes well, tumor eradication willensue. Unfortunately, in cancer patients, this is not the case.
1.1.3

Tumor evasions of dendritic cells surveillance and cytotoxic T

lymphocytes killing mechanisms
Although the immune system is capable of recognizing and eliminating tumor
cells, the inherent genetic instability of the latter frequently interferes with thedevelopment and function of immune responses1, 24. Progressive tumors often
exhibit strategies that promote evasion from immune recognition and/or
killing. These mechanisms include developing poor immunogenic properties
to avoid proper presentation by DCs and to evade CTLs targeting, producing
immunosuppressive cytokines and mediators, as well as piggybacking the proinflammatory immune cells to render DCs and T cells tolerant.
One mechanism to evade immune surveillance is to down-regulate theexpressions of MHC I molecule so as to avoid CTLs inspection. Likewise, theNK cell activating ligands on the tumor cells are usually down-regulated too.
8

As such, they could avoid being targeted by the T cells as well as escaping NK
cells attacks24, 28, 29.
Another scenario by which tumor cells reduce immune stimulation is
the induction of tolerogenic DCs. Indeed, a number of studies have reported
that DCs are dysfunctional in cancer patients30, 31. The accumulation oftolerogenic DCs is attributed to the secretion of SDF-1 (Stromal-derived
Factor-1) and IL6 by tumor cells28, 32. These DCs are defective in antigen
presentation and further induce T and NK cells tolerance through the
production of IL10 or TGFβ28, 33.
Aside from SDF-1, IL10 and TGFβ, other immunosuppressive
mediators which are abundant in tumor microenvironment include adenosine,
prostaglandin E2 and Vascular Endothelial Growth Factor A (VEGF-A)34.
These mediators promote angiogenesis via attraction of endothelial cells andfavor stromal development by recruiting pro-inflammatory immune effectors(such as macrophages and complement components)28, 35. In addition, they
promote the development of the CD4+CD25+ T cell subpopulation known as
Tregs, which suppress CTL responses28, 36. Increased accumulation of Treg
cells in many human malignancies, such as lung, head and neck, ovarian,stomach and skin cancer, is associated with poor prognosis for patients28, 36.
Depletion of Tregs through the administration of anti-CTLA-4 antibody
showed marked therapeutic effects in murine models and clinical trials24, 36.
Undeniably, the understanding of immunosuppressive strategies mediated bytumor cells leads to development of more promising anti-cancer treatments.

91.2 Cancer immunotherapy
Cancer immunotherapy aims to strengthen the cancer patient’s immune
system37. Initial studies on cancer immunotherapy dated back to the late 1800s
when Dr. William Coley concocted a mixture of killed Streptococcus pyogens
and Serratia marcescens. The administration of Coley’s toxins was shown toeliminate

carcinomas11.

Currently,

variousclinicaltrialsemploying

immunotherapy approaches are being conducted. A search on clinicaltrials.gov
using the search terms “cancer immunotherapy” revealed 903 search results
encompassing a wide range of cancer diseases (as of July 2013). Some of the
major approaches in immunotherapy will be discussed below.
1.2.1

Stem cells

ascellulardeliveryvehicleforcancergene

immunotherapy
1.2.1.1 Stem cell candidates for immunotherapy
Stem cells are a population of cells that demonstrate self-renewal capacity anddifferentiation capability. With recent advances in the study of stem cells,
different types of stem cells/progenitors such as mesenchymal stem cells(MSCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs) or
endothelial progenitor cells (EPCs) are believed to be ideal candidates as
vehicles for anti-cancer gene delivery. Tumor microenvironments areabundant with various chemotactic cytokines and inflammatory signals that
attract these progenitor cells. The migration of the cells toward tumor sites ismediated by chemokine receptors such as CXCR4 or CXCL12 on the stem
cells38. Due to their tumoritropic properties, the principal advantage of stem

10

cell-based delivery of anticancer therapeutics is in its potential to achievetumor-specificity, thus enhancing therapeutic effectiveness.
Mesenchymal Stem Cells (MSCs) have been widely proven to be ideal
vehicles for the targeted delivery of anticancer agents. This is attributed totheir easy isolation from bone marrow or adipose tissues and enormous
expansion potential in culture on top of their tumor-tropic capacities38. MSCs
play an important role in maintaining tissue homeostasis by repairing injured
tissues. A tumor microenvironment mimics that of a wound, thus attracting
MSCs which can then be exploited to promote the growth of tumor stroma.
Based on this, MSCs can be employed as cellular vehicles to send immuneinducing agents to the tumor sites with the aim of killing the tumor cells via
immune responses.
Malignant brain tumors such as glioblastoma multiforme remain lethal
and incurable39. Infiltrating immune cells are found in brain tumors, but theirfunctions can be curbed by tumor-derived immunosuppressors as mentioned
above. Recent studies have shown that intracranially or intravenously injected
NSCs migrate toward brain tumors40, 41. Therefore, the NSCs-mediatedimmuno-stimulatory gene delivery system has the potential to significantly
improve clinical outcomes for brain cancer patients.
Another promising candidate for cellular delivery-based therapies is
EPCs due to their involvement in tumor angiogenesis. In order to grow,tumors actively construct new vessels, and in the course of thisneovascularization, circulating EPCs are actively incorporated into the tumor
sites. Therefore, these cells can also be utilized as a ‘Trojan horse’ equipped11with immunostimulatory genes to kill the tumor cells. As Hamanishe et al.
demonstrated, the systemic delivery of EPCs that express a lymphocyte
migrating C-C chemokine ligand (CCL)-19 led to tumor repression in amurine ovarian cancer model. The use of EPCs is especially useful fortargeting remote metastases as angiogenesis is a crucial factor in tumor
metastatic progression.
1.2.1.2 Stem cell delivery of cytokine for cancer immunotherapy
Cytokines are biologic immune modulators produced by and acting on cells 11.
As mentioned above, cytokines play important roles in the regulation of
immune responses and tolerance. Immunological manipulation using
cytokines for cancer therapy has been prevalently attempted. For instance, IL2
and IFNα have been used for the treatment of various cancers in the clinic
such as renal cell carcinoma (RCC), AIDS related Kaposi’s sarcoma,melanoma, renal cancers and chronic myelogenous leukemia (CML)11, 24, 42,43.These cytokines act mainly by skewing the immune responses towards TH1

axis, which in turn promotes CTL anti-tumor activities11. Although theirefficacies have been proven under clinical conditions, the systemic
administration of cytokines requires relatively high doses to obtain clinicaleffects. The non-specific and broad activation of the immune system by thesecytokines can lead to life-threatening toxicities such as liver failure in humanrecipients24. The combination of the cytokine-mediated activation of immune
responses and tumor suppression together with tumor-targeted delivery oftransgene by stem cells is expected to provide therapeutic effects with minimal
off-target toxic effects.

12MSCs have been widely used as a cellular vehicle to deliver immunestimulatory genes to tumors and tumor microenvironments to enhance
antitumor immune responses. It has been shown that MSCs stably transducedwith a retroviral vector expressing IL12 strongly reduced formation of lungmetastases and retarded the growth of pre-established melanoma mousemodel44. In another example, intra-tumor injection of primary mouse NSCs
transduced with a retroviral vector encoding IL4 resulted in the extended
survival of glioma-bearing mice39, 45. Likewise, intracranial injection of NSCs
expressing IL12 into glioma mouse model resulted in the prolonged survivalof the animals46. Moreover, human NSCs expressing IFNβ by adenoviraltransduction reduced metastatic neuroblastoma upon injection47. These
preclinical studies have spurred the idea of using stem cells as an excellent
platform for tumor-specific cytokine-mediated cancer immunotherapies.
1.2.1.3 Immunotherapy via in situ antibodies delivery by stem cells
The current top-selling cancer drugs are monoclonal antibodies (mAbs) suchas trastuzumab, rituximab and bevacizumab27. The anticancer effects of mAbs
are based on multiple immunologic mechanisms, including complementmediated cytotoxicity, antibody dependent cytotoxicity and enhancement ofadaptive immune response27. Trastuzumab (also known as Herceptin), is ahuman epidermal growth factor receptor 2- (HER2-) targeted antibody which
has been shown to significantly improve outcomes for HER2-positive breast
cancer patients48.
Despite the promise shown by antibody-based therapies, the size, halflife and toxic effects of these antibodies pose problems in clinical13

circumstances. The large molecular size and short half-life of antibodies limit
their ability to efficiently penetrate large solid tumors or tumor in obscureplaces, e.g. brain tumors whereby the antibodies are inefficient in crossing theblood brain barrier to exert their therapeutic effects. Moreover, toxic side
effects due to interactions with unintended targets need to be resolved. A
delivery vehicle system using stem cells can be harnessed to overcome these
problems. The inherent tumor tropism of stem cells could be useful to deliver
therapeutic genes to solid tumors and across blood brain barrier.
Studies have shown that NSCs could deliver the tumor-specific antiHER2

antibody(functionallyequivalenttocommerciallyavailable

Trastuzumab) in vivo49, 50. The anti-HER2 immunoglobulin-secreting NSCs
exhibit preferential tropism to tumor cells in vivo and are capable of delivering
tumor-specific antibodies to human breast cancer xenografts in mice50.
Although promising, there are several critical challenges that require
attention before stem cell-based gene immunotherapy is applied for clinicaluse. The first challenge is the choice and source of stem cells used for
different types of cancer. It has been shown that NSCs perform better than
MSCs in delivering an oncolytic adenovirus in a rodent orthotopic glioma
model. Thus, NSCs may be a better candidate to deliver immunotherapeuticgene for brain tumors treatment. Another challenge is that this approach has tobe able to produce the therapeutic agents of effective concentration at thetumor site. In addition to this, finding balance between therapeutic effects andtumor promoting effects as well as fine-tuning the gene expression and safety14

of genetic engineering in the stem cells are important for this approach to be
fully optimized in the clinics.
1.2.2

Dendritic cell-based vaccinations

Vaccines are one application of immunology that has successfully eradicateddiseases such as polio and smallpox. Vaccines work by inducing protective
immunity against the disease. The same principle applies to cancer vaccines inwhich the vaccines aim to stimulate tumor-specific immune responses forprophylactic (preventive) and therapeutic purposes. Two types of prophylactic
vaccines have been approved by FDA, the vaccine against hepatitis B virus toprevent liver cancer and the vaccine against human papillomavirus (HPV) to
prevent cervical cancer (Gardasil® and Cervarix®)34. In contrast, thedevelopment of therapeutic vaccines is more challenging. Recently, Provenge,a DC-based cancer vaccine for prostate cancer treatment, has been approvedby FDA, inciting more studies for the development of next-generation DCbased cancer vaccines.
1.2.2.1 Dendritic cells as an excellent candidate for developing therapeutic
vaccines against cancer
There are several different approaches in cancer vaccines, including viral-,peptide-, vector-, tumor cell- and DC-based, each offering unique advantages
and disadvantages11. DC-based vaccines aside, all these approaches are basedon the presumption that they can stimulate DCs and generate immune
responses in vivo. Unfortunately, these vaccines are likely to have poor
pharmacokinetic properties and may be cleared rapidly before being loaded
onto the DCs due to their short half-lives34. Moreover, as mentioned above,15

the DCs in cancer patients may be functionally defective and induced to
become tolerogenic DCs. Dendritic-cell-activating adjuvants such as GM-CSF
and IL2 cytokines have been used together with these vaccines to boost their
therapeutic efficacies. This underscores the importance of dendritic cells in
regulating immune responses. Therefore, dendritic cells themselves become
the obvious target and adjuvant tool-base for cancer vaccination.
The approach of DC-based vaccines involves the isolation of DCs fromthe patients, followed by loading of antigens ex vivo, activation andconditioning to induce DC maturation, and finally re-infusion of the DCs backinto the patient. The closest approach to DC-based vaccine by definition is
Provenge, the only therapeutic vaccine to have been approved by the FDA todate. This vaccine, which comprises a mixture of the patient’s own PBMCs(Peripheral Blood Mononuclear Cells), prostate antigen PAP (Prostatic Acid
Phosphatase) and GM-CSF supplement (DC growth and differentiation
factor), is used for prostate cancer treatment34.
1.2.2.2 Loading dendritic cells with tumor-specific antigens
The development of DC-based vaccines requires the introduction of antigens
into the DCs which in turn will present them to T cells. The antigens used canbe in various forms and there are several methods to load them to DCs. To
date, studies have used antigens in the form of DNA, peptides/proteins, RNA(mRNA/total RNA), exosomes and whole tumor cells (alive/killed). These
antigens can be loaded into the DCs via viral vector-mediated gene transfer,electroporation, transfection as well as co-culture/mixing.

16

The advantage of using DNA to load antigens to DCs is that the tumor
antigens will be expressed by the DCs and presented via MHC I complex in a
way that mimics endogenous protein presentation51. Some studies have shown
its efficacy in mounting cytotoxic effect against tumors52-54. However, since
usually viral vector-mediated loading is adopted, this method raises safetyconcerns regarding the use of the virus and its effect on the transduced DCs.
Antigen loading using peptides has the advantage of being specific,
hence reducing the unwanted toxicities from non-specific immune responses6,
51. Yet, this specificity is a double-edged sword, as it also limits the target

cells. Moreover, as mentioned previously, the application of peptide-based
vaccine is MHC restricted. However, the efficacy of using peptide-pulsed DCs
for cancer immunotherapies has been proven in several studies and even inclinics55-57.
Targeting a broader spectrum of tumor antigens without the restrictionof MHC typing is sometimes preferred although such approaches are usuallylimited by the minute quantity of cancer antigenic molecules present. The useof proteins, total RNA, exosomes and whole cell-based antigens can achievethis. However, greater technical demands are required to prepare antigens
from proteins, RNA and exosomes especially in large-scale productions and it
is costlier too. Despite this, their efficacies in preclinical and clinical studies
have been proven58, 59. Loading DCs with whole tumor cell antigens can beachieved with tumor lysate prepared by repeated freeze-thaw cycles orapoptotic cells generated by irradiation. This approach is simple yet well-17established in DC-based vaccine immunotherapies and its therapeutic effects
have been reported by many studies60-64.
More research needs to be done since even the success story ofProvenge can only deliver a meager 4-month advantage in overall survival27,
34. A better understanding of DCs generation and differentiation, of immune

tolerance and of tumor antigen choices, formulation and incorporation into theDCs will allow us to design a better cancer vaccine.
1.2.3

Other approaches

1.2.3.1 Adoptive T cells for cancer therapy
Growing cancers contain tumor infiltrating lymphocytes (TILs), indicating thepresence of T cell immune response against cancer. It has been shown that the
prognosis of hepatocellular carcinoma (HCC) cancer patients with marked
TILs is better than that of patients without TILs. Unfortunately, TILs very
often

succumb

totheimmunosuppressiveandtolerogenic

tumor

microenvironment. This is why the idea of providing improved anti-tumor
reactive T cells via adoptive T cell therapy becomes very promising.
The feasibility of adoptive immunity was discovered after a study inmice revealed that specific immunity to tumors could be transferred to normal
mice using lymphocytes from the spleen or peritoneal cavity of immunizeddonors43. The donation of T cells can be allogeneic or autologous. Though it
has been performed with marked therapeutic effects in clinical condition,
allogeneic donation may lead to graft-vs-host disease (GVHD). Thus,
isolation, expansion and reinfusion of autologous tumor reactive T cells into
the patient are more preferred.
18

T cells can be isolated from peripheral blood, tumor effusions or fromdraining the lymph nodes of the patient/donor21. Once obtained, the T cells arestimulated and expanded in vitro via priming with DCs which present thetumor antigens or using CD28/CD3 antibodies11, 21, 24. Regardless of themethods to isolate and expand the T cells, the challenge is to maintain thesurvival and properties of the cells after being re-injected into the patient11, 24.
1.2.3.2 Genetic engineering of T cells
Genetic engineering to confer T cells with a stronger and more specific tumorkilling power has also been adopted. This can be achieved through tumor
antigen-specific TCR gene transfer or introduction of chimeric antigen
receptor (CAR)1. TCR gene transfer has been used in clinical trial formelanoma using melanoma tumor antigen melanin A (MART-1)1. Adoptive T
cell transfer with an HLA-A2 restricted T cell receptor (TCR) specific for NYESO-1, a cancer-testis antigen expressed by human cancers and testis but noother normal adult tissues, resulted in tumor regression in 5 out of 11 patientswith melanoma and 4 of 6 patients with synovial cell sarcoma27. One
drawback of this therapeutic regime is its limitation due to the MHCrestriction.
CARs, single-chain constructs composed of an Ig variable domain
fused to a TCR constant domain, were developed to overcome this21. Whenintroduced into the T cells, they combine the antigen recognition properties ofantibodies with T cell lytic functions. Thus, this method broadens the
spectrum of tumor antigen recognition and is not MHC-restricted. The first
generation of CAR consists of a single signaling domain, CD3ζ. Although the19resulted redirected T cells are functional, they fail to persist in the long term.
In the second generation of CAR, the CD28 signaling domain was added toconfer resistance to the T cells. The newer generations include more costimulatory molecules such as 4-1BB (CD137) domains to enhance T cell
survival and function even further1. Encouraging early clinical results havebeen obtained in patients with lymphoma65. However, the toxicities as well as
the search for the optimal configuration for CAR are the challenges faced bythis immunotherapy1.
1.2.4

Challenges in cancer immunotherapy

Despite surmounting evidence and convincing data in preclinical studies,cancer immunotherapies in clinical trials have failed to deliver promising
results. Clearly, some roadblocks need to be cleared in order to attain successin clinical applications. One such hindrance is the limitations of preclinicalanimal models. The existing models, be it the syngeneic or xenograft models,
have provided us with tools to understand the mechanisms of cancer
immunology and immunotherapy. Yet, they are still far from recapitulating thecomplex and progressive pathophysiological of cancer in patients66.
Improvements in animal models will result in better prediction of therapeutic
efficacies in humans. Naturally occurring cancers have been observed incompanion animals (pet dogs and cats)67-70. Understanding the biology andtreatment of cancer in these animals may bridge the gap between preclinicaland clinical studies closer.
Anothermajor

roadblockhamperingthesuccessofcancerimmunotherapy is the lack of understanding of the overall immune status of20

patients. Mechanisms that suppress the immune system provide a fundamental
reason why immunotherapy fails to induce consistently robust immune
responses in patients71. Prescreening of patients for immune status will help
identifying patients who are more likely to respond to certain therapies.
Eventually, this knowledge will be useful in designing cancer therapy that will
work for certain groups of patients with the same immune parameters.
Development of biomarkers and assays that can test arrays of markers
efficiently would be very valuable. Recently, a new generation of flow
cytometry based on mass spectrometry readout of heavy metal ion-labeledprobes has been developed72-75. This technology allows testing of 40
biomarkers samples at once, which is beyond the ability of traditional
multicolor flow cytometry73.
While the heterogeneity of cancer is considered a critical hurdle,
appreciating this complexity and designing therapeutic combinations holds
great potential in clinics. There is a substantial evidence to suggest that somecombinations of chemo- or radiotherapy and vaccine treatment aresynergistic66, 76, 77. Other emerging therapeutic strategies that can be used in
combination with current available therapies are antibodies targeting theimmune checkpoint blockade (e.g CTLA-4) or antibodies targeting PD-1,
which will lead to depletion of Tregs population. However, more preclinicaldata and trials are required to demonstrate the effectiveness of these
combination therapies71, 78.

21

1.3 Purposes and motivations
The idea that our own immune systems could recognize and kill cancer cells
have been supported by a large amount of evidence. Studies on cancer
immune responses and immune tolerance have led to the emergence of various
potential cancer immunotherapeutic modalities. Yet, only a few could
eventually develop into clinical applications despite all the effort, time andfunding invested in myriad clinical research studies and trials.
One critical hurdle is breaking the immune tolerance barriers createdby the tumor microenvironment. To achieve this, we need to equip immune
effectors with the appropriate signals which not only activate but also maintainthe anti-tumor immune responses long enough to eradicate the tumor cells.
Another crucial challenge is to fine-tune the balance between the specificity
and the efficacy of the cancer therapy. We need to design treatments which
can abrogate the tumor heterogeneous cell population while minimizing theoff-target toxicities against the healthy normal cells. Treatments that canovercome these obstacles will be very valuable in the clinics.
Targeted immuno-gene therapy using the stem cell delivery vehicle is
an attractive approach to create such a treatment. The inherent tumor tropismof the stem cells combined with the immune-stimulating gene can become apowerful cancer treatment modality. Endothelial progenitor cells (EPCs) is
particularly an excellent choice of cellular vehicle, not only due to their tumor
tropism but also their involvement in cancer angiogenesis. However, deriving
sufficient amount of EPCs for effective clinical translation is one of the criticalchallenges. In this study, we sought to explore whether human induced
22

pluripotent stem (iPS) cells could be used as a reliable and accessible cellsource to generate uniform human EPCs with cancer gene therapy potential(Chapter 2).
The presence of TILs indicates that immune cells also possess natural
tumor homing capability. Moreover, DCs, the immune cells which bridge theinnate and adaptive immune arms, are capable of stimulating adaptive immune
responses by presenting tumor antigen to the T cells. Thus, we turn our focus
utilizing and stimulating these cells to overcome the immune tolerance barriers
created by the tumor microenvironment.
Another immense obstacle in finding a cure for cancer is the presenceof cancer stem cells (CSCs). Most of the available cancer treatments including
chemotherapy and radiation therapy, while proven as effective in reducing thetumor burden, cannot eliminate the chance of tumor recurrence in patients.
This is because these treatments failed to target the cancer stem cells (CSCs)
population. The surviving CSCs, with their self-renewal and differentiation
capability, are capable of reestablishing the tumor mass. Thus, we aim todevelop a targeted DC-based immunotherapy approach against this
subpopulation of cancer (Chapter 3).

23

CHAPTER II: ANTITUMOR EFFECTS OF CD40 LIGANDEXPRESSING ENDOTHELIAL PROGENITOR CELLSDERIVED FROM HUMAN IPS CELLS IN A METASTATICBREAST CANCER MODEL24

2.1 Introduction
2.1.1

EPCs

2.1.1.1 Definition, Sources and characterization
Endothelial progenitor cells (EPCs) are a population of CD34+/Flk1+/CD133+
stem cells that can differentiate into endothelial cells (ECs), the cells thatmake up the lining of blood vessels79-82. EPCs are also identified by a set ofmarkers, including the von Willebrand Factor (vWF), CD31 and vascularendothelial (VE)-cadherin80, 83. EPCs play significant roles in vasculogenesis(formation of blood vessels by in situ differentiation of EPCs), angiogenesis(development of pre-existing blood vessels) and the growth of solid tumors.
Over the past decade, the observation that circulating EPCs from the bonemarrow can be recruited into tumor neovessels has promoted many studies
using various sources of endogenous and exogenous EPCs for therapeutictargeting to inhibit tumor neovasculaturization81, 83, 84. The systemic deliveryof therapeutic gene-loaded EPCs can potentially enable efficient therapy formetastatic cancer by targeting multiple metastases in different sites throughcirculation, and this is probably the most attractive feature of EPC cellularvehicles in cancer treatment85, 86.
In vitro, EPCs can be isolated from various sources, such as peripheral
blood, bone marrow, adipose tissue and umbilical cord blood87, 88. However,
the relatively low yield and difficult expansion of these cells pose a challenge
for clinical usage. With the increasing potential to use EPCs as cancer
therapeutics, it is desirable to have a reliable and stable supply of human
EPCs. Pluripotent stem cells, such as embryonic stem (ES) cells, can be25

expanded indefinitely in culture and have the potential to generate different
types of cells, including endothelial lineage cells87, 89-92, in vitro in virtuallyunlimited numbers. Interestingly, mouse embryonic EPCs have been shown tobe able to target lung metastases in an allogenetic setting in mice83, 85, 93. Most
recently, Su and colleagues presented that human ES cell-derived
CD34+/CD133+ endothelial cells armed with herpes simplex virus truncatedthymidine kinase (HSV-ttk) could inhibit MDA-MB-231 breast cancer
growth94.
Induced pluripotent stem (iPS) cells are another type of commonlyused pluripotent stem cells. Human iPS cells appear more attractive forclinical applications since these cells can be generated relatively easily
through reprogramming differentiated somatic cells with transcription factors,
a procedure that circumvents the bioethical controversies associated with thederivation of human ES cells from human embryos95-98. Utilizing iPS cells togenerate EPCs therefore holds great potential for clinical applications.
2.1.1.2 EPCs gene therapy strategies
2.1.1.2.1

Suicide gene therapy

The major challenges of cancer therapy using cytotoxic drugs are the short
drug half-life, insufficient delivery of the drug to tumor and their suboptimal
specificity to malignant tissue. Tumor-targeted suicide gene delivery by stem
cells will alleviate these issues. This therapy involves the stem cell-delivery ofan enzyme which converts an inactive non-toxic pro-drug into a cytotoxic
agent. Subsequently, the toxic drugs kill the cancer cells via bystander effects
through diffusion or formation of gap junctions.
26

The first study to show that embryonic EPCs can be used as cellularvehicles to systemically deliver a suicide gene for cancer therapy was done by
Wei et al99. In this study, cytosine deaminase/5-fluorocytosine (CD/5-FC) wasused to treat mice with metastatic lung cancer. This CD/5-FC enzyme/prodrug
system utilizes the conversion of an inactive non-toxic prodrug into acytotoxic drug metabolite through the enzyme, cytosine deaminase. The 5-FC
prodrug, an antifungal agent, will be converted to a diffusible and highly
cytotoxic DNA synthesis inhibitor compound, 5-fluorouracil (5-FU), upon
injection. Another suicide gene which has been extensively studied is theherpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system. In
this system, HSVtk mediates GCV conversion to GCV 5’-triphosphate, which
inhibits cell DNA polymerase100. HSVtk delivered by EPCs has been used totreat mice models of human ovarian cancer83.
Although suicide gene therapy has been widely used and studied, it has
limitations too. The bystander killing effect of the suicide gene requires time.
Hence, it may not be able to compete with fast progressing tumor cells, ortumor cells in large amounts. Furthermore, the suicide gene vehicle has aninherent disadvantage, since it destroys itself, thus limiting the therapeutic
window time99.
2.1.1.2.2

Antiangiogenic therapy

The growth and progression of solid tumors are dependent on newly formed
blood vessels for cancer cells to get nutrients, as well as for a route via which
the tumor cells can form metastases79, 101. Antiangiogenic therapy follows arationale in which tumor vessels are disrupted, thus starving the tumor102. The
27

prolonged administration of high doses of angiogenic inhibitors such as
endostatin, thrombospondin or angiostatin in vivo is commonly required to
produce antitumor effects due to its short half-life in circulation83, 101. Thus,
EPC cellular delivery-based antiangiogenic gene therapy, which facilitates
gene-encoding such proteins directly into the tumor, may solve thisproblem101.
2.1.1.2.3

Immunotherapy

Several studies have demonstrated the use of EPCs to deliver immunotherapeutic genes for cancer therapy. One such studiy was done by Ojeifo etal., in which the administration of 3 sequential i.v (intravenous) injections of
105 ECs expressing the human IL2 transgene abrogated the tumor metastases
and prolonged the survival of C57Bl/6 mice harboring lung metastases frommelanoma83, 103. In another study, using mouse embryonic EPCs (eEPCs)
which were retrovirally transduced with the mouse CC chemokine ligand-19(CCL19) gene, Hamanishi et al. showed the suppression of growth andmetastasis of murine ovarian cancer in animal models. CCL19 elicits
chemotactic activity for T cells, B cells and mature DCs, which supports thehoming of naïve T cells and DCs into T cell areas of secondary organs. The
tumor repression was accompanied by increased numbers of tumor infiltrating
CD8+ lymphocytes. In contrast, no tumor repression was observed when thesame experiment was done in immunodeficient (SCID) mice, suggesting thatthe crucial role of T cell function in this system104.
The use of immune modulators for cancer therapy has been discussed
in the previous chapter. However, the systemic administration of such immune
28

modulators can evoke toxic effects to the body due to their broad range of
immune effects. Delivering their genes to the tumor sites via stem cells maylead to a more targeted approach to killing tumor cells, thereby attenuating thetoxic effects.
2.1.2

CD40 ligand

CD40 ligand (CD40L) is a 33kDA type II membrane protein, a member of the
TNF gene family. These ligands interact with CD40 receptors, which are
expressed by a wide range of cells including antigen presenting cells (APCs),
B cells, epithelial cells, endothelial cells (ECs) and malignant cells105. Since it
was shown to have an antitumor effect in 1997 by Grossmann et al., many
have studied the potential of CD40L as a cancer therapeutic agent. Binding of
CD40L with CD40 receptors on APCs, such as dendritic cells, will result inthe maturation of APCs. This, in turn, activates antitumoral T cell responses
againstcancer cells106,

107. Interestingly,

studiesusing CD40L in

immunodeficient mice showed delayed tumor progression, which may indicate
other mechanisms than the T cell responses. In B cells, cross-linking CD40
drives differentiation, proliferation and immunoglobulin class switching while
preventing B cell apoptosis107. Meanwhile, Gomez et al. showed that tumor
cells treated with AdCD40L had decreased viability due to apoptosis induction
by CD40L106, 108. Currently, there are several ongoing clinical investigations
utilizing adenoviral-CD40L, involving diseases such as CLL (chronic
lymphocytic leukemia), acute leukemia, urinary bladder cancer and malignant
melanoma 106. Moreover, humanized anti-CD40 monoclonal antibody SGN-40
has been used in a clinical phase I trial for multiple myeloma in the US105.

29

2.1.3

Induced pluripotent stem cells

The iPS cells have provided us with a tool to generate EPCs perpetually.
However this groundbreaking technology would not come about if notsupported by abundant prior studies and knowledge on stem cells. One suchimpactful study is the work on somatic cells nuclear transfer (SCNT)109. This
technique allow the generation of fully-functioned organisms, althoughusually with severe genetic abnormalities, from differentiated cells as nuclear
donor and cytoplasm of an oocyte. Studies have also shown that various cell
types have the capacity to functionally influence or reprogram one anotherfollowing cell fusion110. Interestingly, pluripotent cells (ESCs or embryoniccarcinoma cells) have the superiority to reprogram the differentiated cells 111,
112

. Both methods take advantage of cellular materials, either the nuclear

factors present in the cytoplasm of the enucleated oocyte or in the hESCs
fused to the adult cells, to establish pluripotency in somatic cells113, 114.
Yamanaka and colleagues ingeniously compiled 24 ‘candidate factors’:
genes that were known to be highly associated with pluripotency. Afterremoving the factors one by one, the final Yamanaka reprogramming factors,
Oct4, Sox2, Klf4, and C-Myc, were deduced to be crucial to reprogram adultcells back into pluripotent state110. These genes encode for transcription
factors which activate the expression of genes in the pluripotency network andsuppress those that promote differentiation via chromatin remodeling114. Oct4
has been shown to be indispensable factor in the reprogramming cocktails. It
forms heterodimers with Sox2 to attain a more stable and efficient DNA
binding115, 116. Klf4 or Kruppel like factor 4 is a zinc finger transcription factor
that regulates cell proliferation and differentiation116. Although dispensable,
30

C-Myc has been known to induce changes in histone methylation patterns,
thus providing easier access to the reprogramming factors and enhancing theefficiency of the reprogramming process116.
Although iPS cells hold great promise in cancer and regenerative
medicine, it has to pass critical safety concerns prior entering the clinics. One
concern is the use of viral systems that result in integration of the
reprogramming factors into the host cell genome and subsequently may lead tomutagenesis and tumorigenesis117. Non-integrating approaches using vectorsthat do not integrate into host cell genome or that could subsequently beremoved from the genome are preferred112. Another concern is the oncogenic
nature of all the reprogramming factors. Thus, transient expression of thesefactors is favored117. One potential clinical application of iPS cells is theestablishment of HLA-matched banks of lines. The use of hESCs for thispurpose hits a major ethical block as it requires embryos to derive the celllines. Furthermore, limited availability of hESCs derived from surplus
embryos donated following IVF treatment is unlikely to provide sufficientnumbers to populate a highly selected cell bank118. Collection of adult cells
that can be used to derive iPS cells banks would be more feasible and lesscontroversial.
2.1.4

Objective and Aim of Study

The overall aim of our study is to investigate the anti-tumor effect of CD40L
while utilizing EPCs as the cellular delivery vehicle. Most studies utilized
CD40L expression in DC or tumor cells in order to activate T cell
responses105, 106, 108, 119, 120. However, in this study, we used EPC as a cellular31

vehicle to deliver the CD40L systemically and tested its therapeutic effect on
4T1 metastatic breast cancer model in mice. It has been shown that CD40L
can bind to the receptors located on ECs, which results in the localization ofleukocytes to the tumor endothelium via cytokines and chemokines
production121, thus justifying our selection of EPCs as cellular vehicles for
CD40L as cancer therapy.
The criteria for an excellent cellular delivery vehicle include simple
production and expansion, capability for tumor tropism, feasibility of genetransfer, and ability to exert therapeutic effects. We will investigate whether
our produced EPCs possess these traits in this project. In the first part of thestudy, we investigated the derivation and characterization of EPCs fromhPSCs, both the hESCs and the iPS cells.
We continued by observing the tumor tropisms of EPCs toward cancer
cells. We employed a dual-color imaging system, making use of luminescentbased luciferase and fluorescent-based near-infrared DiR systems. Using thismethod, we tracked both the growth of tumor cells expressing luciferase andthe distribution of DiR-labeled cells after injection into the mice noninvasively over time. The tumor tropisms of EPCs in different mouse tumor
models were investigated: the 4T1-luc mammary pad model, 4T1-luc lungmetastatic model and orthotopic glioma model.
Though attractive, the tumor tropic properties can be a double edged
sword. Mesenchymal stem cells (MSCs) are also favored as cellular vehicles
due to their tumor tropism. Yet, it has been noted that MSCs may contribute totumor growth by promoting angiogenesis, modulating the immune response32against cancer cells and promoting metastasis122-124. Hence, the therapeutic
effects exerted by the transgene expressed via these cellular vehicles must beable to overcome the ability of these cells to sustain cancer cells. We
investigatedthecapabilityof

iPS-EPCs

in

tumor

growthandimmunomodulation alongside iPS-NSCs, another well-known cellular vehicle.
We went on to test the feasibility of gene transfer in hPSCs-derived
EPCs. We focused on the use of baculovirus to facilitate the gene transfer to
EPCs. Baculoviral vectors, which are derived from an insect virus Autographacalifornica multiple nucleopolyhedrovirus, emerged as a potentially safe classof gene delivery vectors due to its inability to replicate or cause toxicity in
mammalian cells125.
Finally, we investigated the cancer immunotherapeutic efficacy of
CD40L delivered by EPCs in a mouse tumor model. We also examined thetherapeutic effects of the HSVtk gene, the golden standard of the suicide genetherapy. Recently, a novel angiogenesis inhibitor, Isthmin (ISM), has beenreported. Originally identified in Xenopus brains, this 60kDa protein could
suppress mouse melanoma tumor growth when overexpressed126, 127. Whether
ISM could inhibit tumor growth in pre-established tumors in mice when
delivered systemically, will be investigated in this study.
2.2 Material and Methods
2.2.1

Cell culture

H1 or H9 hESC lines (WiCell, WI, Madison, USA) were cultured in
mTeSRTM1 medium (Stemcell Technologies Inc, Vancouver, Canada) on
hESC-qualified matrigel (BD Biosciences, San Jose, California, USA). Human
33

iPS cells were generated by utilizing a Cre-excisable polycistronic lentiviral
vector containing Oct4, Klf4, Sox2 and c-Myc genes (Millipore, Bedford,MA) as stated in our previous study128. The generated iPS cells were also
cultured on hESC-qualified matrigel using mTeSRTM1 medium. For
passaging, confluent H1 or iPS cells were washed twice with 1× Dulbecco’s
phosphate-buffered saline (DPBS) without calcium and magnesium (Gibco,
Invitrogen, Carlsbad, CA). Then 1 ml dipase (1 mg/ml; Gibco, Invitrogen) was
added to dissociate the cells in a 37oC incubator for 5 min. The cell colonies
were gently scraped off with a 20 µl pipette tip and seeded onto a new
matrigel-coated 6-well-plate (Nunc, Penfield, New York, USA). A mediumchange was performed every day until the next passaging at day 7.
Mouse 4T1-luc is a breast cancer cell line that stably expresses theluciferase gene (Caliper Life Sciences, Hopkinton, MA, USA). These cells
were passaged every 4 days at a split ratio of 1:5 and maintained in a RPMI1640 medium (Gibco, Invitrogen) supplemented with 10% FBS (fetal bovine
serum,

Hyclone,Waltham,

Massachusetts,

USA)

and

1%

penicillin/streptomycin (P/S; Gibco, Invitrogen). M2-10B4 mouse bonemarrow stroma cells (ATCC, Manassas, Virginia, USA) were also maintainedin this medium, but passaged in 1:10 ratio every 4 days. OP9 cells (ATCC)
were maintained in α-MEM (Gibco, Invitrogen) supplemented with 20% FBS.
OP9 cells were passaged every 4 days at a split ratio of 1:8. T-75 cm2 flasks(Nunc) were coated overnight with 6 ml of 0.1% gelatin A (Sigma-Aldrich, St.
Louis, Missouri, USA) prior to OP9 cell seeding. HUVEC (Lonza, Basel,
Switzerland) were maintained in EGM-2 (Endothelial cell Growth Medium-2,
Lonza).
34

To subculture the cells, the confluent cells were washed twice with 1×
DPBS and treated with 1× trypsin-EDTA (Gibco, Invitrogen) for 5 min in an
37oC incubator. The trypsinized cells were then collected and rinsed with thecell culture medium to neutralize the trypsin. The cells were centrifuged at200-300g for 5 min. Then, the cells were re-suspended in the cell culture
medium and seeded onto a new flask according to the appropriate split ratio.
All the cells were maintained in a 5% CO2 humidified incubator at 37oC.
2.2.2

Stromal-based EPC derivation method

2.2.2.1 OP9 co-culture
To prepare for hematopoietic differentiation of hPSCs, OP9 cells were
cultured in T-175 cm2 flasks (instead of T-75 cm2) for normal culture
maintenance. When they became confluent on day 4, half of the medium waschanged, and the culture dishes were maintained for an additional 4 days. On
day 8, the super-confluent OP9 medium was replaced with 20 ml ofhematopoietic differentiation medium, the α-MEM supplemented with 10%
FBS and 100 μM monothioglycerol (MTG; Sigma Aldrich). Three wells of a
6-well-plate of confluent hPSCs were harvested and added to each dish ofovergrown OP9 cells. The flask was tapped and swirled gently to ensure thatthe hPSCs colonies were well distributed throughout the co-culture before
putting it into the 37oC incubator. The differentiation medium was fully
replaced at day 1, and from day 4 of the co-culture, half of the medium waschanged every 2 days.
The cell co-cultures were harvested at day 8 using collagenase IV
treatment for 10 min, followed by trypsin for 10 min. The cell suspension was
35then filtered through a 70 μM nylon cell strainer (BD Labware, Bedford, MA,
USA). The harvested cells were then magnetically sorted using CD34
microbeads (Miltenyi Biotec, Cologne, Germany). The sorted EPCs wereseeded on fibronectin-coated plates in EGM-2 medium. Fibronectin was usedas the extracellular matrix as it has been shown to improve endothelial celladhesion and seeding efficiency129.
2.2.2.2 M2-10B4 co-culture
To prepare M2-10B4 as stromal cells, the confluent cells were treated withmitomycin C (10ug/ml; Roche, Basel, Switzerland) for not more than 3 h. The
cells were then collected, seeded onto a T-175 cm2 flask (4.5e6 cells/flask) andwere allowed to settle overnight in a 37oC incubator with 5% CO2. The nextday, the hPSC colonies were seeded onto the M2-10B4 stroma as explained
above. The co-culture medium used was RPMI-1640 supplemented with 15%
FBS, 1 mM NEAA (Non-Essential Amino Acids; Invitrogen) and 0.1 mM βmercaptoethanol (Gibco, Invitrogen). The medium change was performed
every 2-3 days. The co-cultures were harvested after 13 days using the same
method as above.
2.2.3

Non-stromal-based EPC derivation method

2.2.3.1 2-D culture
For 2-D monolayer differentiation, the method was drawn from a publishedarticle130, with some modifications. Undifferentiated hPSCs were cultured at ahigher density on matrigel for 7 days. At the start of the differentiation phase,the culture medium was changed to a 2-D differentiation medium, IMDM
supplemented with 15% defined-FBS (Hyclone), 450 µM MTG, 2 mM L36

glutamine (Gibco, Invitrogen), 1× P/S and 0.1 mM NEAA. The medium waschanged every 2-3 days for 10 days. After differentiation, the single-cellsuspensions were prepared by treatment with 2 mg/ml collagenase B (Roche)for 10 min at 37oC. CD34+ cells were then isolated as mentioned above.
2.2.3.2 Embryoid bodies method
The confluent hPSC cultures were treated with 1 mg/ml dispase for 5 min.
After dispase was removed, the cells were washed twice with PBS. The
colonies were then detached by scraping the colonies with a pipette tip. The
scraped colonies were transferred in a low attachment dish in STEMdiffTM
APELTM medium (Stemcell Technologies Inc) to form embryoid bodies (EBs)for 4 days. This animal product-free medium has been shown to result insuperior

reproducibilityofEB

formation

and

high

efficiency

ofdifferentiation131. Afterwards, the formed EBs were plated onto a matrigel
coated 10-cm culture dish and cultured for another 10 days.

Cytokines(Peprotech, Rocky Hill, New Jersey, USA) were added to supplement themedium as follows: 20 ng/ml BMP4 (day 0-7), 10 ng/ml Activin A (day 1-4),
8 ng/ml FGF2 (day 2 onwards), and 25 ng/ml VEGF (day 4 onwards). In
addition, a TGF-β inhibitor, 10 μM SB431542 (Tocris Bioscience, Bristol,UK) was added from day 7 onwards132. On day 14, the cells were harvested bytreatment with collagenase IV (Gibco, Invitrogen) for 10 min, followed bytrypsinisation (trypLE, Invitrogen) for another 10 min. The harvested cells
were then magnetically sorted using CD34 microbeads. The sorted EPCs wereseeded on fibronectin plates in Endothelial Growth Medium-2 (EGM-2

37

medium, Lonza) supplemented with 10 μM SB431542. The cells frompassages 2 and 3 were used for characterization and animal experiments.
2.2.4

Characterization of EPCs

2.2.4.1 Flow cytometry
Generated iPS-EPCs were characterized by flow cytometric analysis using
anti-CD34, CD31, Flk1, CD144, CD45 antibodies (BD Biosciences) and antiCD133/1 antibody (Miltenyi Biotec). Quantitative analyses were performed
using FACSCalibur flow cytometer (BD Biosciences).
2.2.4.2 Immunostaining
Immunofluorescent staining was prepared using primary anti-von Willebrandfactor (vWF; Abcam, Cambridge, UK) and secondary fluorescent Alexa488
conjugated anti-rabbit antibodies (Invitrogen). Antibodies were diluted at
1:200 in 1% BSA/PBS. Hoechst (Invitrogen) was used to stain the nucleus ofcells. Immunofluorescence was visualized and images were captured using an
Olympus image analysis system (Olympus, Tokyo, Japan).
2.2.4.3 Tubulogenesis assay
For tubulogenesis assay, a 48-well-plate was coated with matrigel (150µl/well) and incubated at 37oC for 1 h. EPCs were then seeded on the matrigel
coated plates, 2×104 cells/well, in 250 µl EGM-2 medium and incubated at
37oC overnight. The tube-like structures were photographed under a phasecontrast microscope.

38

2.2.4.4 DiI-Ac-LDL assay
To test the ability of our iPS-EPCs to uptake acetylated-LDL (Ac-LDL), the
cells

were

incubatedwith

1,1’-dioctadecyl

3,3,3’,3’-tetramethylindo

carbocyanine perchlorate (DiI-Ac-LDL; Invitrogen) at a concentration of 10µg/ml in EGM-2 for 4 h at 37oC. After washing, the cells uptaking Ac-LDL
were observed by their fluorescent signal under the microscope andphotographed.
2.2.5

Baculoviral vector preparation

Recombinant baculoviral vectors expressing eGFP, mouse Isthmin, HSVtk or
CD40L were constructed using the BAC-to-BAC baculovirus expression
system according to the manufacturer’s manual (Invitrogen). These genes
were under the control of a CMV promoter with the R segment and part of the
U5 sequence of long terminal repeat from the human T-cell leukemia virus
type 1 at 5’ UTR and the woodchuck hepatitis virus posttranscriptional
regulatory element (WPRE) at 3’ UTR. The constructs were produced byhomologous recombination after co-transfection of Sf9 insect cells with
pBacPAK9 transfer vector containing the expression cassette and linearized
AcMNPV viral DNA (Clonetech, Mountain View, CA, USA).

The

recombinant baculoviruses were propagated in Sf9 insect cells (Invitrogen) at
an MOI of 0.1 in Sf-900 II SFM (Invitrogen) and maintained at 27oC. The
virus-containing supernatant was collected 3 days after virus infection. The
budded viruses in the insect cell-culture medium were centrifuged at 1000 g
for 10min to remove cell debris, and concentrated by a second round ofcentrifugation at 28,000 g for 1 h.
39Virus pellets were re-suspended in 1× PBS and virus titers were
determined by real time PCR for Gp64 expression. Viral DNA was isolatedusing a Viral DNA purification kit (Roche). For real time PCR, the forwardandreverseprimers

used

were

gp64-F1337-S23:

5'-

AAAGCAACCTCATAACCACCATG,

andgp64-R1429-A20:

5'-CCAATTCGCCTTCAGCCATG. The Taqman probe gp64-P1395-A21 waslabeled with FAM (6-carboxy-fluorescein) at the 5’-end and a non-fluorescentdark quencher, BHQ1(Black Hole Quencher), at the 3’-end, 6-FAM-5'CAGACTGGTGCCGACGCCGCC-BHQ1. The PCR sample mix was asfollowed: 100 nM primers and Taqman probes, 2X iQ Supermix (Bio-Rad,
Hercules, California, USA), 2 μl template viral DNA or water as the nontemplate control to a final volume of 25 μL. The thermal cycling included an
initial denaturation step of 3 min at 95°C, followed by 40 cycles of 95°C for
15 sec, and 60°C for 1 min with fluorescence detection. For all real-time
amplifications, triplicates of five 10-fold dilutions of the plasmid DNA
standards, viral DNA samples and non-template water controls were
simultaneously subjected to analysis.
For baculoviral transduction, cells were transduced in at a multiplicity
of infection (MOI) of 100 plaque forming units (pfu) per cell overnight. A fullchange of medium was done the next day to remove the viral particles andstop the transduction. GFP expression was detected under a fluorescencemicroscope and by flow cytometric analysis. To confirm CD40L gene
expression, flow cytometry was performed using anti-CD40L antibody (BDbiosciences).

40

2.2.6

Animal studies

2.2.6.1 Animals
Pathogen-free,

6

to

8

week-old

immunocompetent

BALB/cmice,

immunocompromised athymic nude (nu/nu) BALB/c mice or NSG mice
(NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice from Jackson Laboratory, USA) wereused in this study. All handling and care of animals were carried out according
to the Guidelines on the Care and Use of Animals for Scientific Purposesissued by the National Advisory Committee for Laboratory Animal Research,
Singapore.
2.2.6.2 Dual in vivo imaging system
Mouse 4T1-luc was utilized to facilitate in vivo bioluminescent imaging. To
generate an orthotopic breast cancer model, the cells were injectedsubcutaneously onto the mammary fat pad (1×105 cells in 50 µl PBS). To
generate a breast cancer lung metastasis model, 4T1-luc cells were injected
intravenously via the tail vein of the mice (1×104 cells in 200 µl PBS). To
monitor the bioluminescent signals of 4T1-luc in the mice, isoflurane gasanesthetized animals were injected intraperitoneally with D-luciferin(Promega, Madison, Wisconsin, USA) at 100 mg/kg in PBS.
To observe the biodistribution of iPS-EPCs in the mice non-invasively
over time, a lipophilic, near-infrared fluorescent dye 1,1'-dioctadecyl-3,3,3',3'tetramethyl indotricarbocyanine iodide (DiR; Caliper Life Sciences) was used
to label the cells. Cells were labelled with 50 µg/ml DiR for 30 min in a 37oC
incubator. After labelling, the cells were washed three times with 1× PBS.
Labelled or non-labelled iPS-EPCs cells were injected into mice through the41

tail vein (5×105 in 200µl PBS) or via an intratumor injection (5×105 in 100µl
PBS).
After injections, whole animal imagings were performed at day 1, 3, 7
and 14 to track the migration of the iPS-EPCs and observe the tumor growth.
Briefly, the mice were placed on a warmed stage inside the camera box of the
IVIS imaging system coupled with cool CCD camera (Xenogen, Alameda,
CA, USA). Images and measurements of luminescent signals were acquired
and analyzed with the Xenogen living imaging software v2.5 and quantified as
photons per second.
2.2.6.3 Biodistribution of EPCs in intracranial 2M1 tumor model
Invasive human glioma cell line 2M1 previously established in our lab wasused to test the biodistribution of EPCs in the mouse glioma model133, 134. To
investigate iPS-EPCs distribution in the tumor, 5×105 2M1 cells were labeledwith green fluorescent dye CM-DiO (Invitrogen) and inoculated into the rightside of the nude mouse striatum. One week later, 1×106 iPS-EPCs, which werelabeled with CM-DiI dye (Invitrogen), were injected at the same site of thetumor injection. Brain tissues were collected one week after the EPCs
injection for sectioning and examination.
2.2.6.4 Therapeutic studies of EPCs
To investigate therapeutic effects of iPS-EPC-mediated gene delivery, thebreast cancer lung metastasis model in nude (nu/nu) BALB/c mice describedabove was used. At day 3 post-tail vein injection, mice were randomly divided
into different groups (n = 10 per group). For the CD40L experiment, the42

groupings were as follows: PBS, iPS-EPC, iPS-EPC-Bacpak and iPS-EPCCD40L. For the HSVtk experiment, the groupings were: PBS, iPS-EPC, iPSEPC-HSVtk and iPS-EPC-HSVtk+GCV. Lastly, for the isthmin experiment,
the groupings were: PBS, iPS-EPC, iPS-EPC-Bacpak and iPS-EPC-misthmin.
The iPS-EPCs were transduced with the respective recombinantbaculovirus at an MOI of 100 pfu per cell. Three days after tumor inoculation,baculovirus-transduced iPS-EPCs (1×106 in 200 μl PBS per animal) were
injected into the mice through the tail vein. Tumor growth was monitored
using whole-animal imaging as described above and animal survival rates
were recorded.
2.2.7

Histology

The collected organs from mice were submerged in 4% paraformaldehyde for
24 h. The organs were then transferred into a 30% sucrose solution and were
immersed in 30% sucrose for another 24 h. Subsequently, the tissues wereembedded in Jung tissue freezing medium (Leica Microsystems, Gmbh,
Nussloch, Germany). Organs were then frozen and placed in a -80oC freezer
for storage. Sections 12μm thick were taken with a Leica CM3050S cryostat(Leica Microsystems, Wetzlar, Germany). They were then observed with aconfocal microscope.
2.2.8

Statistical analyses

All data are represented as mean ±SD. The statistical significance ofdifferences was determined by one-way ANOVA. The statistical analysis ofsurvival data was performed using the Gehan-Breslow log rank test followed43

by the Holm-Sidak method for pairwise multiple comparison tests. A p valueof <0.05 was considered to be statistically significant.
2.3 Results
2.3.1

Generation of EPCs from Human Pluripotent Stem Cells

2.3.1.1 OP9 co-culture method
We first derived EPCs from hESC line H1 via co-culture with OP9 mousestromal cells according to a previously published protocol by Vodyanik and
Slukvin (Fig. 2.1A)135. The super-confluent OP9 cells ready for co-culture isshown in Figure 2.1B, whereas Figure 2.1C shows an example of a healthy
undifferentiated H1 colony. The H1 colony was nicely seeded and attached on
top of the OP9 stroma forming a button-like structure (Fig. 2.1D left). On day
4 of the co-culture (Fig. 2.1D middle), the H1 cells differentiated by forming
organized root-like structures that penetrated the OP9 portion of the co-culture
and spread away from the button-like structure. The root-like structures grewthicker over time, as seen in the image of the co-culture on day 6 (Fig. 2.1D
right). After 8 days of co-culture, the cells were harvested and the CD34+ cells
were then sorted by two rounds of selection using the Magnetic Assorted Cell
Sorting (MACS) method. From one T-175 cm2 flask, in which three wells of a
6-well-plate H1 culture were seeded, we could obtain 5×105 to 1×106 CD34+
cells after two rounds of MACS. The cells showed typical cobblestone EPC
morphology, after they were reseeded on a fibronectin-coated plate (Fig.
2.1E).
We observed the percentage of CD34+ cells by using flow cytometry(Fig. 2.1F). Before sorting, the percentage of CD34+ cells varied from 5-10%.
44

After two rounds of sorting via MACS, the purity of the CD34+ cells can reachup to 80-99%. Most of the sorted CD34+ cells also co-expressed the CD31
marker (82.84%) as well as Flk1 (75.92%). On the contrary, the CD45 marker
was expressed at a very low level (3.28%). Furthermore, these cells canmaintain the CD34 and CD31 markers for up to four passages (Fig. 2.1G).
The sorted EPCs formed tube-like formations upon seeding on matrigel (Fig.
2.1H), another in vitro characteristic of EPCs.
EPCs can also be generated from iPS cells (Fig. 2.2A) via the OP9 coculture method. The co-culture of iPS colonies and OP9 cells at day 8 also
displayed outward root-like expansion, similar to that of H1-OP9 co-culture(Fig. 2.2B). The resulting EPCs after sorting had similar morphology (Fig.
2.2C) and expression of endothelial markers shown by flow cytometry (Fig.
2.2D) as that of H1-EPCs. Furthermore, these cells also had the capability of
tubulogenesis upon seeding on matrigel (Fig. 2.2E).

45

Figure 2.1. Derivation of hESC-derived EPCs via mouse stromal OP9 co-culture. A.
Schematic diagram on the OP9 co-culture protocol. H1 colonies were co-cultured withovergrown OP9 culture for 8 days. CD34+ cells were then collected using magnetic cellsorting. B. Super-confluent OP9 at day 8 ready for co-culture. C. H1, hESC cell line
colony. D. Co-culture of H1 and OP9 at different time points (Left to right: day 1, 4 and
6). E. EPCs at day 5 after CD34+ magnetic sorting on fibronectin-coated dish. F.
Expression of EPCs surface markers: CD34+, CD31+, Flk1+ before and after sorting
process via flow cytometry. EPCs expressed very low CD45 markers, hematopoietic
lineage markers. G. Expression of CD34 and CD31 after 2nd passage of EPCs. Most of
the cells still co-expressed these markers (95.93%). H. EPCs form tubule-like formation
when seeded onto a matrigel-coated dish.

46

Figure 2.2. iPS-derived EPCs produced via mouse stromal OP9 co-culture. A.
Colonies of iPS cells. B. iPS co-cultured on OP9 stromal cells after 8 days. C. iPSEPCs after sorting by MACS seeded onto fibronectin coated dish. D. Flow cytometry
showing the percentage of CD34 and CD31 markers in the cells before and after
sorting via MACS. E. iPS – EPCs form tubule-like formation when seeded onto amatrigel-coated dish.

2.3.1.2 M2-10B4 co-culture method
M2-10B4 is a mouse bone marrow stroma cell that can support
hematoendothelial differentiation136, 137. Thus, we also used this cell line
alongside OP9 cells to generate EPCs from hPSCs (Fig. 2.3A). After reachingconfluency (Fig. 2.3B), M2-10B4 cells will be treated with mitomycin C inorder to prepare mitotically inactive feeder cells for the co-culture. The cells
were then washed extensively with PBS before being reseeded onto a T-175
cm2 flask and allowed to settle in a 37oC incubator overnight. The next day,colonies of H1 or iPS cells were seeded onto the M2-10B4 feeder cells (Fig.
2.3C). The M2-10B4 co-culture process took a longer time of 13 days,
compared to OP9 co-culture, which only took 8 days. After two rounds of
MACS, we also obtained around 5×105 to 1×106 CD34+ cells (Fig. 2.3D).
47

Figure 2.3. Derivation of hESC-derived EPCs via mouse stromal M2-10B4 co-culture. A. Scheme
diagram on the M2-10B4 co-culture protocol. H1 colonies were co-cultured with mitomycin-C treatedM2-10B4 cell culture for 13 days. Magnetic sorting was then performed to obtain CD34+ cells. B. M210B4 culture at day 4. C. Co-culture of H1 and M2-10B4 at day 13. D. EPCs at day 3 after CD34+
magnetic sorting on fibronectin-coated dish. E. H1 derived EPCs via M2-10B4 co-culture expressed
endothelial lineage surface markers: CD34+, CD31+, Flk1+ before and after sorting process via flow
cytometry. EPC derived from H1 co-cultured with M2-10B4 cells express CD45 markers albeit at low
level, which is a marker for hematopoietic lineage. F. Expression of von Willebrand Factor as shown by
immunostaining (Left to right: phase-contrast, GFP expression, hoechst nucleus staining). G. hESCEPCs form tubule-like formation when seeded onto a matrigel-coated dish.

48

The expression of EPCs markers were tested via flow cytometry (Fig.
2.3E). We observed that the percentage of CD34+ cells varied from 5-20%
prior to sorting. We also noticed that the co-expressions of the CD34 marker
and other EPC markers such as CD31 and Flk1 were lower compared to OP9co-culture derived EPCs. Furthermore, we noted that the sorted CD34+ cells
had a higher expression of the CD45 marker, compared to the OP9 co-culture
derived EPCs.

Figure 2.4. H9 hESC line-derived EPCs via mouse stromal M2-10B4 co-culture.
A. Co-culture of H9 and M2-10B4 at day 6. B. EPCs at day 6 after CD34+ magnetic
sorting on fibronectin-coated dish. C. Flow cytometry showing expression of CD34
together with other EPCs markers: CD31, Flk1 and CD144 before and after MACS.
D. Expression of von Willebrand Factor as shown by immunostaining (Left to right:
phase-contrast, GFP expression, hoechst staining).

49

We further tested the expression of von Willebrand Factor (vWF), a
glycoprotein constitutively produced in the endothelium, in the sorted CD34 +cells via immunostaining, and the results were positive (Fig. 2.3F). These cells
could also form tube-like formations when seeded onto matrigel (Fig. 2.3G).
However, by observing the matrigel tubulogenesis results qualitatively, we
noticed that tubulogenesis was more efficient for the cells that were produced
via OP9 co-culture, compared to the M2-10B4 co-culture method. We also
tested this method using H9 and iPS cells, and we obtained EPCs with similar
characteristics (Fig. 2.4 – 2.5).

Figure 2.5. iPS cells-derived EPCs via mouse stromal M2-10B4 co-culture. A. Coculture of iPS colonies and M2-10B4 at day 9. B. EPCs at day 6 after CD34+ magnetic
sorting on fibronectin-coated dish. C. Expression of von Willebrand Factor as shown by
immunostaining (Left to right: phase-contrast, GFP expression, hoechst staining). D.
Flow cytometry showing co-expression of CD34 and Flk1 before and after MACS.

50

2.3.1.3 Non-stromal 2-D differentiation method
The above co-culture method can recreate native stem cell niches, thus
providing an inductive environment for the differentiation of hESCs.
However, contamination of animal cells is one concern which needs to be
tackled before this method can be used in clinical settings. Thus, we opted fora non-stromal 2-D differentiation method (Fig. 2.6A) described by Wang etal130. After changing the medium from mTeSR to the differentiation medium,the H1 cells started to spread out, leaving the colonies and filling the entire
culture dish. Interestingly, they also formed root-like structures like thosefound in the co-culture (Fig. 2.6B). From one 10-cm dish of H1 culture, we
obtained around 5×105 to 1×106 CD34+ cells after 2 rounds of MACS (Fig.
2.6C). The EPCs generated via the 2-D differentiation method had a similar
expression of surface markers as those produced by the OP9 co-culture
method. After sorting, most of the CD34+ cells also co-expressed CD31, Flk1
and VE-Cadherin, but not CD45 (Fig. 2.6D).
By using the same method, we can also generate iPS cell-derived EPCs
(Fig. 2.7A-B). The flow cytometry results reveal some differences between
the hESC and iPS cell-derived EPCs (Fig. 2.7C). For H1-derived EPCs, thepurity of CD34+ cells after 2 rounds of MACS was higher (˃90%). Besides,
most of the CD34+ cells were also CD31+, Flk1+ and VE-cadherin+(approximately 90%). However, in the case of iPS cell-derived EPCs,
approximately 50-55% of the cells co-expressed these markers. Yet, thesecells were negative for CD45 expression, similar to those derived from H1.

51

Figure 2.6. H1 hESC-derived EPCs via 2-D differentiation method. A. Protocol
for 2-D differentiation method. H1 medium was changed to differentiation medium
when H1 culture reached confluency. After 10 days, MACS was performed to obtain
CD34+ cells. B. H1 differentiation at day 6. C. Sorted CD34 on fibronectin-coateddish. D. Flow cytometry of the cells before and after sorting via MACS of thedifferent EPCs markers. Most of the CD34+ cells co-expressed CD31, Flk1, and VEcadherin, but 0.35% expressed CD45 marker after sorting.

52

Figure 2.7. iPS cells-derived EPCs via 2-D differentiation method. A. Image
showing iPS at day 6 of differentiation. B. Sorted CD34+ cells plated on fibronectincoated plate. C. Flow cytometry of the cells before and after sorting via MACS of thedifferent EPCs markers. Lower percentage of CD34 as well as other markers ascompared to hESC-EPCs was observed before and after MACS.

2.3.1.4 Human iPS cell-derived EPCs via embryoid bodies formation
A protocol has been published to use well-established human ES cell lines todifferentiate mesoderm cells in EBs into endothelial precursors132. We tested
whether the protocol could be used to derive endothelial precursors fromearly-passage human iPS cells (around p10). EBs formed by human iPS cells
cultured on low attachment dishes were plated onto a matrigel-coated culture
dish and treated with BMP4 (day 0-7), Activin A (day 1-4), FGF2 (day 2
onwards), VEGF (day 4 onwards), and SB431542, a TGF-β inhibitor, (day 7
53

onwards) for 2 weeks (Fig. 2.8A-B). After the EB was replated, we observed
the root-like structure again during the differentiation process (Fig. 2.8B
right). From one 10-cm dish of EB (i.e from one well of a 6-well plate iPS
cell culture), we could obtain approximately 1×106 CD34+ cells with just one
round of MACS (Fig. 2.8C).
The percentage of EPCs markers before sorting was highest using thismethod compared to other methods (Fig. 2.8D). Fluorescence-activated cellsorting (FACS) analysis revealed that the generated cells were a mixed
population of cells containing ≥ 20% cells positive for CD34, a hematopoietic
stem cell marker. The mixed population of cells co-expressed endothelial cellmarkers, including CD31 (17.60%), Flk1 (8.13%), and VE-cadherin (10.71%),
but had a low expression level of the CD45 hematopoietic marker (1.44%).
After undergoing immunomagnetic separation to enrich the CD34+
cells and promote cell expansion in an endothelial growth medium, flow
cytometric analysis showed that ≥ 95% of cells at passage 2 were CD34positive (Fig. 2.8D). A majority of the purified CD34+ cells were also positive
for CD31 (81.29%). While a majority of the purified CD34+ cells were also
positive for CD31 (81.29%), approximately 21% of the CD34+ cells coexpressed Flk1, 48% expressed VE-cadherin, and only 8.98% of these cells
were CD45+. Since CD34 is expressed on both immature EPCs and mature
circulating endothelial cells, we also examined the expression of CD133, a
stem cell marker restricted to immature EPCs, on the sorted cells, and foundthat 30% of the cells were CD133-positive (Fig. 2.8E). As a negative control,54

human fibroblasts used to generate iPS cells were tested and showed noCD133 expression (0.76%).
Furthermore, many of the iPS cell-derived cells were vWF-positive(Fig. 2.9A) and CD31-positive (Fig. 2.9B), as shown by immunostaining.
Biological and functional characterization showed that the derived cells wereable to incorporate the DiI-labelled acetylated LDL (Fig. 2.9C) and readily
formed tubule-like structures upon seeding onto matrigel (Fig. 2.9D).
Therefore, we use the term iPS-EPCs to refer to all endothelial lineage cells
derived from iPS cells in the described method.

55

Figure 2.8. Generation of EPCs from human iPS cells via EB method. A.
Generation of EPC from iPS. Confluent iPS colonies were scraped to generate EBs.
After 4days, the EBs were replated onto matrigel-coated dish and cultured for 10
days. CD34+ cells were then harvested and maintained on fibronectin-coated dish. B.
Left to right: EB, 1 day after the EBs were replated, differentiation day 10. C. EPCs
at passage 3 after MACS. D. Flow cytometry before and after the magnetic cellsorting to show expression of EPCs markers CD34, CD31, Flk1 and VE-cadherin,
and less expression of hematopoietic marker CD45. E. Expression of CD34 andCD133 “stemness” markers on sorted for CD34+ cells. Human fibroblast were used as
negative control. Isotype controls were always set ≤1.

56

Figure 2.9. Characterization of iPS cells-derived EPCs. A. Expression of vonWillebrand Factor as shown by immunostaining (left to right: phase contrast, GFP,
Hoechst staining). B. Expression of CD31 as shown by immunostaining (left to right:
phase contrast, GFP, Hoechst staining). C. iPS-EPCs could uptake AcLDL. Phase
contrast (Left) and red fluorescence (Right) images of Ac-LDL taken up by thegenerated iPS-EPCs. Arrow shows DiI-Ac-LDL being taken up by the cells. D. iPSEPCs could form tube-like formation upon seeded onto matrigel-coated dish.

57

2.3.2Tumor tropism of iPS-EPCs

2.3.2.1 Homing of hPSC-EPCs to 4T1-luc orthotopic breast cancer model
To non-invasively examine the in vivo distribution of hPSCs-derived EPCs inmice over time following systemic injection, we employed IVIS dual colorimaging technology. This system detects and captures luminescent signals
from 4T1-luc cells and fluorescent signals from DiR-labelled iPS-EPCs. Prior
to animal experiments, labeling optimization of iPS-EPCs by DiR was
performed. The cells can be efficiently stained with DiR at a concentration of
50 μg/ml for 30 min at 37oC, a process which in having 95.93% of the cells
positively and uniformly labeled as indicated by the sharp peak (Fig. 2.10A).
To examine the in vivo tumor tropism of the generated hPSCs-EPCs,
we established two mouse breast cancer models in athymic Balb/c nude mice:an orthotopic breast cancer model generated by subcutaneous injection of
luciferase-expressing mouse 4T1 breast cancer (4T1-luc) cells onto themammary fat pad (Fig. 2.10B) and a breast cancer lung metastasis model
generated by intravenous injection of 4T1-luc cells via the tail vein (Fig.
2.10C).
We first tested in vivo tumor tropism of iPS-EPCs in the orthotopic
breast cancer model (Fig. 2.11). After labeling iPS-EPCs with the near-infrared
fluorescent dye DiR, we injected the cells into the tumor-bearing mice throughtail vein injections at day 7 post-tumor inoculation. The Xenogen IVIS Imaging
System was employed for non-invasive dual-color in vivo imaging tolongitudinally monitor the distribution of the labeled iPS-EPCs at days 0, 1, 3,
7, and 14 post-EPC injection in the same sets of animals. Whole-body58bioluminescence imaging showed that tumors grew quickly around the
inoculated mammary fat pad during the 21-day observation period, andluciferase signals outside the primary tumor region were observed at day 21
post-tumor inoculation, indicating metastasis (Fig. 2.11A).

Figure 2.10. Dual in vivo imaging system. A. DiR labeling optimization of EPCs.
EPCs can be efficiently labeled with DiR at concentration 50μg/ml for 30min at 37oC.
B. Schematic illustration of the dual in vivo imaging system utilizing both 4T1luc and
DiR-labeled EPC in mammary pad model. Breast cancer 4T1luc cells are inoculated
into mammary fat pad of the mice. One week after the tumor inoculation, DiR-labeled
EPCs are introduced into the mice via tail vein injection. IVIS imaging are carried out
at day 1, 3, 7, and 14 post tail vein injection. C. Protocol for tumor tropism study inlung metastatic model. The 4T1luc cells are inoculated into the mice via tail vein
injection. Three days after the tumor inoculation, DiR-labeled EPCs are introduced
into the mice via tail vein injection. IVIS imaging are carried out at day 1, 3, 7, and14 post tail vein injection.

59

Figure 2.11. Tumor tropism of iPS cells-derived EPCs in the 4T1 orthotopic mousemodel of breast cancer. A. Whole animal images of Balb/c nude mice of injected with DiRlabeled – iPS-EPCs alone, and DiR-iPS-EPCs with 4T1luc breast cancer cells. EPCs can beseen migrating toward primary tumor site (mammary pad region) as early as day 1 post tailvein injection. After 14 days, more cells are migrating towards the tumor site. B. Percentage
distribution of iPS-EPCs in the primary tumor region over the whole body. Overall average of
12% in the tumor-bearing mice as compared to 3% in animal without tumor over time. Therevalues are statistically significant different as tested by one-way ANOVA (p=0.03, 0.026,
0.006,0.021 for day 1, 3, 7, and 14 respectively. C. Ex vivo image of the different organs:brain, lungs, heart, spleen, liver, intestines, kidneys, spinal cord, femur, mammary pad (left toright). Red circle indicates strong DiR signals in the mammary fat pad of tumor-bearing
animals. No singles were detected in the same region in controls.

60Whole-body DiR fluorescence imaging showed that tail vein-injected
iPS-EPCs were initially distributed predominantly to the lung region. One day
after tail vein injection, the size of the DiR signal-positive area in the thoracic
region significantly increased. Meanwhile, we observed significantly
increased DiR signals in the tumor inoculation site in tumor-bearing mice
(Fig. 2.11A). The DiR signals in this region were significantly higher,
compared with those in mice without tumors, and remained high for at least 14
days, although the overall DiR signal intensity reduced from day 3 onwards(Fig. 2.11A). On average, approximately 12% of the injected iPS-EPCs homed
into the 4T1-luc tumor cell inoculated mammary pad region as calculated by
the percentage of DiR signal in the region over the whole body signal (Fig.
2.11B).
At day 14, the mice were sacrificed and ex vivo images of different
organs were taken (Fig. 2.11C). Here, we can scrutinize the metastases thathave occurred in the mice, manifestations which could not be detected bywhole-body imaging alone. The luc signals indicated that the 4T1 tumor cells
metastasized to the lungs, spleen, liver, intestines, spinal cord and femur.
There were overlaps of luminescent (luc) and fluorescent (DiR) signals in the
primary tumor site, implying the homing property of iPS-EPCs (Fig. 2.11C).

61

Figure 2.12. In vivo migration of hESC-EPCs toward 4T1 breast cancer cells in
4T1 orthotopic immunocompromised NSG mice. A. EPCs migrated towardprimary tumor site as early as day 1 post tail vein injection. After 14 days, more cells
are migrating towards the tumor site. B. Percentage distribution of iPS-EPCs in the
primary tumor region over the whole body. Statistical tests showed significant
difference in the values between the two groups (p=0.021, 0.0009, 0,005 for day 1, 7
and 14 respectively). C. Ex vivo image of the solid tumor mass shows overlap of
luminescent luc and fluorescent DiR signals. This implies that EPCs have migrated tothe tumor.

We also tested the in vivo distribution of hESC-derived EPCs in
immune-compromised NSG mice bearing the 4T1-luc tumor in the mammary62

pad. As early as day 1 post-intravenous injection, the DiR fluorescent signal
can be seen around the mammary pad where the 4T1 tumor had been
inoculated a week before (Fig. 2.12A). Most cells were trapped in the liverand lung area. Over time, more EPCs migrated to the 4T1 primary tumor site.
More than 20% of cells migrated to the mammary pad region, based on thepercentage of DiR signal in that region over the whole body signal at day 14(Fig. 2.12B). The tumor ex vivo image reveals that there was an overlap ofsignals between the fluorescent DiR and luminescent luc, indicating that EPCs
home to 4T1 tumor cells (Fig. 2.12C). Thus, we showed that both iPS cells
and hESC derived EPCs have tumor homing properties.
2.3.2.2 Homing of iPS-EPCs to breast cancer lung metastasis model
Next, we tested in vivo tumor tropism of iPS-EPCs in the breast cancer lungmetastasis model (Fig. 2.13). We injected the DiR-labeled iPS-EPCs into thetumor-bearing mice through tail vein injections at day 3 post-tumor inoculation.
Whole-body bioluminescence imaging showed that tumor growth became
detectable at day 10 post-tumor inoculation (day 7 post-EPC injection). By
day 17 post-tumor inoculation, cancer cell dissemination to other organs wasobserved (Fig. 2.13A). Whole-body DiR fluorescence imaging showed thattail vein-injected iPS-EPCs were distributed predominantly to the lung region
and decreased over time from day 1 to day 14 post-EPC injection (Fig.
2.13A). When compared with DiR signals in normal Balb/c mice that were
injected with only the labeled iPS-EPCs without 4T1 tumor cell inoculation, it was
found that DiR signals in the tumor-bearing mice were relatively stronger in63the lung region and a statistically significant difference between the twogroups of mice was observed at day 14 (Fig. 2.13B).

Figure 2.13. Tumor tropism of iPS cells-derived EPCs in the 4T1 breast cancer
lung metastasis model. A. Whole body images showing the EPCs are trapped in thelungs regions, for both the control group and tumor bearing animal group. B. The DiR
signal over time in both groups showed similar trend.

642.3.2.3 Tumor tropism of iPS-EPCs to 2M1 invasive glioma model
We also investigated the distribution of EPCs after intratumor injection into a
2M1 invasive glioma orthotopic tumor model (Fig. 2.14A). In the primarytumor site, we can see that the EPCs infiltrated the tumor mass, migrating
towards the tumor vessels (Fig. 2.14B-C). More importantly, EPCs can beseen infiltrating successfully into the tumor blood vessels, as shown in theyellow circle (Fig. 2.14D). Interestingly, EPCs can also be found in tumor
satellites located in the lateral ventricle, away from the primary tumor site
(Fig. 2.15A-C). This implies that EPCs can target secondary metastatic tumor
sites.

65

Figure 2.14. Biodistribution of EPCs
in intracranial 2M1 glioma model atprimary tumor mass. A. Protocol of thebiodistribution experiment on mice
glioma model. B. Primary DiO-labeled
2M1 tumor mass, infiltrated by DiIlabeled EPCs. White line: tumor borderregion. C. Magnified view of B, showing
the tumor blood vessels. The EPCs
infiltrate the tumor region, toward thetumor blood vessels. White line: tumor
border region, yellow lines: the tumor
blood vessels. D. EPCs incorporated into
the tumor blood vessel (yellow line).
White line: tumor border.

66

Figure 2.15. Biodistribution of EPCs
in intracranial 2M1 glioma model atsecondary tumor foci. A. DiOlabeled 2M1 cells found at the lateral
ventricle, infiltrated by DiI-labeled
EPCs. B. Magnified view of A. EPC
can target secondary tumor sites. C.
Different secondary tumor site, where
EPCs were also found.

2.3.3

Effects of iPS-EPCs on tumor development and metastasis

The potential role of EPCs in promoting tumor growth is a concern when thesecells are used as delivery vehicles for cancer therapy138. To investigate
whether iPS-EPCs alone affect tumor progression and metastasis in the above
orthotopic breast cancer model, we injected iPS-EPCs intratumorally at day 7
post-inoculation of 4T1-luc breast cancer cells (Fig. 2.16A). Intratumor
injections of iPS cells, fibroblasts, and PBS were included as controls. Anotherwell-established cellular delivery vehicle, iPS-NSCs, was also injected
intratumorally for comparison (Fig. 2.16A). The tumor progression is similar
throughout the different groups and comparable with the biodistribution

67

results (Fig. 2.11). The average tumor signals of the four groups were similar
up to day 7 (Fig. 2.16B). Up to day 14, the tumor volume for the iPS-EPC
group remained low compared to the rest of the groups. However, by day 21
the luc signal is comparable among all the groups. However, this can be due tothe deaths of animals from the iPS group, which might explain the sudden
drop in the luc signal at day 21 (Fig. 2.16B). The animals were sacrificed atday 21 and their organs were collected for ex vivo bioluminescence imaging(Fig. 2.17A). The 4T1-luc signal intensities in the examined organs werecomparable in all groups (Fig. 2.17B). Although the tumor signal in theintestine was higher in the iPS-EPC group compared to the other groups,
statistical analysis revealed that the differences were not significant.
Possible effects of iPS-EPCs on tumor development were further
investigated in the breast cancer lung metastasis model. Taking advantage ofthe quick animal death in this model, we focused on the survival of tumorbearing animals in this experiment. The mice were separated into five groups:
PBS, iPS, human fibroblast, iPS-NSCs and iPS-EPCs. The different cells were
injected intravenously via the tail vein 3 days after intravenous injection of
4T1-luc cells. Tumor growth was monitored by whole-body bioluminescentimaging of 4T1-luc cells. Whole body imaging of three mice from each groupwas shown as representatives (Fig. 2.18A). The bioluminescence intensities,
indicative of tumor volume, demonstrated that there were no significant
differences among the PBS group, iPS cell group, and iPS-EPC group, although asignificantly higher level of 4T1-luc signal was observed in the HFF group at day
21 (Fig. 2.18B).

68

Mice started to die at day 17 in all groups. The median survival times were26.2, 26.8, 25.6, and 27.6 days for the PBS, fibroblast, iPS cell and iPS-EPC
groups respectively. There was no statistically significant difference among
the four groups in survival rate (p = 0.946) (Fig. 2.18C). Our findings indicatethat iPS-EPCs did not obviously promote tumor growth in the 2 tested tumor
models.

Figure 2.16. Effects of iPS-EPCs on 4T1 tumor development and metastasis in
the 4T1 orthotopic mouse model of breast cancer. A. Whole body imagings of 3
representative animals from each groups: PBS, HFF, iPS and iPS-EPCs. Similar
tumor progression as shown by the bioluminescent signals. Metastases can bedetected at 2 weeks time point onwards. B. Tumor volume average from each groups.
iPS-EPCs remained low.

69

Figure 2.17. Effect of iPS-EPCs on tumor development and metastasis in 4T1
mammary pad model. A. Representative ex vivo images of different organs
collected at day 21 post-intratumor injection: lungs, liver, spleen, kidneys, heart,brain, intestines, spinal cord, femur, primary tumor mass (from left to right). Images
on iPS-EPCs group showed comparable metastatic progression as the other control
groups. B. Luc signal of different organs. ANOVA statistics revealed that no
statistically significant difference in the signal of the different organs among thegroups.

70

Figure 2.18. Effect of iPS-EPCs on 4T1luc tumor development in the 4T1 breast cancer
lung metastasis model. A. Whole body imagings of 3 representative animals from eachgroups: PBS, human foreskin fibroblast (HFF), iPS and iPS-EPCs. Bioluminescent signal of
4T1luc show the tumor development over time after tail vein injection of different cells. At
day 21, animals from iPS-EPCs group show less intense signal and less metastatic indication.
B. Average tumor volume in each group represented by the bioluminescent luc signal. At day
21, there is a statistically significant difference in the luc signal among the groups (p=0.004).
Value in HFF group is significantly different from PBS, iPS-NSCs and iPS-EPCs groups(p=0.00301, p=0.0007 and p=0.001 respectively). C. Survival curves of tumor-bearing mice.
No statistically significant difference in the survival rates among the groups (p=0.946).

71

2.3.4

Genetic modification of EPCs

Figure 2.19. Genetic modifications of EPCs. A-B. Baculovirus transduction in EPCs
(A.) Flow cytometry showing the percentage of EGFP in iPS-EPCs after transduction
with baculoviral vector expressing EGFP. (B.) Cells expressing green eGFP after
transduction with baculovirus (left) and the phase-contrast picture (right). C. H1-BVRMCE-EGFP in M2-10B4 co-culture. H1 colonies maintained the EGFP expression
throughout the differentiation process. D-E. H1-BV-RMCE-EGFP in 2D EPC
differentiation. (D.) H1 colonies, CD34+ cells after sorting and replating on fibronectincoated dish expressed EGFP as shown by the picture (E.) and flow cytometry of EGFP.

72

A high level of permissiveness to gene transfer is a vital requirement thatallows the use of cellular vehicles for cancer gene therapy. Baculoviral
vectors, which are derived from an insect virus Autographa californica
multiple nucleopolyhedrovirus, emerge as a potentially safe class of genedelivery vectors due to their inability to replicate or cause toxicity in
mammalian cells125. We showed that baculoviral vectors are capable oftransducing iPS-EPCs. By using a baculoviral vector which expresses EGFP,
we showed that 85.93% of the cells are EGFP positive, 60.99% of which are
CD34+ (Fig. 2.19A). The expression of EGFP in the cells were strong andbright (Fig. 2.19B).
Recently, our lab has successfully established a system to geneticallymodify hESCs specifically at AAVS1 locus139. The system involves a two-stepprocess combining homologous recombination with baculovirus-mediated(BV) Cre-RMCE (Cre recombinase-mediated cassette exchange). The
generated master loxP-hESC line produced in the first step provides rich
opportunities for readily introducing transgenes into AAVS1 through efficient
BV-Cre-RMCE. By using the two-step process combining homologous
recombination with BV-Cre-RMCE, we can produce EGFP expressing EPCs
from the loxP-hESC line (Fig. 2.19C-D). The stable EGFP-expressing H1
colonies could be differentiated into EPCs. In Fig. 2.19C, we see that the H1colonies maintain their EGFP expression throughout the M2-10B4 co-culture
period. The EGFP expressing H1 could also be differentiated via the 2-D
differentiation method without any loss of EGFP expression (Fig. 2.19D).
After sorting, all the CD34+ cells expressed EGFP (Fig. 2.19D), and this was
further confirmed by flow cytometric analysis (Fig. 2.19E).
73

2.3.5EPCs therapeutic effects

2.3.5.1 iPS-EPC expressing CD40L impede tumor development in a breast
cancer lung metastasis model
To assess the prospects of iPS-EPCs as the cellular delivery vehicle fortherapeutic genes, we transduced the cells with baculovirus expressing CD40L
gene, a member of the tumor necrosis factor (TNF) gene family108. Flowcytometric analysis indicated that up to 82.23% of iPS-EPCs could betransduced to express detectable levels of CD40L (Fig. 2.20B). The
transduced cells were injected via the tail vein into athymic Balb/c nude micebearing 4T1-luc breast cancer lung metastases. Tumor growth was monitored bywhole-body bioluminescent imaging of 4T1-luc cells. Figure 2.20A shows
representative 4T1 cell-bearing mice from different groups at 4 different time
points. The bioluminescence intensities, indicative of tumor volume, demonstratethat the inhibitory effect of CD40L-expression iPS-EPCs on tumor growth was
visible at day 14. Compared to the control groups, a statistically significant lower
level of 4T1 cell-related luciferase signals was observed in the treated mice by
day 21 post-tumor inoculation (Fig. 2.20C). Survival of the tumor-bearing micein this group was significantly prolonged, and this was attributed to the effect of
CD40L-expression of iPS-EPCs (Fig. 2.20D). On day 35, while all the mice in thethree control groups had died, 60% of animals in the treated group were still alive(Fig. 2.20E). The median survival times were 25 to 29 days in the control
groups, and 34 days in the treated group. The difference was statisticallysignificant (p = 0.004 in Holm-Sidak test). There was no statisticallysignificant difference among the control groups in median survival time (p >0.05).
74

75

Figure 2.20. Therapeutic effects of CD40L-expressing iPS-EPCs in the 4T1
breast cancer lung metastasis model. A. Bioluminescent images of tumor growth inrepresentative animals from each group at days 1, 7, 14 and 21 after iPS-EPC
injection. Tumor signal is weaker in animals belong to iPS-EPCs-CD40L. B. CD40L
expression in iPS-EPCs after transduction with a baculoviral vector containing
CD40L gene under the control of the CMV promoter. C. Quantitative analysis ofbioluminescent signals. Up to week 2, the average tumor signal is comparable. At day
21, there is a significant difference in the tumor volume of iPS-EPCs-CD40L groupagainst the other control groups (P<0.001). Bar: s.d. * p < 0.05 vs the group of mice
injected with iPS-EPC/BV-CD40L by analysis of variance. D. Survival curves of 4T1
tumor-bearing mice. CD40L expression prolongs the life of 4T1 tumor-bearing
mice.The Gehan-Breslow statistic for the survival curves is greater that would be
expected by chance; there is a statistically significant difference between survival
curves (P = 0.004). E. Graph showing the values of median survival times of animals
from different groups. Statistical analysis by the log rank test reveals the significantly
improved survival in the group of mice injected with iPS-EPC/BV-CD40L as
compared with other groups.

2.3.5.2 iPS-EPCs expressing HSV-tk
We also tested the therapeutic effect of EPCs expressing the HSV-tk suicide
gene. According to the images of the whole animal (Fig. 2.21A) and theaverage luc signal reading (Fig. 2.21B), the HSV-tk+GCV group has a lower
tumor burden compared to the control groups (PBS, EPC alone, EPC-HSVtk
without GCV) at day 14 and 21. However, when we ran the one way ANOVA
tests, it was found that these differences are not statistically significant.
Furthermore, the survival of the mice is only slightly prolonged and thesurvival curve (Fig. 2.21C) shows no significant differences among the groups
according to the Gehan-Breslow test (P=0.084).

76

Figure 2.21. Therapeutic effect of iPS-EPCs expressing HSV-tk in 4T1luc lungmetastatic Balbc/nude mice. A. Whole images of 3 representative animals fromeach group: PBS, iPS-EPCs, iPS-EPCs express empty baculoviral vector, and iPSEPCs express baculoviral vector containing HSVtk gene. B. Tumor volume of thedifferent groups of mice as shown by the luc signal. C. Survival curve of the animals.
The Gehan-Breslow statistic for the survival curves is not great enough to exclude thepossibility that the difference is due to random sampling variability; there is not a
statistically significant difference (P = 0.084).

2.3.5.3 iPS-EPCs expressing Isthmin
Isthmin (ISM) is a 60kDa anti-angiogenesis protein that is secreted at highlevels in the hindbrain-midbrain organizer in Xenopus embryos126, 127. We
investigated the cancer therapeutic effect of this anti-angiogenic protein when
it is expressed through EPCs. We constructed a baculoviral vector which77

expresses mouse isthmin (BV-CMV-mISM-WPRE). The mouse isthmin
plasmid was generously given by Prof Ge Ruowen’s lab (Department of
Biological Sciences, National University of Singapore).
BV-CMV-mISM-WPRE was tranduced into EPCs and its expression
was confirmed through PCR (expected band size of 485 bp) (Fig. 2.21A). We
then proceeded to test the anti-angiogenic activity of isthmin in vitro via
matrigel tubulogenesis experiments (Fig. 2.21B-F). Figure 2.21B shows theresults of matrigel tubulogenesis using EPCs which have been transduced with
BV-CMV-EGFP-WPRE. We can see that the tubulogenesis was not affected,
compared to the control set (Fig. 2.21F). Likewise, when we used theconditioned medium of EPCs transduced with EGFP, and ran the
tubulogenesis experiment using freshly prepared EPCs, the tubulogenesis was
not affected too (Fig. 2.21C). Unfortunately, when we tested the tubulogenesis
of EPCs transduced with BV-CMV-mISM-WPRE (Fig. 2.21D) or fresh EPCs
with a conditioned medium of EPCs transduced with isthmin (Fig. 2.21E), thetube-like formations were not affected as well.
Interestingly, when HUVEC is transduced with isthmin before matrigel
tubulogenesis was performed, the same results were observed whereby thetubulogenesis was not affected. However, when the isthmin-conditioned
medium was concentrated 100× and used for the matrigel tubulogenesis for
fresh HUVECs, tubulogenesis failed to occur (data not shown, performed in
Prof Ge Ruowen’s lab, DBS, NUS). Thus, we proceeded to test whether
isthmin-expressing EPCs have a therapeutic effect in 4T1-luc tumor bearing
mice.

78

Figure 2.22. Therapeutic effects of EPCs encoding mIsthmin. A. In vitro
expression of BV-CMV-mISM-WPRE in iPS-induced EPCs. B. matrigel
tubulogenesis performed using EPCs transduced with BV-CMV-EGFP-WPRE in
fresh EPC medium (left: phase contrast, right: EGFP expression). C. Negative
control; Fresh EPCs in fresh EPC medium. D. matrigel tubulogenesis performed
using EPCs transduced with BV-CMV-mISM-WPRE in fresh EPC medium. E.
Matrigel tubulogenesis performed using fresh EPCs in BV-CMV-EGFP-WPRE
transduced EPCs conditioned medium. F. Matrigel tubulogenesis performed using
fresh EPCs in BV-CMV-mISM-WPRE transduced EPCs conditioned medium. G.
Tumor volume of the different groups of mice as shown by the luc signal. One way
ANOVA tests showed no significant difference among the groups. H. Survival curveof the animals. The Gehan-Breslow statistic for the survival curves is not great
enough to exclude the possibility that the difference is due to random sampling
variability; there is not a statistically significant difference P=0.401.

79

The 4T1-luc cells were injected intravenously to create lung metastatic
model mice. Three days later, 5×105 iPS-EPCs transduced with BV-CMVIsthmin-WPRE were injected intravenously into the tumor bearing mice. As
controls, PBS, iPS-EPCs, and iPS-EPCs transduced with empty baculoviral
vectors were injected into the mice. From both the average luc signal reading(Fig. 2.21G) and the survival rate of the animals (Fig. 2.21H), we were notable to observe any obvious therapeutic effect in the tumor-bearing mice.
2.4 Discussion
2.4.1

Derivation of EPCs

The hPSCs provide an unlimited source of cells for cell therapy. Moreover,
hPSCs provide a method of studying basic human developmental biology
without trespassing on ethical boundaries. During embryogenesis, thedevelopmental progression of hematopoietic and endothelial lineages isclosely associated. Thus, endothelial and hematopoietic progenitors sharecommon markers such as CD34, CD31 and Flk1 79. Hematopoietic cells werethen distinguished from its endothelial counterpart by the expression of CD45,
a common leukocyte antigen which is not expressed in endothelial cells80, 130.
Our EPCs expressed CD34, CD31 and Flk1 but not the CD45 hematopoietic
marker. After further culture in an EGM2 endothelial cell medium, these cells
were positive for other characteristics associated with EC lineage markers
such as VE-cadherin, von Willebrand Factor, uptake of Ac-LDL. In addition
to this, the cells could form tubular structures when seeded onto matrigel79, 83.
In our study, we initially utilized the mouse stromal cells, OP9 andM2-10B4, to produce EPCs from hESC and iPS cells. These cells have been
80

shown to support hematoendothelial differentiation of hESCs due tosoluble/secreted

factorsthatareimportantforhematoendothelial

development140. Our generated EPCs via these co-culture methods werecomparable to previous reports 141, 142. However, the M2-10B4 co-culture
process took a longer time to generate EPCs (13 days instead of 8 days forOP9 co-culture). Moreover, although Tian et al. showed that CD45+ cells
started to appear only in approximately 17-21 days of culture, we noticed thatour produced EPCs expressed the CD45 marker earlier (we harvested at day
13-14). This CD45 expression is higher as compared to that of the OP9 coculture derived EPCs. Furthermore, by observing the matrigel tubulogenesis
results qualitatively, we noticed that tubulogenesis was more efficient for thecells that were produced via OP9 co-culture as compared to the M2-10B4 coculture. Thus, we conclude from our studies that OP9 co-culture is morerobust in producing EPCs than M2-10B4 co-culture.
The co-culture methods can recreate native stem cell niches, thus
providing an inductive environment for the differentiation of hESCs. The
ability to modify stromal cells to express various components that may
promote or inhibit lineage-specific differentiation also provides a useful means
to define specific niche factors that regulate human hematopoiesis137, 140. For
example, M2-10B4 stromal cells expressing Wnt1 or Wnt could enhance EPC
differentiation from hESCs137. However, avoidance of xenogeneic tissue, suchas murine cells that are used for the maintenance of undifferentiated hPSCs or
stromal cell lines that are commonly utilized to induce differentiation, ispreferred in clinical settings140.

81Interestingly, in all the differentiation methods, we observed similarmorphological changes during the differentiation process. The initial buttonlike structure of H1 or iPS grew into root-like structure be it in the stromal coculture or stromal-independent culture. Previously, our lab has investigated the
localization of CD34+ cells in OP9 co-culture via live CD34+ cell
immunostaining of differentiated hESC colonies. We found that the main
population of CD34+ cells is located in the root-like structures that penetrate
the OP9 portion of the co-culture. Thus, since we also observed similar
structures in the other methods, we speculate further that these structures
might mimic the hematopoietic niche in the bone marrow. However, further
studies need to be done to prove this.
Our produced EPCs both from iPS cells and hESCs express markers insimilar manners. However, in the 2-D differentiation method, we noted somedifferences in between the hESC and iPS cell-derived EPCs in terms of thepurity of CD34+ cells after sorting and the co-expressions of the EPCs
markers. Such differences have also been reported previously143, 144. White andcolleagues previously have shown that there are differences in the gene
expression between ECs derived from hESC and iPS cells, notably genes
related cell cycle and adhesion 92. However, despite the gene expression
heterogeneity, they concluded that ECs derived from both pluripotent cell
types behave very similarly 92.
The yield and purity of EPCs derived using EB method was superior
compared to the other methods. The differentiation medium was supplemented
with BMP4, Activin A, bFGF, VEGF cytokines and TGF-β inhibitor82

(SB431542). BMP4 has been shown to promote mesodermal lineage
differentiation from hPSCs 88, 143. Addition of VEGF and bFGF has beenshown to enhance the expression of CD34 during the hematoendothelial
differentiation 143. Studies have shown that TGFβ pathway is important forhematopoietic development and that TGFβ inhibition maintains theproliferation and identity of endothelial cells in vitro 132, 140.
CD133 (AC133, prominin-1) is a transmembrane domain cell surface
glycoprotein which serves as a marker of asymmetric division, lineage
plasticity, tumor cell dormancy and inherent embryonic gene expression 80, 145.
CD133 have been repeatedly used as a marker for early endothelial lineage
cells 81, 82, 146. EB derived EPCs expressed CD133 stem cell marker indicatingthat they are still in early phase of endothelial differentiation. Moreover, these
iPS cell-derived cells are positive for the above-mentioned endothelial
progenitor markers and display other characteristics associated with EPCs,
including VE-cadherin, von Willebrand Factor, uptake of AcLDL andformation of tubule structure upon seeding onto matrigel 79, 83.
In our study, the produced EPCs could maintain the markers andproperties only for up to 5 passages. However, the self-renewing hPSCs
possess unlimited proliferation capacity, thus can be used as a reliable source
to generate vast amounts of human EPCs for cell therapeutics purpose. The
use of human iPS cells especially garners attention since it can avoid theethical concerns regarding its derivation, in contrast to the hESC counterpart.
iPS cells are a unique type of biological material that can be used togenerate either autologous or allogeneic cells. If cells to be used are the83differentiated progeny of iPS cells reprogrammed from the patient’s own cells,
these cells are viewed as “autologous” cells. The likelihood of immune
rejection towards them after transplantation will be significantly reduced. In
the case that a human iPS bank consisting of various human leukocyte antigen(HLA) types is established, iPS cells selected from the cell bank can be used
as an alternative to generate semi-allogeneic differentiated cells that sharesome of the HLA alleles with recipients. The third method of using iPS cells is
to generate differentiated allogeneic cells suitable for applications that do not
require the long-term survival of transplanted cells, such as when using stem
cells as cellular vehicles for targeted cancer therapy. While long-term
immunosuppressive treatment is required for many regenerative medicine
applications of allogeneic stem/progenitor cells, it will not be necessary when
iPS cell-derived cells are used as exogenous cellular vehicles for cancer
treatment, since the application does not really require stem cell engraftment.
Using allogeneic cells derived from iPS cells as cellular vehicles forcancer therapy can be justified for several reasons. Firstly, it is possible to use
genetic engineering to introduce a therapeutic gene into iPS cells, in order togenerate a master cell line for cell therapeutic production. Secondly, theallogeneic cell approach allows large-scale mass production of celltherapeutics, a prerequisite for widespread application of cell therapy suitable
for repeated patient treatments. Large-scale mass production will also increasecost-effectiveness by reducing labor costs and simplifying the logistics of cell
culture operations. In addition, it becomes possible to prepare cryopreserved
derived cells as commercial products in ready-to-go format. Thirdly, theallogeneic cell approach allows standardized manufacture, which should be84

helpful in eliminating variability in the quality of cellular products, thus
facilitating reliable comparative analysis of clinical outcomes.
Despite the advantages of iPS cells, more extensive studies need to bedone to prove that the differentiated cells from iPS cells and hESCs areinterchangeable. One concern about using iPS cells to derive downstream cells
is the maintenance of genetic and epigenetic states of the iPS cells such as the
methylation pattern. It has been shown that the reprogrammed stem cells carryan epigenetic memory of the original tissues which lingered even after the iPS
cells had been differentiated into new tissues147. In addition to this, anotherimportant concern regarding the use of hPSCs derived cells is the ability ofhPSCs to develop teratomas. This safety concern applies to iPSCs too, as they
can also form teratomas140.
2.4.2

Tumor tropism of iPS-EPCs

Our biodistribution experiments have clearly demonstrated the tumor tropismof the generated iPS-EPCs. However, our study also showed that majority of
intravenously injected iPS-EPCs were trapped in the lungs in the orthotopic
breast cancer model, possibly due to the narrow diameters of lung capillaries.
After initial pulmonary trapping, many intravenously injected cells tended tobe re-distributed from the lungs to the liver and spleen. Comparable results
were shown by Wei et al., in which tail vein injections of mouse embryonic
EPCs into C3H mice resulted in the injected cells being predominantly
sequestered in the lung and spleen85. Our previous biodistribution studies
using human neural stem cells (NSCs) also showed a similar cell distribution
pattern128. This distribution pattern is useful for eliminating metastatic cancer85

cells in the lungs, liver and spleen and has provided a rationale for us to test
the therapeutic effects of intravenously injected iPS-EPCs in the breast cancer
lung metastasis model in the current study. This cell distribution pattern,
however, also emphasizes the importance of minimizing off-target transgene
expression if the cell delivery aims to treat primary breast cancer.
In the invasive 2M1 glioma model, we showed that EPCs can beincorporated into the 2M1 tumor blood vessels. These results corroborate thestudy by Moore et al. which demonstrated systemically transplanted cord
blood-derived EPCs in vivo tropism for intracranial gliomas148. Furthermore,
EPCs were also found in tumor satellites away from the primary tumor site,
suggesting that there is potential for cell delivery of gene therapy to metastatic
sites.
2.4.3

Effect of iPS-EPCs in cancer growth and metastasis

Circulating bone-marrow-derived EPCs might follow the gradients of growth
factors and cytokines that are released into circulation by tumors andcontribute to tumor neovascularization, tumor growth, and metastasis149.
However, this contribution remains controversial, since the existence of these
EPCs during tumor progression vascularization has been established in certain
models and disproved in others, depending on the tested tumor models andmethodologies150. NSCs have also been exploited as cellular vehicles in many
preclinical and clinical studies for targeted delivery of therapeutic genes forcancer therapies39, 128, 151. However, our group has demonstrated that hESCderived NSCs suppress the maturation of DCs and impair the functional
capacity of DCs to stimulate alloreactive T cells152. These immunosuppressive86

effects exerted by stem cells will have a negative impact on cancer treatments
when they are used as cellular gene therapy delivery vehicles. Thus, in thisstudy, we also included iPS-NSCs alongside our iPS-EPCs to study thedifferences between different cellular vehicles.
We did not observe any obvious contributions of iPS-EPCs and iPSNSCs to breast cancer growth and metastasis in both the primary andmetastatic tumor models. Interestingly, in the lung metastatic cancer model,
injection of fibroblasts alone resulted in an increased tumor volume at day 21,
although there was no significant difference in animal survival by the end of
the experiment. Carcinoma-associated fibroblasts (CAFs), the activatedfibroblasts, are the primary type of host cells in tumor microenvironment. The
origin of CAFs is not conclusively established and remains controversial153.
Whether the systemically injected fibroblasts tested in the current study wererecruited by the tumor and activated in situ to become CAFs is worth pursuingin future research.
2.4.4

Immunotherapy of EPCs using CD40L

To employ iPS-EPCs as donor cells to deliver the CD40L gene or othertherapeutic genes for cancer therapy, it is essential that these cells can be
genetically modified in a safe and effective way to carry therapeutic payloads
of interest over the period of time crucial to the therapy. In the current study,
we demonstrated for the first time that baculoviral vectors can be used forgenetic modification of human EPCs to provide a window of transgene
expression suitable for cancer therapy. Large cloning capacity, ease of virus
production and low cytotoxicity to transduced cells are the obvious advantages
87

associated with the emergence of baculoviral vectors in the gene delivery
field. Insect baculovirus also bypasses the risk of virus replication andpotential viral infection in host cells, risks borne by conventionally-usedanimal viruses such as adenovirus, retrovirus and adeno-associated virus. The
pre-existing host immune response against adenoviral vectors, a type ofvectors commonly used for transient transgene expression, is another concernthat restricts the use of these vectors154, 155. Unlike adenovirus, baculovirus is
not targeted by pre-existing immunity in humans156.
CD40L was used as a model therapeutic agent to test iPS-EPCmediated gene delivery. CD40L, also called CD154, is a 33-kDa type IImembrane protein belonging to the TNF gene family and is mainly expressedon activated CD4+ T cells. It interacts with CD40 expressed on a wide range ofantigen-presenting cells (APCs) including dendritic cells, macrophages, and B
lymphocytes, as well as non-immune cells such as epithelial cells, endothelial
cells and malignant cells105. CD40L-CD40 interaction induces activation in
APCs in association with T cell receptor stimulation by MHC molecules on
the APCs, ensuring the generation of antigen-specific cytotoxic T lymphocytes(CTLs). Increased antigen presentation by APCs can lead to increased
cytokine production, expression of other co-stimulatory molecules, andinduction of cytotoxicity. CD40L can also exert antitumor effects by inducing
tumor cell apoptosis via autocrine/paracrine induction of death ligands such as
TNFa, FasL, and TRAIL105. Local CD40L gene delivery to directly transduce
tumor cells can mediate multiple antitumor effects, including the induction ofT-cell responses, upregulation of TH1 cytokines and induction of apoptosis,
which effectively inhibit tumor growth108, 119, 157.
88

Our results showed that CD40L delivered by systemically injected iPSEPCs could reduce the tumor burden and eventually prolong the survival oftumor-bearing animals. To test human EPCs, immunocompromised Balb/cnude mice were used to produce tumor models in the current study. This strainof mice lacks T cells, but still acquires B cells and innate immune cells such as
NK cells and complement immune system. Thus, the upregulation of TH1
cytokines and the induction of apoptosis, but not induction of T-cell responses,
might play more important roles in the observed antitumor action. Our
approach is different from the strategy of direct transduction of tumor cellswith the CD40L gene, as it uses baculoviral CD40L transduction of iPS-EPCs
and the tumor tropism property of the cells to activate a local CD40L-CD40
interaction in tumors. This is most likely achieved through a bystander
mechanism of intercellular transfer of the CD40L protein from donor iPSEPCs to tumor cells. It has been established that CD40L protein transfer totumor cells occurs in transduced fibroblasts, epithelial cells and bone marrowstromal cells158.
We also studied the therapeutic effect of EPCs expressing HSV-tk
using the same settings. However, we found that the effect is less pronounced
compared to EPCs that express CD40L as the therapeutic gene. This may be
due to the multiple antitumor mechanisms that CD40L has. Besides, homed
EPCs might trigger tumor growth until the suicide gene system is activated as
the suicide gene needs time to kill using the bystander effect. Furthermore, thesuicide gene vehicle has an inherent disadvantage in that it destroys itself,
limiting the amount of time it has in which to work99. Due to these reasons, it

89

is possible that the therapeutic effects exerted by HSVtk cannot overcome thefast growing tumor mass and metastasis.
We also investigated the therapeutic effect of a novel angiogenic
inhibitor, Isthmin (ISM). This 60kD protein, which was first identified in the
Xenopus brain, has been shown to inhibit endothelial cell (EC) capillary
network formation on Matrigel and induce EC apoptosis126, 127. Although it has
been shown that overexpression of ISM in mouse B16 melanoma resulted ininhibition of the tumor growth upon subcutaneous injection into the mice 126,
our results showed that ISM could not inhibit the growth of pre-establishedtumour in mice when delivered systematically. One plausible reason is that ahigh concentration of ISM may be needed to exert the antiangiogenic effect.
When overexpressed in the tumor, the tumor angiogenesis is inhibited fromthe start, thus making a more distinct impact on tumor growth. In a preestablished tumor, a higher concentration and more prolonged exposure of
ISM may be required to exert the same effect. Moreover, studies have shownthat only soluble ISM has antiangiogenic effects, whereas immobilized ISM
loses its antiangiogenic function and instead promotes EC adhesion, survival
and migration through avb5 integrin127. Further studies are needed toinvestigate whether the isthmin delivered by the EPCs are immobilized in the
ECM of a tissue or soluble in circulation.
2.4.5

Challenges and future direction

In this study, while the term “iPS-EPC” was used for these iPS cell-derived
cells, we do realize that the population could contain cells at different levels ofdifferentiation along the endothelial lineage. A further purification step, e.g.
90isolation of CD133+ cells, can be added to remove relatively matured cells.
For clinical applications, more robust and cost-effective cell expansion
technologies will be required. In this aspect, expansion methods such as
automated bioreactors and rotary culture machines hold promise forgenerating iPS cell derivatives in bulk159.
An important concern regarding the use of therapeutic cells derived
from iPS cells is the potential of the latter to form teratoma. Incomplete
differentiation could bear a risk of teratoma development in mice or patients.
Thus, an experiment to test teratoma formation in animal models after
injection of iPS-EPCs can be performed to ensure the safety of this therapeuticmode.
One issue that needs to be resolved is the degree of the EPCs tumor
tissue selectivity, to reduce potential toxicity due to off-target organ failures.
Several strategies can be used to enhance the tumor tissue-specific transgene
expression for cancer gene therapy. A popular method is the use of tumor
environment-specific promoters including hypoxia response elements, aproendothelin-1 promoter, or a survivin promoter93, 160-162. Given that thevesicular stomatitis virus glycoprotein (VSV-G) can promote the formation ofmultinucleated syncytia to kill cells in a pH-dependent manner, we have generated
a VSV-G mutant that efficiently promotes syncytium formation at the tumor
extracellular pH but not at pH 7.4163. Using transduced NSCs derived from iPS
cells to deliver the VSV-G mutant into mice with metastatic breast cancers in
the lung through tail vein injections, we observed tumor-selective killingwithout obvious toxicity to normal non-targeted organs. We are also working

91

on the development of regulatory systems that regulate the transgene
expression in the cellular vehicle in an inducible manner in response to tumor
hypoxia.
The transient gene expression mediated by baculovirus of nonintegrating nature has been noted to have limits to its efficacy in studies that
require long-term transgene expression. One way to improve the survival rateof the animals is through multiple rounds of iPS-EPCs-CD40L injections. Thisway, the CD40L expression in the animal can be periodically boosted, which
in turn will enhance the immune reaction against the tumor in the animals.
Another way to overcome the limitation is by ensuring longer expression of
the transgene in the iPS-EPCs. Our lab has developed several baculoviraltransduction-based approaches using either recombinase-mediated cassette
exchange (RMCE), zinc finger nuclease (ZFN) technology or transcription
activator-like effector nuclease (TALEN) technology for site-specificintegration, without random integration, of a transgene in human pluripotent
cells 98, 139, 164, 165. We are in the midst of establishing iPS cell lines to achieve
stable expression of transgenes in the differentiated progeny of the iPS cells,
including EPCs.
In conclusion, we have demonstrated the feasibility of generating EPCs
from early-passage human iPS cells. We further showed the tumor tropism of
the generated EPCs and the effectiveness of using intravenously injected EPCs todeliver CD40L to inhibit metastatic breast cancer growth in the lung, possibly
through an intercellular transfer mechanism. The potential of this approach is
not limited to metastatic breast cancer therapy but can likely be extended to92treat primary lung cancer and many other types of pulmonary metastases. In
this chapter we demonstrated that an immune-modulatory gene, CD40L, could
result in cancer therapeutic effects after being delivered via EPCs. In the next
chapter, we explored another targeted approach in immunotherapy.

93

CHAPTER III: TARGETED CANCER THERAPY USINGCYTOTOXIC T LYMPHOCYTES ACTIVATED BY
DENDRITIC CELLS PULSED WITH CANCER STEM
CELL-LIKE CELLS

94

3.1 Introduction
3.1.1

Cancer stem cells

Cancer stem cells (CSCs) are a small population of cells in the tumor thatpossesses the ability to self-renew and drive the tumorigenesis166, 167. Studies
have shown that CSCs express similar gene expression as normal embryonicstem cells, including STAT3, Oct3/4 and nanog, which explains their similar
properties in self-renewal and differentiation properties166, 168. However,isolation and further observations have been hampered by the small number ofcells since CSCs are a minority population just like normal stem cells.
Efficient methods to enrich this cell population would alleviate this problem
significantly. The non-adherent, 3-D tumor spheres culture method andreprogramming approach have been shown to enrich CSCs in vitro169, 170.
Taking advantage of sphere culture, surface markers analyses,
functional studies in vitro (spheres formation and in vitro differentiation
assays) and in vivo tumor initiation models, the existence of CSCs has been
demonstrated in several cancers including breast, ovarian and prostate cancers,
melanomas, gliomas, head and neck squamous carcinoma and many more 166,
167, 171. The tumor-initiating cells were first isolated from a breast cancer by

Al-Hajj et al. and identified as CD44+/CD24-/low population167, 172. Other
well-studied cell surface markers for CSCs include CD133, epithelial celladhesion molecule (epCAM), THY1, and ATP-binding cassette B5(ABCB5)166.
The

four

factors(Oct4,

Sox2,

Klf4

and

Nanog)

in

Takahashi/Yamanaka reprogramming iPS cocktails are oncogenes by95

definition, and their over-expression has been associated with cancers173-175.
Thus, many researchers have turned their focus to the use of reprogramming
approach to enrich CSCs by introducing at least one of these factors170, 175.
Hypoxic conditions have also been utilized to reprogram glioblastoma cells
into CSC-like cells176. The acquired stem cell phenotypes have been shown tobe more tumorigenic and resistant to chemotherapeutic drugs170, 176. Thus,
these cells are very promising novel targets for tumor therapy.
3.1.2

Objective

DC-based vaccines have been perceived as a powerful immunotherapy
approach against cancer. This is because DCs hold a very important place inthe stimulation of immune responses, especially CTLs, powerful immune
killer cells to eradicate cancer cells. Depending on the antigens loaded onto
and presented by the DCs, T cells will target populations of cells that express
them. Thus, the choice of target antigen is crucial. Conventional chemotherapy
and radiation therapy, which target rapidly growing tumor cells, are incapableof destroying the CSCs166, 167, 170. The quiescent state of CSCs, efficientmechanisms of DNA repair, intrinsic expression of anti-apoptotic mechanisms
and expression of efflux transporters are some strategies to build thisresistance166, 167. The survival of CSCs explains the tumor recurrence inpatients. Therefore, CSCs emerge as a promising therapeutic target for
anticancer therapies.
Our lab has successfully developed several baculoviral transductionbased approaches using zinc finger nuclease (ZFN) technology for sitespecific integration, without random integration, of reprogramming factor
96

genes to facilitate the generation of human pluripotent cells177. Zinc Finger
Nucleases (ZFNs) technology facilitates site-specific integration of thetransgene encoding the four reprogramming factors at adeno-associated virus
type 2 target region (AAVS1 locus) on chromosome 19. This site is known asa ‘safe-harbor’ human genomic site for exogenous DNA insertion98, 139. Using
the same method, we have also successfully reprogrammed various cancer celllines such as glioma and colorectal cancer (CRC) into CSC-like cell states.
Our lab showed that CRC-CSC-like possess higher propensities to form
tumorspheres in vitro as compared to wild-type CRC. Moreover they express
significantly higher CSC markers such as CD133 and CD24 (data not shown,
performed by lab members).
The conceptual flow of the project can be seen in Figure 3.1. Wholetumor lysates were produced from the CRC and glioma CSC-like cells. We
then used these whole lysate antigens to pulse PBMC-derived DCs. We
believed that the four reprogramming factors are also included in the lysate
and we hypothesized that these antigens can be presented by DCs to the Tcells and subsequently results in the activation of CTL which can target the
CSCs population.
The key factors in this project were the viability of the DCs used andthe proper assessment of the T cells activation. In the first part of our study,
we focused on producing a sufficient quantity of highly functional DCs from
PBMCs. We relied on flow cytometry which has been widely used tocharacterize the PBMC-derived DCs.

97

In the second part, T naïve cells isolation and characterization werecarried out. In this project, naïve T cells were isolated and memory T cells
were depleted. This is because memory T cells have been shown to have lower
activation threshold, thus can result in non-specific expansion178. Flowcytometry to characterize the co-stimulatory as well as inhibitory markers of T
cells will be tested.
In the last section, the level of CTL activity in terms of functionality
and specificity will be examined. A functional assessment of T cells in termsof the production of cytokines such as IFNγ will be performed. The ELISPOT
assay is a highly sensitive antibody-capture-based method for enumerating
specific T cells that can secrete cytokines. Thus, this technique will be adopted
in this project. We aimed to test whether IFNγ production by the T cells was
specific to the target cells that expressed the CSC-like antigens. Ultimately,
we postulated that a CSC-specific T cell response from DC vaccination using
CSC-like antigens can be generated.

Figure 3.1. Antigen presentation and CTL activation. CSC-like antigens
presentation by the DCs leads to CTL activation and eventually targeting of cells
expressing the antigens.

983.2 Material and methods
3.2.1

DCs and naïve T cells derivation from PBMC

DCs and naïve T cells were derived from human peripheral bloodmononuclear cells (PBMCs), both HLA-A2 positive and negative (AllCells,
Stemcell Technologies Inc) based on previous studies 178. To generate DCs,
1×108 human PBMCs were seeded onto a T-175 cm2 flask and incubated for 3
h at 37°C in Cellgro® DC medium (CellGenix, GmbH, Freiburg, Germany)
supplemented with 5% human AB serum and 1×P/S. After 3 h, 20 ml of DC
differentiation medium (Cellgro DC medium + 5% human serum + 1×P/S +50 ng/ml GM-CSF + 100 ng/ml IL4) was added to the attached cells. Half ofthe medium was changed 2 days later. As most of the cells have detached at
this point, the medium change was performed as follows: take out 10 ml
medium, spin down 300 g for 5 min, re-suspend with 10 ml fresh medium andput the re-suspended cells back to the flask. On day 4, the immature DCs werecollected for DC pulsing and maturation or for DC characterization by flow
cytometry. Meanwhile, the non-attached cells were collected and cultured
separately in Cellgro medium as above with additional supplementation of IL7(5 ng/ml). Prior to T cell priming, selection of CD8+CD45RO-CD57- naïve T
cell population from the PBMC was carried out via MACS using CD8,
CD45RO and CD57 microbead kit (Miltenyi Biotec). All the cytokines werepurchased from Peprotech or Miltenyi Biotec.
3.2.2

Tumor lysate preparation

To prepare for the tumor lysate, wild type and 4F-BV-ZFN reprogrammed
glioma and colorectal cancer cells were used. Tumor cells were harvested and99

re-suspended at 1×106 cells/ml in PBS. The cells were then subjected to six
cycles of freeze-thaw treatment (freezing with liquid nitrogen for 20 min andthawing 5 min at 37oC water bath) to produce the whole tumor lysate.
3.2.3

DCs pulsing with tumor lysate and maturation

The dendritic cells were harvested and separated into different groups: DC
pulsed with 4F-BV-ZFN-tumor, DCs pulsed with WT tumor and unpulsed
DCs. The DCs were then mixed with the whole tumor lysates with a cell ratioof 1:1 according to the groupings. After a 16 h incubation, 60 EU/ml of LPS(Escherichia coli; Sigma) and 2000 IU/ml of IFNγ were added into the culture
to stimulate DC maturation for a further 16 h at 37°C, 5% CO2.
3.2.4

CTL stimulation and expansion

The naïve T cells were then primed with the pulsed and matured DCs at 1:4
DC: T cell ratio. Prior to the priming, the DCs were subjected to mitomycin C
treatment in order to inhibit cell proliferation including the contaminants NK
cells and monocytes. The cell mixtures were cultured and expanded in a
Cellgro medium supplemented with 5% human serum, 1×P/S, IL21 (30ng/ml), IL7 (5 ng/ml) and IL15 (5 ng/ml) for 10 – 11 days.
3.2.5

Flow cytometry

For DCs characterization, flow cytometric analyses were performed using theantibodies: CD11c, CD86, DC-SIGN, CD80, CD40, CD83 and HLA-DR (BDBiosciences). For T cell characterization flow cytometric analyses were
performed using these antibodies: CD8, CD4, CD62L, CCR7, CD27, CD28,100

CD45RO, CD45RA, CD57, CD25, CD127, HLA-ABC and HLA-A2 (BDBiosciences).
3.2.6

ELISPOTTo detect the cytokine (IFNγ) production by the stimulated CTL, we
performed ELISPOT assay (MabTech AB, Sweden). As target cells, T2 cells
or 0.05% glutaraldehyde-fixed DCs were used when the PBMCs were HLAA2 positive or HLA-A2 negative respectively. The pulsed or unpulsed DCs
were fixed with 0.05% glutaraldehyde for 30 seconds. The T2 cells wereincubated with 3 µg/ml Oct4 or EGFP peptide at room temperature for 3 h.
The T responder cells were then co-cultured with fixed DCs or T2
target cells at responder: target = 10: 1 in Cellgro medium using 96-well Ubottom plates. After 24 h of incubation, the cells were then transferred to theantibody-coated microtiter plates. The antigen-specific T cells were detectedby the sequential addition of biotinylated mouse anti-human IFNγ (2 h at room
temperature), five washes with DPBS, alkaline phosphatase-conjugatedstreptavidin (1 h at room temperature), five further washes with DPBS, andsubstrates for streptavidin. Spots were then developed and stopped after 10
min by washing vigorously with tap water. The spots corresponding to the
IFNγ producing cells were then scanned and analyzed using an automatic plate
reader CTL – Biospot® S6 UV analyzer (Cellular Technology Limited, Ohio,
USA).

101

3.2.7

Statistical analyses

All data are represented as mean ±SD. The statistical significance ofdifferences was determined by one-way ANOVA followed by the Holm-Sidakmethod for pairwise multiple comparison tests. A p value of <0.05 wasconsidered to be statistically significant.
3.3 Results
3.3.1

DCs derivation and characterization

The production of sufficient and functional DCs is crucial in DC-based cancer
immunotherapy. In this study, DCs were derived from PBMCs, with Figure
3.2A showing the schematic diagram of the protocol. The PBMCs wereseeded and incubated for 3 h. The attached cells were the monocytes which
will be differentiated to DCs whereas the floating cells were collected for Tnaïve cell sorting. To differentiate DCs from PBMCs, IL4 and GM-CSF wereadded to the differentiation medium. Thereafter, the attached monocyte-DCs
become floating or loosely attached DCs (Fig. 3.2B, C). Furthermore, we
observed morphological changes in the cells over time. In the beginning, mostof the cells are spherical. However, at day 4, most of the cells are irregularshaped with dendritic arms starting to develop (Fig. 3.2C). We can produce up
to 8×106 DCs from 1×108 PBMCs.
Our lab has established the CSC-like cells by reprogramming cancer
cells such as colorectal cancer (CRC) and U87 glioma cells by using thebaculoviral vector expressing four reprogramming factors (Oct4, Sox2, Klf4
and C-Myc), zinc finger and EGFP (4F-ZF-EGFP). The increased expressionof the four factors has been analysed using RT-PCR, real time PCR and102

western blot (data not shown). These reprogrammed cancer cells were used tomake the cell lysates. CRC and U87 wild type cells were used as controls, aswell as PBS as unpulsed control. After pulsing with the lysates, the cells were
subjected to IFNγ and LPS in order to induce the DC maturation. Interestingly,
after pulsing followed by maturation, most of the DCs became tightly attachedcells (Fig. 3.2D).

Figure 3.2. Dendritic Cells and T cells derivation from PBMC. A. DCs
differentiation and naïve T cells selection from human PBMC. B. Differentiated DCs
at day 1. C. DC differentiation at day 4. D. DC after pulsing and maturation.

103

Evaluation of immunological surface markers is a common yet
important method to evaluate the identity and functionality of the DCs. At
first, we performed flow cytometry of the whole population of PBMCs to
examine the profile of the cells available (Fig. 3.3). We could see that all thecells were positive for CD45 marker which is the leukocyte common antigen(99.95%). High expressions of lymphocytes markers were found. The cells
were positive for cytotoxic and helper T cells markers, CD8 (34.98%) andCD4 (50.35%) respectively. They were also positive for other lymphocytes
markers CD3 (80.31%) and CD7 (73.24%). HSC marker CD34 was negative(0.2%) but 75.34% of the cells were positive for CD31 marker. Furthermore, asmall but distinct population of the PBMC expressed NK cell markers: CD16
(4.52%), CD94 (3.02%) and CD56 (0.67%).
We then evaluated the expression of DC markers in the immature andmature DCs (Fig. 3.4). The immature DCs were positive for DC markers suchas CD11c, CD86 and DC-SIGN with expression of more than 90%. However,
the expression of CD40 was dimmed (48.97%) and no expression of CD83
was detected (0.96%). Upon maturation, the expression of CD83 and CD40
increased considerably, from 0.96% to 98.76% and from 48.97% to 99.30%
respectively. Furthermore, the expressions of the other DC markers weremaintained at high levels. In addition to this, the DCs were positive for MHC I
and II (HLA-ABC & HLA-DR), with the percentage expression of 99.93%
and 98.12% respectively.

104

Figure 3.3. Characterization of PBMCs by flow cytometry. The cells are positive
for cytotoxic and helper T cells markers, CD8 (34.98%) and CD4 (50.35%)
respectively. They are also positive for other lymphocytes markers CD3 (80.31%)
and CD7 (73.24%). All the cells are positive for leukocyte common antigen, CD45.
The cells are negative for HSC marker CD34 (0.2%) but 75.34% positive for CD31
marker. Small population of the PBMC expressed NK cell markers: CD16 (4.52%),
CD94 (3.02%) and CD56 (0.67%).

105

Figure 3.4. Characterization of DCs by flow cytometry before and after pulsing
and maturation. The DCs are positive for MHC I and II (HLA-ABC & HLA-DR).
The DCs are also positive for DC markers such as CD11c, CD86, DC-SIGN withexpression of more than 90%. Upon maturation, the expression of CD83 and CD40
increased considerably, from 0.96% to 98.76% and from 48.97% to 99.30%
respectively.

106

3.3.2Naïve T cells selection and characterization

We investigated whether tumor antigen-specific CTL can be mounted from thenaïve T cells. Thus, we selected the naïve T cells from the PBMC derived
lymphocytes repertoire by using MACS (Fig. 3.2A). First we selected theCD8+ T cells from the whole floating PBMC-lymphocytes, followed bydepletion of CD45RO+CD57+ memory T cells. From the flow cytometry
results, we observed that the proportion of CD8+ cells out of the whole
PBMC-lymphocytes was approximately 30% on average (28.3% as shown in
Figure 3.5A). After cell sorting, the purity of the CD8+ cells reached 98.2%.
We tested the memory T cell markers and the results showed that 28.57% of
the cells expressed CD45RO (Fig. 3.5A). The CD57 memory T marker
expression was lower than that of CD45RO, 9.37% (Fig. 3.5A). However,after depletion of memory T cells by magnetic sorting, expression of thesemarkers was not detected (Fig 3.5A). On average, we obtained 1×107 naïve T
cells from 1×108 PBMCs. We also checked the expressions of T cell costimulatory molecules such as CD62L, CCR7, CD27 and CD28 before andafter priming with the DCs (Fig. 3.5B). All markers were expressed before
and after DC activation.
Tregs inhibit the CTL activation and antitumor effect. Thus, wechecked the expression of Treg markers in the sorted naïve T cells population.
Treg cell population is normally identified as CD4+CD25+CD127- cells. From
Figure 3.5C, we observed that our sorted T cells were negative for Treg
markers as the expression of CD4 marker was very low (0.87%). Moreover,107the CD25 marker albeit expressed at 9.66%, showed a dimmed expression. In
addition, almost all of the cells (97.56%) were positive for CD127.

Figure 3.5. Characterization of CTL by flow cytometry before and after primingwith DCs. A. Naïve T cells characterization before and after sorting. CD8 expression
before sorting was 28.3% and after sorting, the purity of CD8 + cells was 98.2%. The
expression of memory T cells markers was 28.57 and 9.37% for CD45RO and CD57
respectively. After depletion of memory T cells, the memory T cells markers
expression were very low (1.1%). B. Expression of costimulatory molecules before
and after DC priming as shown by flow cytometry. C. Flow cytometry for Treg
markers: CD4 (0.87%), CD25 (9.66%) and CD127 (97.56%).

108

3.3.3IFNγ production of CTL activated by CSC-like-CRC-pulsed DC

After the activation and expansion period, the T cells were mixed with thetarget cells and their IFNγ production was evaluated. Figure 3.6 shows thecytokine production by CTL activated by CSC-like CRC lysate-pulsed DCs.
The groupings of the cells are shown in Table 3.1. T2 cells were used as thetarget cells as the PBMCs were HLA-A2 positive. These cells were pulsedwith Oct4, EGFP peptides or PBS as control. T2 target cells alone without theaddition of effector T cells were used as a negative control, as these T2 cells
should not produce IFNγ. As a background control, spontaneous production of
IFNγ from naïve T cells alone was measured. On the other hand, the cytokine
productions from naïve T cells which have been activated by CD3 antibody
were used as positive control.
As expected, T2 cells alone did not release any IFNγ, but the T cells
alone spontaneously released IFNγ at low levels with an average of 61.67 T
cells/ 50,000 cells. More T cells produced IFNγ after activation by CD3
antibody, 96.5 T cells/50,000 cells. Production of IFNγ is the highest in CTLs
which have been primed with DCs pulsed with 4F-ZF-EGFP CRC cells mixed
with T2 target cells pulsed with Oct4 or EGFP. The ANOVA and the HolmSidak pairwise comparison tests showed that the differences between these
two groups are not significant. However, when either of this group wascompared to the rest of the groups, then the differences are very significant.
Furthermore, statistical tests comparing the WT-T, UP-T, T alone and 3.11-TT2 groups showed no significant differences.

109

Table 3.1. Grouping of ELISPOT for T cells after activation by DCs pulsed with
CRC stem cell-like cells.

Figure 3.6. Production of IFNγ by CTL after activation by DCs pulsed withreprogrammed CRC cell lysate. Production of IFNγ is the highest in T cells whichhave been primed with DC pulsed with 4F-ZF-EGFP CRC cells mixed with T2 target
cells pulsed with Oct4 or EGFP (Anova P value =˂0.001).

3.3.4

IFNγ production of CTL activated by CSC-like-glioma-pulsed DC

We have also established CSC-like glioma cells by transducing U87 glioma
cells with 4F-EGFP-ZF baculovirus. We then utilized this CSC-like glioma to
produce a cell lysate that contains all the tumor antigens including the proteinsof the four factors. Subsequently, this lysate was used to pulse the DCs which110

in turn were used to prime the autologous naïve T cells. We then examined the
production of IFNγ by these activated CTLs upon mixing with target cells. As
the PBMCs from which the DCs and T cells were derived from are HLA-A2
negative, peptide pulsed T2 cells were not used as the target cells. Instead,dendritic cells pulsed with the CSC-like U87 lysate and subsequently fixed byglutaraldehyde were used as target cells. The groupings of the cells wereshown in Table 3.2.
As shown in Figure 3.7, IFNγ production was the highest in the T cells
group that had been pulsed with DCs and mixed with the glutaraldehyde-fixed
pulsed-DC. The ANOVA and Holm-Sidak tests showed a significant
difference between this group and the rest of the groups. On the other hand,when comparing the other groups, no statistically significant differences wereobserved.
Table 3.2. Grouping of ELISPOT for T cells after activation by DCs pulsed with U87
glioma stem cell like cells.

111

Figure 3.7. Production of IFNγ by CTL after activation by DCs pulsed withreprogrammed glioma cell lysate. Production of IFNγ is the highest in T cells
which have been primed with DC pulsed with 4F-ZF-EGFP U87 glioma cell lysate
mixed with fixed-DC target cells pulsed with the lysate; ANOVA P = <0.001.

3.4 Discussion
3.4.1

DC differentiation and characterization

Dendritic cells are the center of innate and adaptive immune axes. Numerous
studies have been done to investigate their derivation, differentiation andfunctions in immunology and immunotherapies. Human DCs can bedifferentiated from various cellular sources, such as CD34+ progenitor cells
from bone marrow, cord blood and pluripotent stem cells, or from circulating
monocytes from PBMC, and using various protocols and cytokine cocktails to
skew the differentiation towards DCs178-181. The most widely used DCs forclinical trials are monocytes-derived DCs from PBMC in the presence of GMCSF and IL4179.
In this study, we adopted a method from the study by Wolfl et al.178 togenerate both DCs and naïve T cells from PBMCs. The profiles of the whole
PBMC we used are similar to previous data using freshly isolated human
PBMCs except that lower NK cell markers were detected182, 183. These112

differences can be due to batch-to-batch variations of the PBMCs used.
Likewise, the derived DCs are comparable to previous reports which used
GM-CSF and IL4 supplements179,

184-186. Lysate pulsing represents amaturation inducer for the DCs. Moreover, this maturation process was further
supported by LPS and IFNγ supplementations. Other maturation conditions
have been described by others, such as the use of TNFα. However, thecombination of LPS and IFNγ has been shown to stimulate IL12 production by
the DCs, which is necessary for skewing towards a Th1 activation andaugmenting CTL responses6, 178, 179. Indeed, upon pulsing with lysate, there
was upregulation in CD83 and CD40 DC maturation markers expression,comparable to previous studies179, 185, 186.The upregulation of these costimulatory molecules is necessary to enhance the capacity for antigen
presentation and T cell stimulation, which most probably occurred at the costof antigen uptake capacity4. Moreover, the DCs were positive for MHC I
which is crucial for antigen presentation process. In addition to this,morphological changes were also detected. The mature DCs developed longarms of dendrites as opposed to small irregular shaped appendages on theimmature ones. These long dendritic processes have been known to increase
the chance of T capture and interaction34.
Thus, using this protocol and based on the surface markers detection,
we have generated sufficient numbers of functional DCs. These lysate-pulsedDCs were used to prime the naïve T cells. Aside from detection of surface
markers, DCs can be characterized by their phagocytic capability (immaturestate) as well as their capacity to produce cytokines (mature state) such as
IL12, IL6. These are areas that we can explore further.
113

3.4.2

Activated CTLs display appropriate co-stimulatory molecules andantigen-specific targetingNaïve T cells are T cells that have not come across antigen activation. Once
stimulated, these cells will differentiate into CTLs and memory T cells
although the exact process of differentiation has not been elucidated yet21. The
memory T cells are responsible for generating responses if the same antigen is
detected again. When this happens, the duration of the immune response
mounted is shorter as memory T cells have a lower activation threshold178.
This is the reason why these cells need to be depleted. Similar to what wasreported by Wolfl et al., non-specific T activation shown by higher variations
amongst the elispot wells were detected when no memory T cell depletion was
performed.
The flow cytometry results of our isolated naïve T cells werecomparable with results by Wolfl et al178. We observed that the naïve T cells
could not survive past 10 days without stimulation by DCs. Interestingly, thesurvival of the cells improved greatly when no sorting was carried out. With
this in mind, we performed sorting just prior to DC pulsing to minimize the Tcell deaths. This implies that CD4+ cells support the survival of the CD8+naïve T cells. This may have great implications for adoptive T cell therapy.
While it is very tempting to select only CD8+ T cells, exclusion of othersubtypes of T cells may lead to deleterious effects for the T cells.
As mentioned in Chapter I, antigen recognition alone is not sufficientfor T cell activation. Interactions of co-stimulatory molecules on the T cellsurface are critical for T cell survival and functions. Our flow results show that114

our T cells maintained the expressions of the necessary co-stimulatory
molecules comparable to previous findings178, 183. CD28, as mentioned, isneeded to activate the CTLs via amplification of the signals from TCR.
Moreover, CD28 stimulation also maintains T cell telomeric length which may
improve their survival21. CD27, on the other hand, has been shown to support
antigen specific expansion of naïve T cells and to complement CD28
activity187, 188. In addition, CD11c and CD96 play a role in the homeostasis of
T cells189, 190.
Monitoring chemokine receptor expression provides information
regarding the status of T cells. After maturation, there seemed to be adownregulation of CCR7 expression, but not CD62L. Although this result is
not indicated in Wolfl’s study, Dumortier et al. demonstrated thedownregulation of both CCR7 and CD62L expression on T cells after LPSmatured DCs priming. Both CD62L and CCR7 are lymph node homing
receptors6, 178, 191, 192. By downregulating these markers, the T cells possessmigratory capacity towards the target site192-194. As to why the downregulation
was only detected in CCR7 but not CD62L in our case, the reason is unclear
and requires more investigations.
While CD8+ cells represent the CTLs which are involved in the tumor
killing, the CD4+ T lineages are more complex. They comprise of T helpers(Th1 and Th2) which are important in assisting antibodies production by B
cell and T cells proliferation through cytokine signaling. However, a different
subpopulation of CD4+ named Tregs has been known to attenuate immune
response. Thus, it is important to exclude this subpopulation of cells from the115

activated CTLs. Treg cells make up 5-10% of naïve CD4+ T cells in the
periphery36. By the end of the expansion phase, the expression of Treg
markers on the T cell population was low; hence we expected little inhibitionin CTL activity.
The main cytolytic components from CTL are granzyme B and IFNγ.
We have shown that the production of IFNγ is the highest on the target cells
that express the antigen (T2 pulsed with Oct4 or lysate-pulsed-DCs fixed withglutaraldehyde). Therefore, based on the flow cytometric analyses and the
ELISPOT results of the activated CTLs, we have demonstrated that the DCs
pulsed with CSC-like cells antigens can stimulate specific CTL responses.
Although these results are still at a preliminary stage, the potential of CTLs
targeting CSC population could already be perceived.
3.5 Future Direction
As this is an ongoing project, there are several future works which need to be
tackled. We are in the midst of testing the antigen-specificity targeting of
CTLs by investigating granzyme B production of the cells. Moreover, we will
also assess the CTLs killing effect using the CSC-like cells as well as primarytumor cells as the target cells, to verify this concept under more practicalconditions in a preclinical experimental setting. In addition to this, we are
eager to evaluate the CSC-like targeting using DC-based vaccine approach inother cancer lines which have been reprogrammed in our lab (for example,
melanoma).
The limitation in the number of monocytes that can be obtained from
PBMCs and the variability of DC-differentiation potential from different
116patients/donors lead to a demand for a better source of DCs for vaccination
purposes. An alternative approach to this is the use of human iPS cells as anunlimited source to generate human DCs of consistent quality195, 196. As
already discussed in the previous chapter, the use of iPS cells has many
advantages, including ease of genetic engineering and the feasibility of largescale mass production of cell therapeutics. An especially fascinating directionis the possibility of generating human iPS banks with various human HLA
types. This way, donor iPS cells selected from the cell bank can be matchedwith the HLA alleles of the recipients and used to produce semi-allogeneic
differentiated DC vaccines.
We have demonstrated that the combination of baculoviral vector andzinc finger nucleases technologies can be used to introduce stably site-specificintegration of transgene164, 165, 177. Thus, using this method, we can overexpress
tumor antigenic genes or DC activation/co-stimulatory molecules into the iPS
cells, then differentiate the iPS into the DCs. The DCs expressing the antigens
could then be used to activate CTLs responses. Our lab has also shown that
hESCs stably modified with CD1d (a glycolipid MHC-related molecule197)
gene may serve as a convenient, unlimited and competent DC source for iNKT
cell activation, a unique subpopulation of lymphocytes which has the ability tolyse iNKT cell-sensitive tumor cells 180, 198, 199. However, the safety of viral
vectors has always been one significant problem of DNA-based DCs vaccines.
By using site-specific integration into the AAVS1 site, the ‘safe-harbor’
human genomic site for exogenous DNA insertion, this problem can be
circumvented.

117

Another interesting approach will be the use of iPS cells to generate
activated T cells, thus bypassing the requirement of DCs stimulation. As
mentioned above, genetic modification of T cells through tumor antigenspecific TCR gene transfer or development of chimeric antigen receptor(CAR) T cells for adoptive therapy purpose also represents encouraging
modalities in immunotherapy. One possibility is to generate geneticallymodified iPS cells which can be used as a master cell line to produce asufficient amount of active CTLs for cancer therapy.
To summarize, the DCs that have been loaded with CSC-like tumor
lysate were able to activate CTLs. Subsequently the activated CTLs could
specifically target the cells expressing the CSC-related antigens, which are thereprogramming factors used to enrich the CSC population. We believe that ourapproach in targeting CSCs via immunotherapy would be profoundly valuable
in clinics. If it is used in combination with other therapies that target the bulkof tumor mass, such as surgical resection, chemotherapy or radiotherapy, thenthe complete eradication of tumors is a possibility within reach.

118

CHAPTER IV: CONCLUSION

119

In the preceding chapters, we have described the intricate mechanisms of ourin-built immune system for fighting against cancerous cells. Elaborate
interplays among innate and adaptive immune effectors are necessary torecognize and eventually eradicate the tumor cells. The innate immune systemis fast in responding against cancer, with the killing effect mainly performedby NK cells. The other components of innate effectors, the APCs (dendritic
cells and macrophages) are responsible for relaying the ‘danger signal’ to theadaptive arms of the immune system. The main killer of the tumor cells is the
CTL. However, their survival and activities depend on the other adaptive
immune cell populations, the helper T cells. The helper T cells produce
cytokines and activate B cells to produce antibodies against tumor cells. When
all the parts are functional, tumor eradication will ensue.
Unfortunately, in cancer patients, the tumor cells have evolved toevade the immune system, or worse, to impair the immune mechanisms. Thisis the reason why cancer immunotherapy holds great potential in the clinics,
by providing repair of or a new source of missing immune effectors. In thesubsequent chapters, we adopted two approaches of immunotherapies: thestem cell- and DC- based therapies.
In chapter 2, we experimented with cancer immune gene therapy byutilizing the CD40L gene delivered using iPS-EPCs to treat lung metastatic
mice cancer model. We have shown that human pluripotent stem cells, hESC
and iPS cells could serve as unlimited sources for the production of EPCs. We
further showed the tumor tropism of the generated hPSCs-EPCs in different
animal models: 4T1 mammary pad and lung metastatic models as well as

120

intracranial glioma model. Moreover, our findings add more value tobaculovirus as a means to genetically modify mammalian cells effectively
because we showed the efficient genetic modification of hPSCs-EPCs.
Most importantly, we demonstrated the capability of EPCs to be an
excellent cellular vehicle for cancer gene therapy. The intravenously injected
EPCs can deliver CD40L to inhibit metastatic breast cancer growth in the lungand prolong the survival of tumor-bearing mice, possibly through anintercellular transfer mechanism. Interestingly, compared to other therapeutic
genes (HSV-tk and isthmin), CD40L has a more effective therapeutic effectagainst the fast growing 4T1 metastatic breast cancer cells in an
immunodeficient mice model, which may be due to the multiple antitumor
effects possessed by CD40L. These effects include upregulation of TH1
cytokines, NK cell activation, antibodies production and apoptosis. The exact
mechanism of each effect is beyond this scope of study and will require
further investigations to be unraveled. The inherent tumor tropism of EPCs
combined with the immuno-modulation capacities of CD40L provide
synergistic antitumor effects which are not only limited to metastatic breast
cancer therapy but can potentially be extended to treat primary lung cancer
and many other types of pulmonary metastases.
In Chapter 3, we adopted the DC-based vaccine to target cancer stem
cell populations. As most of the available cancer treatments have been shownto spare CSCs population, these cells are capable of reestablishing tumor
mass, resulting in the tumor recurrence in patients. Our lab has establishedreprogrammed human glioma and colorectal cancer CSC-like cells by utilizing

121the baculovirus zinc-finger technology. We used whole cell tumor lysate
generated via free-thaw-cycles of these enriched CSCs as a source of tumor
associated antigens.
The crucial factor in this project is to produce sufficient viable andfully functional DCs which can present the tumor associated antigens into the
T cells. We showed that we could obtain sufficient functional DCs which
expressed MHC I and co-stimulatory markers such as CD86, CD83 and CD40
which are important for the antigen presentation process. Upon pulsing with
the CSC-like cell lysate, these DCs were capable of stimulating naïve T cells
into CTLs. The stimulated CTLs were capable of producing IFNγ cytokine.
Moreover, the highest cytokine production is observed in target cells that
expressed the tumor associated antigens. Thus, in this study we havedeveloped a DC-based immunotherapy approach which targets the CSC-likecell population. This CSC-targeting DC-based vaccine approach is not limited
only to glioma and CRC, which makes it more promising for clinicalapplications. We believe that in combination with other therapies that target
the bulk of tumor mass, such as surgical resection, chemotherapy orradiotherapy, it is possible to achieve tumor eradication.
Overall, our work has emphasized the importance of immunotherapy inthe field of cancer research. By stimulating the patient’s own immune system,
be it by delivering therapeutic agents using stem cells or vaccination via tumor
antigen-loaded dendritic cells, tumor development can be abrogated. We
believe that our concepts and findings are of substantial translational potency.

122

Furthermore, we expect this work to spur further studies pertaining to thedevelopment of efficacious cancer treatments.

123CHAPTER V: BIBLIOGRAPHY

124

1.

2.

3.

4.
5.

6.
7.
8.
9.

10.

11.
12.

13.
14.
15.

16.

17.
18.

Marr LA, Gilham DE, Campbell JD, et al. Immunology in the clinic
review series; focus on cancer: double trouble for tumours: bifunctional and redirected T cells as effective cancer immunotherapies.
Clinical and experimental immunology. 2012;167:216-225.
Zanoni I, Granucci F. Regulation of antigen uptake, migration, andlifespan of dendritic cell by Toll-like receptors. Journal of molecular
medicine. 2010;88:873-880.
Granucci F, Zanoni I, Ricciardi-Castagnoli P. Central role of dendritic
cells in the regulation and deregulation of immune responses. Cellularand molecular life sciences : CMLS. 2008;65:1683-1697.
Mellman I, Steinman RM. Dendritic cells: specialized and regulated
antigen processing machines. Cell. 2001;106:255-258.
McDonnell AM, Robinson BW, Currie AJ. Tumor antigen crosspresentation and the dendritic cell: where it all begins? Clinical &developmental immunology. 2010;2010:539519.
Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines
against cancer. Nat Rev Immunol. 2005;5:296-306.
Petersdorf EW. Genetics of graft-versus-host disease: the major
histocompatibility complex. Blood reviews. 2013;27:1-12.
Trowsdale J. Genomic structure and function in the MHC. Trends ingenetics : TIG. 1993;9:117-122.
Wei MQ, Metharom P, Ellem KA, et al. Search for "weapons of mass
destruction" for cancer -- immuno/ gene therapy comes of age. Cell
Mol Immunol. 2005;2:351-357.
Bhargava A, Mishra D, Banerjee S, et al. Dendritic cell engineering fortumor immunotherapy: from biology to clinical translation.
Immunotherapy. 2012;4:703-718.
Alatrash G, Jakher H, Stafford PD, et al. Cancer immunotherapies,
their safety and toxicity. Expert opinion on drug safety. 2013.
Hagn M, Sontheimer K, Dahlke K, et al. Human B cells differentiate
into granzyme B-secreting cytotoxic B lymphocytes upon incomplete
T-cell help. Immunology and cell biology. 2012;90:457-467.
Walzer T, Dalod M, Robbins SH, et al. Natural-killer cells anddendritic cells: "l'union fait la force". Blood. 2005;106:2252-2258.
Waldhauer I, Steinle A. NK cells and cancer immunosurveillance.
Oncogene. 2008;27:5932-5943.
Castriconi R, Daga A, Dondero A, et al. NK cells recognize and kill
human glioblastoma cells with stem cell-like properties. Journal of
immunology. 2009;182:3530-3539.
Pellegatta S, Eoli M, Frigerio S, et al. The natural killer cell response
and tumor debulking are associated with prolonged survival inrecurrent glioblastoma patients receiving dendritic cells loaded withautologous tumor lysates. Oncoimmunology. 2013;2:e23401.
Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer
cells. Nat Immunol. 2008;9:503-510.
Fehniger TA, Cooper MA, Nuovo GJ, et al. CD56bright natural killer
cells are present in human lymph nodes and are activated by T cellderived IL-2: a potential new link between adaptive and innate
immunity. Blood. 2003;101:3052-3057.

125

19.
20.
21.
22.
23.

24.
25.
26.

27.

28.

29.

30.

31.

32.

33.
34.
35.

36.
37.

Alli RS, Khar A. Interleukin-12 secreted by mature dendritic cells
mediates activation of NK cell function. FEBS letters. 2004;559:71-76.
Gill S, Kalos M. T cell-based gene therapy of cancer. Transl Res.
2013;161:365-379.
June CH. Principles of adoptive T cell cancer therapy. J Clin Invest.
2007;117:1204-1212.
Bonilla FA, Oettgen HC. Adaptive immunity. The Journal of allergy
and clinical immunology. 2010;125:S33-40.
Prlic M, Williams MA, Bevan MJ. Requirements for CD8 T-cellpriming, memory generation and maintenance. Current opinion in
immunology. 2007;19:315-319.
Blattman JN, Greenberg PD. Cancer immunotherapy: a treatment forthe masses. Science. 2004;305:200-205.
Williams MA, Bevan MJ. Effector and memory CTL differentiation.
Annu Rev Immunol. 2007;25:171-192.
Pavoni E, Monteriu G, Santapaola D, et al. Tumor-infiltrating B
lymphocytes as an efficient source of highly specific immunoglobulins
recognizing tumor cells. BMC Biotechnol. 2007;7:70.
Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comesof age. Journal of clinical oncology : official journal of the American
Society of Clinical Oncology. 2011;29:4828-4836.
Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive
strategies that are mediated by tumor cells. Annu Rev Immunol.
2007;25:267-296.
Eisele G, Wischhusen J, Mittelbronn M, et al. TGF-beta andmetalloproteinases differentially suppress NKG2D ligand surface
expression on malignant glioma cells. Brain : a journal of neurology.
2006;129:2416-2425.
Onishi H, Morisaki T, Baba E, et al. Dysfunctional and short-lived
subsets in monocyte-derived dendritic cells from patients withadvanced cancer. Clinical immunology. 2002;105:286-295.
Satthaporn S, Robins A, Vassanasiri W, et al. Dendritic cells are
dysfunctional in patients with operable breast cancer. Cancer Immunol
Immunother. 2004;53:510-518.
Alshamsan A. Induction of tolerogenic dendritic cells by IL-6secreting CT26 colon carcinoma. Immunopharmacology andimmunotoxicology. 2012;34:465-469.
Ma Y, Shurin GV, Peiyuan Z, et al. Dendritic cells in the cancer
microenvironment. Journal of Cancer. 2013;4:36-44.
Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes ofage. Nature. 2011;480:480-489.
de Visser KE, Eichten A, Coussens LM. Paradoxical roles of theimmune system during cancer development. Nat Rev Cancer.
2006;6:24-37.
Nizar S, Copier J, Meyer B, et al. T-regulatory cell modulation: thefuture of cancer immunotherapy? Br J Cancer. 2009;100:1697-1703.
Finn OJ. Immuno-oncology: understanding the function anddysfunction of the immune system in cancer. Annals of oncology :official journal of the European Society for Medical Oncology /ESMO. 2012;23 Suppl 8:viii6-9.
126

38.
39.
40.

41.

42.

43.
44.

45.
46.

47.

48.
49.

50.

51.

52.

53.

54.

Cihova M, Altanerova V, Altaner C. Stem cell based cancer genetherapy. Mol Pharm. 2011;8:1480-1487.
Kim SU. Neural stem cell-based gene therapy for brain tumors. Stem
Cell Rev. 2011;7:130-140.
Lee EX, Lam DH, Wu C, et al. Glioma gene therapy using induced
pluripotent stem cell derived neural stem cells. Molecularpharmaceutics. 2011;8:1515-1524.
Zhao Y, Wang S. Human NT2 neural precursor-derived tumorinfiltrating cells as delivery vehicles for treatment of glioblastoma.
Hum Gene Ther. 2010;21:683-694.
Narumi K, Udagawa T, Kondoh A, et al. In vivo delivery of interferonalpha gene enhances tumor immunity and suppresses immunotolerance
in reconstituted lymphopenic hosts. Gene Ther. 2012;19:34-48.
Rosenberg SA. Progress in human tumour immunology andimmunotherapy. Nature. 2001;411:380-384.
Elzaouk L, Moelling K, Pavlovic J. Anti-tumor activity ofmesenchymal stem cells producing IL-12 in a mouse melanoma model.
Experimental dermatology. 2006;15:865-874.
Benedetti S, Pirola B, Pollo B, et al. Gene therapy of experimental
brain tumors using neural progenitor cells. Nat Med. 2000;6:447-450.
Ehtesham M, Kabos P, Kabosova A, et al. The use of interleukin 12secreting neural stem cells for the treatment of intracranial glioma.
Cancer Res. 2002;62:5657-5663.
Dickson PV, Hamner JB, Burger RA, et al. Intravascularadministration of tumor tropic neural progenitor cells permits targeted
delivery of interferon-beta and restricts tumor growth in a murine
model of disseminated neuroblastoma. Journal of pediatric surgery.
2007;42:48-53.
Jelovac D, Emens LA. HER2-directed therapy for metastatic breast
cancer. Oncology (Williston Park). 2013;27:166-175.
Frank RT, Najbauer J, Aboody KS. Concise review: stem cells as an
emerging platform for antibody therapy of cancer. Stem cells.
2010;28:2084-2087.
Frank RT, Edmiston M, Kendall SE, et al. Neural stem cells as a novel
platform for tumor-specific delivery of therapeutic antibodies. PLoS
One. 2009;4:e8314.
Zhou Y, Bosch ML, Salgaller ML. Current methods for loading
dendritic cells with tumor antigen for the induction of antitumor
immunity. Journal of immunotherapy. 2002;25:289-303.
Wei J, Gao W, Wu J, et al. Dendritic cells expressing a combined
PADRE/MUC4-derived polyepitope DNA vaccine induce multiple
cytotoxic T-cell responses. Cancer biotherapy & radiopharmaceuticals.
2008;23:121-128.
Yamanaka R, Zullo SA, Tanaka R, et al. Enhancement of antitumor
immune response in glioma models in mice by genetically modified
dendritic cells pulsed with Semliki forest virus-mediatedcomplementary DNA. J Neurosurg. 2001;94:474-481.
Steitz J, Tormo D, Schweichel D, et al. Comparison of recombinantadenovirus and synthetic peptide for DC-based melanoma vaccination.
Cancer Gene Ther. 2006;13:318-325.
127

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

Sadanaga N, Nagashima H, Mashino K, et al. Dendritic cell
vaccination with MAGE peptide is a novel therapeutic approach forgastrointestinal carcinomas. Clin Cancer Res. 2001;7:2277-2284.
Yamaguchi S, Tatsumi T, Takehara T, et al. Immunotherapy of murine
colon cancer using receptor tyrosine kinase EphA2-derived peptidepulsed dendritic cell vaccines. Cancer. 2007;110:1469-1477.
Koski GK, Koldovsky U, Xu S, et al. A novel dendritic cell-based
immunization approach for the induction of durable Th1-polarizedanti-HER-2/neu responses in women with early breast cancer. Journalof immunotherapy. 2012;35:54-65.
Gholamin M, Moaven O, Farshchian M, et al. Induction of cytotoxic T
lymphocytes primed with tumor RNA-loaded dendritic cells inesophageal squamous cell carcinoma: preliminary step for DC vaccine
design. BMC Cancer. 2010;10:261.
Kim BR, Yang EK, Kim DY, et al. Generation of anti-tumour immune
response using dendritic cells pulsed with carbonic anhydrase IXAcinetobacter baumannii outer membrane protein A fusion proteins
against renal cell carcinoma. Clinical and experimental immunology.
2012;167:73-83.
Lasky JL, 3rd, Panosyan EH, Plant A, et al. Autologous tumor lysatepulsed dendritic cell immunotherapy for pediatric patients with newly
diagnosed or recurrent high-grade gliomas. Anticancer Res.
2013;33:2047-2056.
Hwang EC, Lim MS, Im CM, et al. Generation of potent cytotoxic T
lymphocytes against castration-resistant prostate cancer cells bydendritic cells loaded with dying allogeneic prostate cancer cells.
Scandinavian journal of immunology. 2013;77:117-124.
Hong S, Li H, Qian J, et al. Optimizing dendritic cell vaccine for
immunotherapy in multiple myeloma: tumour lysates are more potent
tumour antigens than idiotype protein to promote anti-tumour
immunity. Clinical and experimental immunology. 2012;170:167-177.
Barth RJ, Jr., Fisher DA, Wallace PK, et al. A randomized trial of exvivo CD40L activation of a dendritic cell vaccine in colorectal cancer
patients: tumor-specific immune responses are associated withimproved survival. Clin Cancer Res. 2010;16:5548-5556.
Xu Q, Liu G, Yuan X, et al. Antigen-specific T-cell response fromdendritic cell vaccination using cancer stem-like cell-associatedantigens. Stem cells. 2009;27:1734-1740.
Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptormodified T cells for acute lymphoid leukemia. N Engl J Med.
2013;368:1509-1518.
Fox BA, Schendel DJ, Butterfield LH, et al. Defining the criticalhurdles in cancer immunotherapy. Journal of translational medicine.
2011;9:214.
Hahn KA, Bravo L, Adams WH, et al. Naturally occurring tumors indogs as comparative models for cancer therapy research. In vivo.
1994;8:133-143.
Khanna C, Hunter K. Modeling metastasis in vivo. Carcinogenesis.
2005;26:513-523.

128

69.

70.

71.

72.

73.

74.

75.

76.
77.
78.
79.

80.
81.

82.
83.
84.

85.

86.

Khanna C, Vail DM. Targeting the lung: preclinical and comparativeevaluation of anticancer aerosols in dogs with naturally occurring
cancers. Current cancer drug targets. 2003;3:265-273.
Vail DM, MacEwen EG. Spontaneously occurring tumors ofcompanion animals as models for human cancer. Cancer investigation.
2000;18:781-792.
Copier J, Dalgleish AG, Britten CM, et al. Improving the efficacy ofcancer immunotherapy. European journal of cancer. 2009;45:14241431.
Bandura DR, Baranov VI, Ornatsky OI, et al. Mass cytometry:technique for real time single cell multitarget immunoassay based on
inductively coupled plasma time-of-flight mass spectrometry.
Analytical chemistry. 2009;81:6813-6822.
Chang S, Kohrt H, Maecker HT. Monitoring the immune competenceof cancer patients to predict outcome. Cancer immunology,
immunotherapy : CII. 2014.
Ornatsky O, Bandura D, Baranov V, et al. Highly multiparametricanalysis by mass cytometry. Journal of immunological methods.
2010;361:1-20.
Tanner SD, Baranov VI, Ornatsky OI, et al. An introduction to mass
cytometry: fundamentals and applications. Cancer immunology,
immunotherapy : CII. 2013;62:955-965.
Finn OJ. Cancer immunology. The New England journal of medicine.
2008;358:2704-2715.
Zitvogel L, Apetoh L, Ghiringhelli F, et al. Immunological aspects ofcancer chemotherapy. Nature reviews. Immunology. 2008;8:59-73.
McDermott DF, Atkins MB. PD-1 as a potential target in cancer
therapy. Cancer medicine. 2013;2:662-673.
Zhang H-r, Chen F-l, Xu C-p, et al. Incorporation of endothelial
progenitor cells into the neovasculature of malignant glioma xenograft.
Journal of neuro-oncology. 2009;93:165-174.
Yoder MC. Human endothelial progenitor cells. Cold Spring Harbor
perspectives in medicine. 2012;2:a006692.
Peichev M, Naiyer AJ, Pereira D, et al. Expression of VEGFR-2 andAC133 by circulating human CD34(+) cells identifies a population offunctional endothelial precursors. Blood. 2000;95:952-958.
Gehling UM. Hemangioblasts and their progeny. Methods inenzymology. 2006;419:179-193.
Debatin KM, Wei J, Beltinger C. Endothelial progenitor cells forcancer gene therapy. Gene therapy. 2008;15:780-786.
De P, Venneri MA, Naldini L. In vivo targeting of tumor endothelial
cells by systemic delivery of lentiviral vectors. Human gene therapy.
2003;14:1193-1206.
Wei J, Blum S, Unger M, et al. Embryonic endothelial progenitor cells
armed with a suicide gene target hypoxic lung metastases after
intravenous delivery. Cancer Cell. 2004;5:477-488.
Arap W, Pasqualini R. Engineered embryonic endothelial progenitor
cells as therapeutic Trojan horses. Cancer cell. 2004;5:406-408.

129

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.
98.
99.

100.

101.

102.

Park SJ, Moon SH, Lee HJ, et al. A comparison of human cord bloodand embryonic stem cell-derived endothelial progenitor cells in thetreatment of chronic wounds. Biomaterials. 2013;34:995-1003.
Menendez P, Bueno C, Wang L. Human embryonic stem cells: A
journey beyond cell replacement therapies. Cytotherapy. 2006;8:530541.
Slukvin, II. Deciphering the hierarchy of angiohematopoietic
progenitors from human pluripotent stem cells. Cell cycle.
2013;12:720-727.
Murry CE, Keller G. Differentiation of embryonic stem cells toclinically relevant populations: lessons from embryonic development.
Cell. 2008;132:661-680.
Li Z, Han Z, Wu JC. Transplantation of human embryonic stem cellderived endothelial cells for vascular diseases. J Cell Biochem.
2009;106:194-199.
White MP, Rufaihah AJ, Liu L, et al. Limited gene expression
variation in human embryonic stem cell and induced pluripotent stem
cell-derived endothelial cells. Stem cells. 2013;31:92-103.
Dudek AZ. Endothelial lineage cell as a vehicle for systemic deliveryof cancer gene therapy. Translational research : the journal oflaboratory and clinical medicine. 2010;156:136-146.
Su W, Wang L, Zhou M, et al. Human Embryonic Stem Cell-Derived
Endothelial Cells as Cellular Delivery Vehicles for Treatment ofMetastatic Breast Cancer. Cell transplantation. 2012.
Narsinh KH, Plews J, Wu JC. Comparison of human induced
pluripotent and embryonic stem cells: fraternal or identical twins? Mol
Ther. 2011;19:635-638.
Nishikawa S, Goldstein RA, Nierras CR. The promise of humaninduced pluripotent stem cells for research and therapy. Nature
reviews. Molecular cell biology. 2008;9:725-729.
Patel M, Yang S. Advances in reprogramming somatic cells to induced
pluripotent stem cells. Stem Cell Rev. 2010;6:367-380.
Simara P, Motl JA, Kaufman DS. Pluripotent stem cells and genetherapy. Transl Res. 2013;161:284-292.
Wei J, Blum S, Unger M, et al. Embryonic endothelial progenitor cells
armed with a suicide gene target hypoxic lung metastases after
intravenous delivery. Cancer cell. 2004;5:477-488.
Keung EZ, Nelson PJ, Conrad C. Concise review: geneticallyengineered stem cell therapy targeting angiogenesis and tumor stroma
in gastrointestinal malignancy. Stem cells. 2013;31:227-235.
Liu LL, Smith MJ, Sun BS, et al. Combined IFN-gamma-endostatin
gene therapy and radiotherapy attenuates primary breast tumor growthand lung metastases via enhanced CTL and NK cell activation andattenuated tumor angiogenesis in a murine model. Annals of surgicaloncology. 2009;16:1403-1411.
Mizukami Y, Sasajima J, Ashida T, et al. Abnormal tumor vasculaturesand bone marrow-derived pro-angiogenic cells in cancer. Int J
Hematol. 2012;95:125-130.

130

103.

104.

105.

106.
107.

108.

109.

110.

111.
112.

113.

114.
115.

116.

117.

118.

Ojeifo JO, Lee HR, Rezza P, et al. Endothelial cell-based systemic
gene therapy of metastatic melanoma. Cancer gene therapy.
2001;8:636-648.
Hamanishi J, Mandai M, Matsumura N, et al. Activated local immunity
by CC chemokine ligand 19-transduced embryonic endothelial
progenitor cells suppresses metastasis of murine ovarian cancer. Stemcells. 2010;28:164-173.
Loskog A, Tötterman TH. CD40L - a multipotent molecule for tumor
therapy. Endocrine, metabolic & immune disorders drug targets.
2007;7:23-28.
Ullenhag G, Loskog AS. AdCD40L--crossing the valley of death?
International reviews of immunology. 2012;31:289-298.
Kuwashima N, Kageyama S, Eto Y, et al. CD40 ligand immunotherapy
in cancer: an efficient approach. Leukemia & lymphoma.
2001;42:1367-1377.
Gomes EM, Rodrigues MS, Phadke AP, et al. Antitumor activity of an
oncolytic adenoviral-CD40 ligand (CD154) transgene construct inhuman breast cancer cells. Clin Cancer Res. 2009;15:1317-1325.
Gurdon JB. The developmental capacity of nuclei taken from intestinal
epithelium cells of feeding tadpoles. Journal of embryology andexperimental morphology. 1962;10:622-640.
Lensch MW, Mummery CL. From Stealing Fire to Cellular
Reprogramming: A Scientific History Leading to the 2012 Nobel
Prize. Stem cell reports. 2013;1:5-17.
Do JT, Scholer HR. Nuclei of embryonic stem cells reprogram somatic
cells. Stem cells. 2004;22:941-949.
Stadtfeld M, Hochedlinger K. Induced pluripotency: history,
mechanisms, and applications. Genes & development. 2010;24:22392263.
Cao S, Loh K, Pei Y, et al. Overcoming barriers to the clinicalutilization of iPSCs: reprogramming efficiency, safety and quality.
Protein & cell. 2012;3:834-845.
Ma T, Xie M, Laurent T, et al. Progress in the reprogramming ofsomatic cells. Circulation research. 2013;112:562-574.
Gunaseeli I, Doss MX, Antzelevitch C, et al. Induced pluripotent stem
cells as a model for accelerated patient- and disease-specific drug
discovery. Current medicinal chemistry. 2010;17:759-766.
Walia B, Satija N, Tripathi RP, et al. Induced pluripotent stem cells:
fundamentals and applications of the reprogramming process and its
ramifications on regenerative medicine. Stem cell reviews.
2012;8:100-115.
Robbins RD, Prasain N, Maier BF, et al. Inducible pluripotent stem
cells: not quite ready for prime time? Current opinion in organtransplantation. 2010;15:61-67.
Taylor CJ, Bolton EM, Bradley JA. Immunological considerations forembryonic and induced pluripotent stem cell banking. Philosophicaltransactions of the Royal Society of London. Series B, Biologicalsciences. 2011;366:2312-2322.

131

119.

120.
121.
122.

123.

124.

125.
126.

127.

128.

129.

130.

131.

132.

133.

134.

Diaconu I, Cerullo V, Hirvinen MLM, et al. Immune response is an
important aspect of the antitumor effect produced by a CD40Lencoding oncolytic adenovirus. Cancer research. 2012;72:2327-2338.
Vonderheide RH, Glennie MJ. Agonistic CD40 Antibodies and Cancer
Therapy. Clin Cancer Res. 2013;19:1035-1043.
Elzey BD, Ratliff TL, Sowa JM, et al. Platelet CD40L at the interfaceof adaptive immunity. Thrombosis research. 2011;127:180-183.
Karnoub AE, Dash AB, Vo AP, et al. Mesenchymal stem cells withintumour stroma promote breast cancer metastasis. Nature.
2007;449:557-563.
Galderisi U, Giordano A, Paggi MG. The bad and the good of
mesenchymal stem cells in cancer: Boosters of tumor growth andvehicles for targeted delivery of anticancer agents. World journal ofstem cells. 2010;2:5-12.
Klopp AH, Gupta A, Spaeth E, et al. Concise review: Dissecting a
discrepancy in the literature: do mesenchymal stem cells support or
suppress tumor growth? Stem cells (Dayton, Ohio). 2011;29:11-19.
Wang S, Balasundaram G. Potential cancer gene therapy bybaculoviral transduction. Current gene therapy. 2010;10:214-225.
Xiang W, Ke Z, Zhang Y, et al. Isthmin is a novel secreted
angiogenesis inhibitor that inhibits tumour growth in mice. Journal ofcellular and molecular medicine. 2011;15:359-374.
Zhang Y, Chen M, Venugopal S, et al. Isthmin exerts pro-survival anddeath-promoting effect on endothelial cells through alphavbeta5
integrin depending on its physical state. Cell death & disease.
2011;2:e153.
Yang J, Lam DH, Goh SS, et al. Tumor Tropism of Intravenously
Injected Human Induced Pluripotent Stem Cell-Derived Neural StemCells and Their Gene Therapy Application in a Metastatic Breast
Cancer Model. Stem cells (Dayton, Ohio). 2012:10.
van der Zijpp YJT, Poot AA, Feijen J. ICAM-1 and VCAM-1
expression by endothelial cells grown on fibronectin-coated TCPS and
PS. Journal of biomedical materials research. Part A. 2003;65:51-59.
Wang ZZ, Au P, Chen T, et al. Endothelial cells derived from humanembryonic stem cells form durable blood vessels in vivo. Naturebiotechnology. 2007;25:317-318.
Ng ES, Davis R, Stanley EG, et al. A protocol describing the use of arecombinant protein-based, animal product-free medium (APEL) for
human embryonic stem cell differentiation as spin embryoid bodies.
Nature protocols. 2008;3:768-776.
James D, Nam HS, Seandel M, et al. Expansion and maintenance ofhuman embryonic stem cell-derived endothelial cells by TGFbeta
inhibition is Id1 dependent. Nat Biotechnol. 2010;28:161-166.
Choudhury Y, Tay FC, Lam DH, et al. Attenuated adenosine-toinosine editing of microRNA-376a* promotes invasiveness ofglioblastoma cells. The Journal of clinical investigation.
2012;122:4059-4076.
Lin J, Teo S, Lam DH, et al. MicroRNA-10b pleiotropically regulates
invasion, angiogenicity and apoptosis of tumor cells resembling

132

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.
146.

147.
148.
149.

mesenchymal subtype of glioblastoma multiforme. Cell Death Dis.
2012;3:e398.
Vodyanik MA, Slukvin II. Hematoendothelial differentiation of humanembryonic stem cells. Current protocols in cell biology / editorial
board, Juan S. Bonifacino ... [et al.]. 2007;Chapter 23:Unit 23.26.
Woll PS, Morris JK, Painschab MS, et al. Wnt signaling promotes
hematoendothelial cell development from human embryonic stem
cells. Blood. 2008;111:122-131.
Hill KL, Obrtlikova P, Alvarez DF, et al. Human embryonic stem cellderived vascular progenitor cells capable of endothelial and smoothmuscle cell function. Exp Hematol. 2010;38:246-257 e241.
Resch T, Pircher A, Kahler CM, et al. Endothelial progenitor cells:
current issues on characterization and challenging clinical applications.
Stem Cell Rev. 2012;8:926-939.
Ramachandra CJA, Shahbazi M, Kwang TWX, et al. Efficient
recombinase-mediated cassette exchange at the AAVS1 locus inhuman embryonic stem cells using baculoviral vectors. Nucleic acids
research. 2011.
Kaufman DS. Toward clinical therapies using hematopoietic cells
derived from human pluripotent stem cells. Blood. 2009;114:35133523.
Vodyanik MA, Bork JA, Thomson JA, et al. Human embryonic stem
cell-derived CD34+ cells: efficient production in the coculture with
OP9 stromal cells and analysis of lymphohematopoietic potential.
Blood. 2005;105:617-626.
Tian X, Hexum MK, Penchev VR, et al. Bioluminescent imaging
demonstrates that transplanted human embryonic stem cell-derived
CD34(+) cells preferentially develop into endothelial cells. Stem cells(Dayton, Ohio). 2009;27:2675-2685.
Park S-W, Jun Koh Y, Jeon J, et al. Efficient differentiation of humanpluripotent stem cells into functional CD34+ progenitor cells by
combined modulation of the MEK/ERK and BMP4 signaling
pathways. Blood. 2010;116:5762-5772.
Niwa A, Umeda K, Chang H, et al. Orderly hematopoietic
development of induced pluripotent stem cells via Flk-1(+)
hemoangiogenic progenitors. Journal of cellular physiology.
2009;221:367-377.
Yu X, Lin Y, Yan X, et al. CD133, Stem Cells, and Cancer Stem Cells:
Myth or Reality? Current colorectal cancer reports. 2011;7:253-259.
Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation ofendothelial cells from AC133-positive progenitor cells. Blood.
2000;95:3106-3112.
Dolgin E. Flaw in induced-stem-cell model. Nature. 2011;470:13.
Moore XL, Lu J, Sun L, et al. Endothelial progenitor cells' "homing"
specificity to brain tumors. Gene therapy. 2004;11:811-818.
Mancuso P, Calleri A, Bertolini F. Circulating endothelial cells andcirculating endothelial progenitors. Recent results in cancer research.
Fortschritte der Krebsforschung. Progres dans les recherches sur lecancer. 2012;195:163-170.

133

150.

151.

152.

153.
154.

155.

156.

157.

158.

159.

160.

161.
162.

163.

164.

165.

Patenaude A, Parker J, Karsan A. Involvement of endothelial
progenitor cells in tumor vascularization. Microvasc Res. 2010;79:217223.
Zhao Y, Lam DH, Yang J, et al. Targeted suicide gene therapy forglioma using human embryonic stem cell-derived neural stem cells
genetically modified by baculoviral vectors. Gene therapy.
2012;19:189-200.
Shahbazi M, Kwang TW, Purwanti YI, et al. Inhibitory effects ofneural stem cells derived from human embryonic stem cells on
differentiation and function of monocyte-derived dendritic cells. J
Neurol Sci. 2013;330:85-93.
Haviv I, Polyak K, Qiu W, et al. Origin of carcinoma associatedfibroblasts. Cell cycle. 2009;8:589-595.
Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to genetherapy vectors: influence on vector function and effector mechanisms.
Gene Ther. 2004;11 Suppl 1:S10-17.
Jooss K, Chirmule N. Immunity to adenovirus and adeno-associatedviral vectors: implications for gene therapy. Gene Ther. 2003;10:955963.
Ong ST, Li F, Du J, et al. Hybrid cytomegalovirus enhancer-h1
promoter-based plasmid and baculovirus vectors mediate effective
RNA interference. Hum Gene Ther. 2005;16:1404-1412.
Fernandes MS, Gomes EM, Butcher LD, et al. Growth inhibition ofhuman multiple myeloma cells by an oncolytic adenovirus carrying theCD40 ligand transgene. Clin Cancer Res. 2009;15:4847-4856.
Biagi E, Yvon E, Dotti G, et al. Bystander transfer of functional humanCD40 ligand from gene-modified fibroblasts to B-chronic lymphocytic
leukemia cells. Hum Gene Ther. 2003;14:545-559.
Fong C-Y, Gauthaman K, Bongso A. Teratomas from pluripotent stem
cells: A clinical hurdle. Journal of cellular biochemistry.
2010;111:769-781.
Modlich U, Pugh CW, Bicknell R. Increasing endothelial cell specificexpression by the use of heterologous hypoxic and cytokine-inducible
enhancers. Gene therapy. 2000;7:896-902.
Varda-Bloom N, Shaish A, Gonen A, et al. Tissue-specific genetherapy directed to tumor angiogenesis. Gene therapy. 2001;8:819-827.
Yang L, Cao Z, Li F, et al. Tumor-specific gene expression using thesurvivin promoter is further increased by hypoxia. Gene therapy.
2004;11:1215-1223.
Zhu D, Lam DH, Purwanti YI, et al. Systemic Delivery of Fusogenic
Membrane Glycoprotein-expressing Neural Stem Cells to Selectively
Kill Tumor Cells. Mol Ther. 2013.
Tay FC, Tan WK, Goh SL, et al. Targeted transgene insertion into the
AAVS1 locus driven by baculoviral vector-mediated zinc finger
nuclease expression in human-induced pluripotent stem cells. The
journal of gene medicine. 2013;15:384-395.
Zhu H, Lau CH, Goh SL, et al. Baculoviral transduction facilitates
TALEN-mediated targeted transgene integration and Cre/LoxP cassette
exchange in human-induced pluripotent stem cells. Nucleic acids
research. 2013;41:e180.
134

166.
167.
168.

169.

170.

171.

172.

173.
174.

175.

176.

177.

178.

179.

180.

181.

Lobo NA, Shimono Y, Qian D, et al. The biology of cancer stem cells.
Annu Rev Cell Dev Biol. 2007;23:675-699.
Ailles LE, Weissman IL. Cancer stem cells in solid tumors. Current
opinion in biotechnology. 2007;18:460-466.
Ben-Porath I, Thomson MW, Carey VJ, et al. An embryonic stem celllike gene expression signature in poorly differentiated aggressive
human tumors. Nature genetics. 2008;40:499-507.
Wei B, Han XY, Qi CL, et al. Coaction of spheroid-derived stem-likecells and endothelial progenitor cells promotes development of coloncancer. PLoS One. 2012;7:e39069.
Kumar SM, Liu S, Lu H, et al. Acquired cancer stem cell phenotypes
through Oct4-mediated dedifferentiation. Oncogene. 2012;31:48984911.
Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a
subpopulation of cells with cancer stem cell properties in head andneck squamous cell carcinoma. Proc Natl Acad Sci U S A.
2007;104:973-978.
Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective
identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U
S A. 2003;100:3983-3988.
Liu SV. iPS cells: a more critical review. Stem Cells Dev.
2008;17:391-397.
Leng Z, Tao K, Xia Q, et al. Kruppel-like factor 4 acts as an oncogene
in colon cancer stem cell-enriched spheroid cells. PLoS One.
2013;8:e56082.
Palla AR, Piazzolla D, Abad M, et al. Reprogramming activity of
NANOGP8, a NANOG family member widely expressed in cancer.
Oncogene. 2013.
Heddleston JM, Li Z, McLendon RE, et al. The hypoxic
microenvironment maintains glioblastoma stem cells and promotes
reprogramming towards a cancer stem cell phenotype. Cell cycle.
2009;8:3274-3284.
Phang RZ, Tay FC, Goh SL, et al. Zinc finger nuclease-expressing
baculoviral vectors mediate targeted genome integration of
reprogramming factor genes to facilitate the generation of humaninduced pluripotent stem cells. Stem cells translational medicine.
2013;2:935-945.
Wolfl M, Merker K, Morbach H, et al. Primed tumor-reactive
multifunctional CD62L+ human CD8+ T cells for immunotherapy.
Cancer Immunol Immunother. 2011;60:173-186.
Chiang CL, Hagemann AR, Leskowitz R, et al. Day-4 myeloid
dendritic cells pulsed with whole tumor lysate are highly immunogenic
and elicit potent anti-tumor responses. PLoS One. 2011;6:e28732.
Zeng J, Shahbazi M, Wu C, et al. Enhancing Immunostimulatory
Function of Human Embryonic Stem Cell-Derived Dendritic Cells by
CD1d Overexpression. Journal of immunology (Baltimore, Md: 1950).
2012.
Tseng S-Y, Nishimoto KP, Silk KM, et al. Generation of immunogenic
dendritic cells from human embryonic stem cells without serum andfeeder cells. Regenerative medicine. 2009;4:513-526.
135

182.

183.

184.

185.

186.

187.

188.

189.
190.
191.

192.

193.

194.

195.

196.

Segain JP, Rolli-Derkinderen M, Gervois N, et al. Urotensin II is a new
chemotactic factor for UT receptor-expressing monocytes. Journal of
immunology. 2007;179:901-909.
Distler E, Bloetz A, Albrecht J, et al. Alloreactive and leukemiareactive T cells are preferentially derived from naive precursors inhealthy donors: implications for immunotherapy with memory T cells.
Haematologica. 2011;96:1024-1032.
Morse MA, Zhou LJ, Tedder TF, et al. Generation of dendritic cells in
vitro from peripheral blood mononuclear cells with granulocytemacrophage-colony-stimulating factor, interleukin-4, and tumor
necrosis factor-alpha for use in cancer immunotherapy. Annals ofsurgery. 1997;226:6-16.
Mahdian R, Kokhaei P, Najar HM, et al. Dendritic cells, pulsed withlysate of allogeneic tumor cells, are capable of stimulating MHCrestricted antigen-specific antitumor T cells. Medical oncology.
2006;23:273-282.
Ho WY, Nguyen HN, Wolfl M, et al. In vitro methods for generating
CD8+ T-cell clones for immunotherapy from the naive repertoire.
Journal of immunological methods. 2006;310:40-52.
Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T
cells and complements CD28 in generation and establishment of theeffector T cell pool. J Exp Med. 2003;198:1369-1380.
Hendriks J, Gravestein LA, Tesselaar K, et al. CD27 is required forgeneration and long-term maintenance of T cell immunity. Nat
Immunol. 2000;1:433-440.
Vinay DS, Kwon BS. CD11c+CD8+ T cells: two-faced adaptive
immune regulators. Cellular immunology. 2010;264:18-22.
Paulsen M, Janssen O. Pro- and anti-apoptotic CD95 signaling in T
cells. Cell communication and signaling : CCS. 2011;9:7.
Campbell JJ, Murphy KE, Kunkel EJ, et al. CCR7 expression andmemory T cell diversity in humans. Journal of immunology.
2001;166:877-884.
Dumortier H, van Mierlo GJ, Egan D, et al. Antigen presentation by animmature myeloid dendritic cell line does not cause CTL deletion invivo, but generates CD8+ central memory-like T cells that can be
rescued for full effector function. Journal of immunology.
2005;175:855-863.
Egawa H, Ozawa K, Takada Y, et al. Coupled regulation ofinterleukin-12 receptor beta-1 of CD8+ central memory and CCR7negative memory T cells in an early alloimmunity in liver transplant
recipients. Clinical and experimental immunology. 2010;160:420-430.
Yang S, Liu F, Wang QJ, et al. The shedding of CD62L (L-selectin)
regulates the acquisition of lytic activity in human tumor reactive T
lymphocytes. PLoS One. 2011;6:e22560.
Senju S, Haruta M, Matsumura K, et al. Generation of dendritic cells
and macrophages from human induced pluripotent stem cells aiming atcell therapy. Gene Ther. 2011;18:874-883.
Senju S, Hirata S, Motomura Y, et al. Pluripotent stem cells as sourceof dendritic cells for immune therapy. Int J Hematol. 2010;91:392-400.

136

197.

198.

199.

Joyce S. CD1d and natural T cells: how their properties jump-start theimmune system. Cellular and molecular life sciences : CMLS.
2001;58:442-469.
Corgnac S, Perret R, Derre L, et al. CD1d-antibody fusion proteinstarget iNKT cells to the tumor and trigger long-term therapeutic
responses. Cancer Immunol Immunother. 2013;62:747-760.
Zeng J, Wang S. Human dendritic cells derived from embryonic stem
cells stably modified with CD1d efficiently stimulate antitumor
invariant natural killer T cell response. Stem cells translational
medicine. 2014;3:69-80.

137

APPENDICES
Suppl. Table 1. Overview of iPS-EPCs derivation methods.

138

Supplementary figure 1. iPS-EPCs tumor tropism to 2M1 glioma intracranialmodel. A. Schematic diagram of injection and location of the cells. Injection was
performed 2mm right of bregma point. B-E. Primary DiO-labeled 2M1 tumor massinfiltrated by DiI-labeled EPCs. F-I. iPS-EPCs can be observed at 2M1 secondary
tumor site.

139