Phân lập, xác định đặc điểm của tế bào ung thư vú người việt nam và bước đầu ứng dụng điều trị thực nghiệm

VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY UNIVERSITY OF SCIENCE PHAM VAN PHUC ISOLATION, CHARACTERISATION OF VIETNAMESE BREAST CANCER STEM CELLS AND INITIAL EXPERIMENTAL RESEARCH

VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

UNIVERSITY OF SCIENCE

PHAM VAN PHUC

ISOLATION, CHARACTERISATION OF VIETNAMESE BREAST CANCER STEM CELLS AND INITIAL EXPERIMENTAL RESEARCH ON BREAST CANCER TREATMENT

Specialty: Animal and Human Physiology

Code: 62 42 30 01

Reviewer 1 Tran Linh Thuoc, Professor, PhD Reviewer 2 Nguyen Sao Trung, Professor, PhD

Independent reviewer 1 Tran Cat Dong, Associate Professor,

PhD Independent reviewer 2 Nguyen Dang Quan, PhD

SUPERVISORS:

1 Truong Dinh Kiet, Professor, PhD

2 Le Van Dong, PhD., MD

ĐẠI  HỌC  QUỐC  GIA  TP.HCM

TRƯỜNG  ĐẠI  HỌC  KHOA  HỌC  TỰ  NHIÊN

PHẠM  VĂN  PHÚC

PHÂN  LẬP,  XÁC  ĐỊNH  ĐẶC  ĐIỂM  CỦA  TẾ  BÀO  GỐC  UNG  THƯ  

VÚ  NGƯỜI  VIỆT  NAM  VÀ  BƯỚC  ĐẦU  ỨNG  DỤNG  ĐIỀU  TRỊ  

Phản  biện  độc  lập 1 PGS.TS  Trần  Cát  Đông Phản  biện  độc  lập 2 TS  Nguyễn  Đăng  Quân

Cán  bộ  hướng  dẫn:

1 GS.TS  Trương  Đình  Kiệt  

2 TS.BS  Lê  Văn  Đông  

TP  Hồ  Chí  Minh  – 2012

ACKNOWLEDGMENTS

The research could not have been completed without the significant contributions made by Professor, Doctor Truong Dinh Kiet and Doctor Le Van Dong I thank my teacher - Phan Kim Ngoc for his help and support in and out of the laboratory

I also thank all members of my Laboratory of Stem Cell Research and Application, Department of Animal Physiology and Biotechnology for their continuous support

and feedback throughout the progress of this project

I extend my appreciation to all members of the Oncology Hospital, Hung Vuong Hospital, Department of Anatomic Pathology, Ho Chi Minh City Medicine and Pharmacy University for their support in supplying breast tumors and umbilical cord blood and in analyzing the tumor histochemistry, respectively

TABLE OF CONTENTS

TABLE OF CONTENTS i

LIST OF ABBREVIATIONS vi

LIST OF TABLES ix

LIST OF FIGURES x

INTRODUCTION 1

Chapter 1: LITERATURE REVIEW 1.1 STEM CELLS AND CANCER STEM CELLS 3

1.1.1 Stem cells 3

1.1.2 Cancer stem cells 4

1.1.2.1 Tumor contains cancer cells with SC properties 4

1.1.2.2 Cancer stem cell hypothesis 6

1.2 BREAST CANCER AND BREAST CANCER STEM CELLS 8

1.2.1 Breast cancer 8

1.2.2 Breast cancer stem cells 9

1.2.2.1 Markers, identification and isolation 9

1.2.2.2 Important characteristics of BCSCs 12

1.3 BREAST CANCER STEM CELLS TARGETING THERAPY 15

1.3.1 Targeting on stemness of BCSCs 15

1.3.1.1 Directly targeting on BCSC self-renewal 15

1.3.1.2 Indirectly targeting on BCSC microenvironment 17

1.3.2 Killing BCSCs by specific markers 17

1.3.2.1 Chemotherapy causes differentiation or apoptosis of BCSCs 17

1.3.2.2 Immune cell based immunotherapy 18

1.3.2.3 Oncolytic virus 18

1.4 KNOCK DOWN GENE THERAPY AND IMMUNOTHERAPY 19

1.4.1 Knock down gene therapy for cancer 19

1.4.1.1 General introduction 19

1.4.1.2 Non-viral vector vs viral vector 21

1.4.1.3 siRNA strategies in cancer treatment 22

1.4.2 Immunotherapy for cancer by dendritic cells 24

1.4.2.1 Immunotherapy 24

1.4.2.2 Immunotherapy for cancer 24

1.4.2.3 Dendritic cells based immunotherapy 25

1.4.2.4 Breast treatment by dendritic cell therapy 26

Chapter 2: MATERIALS - METHODS 2.1 MATERIALS 29

2.1.1 Instruments 29

2.1.2 Chemicals and Consumables 30

2.1.3 Solutions, cell culture medium, growth factors and antibodies 30

2.1.4 Kits 32

2.1.5 Biological samples 32

2.2 METHODS 33

2.2.1 Cell culture 33

2.2.1.1 Primary cell culture 33

2.2.1.2 Sub-culture 34

2.2.1.3 Sphere formation culture 34

2.2.2 GFP transgenesis and establishment of GFP expressing cells 34

2.2.3 Cell sorting 35

2.2.4 Immunophenotype analysis by flow cytometry 36

2.2.5 Immunophenotype analysis by immunocytochemistry 37

2.2.6 Knock-down of CD44 on BCSCs 37

2.2.6.1 CD44 down regulation by siRNA 37

2.2.6.2 CD44 down regulation by shRNA 38

2.2.7 Gene expression analysis 38

2.2.7.1 RNA total isolation 38

2.2.7.2 RT-PCR 39

2.2.7.3 Real-time RT PCR 39

2.2.7.4 GeXP PCR 39

2.2.8 Cell bioassays 44

2.2.8.1 Anti-tumor drug resistant assay 44

2.2.8.2 Apoptosis and cell cycle analysis 44

2.2.8.3 Cell proliferation assay 44

2.2.9 In vivo tumorigenesis assay 44

2.2.10 Experimental treatment of breast cancer by knowdown of CD44 45

2.2.11 Cell culture and differentiation of monocytes into dendritic cells 46

2.2.12 Dendritic cell characterization 47

2.2.12.1 Dextran-FITC uptake assay 47

2.2.12.2 Stimulation of CD4+ T lymphocyte proliferation 47

2.2.12.3 Quantity of production of cytokines/chemokines 47

2.2.13 Experiment treatment of breast cancer by dendritic cells primed with BCSC extract therapy 48

2.2.13.1 Animals models 48

2.2.13.2 BCSC antigen production 48

2.2.13.3 DCs primed with BCSC extract 48

2.2.13.4 Mice treatment schedule 49

2.2.14 Mycoplasma detection 49

2.2.15 Statistical analysis 50

Chapter 3: RESULTS 3.1 ISOLATION OF BREAST CANCER CELL LINE VNBRC1 AND VNBRC2 51

3.1.1 Primary cell culture 51

3.1.2 Isolation of breast cancer cell candidates 52

3.1.3 Characterization of breast cancer cell candidates 54

3.1.3.1 Purity of breast cancer cell candidates 54_Toc343253993 3.1.3.2 Gene expression characteristics of VNBRC 55

3.1.3.3 In vivo tumor formation 56

3.1.3.4 Mycoplasma contamination 57

3.2 ISOLATION OF BREAST CANCER STEM CELL LINE BCSC1 AND BCSC2 58

3.2.1 Existence of BCSC sub-population in primary cells 58

3.2.2 Characteristics of BCSC 59

3.2.2.1 Expression of BCSC markers CD44+CD24- 59

3.2.2.2 In vitro self renewal 60

3.2.2.3 In vivo tumor formation at low dose of BCSC and the tumors are caused by injected BCSC 61

3.2.2.4 BCSC population is resistant with anti-cancer drugs 62

3.2.2.5 Mycoplasma contamination 63

3.2.2.6 BCSCs maintained the phenotype after proliferating 63

3.3 CD44 KNOCK DOWN GENE THERAPY 64

3.3.1 CD44 down regulation and anti-doxorubicin resistance of BCSCs 64

3.3.1.1 Expression of CD44 in CD44 knocked down BCSCs 64

3.3.1.2 Characteristics of BCSC following CD44 down regulation and treatment with doxorubicin 66

3.3.2 Characteristics of CD44 knocked down BCSCs by shRNA combined with puromycin selection 69

3.3.2.1 Preparation of BCSCs and non-BCSCs 69

3.3.2.2 Expression of CD44 after down-regulation in BCSCs 70

3.3.2.3 Gene expression of CD44 knocked down BCSCs compared with BCSCs and non-BCSCs 71

3.3.2.4 Cell cycle in CD44 knockdown BCSCs compared with BCSCs and non-BCSCs 73

3.3.2.5 Tumorigenesis of CD44 knockdown BCSCs compared with BCSCs and non-BCSCs in NOD/SCID mice 74

3.3.3 Experimental treatment of breast cancer in NOD/SCID mice by CD44 shRNA gene therapy combined with doxorubicin 76

3.3.3.1 In vitro CD44 down regulation by the CD44 shRNA lentiviral vector 76

3.3.3.2 Tumor size and weight 76

3.4 TARGETING BCSCS BY DENDRITIC CELLS BASED IMMUNOTHERAPY 77

3.4.1 Successful isolation and differentiation of monocytes into functional dendritic cells murine bone marrow 77

3.4.1.1 Induced monocytes express dendritic cells (DCs) phenotype 77

3.4.1.2 Differentiated DCs from monocytes were in vitro functional 79

3.4.2 Effects of BCSC extract primed DC transplantation on breast cancer tumor murine models 81

3.4.2.1 Induction of host protective immunity against tumor by BCSC-Ag-loaded DCs 81

3.4.2.2 Migratory ability of BCSC-Ag-loaded DCs 82

3.4.2.3 Immune response after i.v injection of BCSC-Ag-loaded DCs 83

Chapter 4: DISCUSSION 4.1 SUCCESSFUL ISOLATION BREAST CANCER CELLS FROM VIETNAMESE MALIGNANT BREAST TUMORS 86

4.2 SUCCESSFUL ISOLATION OF BCSCs FROM VIETNAMESE BREAST CANCER CELLS 88

4.3 CD44 IS A POTENTIAL TARGET FOR BREAST CANCER TREATMENT 90

4.3.1 CD44 down-regulation reduced the anti-doxorubicin resistance of BCSCs 90

4.3.2 CD44 down-regulation cause differentiation of BCSCs 93

4.3.3 CD44 gene therapy suppressed the breast tumors on NOD/SCID mice 98

4.4 DENDRITIC CELL THERAPY IS POTENTIAL THERAPY FOR BREAST CANCER TREATMENT 101

CONCLUSIONS AND SUGGESTIONS 106

FUTURE DIRECTIONS 109

LIST OF PUBLICATIONS ON WHICH THESIS IS BASED 110

REFERENCES 112

LIST OF ABBREVIATIONS

ABC ATP-binding cassette

ABCG2 ATP-binding cassette sub-family G member 2

AML Acute myeloid leukaemia

APC Antigen presenting cell

ATM signaling Ataxia Telangiectasia Mutated signaling

BCL-XL B-cell lymphoma-extra large

BCSC Breast cancer stem cell

bFGF Basic fibroblast growth factor

BRCA Breast cancer protein

BRUCE Baculoviral IAP repeat-containing protein 6 BSA Bovine serum albumin

CD Cluster of differentiation

CDK Cyclin-dependent kinase

CK19 Cytokeratin 19

CKI Cyclin-dependent kinase inhibitor

CSC Cancer stem cell

CTL Cytotoxic T lymphocyte

CXCR C-X-C chemokine receptor

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal growth factor

ELISA Enzyme-Linked ImmunoSorbent Assay

ESA Epithelial surface antigen

FACS Fluorescent activated cell sorting

FBS Fetal bovine serum

FITC Fluorescein isothiocyanate

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GFP Green fluorescent protein

GM-CSF Granulocyte-macrophage colony-stimulating factor GVHD Graft versus host disease

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HER2 Human Epidermal growth factor Receptor 2

HLA Human leukocyte antigen

HSC Hematopoietic stem cell

IAP1 Inhibitor of Apoptosis Protein 1

LAK Lymphokine-activated killer cell

MACS Magnetic activated cell sorting

MCF-7 Michigan Cancer Foundation - 7

MCM Monocyte conditioned medium

MEGS Mammary Epithelial Growth Supplement

MHC Major histocompatibility complex

MLV Murine leukemia virus

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAIP NLR family, apoptosis inhibitory protein

NOD Non-obese diabetic

NSCLC Non-small-cell lung carcinoma

PCR Polymerase Chain Reaction

PDGF Platelet-derived growth factor

ROS Reactive oxygen species

TAC Transit-amplifying cells

TGF Transforming growth factor

TNF-α     Tumor necrosis factor alpha

UCB Umbilical cord blood

VEGF Vascular endothelial growth factor

VNBRC Vietnamese breast cancer cell

XIAP X-linked inhibitor of apoptosis protein

LIST OF TABLES

Table 2.1 Primers for Brca1 gene expression analysis 39

Table 2.2 Primer sequences for GeXP-PCR 41

Table 3 1 Result of bcra1 gene expression on MCF-7 and VNBRC1* 56

LIST OF FIGURES

Figure 1 1 SC division and differentiation 4

Figure 1 2 Cell surface markers of CSCs in some varieties of cancers 5

Figure 1 3 CSCs and tumor progression 7

Figure 1 4 Results of Al-Hajj et al (2003) about BCSCs 10

Figure 1 5 SP profile for a fine needle aspirate taken from a male breast cancer patient 11

Figure 1 6 CSCs and tumor hypoxia 13

Figure 1 7 Differences in CSCs targeting therapy and traditional cancer therapy in breast cancer treatment 15

Figure 1 8 Targeting signal transduction pathways in BCSCs 16

Figure 1 9 Some gene knock-down strategies 20

Figure 1 10 Diagrams of three general ways of encoding siRNA in a plasmid or viral vector [66] 21

Figure 1 11 siRNA and shRNA activity [120] 22

Figure 1 12 Generation of DCs and DC therapy in a patient 26

Figure 2 1 Work-flow chart of the whole research 28

Figure 2 2 Mouse manipulation system 33

Figure 2 3 Three sorter systems used in the research 36

Figure 2 4 The work-flow of mycoplasma detection used e-Mycoplasma PCR detection kit (according to Intron Biotechnology Inc.) 49

Figure 3 1 Primary and secondary culture of breast cancer cells from M7 tumor sample 51

Figure 3 2 CD24 expression of primary cells 52

Figure 3 3 CD90 expression of CD90-positive cell depleted primary cells 53

Figure 3 4 Cell morphology of primary cells before and after CD90 depletion 54

Figure 3 5 CD24 expression of MCF-7 cells and breast cancer cells 54

Figure 3 6 Gene profiling of breast cancer cell candidate, MCF-7 and normal breast cells 55 Figure 3 7 Tumor was created in NOD/SCID mouse by cancer cell candidates 57 Figure 3 8 The existence of BCSC population in breast cancer cell population 59 Figure 3 9 CD44 and CD24 expression of BCSCs confirmed by immunocytochemistry 59 Figure 3 10 The BSCS1s were confirmed with CD44+CD24-/dim phenotype by flow cytometry 60 Figure 3 11 Mammosphere formation of BCSCs 60 Figure 3 12 GFP expressed BCSC cells were analysed by flow cytometry before (A) and after (B) selecting with puromycin They exhibited green in color when they excited in fluorescent microscope at FITC filter (C,D) 61 Figure 3 13 A tumor produced in the mouse model 62 Figure 3 14 Results of apoptosis analysis by flow cytometry techniques using kit annexin-V and PI 63 Figure 3 15 BCSCs maintained the phenotype after proliferating 64 Figure 3 16 Expression of CD44 was markedly decreased after transfection with CD44 siRNA 65 Figure 3 17 Expression of CD44 was decreased after transfection with CD44 siRNA 66 Figure 3 18 CD44 knocked down BCSCs slowly proliferated in comparison with control 67 Figure 3 19 Cell cycle of BCSCs and CD44 knocked down BCSCs 68 Figure 3 20 Expression of CD44 and CD24 in three populations of non-BCSCs 69 Figure 3 21 Colony shape of three populations of non-BCSCs and BCSCs 70 Figure 3 22 Expression of CD44 after down-regulation in BCSCs 71 Figure 3 23 Gene expression of CD44 knocked down BCSCs compared with BCSCs and non-BCSCs 72 Figure 3 24 Graphs of gene expression of CD44 knocked down BCSCs compared with BCSCs and non-BCSCs 73

Figure 3 25 Cell cycle in CD44 knockdown BCSCs compared with BCSCs and

non-BCSCs 74

Figure 3 26 Tumorigenic capacities of CD44 knockdown BCSCs, BCSCs and non-BCSCs in NOD/SCID mice 75

Figure 3 27 In vitro CD44 down regulation using the CD44 shRNA lentiviral vector with doses of IFUs to BCSCs at ratios 1:0 (A and E), 2:1 (B and F), 1:1 (C and G) and 1:2 (D and H) 76

Figure 3 28 Tumor size and weight in experiment and control groups 77

Figure 3 29 Monocytes were obtained from murine bone marrow before (A) and after culture 6 days (C) and 12 days (D) 78

Figure 3 30 Results of DC marker analysis by flow cytometry 78

Figure 3 31 Percentage of induced monocytes consumed dextran-FITC 79

Figure 3 32 Stimulation of lymphocyte proliferation by DCs 80

Figure 3 33 IL-12 concentration of groups 81

Figure 3 34 Presence of a tumor after the injection of the (106) BCSCs subcutaneously on day 18 82

Figure 3 35 BCSC-Ag loaded DCs were stained with Vybrant Dil CM (A); No presence of any labeled DCs in control (B); Existence of labeled DCs in mice spleen after treating (C) 83

Figure 3 36 Presence of labeled DCs in the spleen, in experimental mice group (A and B) while there are no such cells in control (C and D) 83

Figure 3 37 Flow cytometry analysis of the CD45 and cytotoxic CD8 T cell activity after i.v injection of the vaccine (F and M), control (E and L) and normal mice (D and K) A,B,C,D,G,H and I were isotype controls of samples 85

INTRODUCTION

Breast cancer is a complicated problem Many studies have been approaching to find out its cause and how to cure this disease In the past 20 years, there was a significant reduction in mortality from breast cancer all over the world This reduction has been largely due to the improvement in early detection methods and the development of more effective therapies, including adjuvant therapies However, more than 50% of breast tumors do not response, even resist, to these therapies and more 70% of patients relapse after 5 years The reason has been identified as an existence of breast cancer stem cells (BCSCs) in all malignant breast tumors These BCSCs were considered as the origin of tumors and contributed to the metastasis and relapse processes in breast cancer patients Hence, BCSC targeting therapy is considered a new and promising therapy

For 5-10 years ago many properties of BCSCs have been discovered and used   as   targets   of   new   targeting   strategies   Several   the   BCSC’s   targets   have   been  used in clinical trials; among them, some have become conventional indications for breast cancer treatment However, because of complexity in breast cancer phenotype among races and geographic locations, the efficacy of these BCSC targeting therapies seem to be low Although no data about the efficacy of existing therapies in Vietnamese breast cancer treatment has been reported, researches from various nationals showed that up to more than 50% of tumors do not express present targets that therapies will attack as well as drug resistant [54];[71];[72];[156];[214];[343] Since then, study on properties of the BCSCs isolated from Vietnamese breast malignant tumors as well as seeking novel therapies with newly identified targets are demands in the present that can contribute to improve the efficacy in breast cancer treatment for Vietnamese population

From current knowledge, it is recognized that gene therapy and immunotherapy are two suitable directions that can be used in targeting BCSCs As

such,   the   research   of   this   thesis   entitled   “Isolation, characterisation of Vietnamese breast cancer stem cells and initial experimental research on

breast cancer treatment” aim to develop new strategies to attack Vietnamese

BCSCs based on gene therapy and immunotherapy was conducted

The objective of research The main objectives of this study are:

- To isolate and culture breast cancer cells from some malignant

breast tumors of Vietnamese women

- To isolate and establish breast cancer stem cell lines from breast

cancer cell populations

- To establish some fundamental steps of gene therapy and

immunotherapy targeting to breast cancer stem cells experimentally

The significances of this study are the establishment of new Vietnamese BCSCs which are fundamental materials for further researches also the demonstration of the proof of principle of gene therapy and immunotherapy targeting to BCSC for the treatment of breast cancers

1.1 STEM CELLS AND CANCER STEM CELLS

1.1.1 Stem cells

Stem cell (SC) is an unspecialized cell that gives rise to a specialized cell such as a blood cell The term "stem cell" was proposed for scientific use by the Russian histologist Alexander Maksimov (1874–1928) at the Congress of Hematologic Society in Berlin in 1908 It postulated the existence of hematopoietic stem cells (HSCs)

For example, SCs are derived from bone marrow (BM) such as HSCs cannot transport oxygen, but when they are differentiated into red blood cells, they can deliver  oxygen…  SCs  hold  vital  roles  in  regeneration  of  the  human  body  Each  day  there   are   many   millions   of   cells   such   as   blood   cells,   skin   cells…   die   and   are  replaced with new cells that differentiated from SCs Up to date SCs were identified and isolated in almost tissue in the human organism, including BM [123];[205]; [240];[250];[261];[295];[313];[333], adipose tissue [98];[164], peripheral blood [163], umbilical cord blood [97];[148];[269], banked umbilical cord blood [252], umbilical cords [83], umbilical cord membranes [87], umbilical cord veins [280], Wharton's jelly from the umbilical cord, placenta [217];[253], decidua basalis [201];[204], the ligamentum flavum [73], amniotic fluid [103], amniotic membrane [65];[211], dental pulp [24];[160], chorionic villi from human placenta [255], foetal membranes [296], menstrual blood [177];[216], and breast milk [246]…

With a strictly corrective definition, SCs have two characteristics: (1) renewal that the ability to go through various cycles of cell division while maintaining the undifferentiated state There are two mechanisms to ensure that the

self-SC population There are asymmetric division that a self-SC divides into one father cell that is identical to the original SC and another daughter cell that is differentiated; and symmetric division that when one SC develops into two differentiated daughter

cells or two SCs identical to the original (Fig 1.1A) (2) Potency: the capacity that SCs differentiate into varieties of specialized cell types (Fig 1.1B)

Figure 1 1 SC division and differentiation

(A) X: SC; Y: progenitor cell; Z: differentiated cell; 1: symmetric SC division; 2: asymmetric SC division; 3: progenitor division; 4: terminal differentiation; (B) Pluripotent, embryonic SCs originate as inner mass cells within a blastocyst The SCs can become any tissue in the body, excluding a placenta Only the morula's cells are totipotent, able to become all tissues and a placenta

1.1.2 Cancer stem cells

1.1.2.1 Tumor contains cancer cells with SC properties

The relation of SCs and cancer was hypothesized for a long time ago More than 150 years ago, Durante (1874) proposed that cancer origin from a rare normal cell population with SC properties After that a year, Cohnheim (1875) gave a hypothesis that SCs could be misplaced during embryonic development and become the source of tumors that would be formed later in during life This hypothesis was considered for a long time by many researches to confirm about the existence of CSCs In 1926, Bailey and Cushing proposed that cancer was initiated and maintained by a small number of transformed precursor cells [41]

This hypothesis confirmed is true when some researchers showed that some cancer cells from ascites fluid in rats, teratocarcinomas and leukaemias in mice

could give rise new tumors in heterogeneous phenotype [57];[170];[206] This

hypothesis was made clearly when Park et al (1971) and Hamburger and Salmon

(1977) showed that some cancers contain a small cell population with properties of normal SCs, particularly myeloma cancer [129];[244]

Figure 1 2 Cell surface markers of CSCs in some varieties of cancers

(According to Morrison et al 2011 [223])

Nearly Lapidot et al (1994) transplanted the cancer cells derived from

human AML that expressed HSCs into NOD/SCID mice and showed that this transplantation initiated leukaemia while if cancer cells also isolated from AML that were negative with HSCs markers could not cause cancer in mice [179] From this research, causing tumor in NOD/SCID mice was considered a standard method to confirm whether cancer cells or SCs are cancer stem cells (CSCs) To define a CSC, many researches tried to look for differences in surface proteins so-called surface markers Based on these markers, CSCs were isolated easily by monoclonal antibody based sorting instruments The first discovery about CSC marker belongs

to leukaemia CSCs They were confirmed as CD34+CD38- in phenotype [52]

Bonnet and Dick showed that all leukaemia contains leukaemia CSCs with range 0.1-1% of the total cell population

Using the same technique, CSCs were demonstrated the existence in many others tumor types including brain, breast, colon, pancreas, prostate, lung, liver,

skin and head and neck cancer [34];[79];[84];[96];[167];[187];[260];[285] (Fig 1.2)

1.1.2.2 Cancer stem cell hypothesis

In genetic basic, tumor or cancer is created by malignant transformation due

to mutations or genetic instability However, which cells can become cancer cells and cause tumors when it got mutations CSC hypothesis considers only SCs or other differentiated cells that acquired the self-renewal ability (the property of SCs) tend to accumulate genetic alterations and evade the strict control of their microenvironment will cause cancer [292] According to this hypothesis, CSCs are not only origin from SCs but also from differentiated cells However, the genetic alteration in differentiated cells that must be enough to help them to be able to self-renewal and lost in microenvironment control is hard CSC model considers that

tumor progression, metastasis and recurrence of cancer are driven by CSCs (Fig 1.3)

In histopathology CSC hypothesis can help to explain how and why changes

in histochemistry of tissue reflect the malignant grades of tumors With two properties similar to SCs, CSCs can cause strong evasion (because of self-renewal) and tumor phenotypic heterogeneity or strongly differentiated status in tumor (because of differentiating potential) [159];[288]

Figure 1 3 CSCs and tumor progression

(According to Dean et al 2005 [85])

Adult SCs usually proliferate more slowly than their differentiated progeny,

so they can increase the longevity For this reason, they are exposed to more damaging agents than more differentiated cells over time; thus, they accumulate more mutations And these mutations can be transmitted into progeny (so-called rapidly proliferating cells or transmitted amplified cells) [90] That means it is said that progeny of normal adult SCs can be CSCs If we can isolate both normal SCs and CSCs in the same tumor, CSCs will inherit many properties of normal SCs In fact, some researchers showed that CSCs share some signaling pathways with SCs

as well as some markers For example, Notch signaling pathway expressed strongly

in breast CSCs and mammary SCs [101]

The main hallmarks of CSCs are their ability to generate tumors from remarkably few cells so it is easy to make a recurrence, and their strong resistance

to radio- and chemotherapy High recurrence is due to self-renewal and strong resistance to radio and chemotherapy Because of SC properties CSCs can differentiate into many kinds of cells and generate wide heterogeneity [63] Although mature cells lack the self-renewal capacity and low proliferation potential [113], both mature cells and SCs also can transform into CSCs [90] In fact, the

mature cells or progenitors also become CSCs through a process so-called defferentiation - mature cells restore the SCs properties [77]

de-1.2 BREAST CANCER AND BREAST CANCER STEM CELLS

1.2.1 Breast cancer

Breast cancer is the most common cancer in women and the most common reason of cancer-related mortality among women worldwide, with more than 1,000,000 new cases and more than 410,000 deaths each year According to The International Agency for Cancer Research, breast cancer is accounted for 21% of all types of cancer in women all over the world In 1998, the incidence of breast cancer per 100,000 people is 92.04 in European and 67.48 all over the world Even in developed countries there were still 130,000 deaths in Europe [53] and 40,000 deaths in US during 2004 because of breast cancer Breast cancer is becoming common in a developing country In Vietnam, women breast cancer is the highest frequency with age-standardized rate 20.3/100,000 (in Ha Noi, 1998), and age-standardized rate 16.0/100,000 (in Ho Chi Minh city, 2004) [9] Breast cancer was considered as the most or the second common cancer in woman in Vietnam [4];[6];[18] Especially the age range (from 40-49) is accounted for 35.2% of cases [11]; 52.2% of cases [6], 47.8% of cases [4], 30.17% of cases [18] depending on countries

At the present, breast cancer is mainly treated by surgery, cytotoxic, hormonal, and immunotherapeutic agents In general, these methods have response rates ranging from 60% to 80% for primary breast cancers and about 50% of metastases [118];[124] However, up to 20% to 70% of patients relapsed within 5 years [78];[262] Commonly when recurrence appears, resistance to therapy will increase the risk of death [118] In Vietnam, there are 21.7% of patients relapsed within 5 years when were treated by modified radical mastectomy of Scanlon [20]

Ten years ago, in Vietnam there are many researches and applications that significantly improved the treatment efficacy as well as found out some characteristics of Vietnamese breast cancer Especially, in combination of radiation therapy and cytotoxic therapy with Arimidex, the 4 year survival rate is 91.6% [16]

In metastases, pegylated liposomal doxorubicin induced response rates ranging from 27.27% in stable disease to 45.46% in progressive disease [1] Response rates and toxicities of TAC (Taxotere, Doxorubicin, Cyclophosphamide) regimen in metastatic breast cancer positive hormonal receptor also recognized with an overall response rate 70.1%, complete response rate 51.1%, partial response rate 20.0% [15] Some mutations as well as dis-regulation of gene expression also were recognized in Vietnamese breast cancer such as over-expression of heparansulfate interacting protein [12];[13], mutations in p53 associating with high histological grade and lymph node positive breast carcinoma [2], mutations in mitochondrial D-loop [1], there are not

any mutations in BRCA1 and BRCA2 in 95 investigated breast cancer patients [17]

Some translational research also carried out that used natural substance such as curcumin [7] or semi-synthetic substance 3', 5, 7-triacetyl-4'-methoxyflavanon [5]

to cause apoptosis in MCF-7 cells About pathohistology, some research showed that the higher histologic grade, the worse breast cancer prognosis [10];[14];[19] ER and PR status did not correlate with groups of age, but was significantly related with histological grades [3]

1.2.2 Breast cancer stem cells

1.2.2.1 Markers, identification and isolation

The first discovery of CSCs in human breast tumors was reported in 2003 by Al-Hajj and collaborators They discovered a cellular population characterized by cell-surface CD44+CD24-/lowESA+ markers, and lin- (lack of expression of CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b) As few as 200 of these cells were able to form tumors when injected into NOD/SCID mice while tens of

thousands of other cells could not cause [33] (Fig 1.4) The tumors that were

generated recapitulated the phenotypic heterogeneity of the initial tumor contained a minority of CD44+CD24-/dimlin- cells that can be serially passaged to form new tumors [33] After that, the CD44+CD24- phenotype has been used to identify and isolate cancer cells with increased tumorigenicity

Figure 1 4 Results of Al-Hajj et al (2003) about BCSCs

(A) A representative tumor in a mouse at the CD44 + CD24 -/low Lin - injection site, but not at the CD44 + CD24 + Lin - injection site (B) Flow cytometry determined the existence of BCSCs in human breast tumors [33]

Using CD44+CD24-/dim BCSCs can be isolated from patient samples after in

vitro primarily cell culture in year 2005 [259] or from previously established breast

cancer cell lines in year 2008 [106] The BCSCs can form mammospheres in culture Mammosphere culture is used to allow mammary epithelial cells in an undifferentiated state [90];[91] Accordingly, mammospheres demonstrated that they are early progenitor/SCs That means they have the ability to differentiate into all three mammary epithelial lineages [90];[91]

In fact, mammospheres derived from breast cancer cells are enriched in cells with the CD44+CD24-/dim-or-low phenotype and these cells can cause tumors when injected into NOD/SCID mice However, a few BCSCs are able to form secondary mammospheres [259] Moreover, cancer cell lines with 90% BCSCs are not more tumorigenic than cell lines with 5% of them with the same phenotype [106], indicating that only a subgroup within the BCSCs is self-renewing

Based on CD44+CD24-/low phenotype of BCSCs, some other markers were discovered Aldehyde dehydrogenase (ALDH) family of cytosolic isoenzymes is

responsible for oxidizing intracellular aldehydes, leading to the oxidation of retinol

to acid, an event that occurs in early SC differentiation ALDH1 is the predominant ALDH isoform in mammalian cells Firstly ALDH activity is high in HSCs It also strongly acts in many kinds of CSCs [80];[138] Some research used Aldefluor staining to identify breast CSCs [74] Breast cancer cells that were positive with ALDH can highly cause tumors in NOD/SCID mice And it is suggested that ALDH+ pool contain the CSC population [113] Combination of phenotype CD44+CD24- and ALDH+ phenotype to select CSCs showed that cells with this phenotype hold higher tumorigenicity in comparison with CD44+CD24- or ALDH+cells [113] New other strategies to improve the identification and isolation efficiency of BCSCs have been recently reported [75];[249];[277]

Figure 1 5 SP profile for a fine needle aspirate taken from a male breast cancer patient

(A) A SP population of 0.6% of the total cell population was observed when cells were incubated with 2.5µg/mL Hoechst 33342 dye; (B) The efflux of Hoechst dye

by the SP population was partially inhibited by the addition of a combination of 10

µM FTC and 50 µM verapamil prior to the Hoechst incubation

Recently a different approach was reported by Sajithlal and collaborators [277] They labeled the CSC population with GFP under the control of the Oct3/4 promoter In MCF-7 cells, there is only 1% of the population expressed GFP and almost cells were CD44+CD24- As predicted, the GFP+ cells were 100-300 times more tumorigenic than others and displayed resistance to cytotoxic drugs

In general, BCSCs can be identified using several methods [31];

- Mammosphere—forming ability [207][195];

- Flow cytometry—analysis of BCSC markers including CD44 [33], CD24 [290], CD49f [256] and ALDH1 [113] Cells are then sorted and characterised

analyzed by clonogenicity, proliferation, differentiation and in vivo tumorigenicity

- Functional assays—SP cells which have an increased ability to efflux Hoechst 33342 dye [120] and ALDH-positive cells that are identified using the

Aldefluor assay which identifies cells with high ALDH activity [113] (Fig 1.5) 1.2.2.2 Important characteristics of BCSCs

a Chemoresistance

Mammosphere formation was 14-fold higher in tumor cells from the patients that had been treated by chemotherapy [344] Another evidence from mouse models showed that exposure to chemotherapeutic agents elicits a selective pressure as well

as prevents differentiation of CSCs And this is a reason to increase the proportion

of CSCs in the tumors Yu and collaborators showed that in tumor bearing mice receiving epirubicin those tumors were highly enriched in CD44+CD24-lin- cells and were able to form 20-fold more mammospheres than cells isolated from tumors with parental cell line [344]

In another study, Shafee et al (2008) showed that the proportion of

CD29hiCD24med cells (tumorigenic cells) in tumors arising after they were treated with cisplatin (4-fold greater) in comparison to untreated primary tumors [286] The chemoresistance in BCSCs is caused partially by the expression of ABC transporters A subpopulation of breast cancer cells with the capability to extrude the dye Hoechst 33342 (a measurement of ABC transporters activity) is enriched in CSCs [247];[74];[334]  This  subpopulation,  called  “side  population”  exhibited  a  30-fold increased in ABCG2 mRNA expression in comparison to unsorted cells [74]

Thus, the expression of ABCG2 and the ability to efflux drugs is lost during differentiation of CSCs to cancer cells These data can explain why primary chemotherapy resistance of BCSCs is

b Radiation resistance

The mechanisms related resistance of BCSCs to radiation are essential to overcome the barriers resistance poses to more effective cancer treatment Recent data showed that CSCs implicate low levels of reactive oxygen species (ROS) and

decreased levels of cellular defenses against oxidative stress [251] (Fig 1.6) From

analysis  of  the  survival  curves  for  radiated  breast  cancer  cells  showed  a  “differential  shoulder   region”   suggestive   of   a   difference   in   DNA   repair   between   BCSCs   and  non-BCSCs Therefore, capacity of DNA repair is a mechanism to enhance BCSCs resistance to radiation

Figure 1 6 CSCs and tumor hypoxia

Like long-term repopulating HSCs, quiescent CSCs may exist in a nonperivascular hypoxic niche, relatively protected from ionizing radiation Activated, and thus cycling, CSCs are found in a peri-vascular niche with confers increased radiation sensitivity and the dependence of CSCs on that niche makes them vulnerable to anti- angiogenic strategies, which target endothelial cells, thereby destroying the CSC niche Re-oxygenation of the hypoxic CSC niche during radiation fractionation

redistributes quiescent CSCs as increasing oxygen levels will modify the niche conditions to render those found in peri-vascular regions and may cause the transition from a quiescent into an activated, proliferative CSC state [242]

In a recent study, Hong Yin and Jonathan Glass (2011) showed that activation of ATM signaling was significantly increased in CD44+CD24−/low cells compared to non CD44+CD24−/low cells in both from breast cancer cell lines and primary human breast cancer cells And if BCSCs were treated an ATM inhibitor, radiation resistance of CD44+CD24−/low effectively decreased This result suggested that ATM signaling abolish the radiation resistance of breast cancer [342]

c Metastasis

In  1889,  Paget  devised  the  “seed  and  soil  hypothesis”  of  cancer  metastasis in which he suggested that the seeds of cancer (the tumor cells) have an ability to survive in specific soils (organs) that are conducive to their growth [241] This hypothesis could easily refer to the properties of CSC

CSCs causing metastasis had been identified [188] Accordingly, several authors have proposed a model in which CSCs appear as the active source of metastatic spread [122];[188];[317];[330] In that model, a sub-population of tumor cells expressing SC markers has been identified in metastatic breast cancer patients and a high percentage of BCSCs have been found in metastases [29];[43];[310]

The ability of BCSCs to invade and proliferate at the metastatic sites has

been studied both in vitro and in vivo ALDH+ cells isolated from breast cancer cell lines were more migratory and invasive than the ALDH- cells [81] Intra-cardiac injection of ALDH+ cells isolated from human breast cancer cell lines to NOD/SCID mice generated metastases at distinct organs; in contrast, ALDH- cells produced only occasional metastases limited to lymph nodes [66];[67]

1.3 BREAST CANCER STEM CELLS TARGETING THERAPY

1.3.1 Targeting on stemness of BCSCs

1.3.1.1 Directly targeting on BCSC self-renewal

There are some therapies to target the stemness of BCSCs All therapies aim

to differentiate BCSCs into non-BCSCs That means these therapies lose the renewal capacity of BCSCs In fact once they lost their self-renewal, they can lose the resistant potential to radiotherapy or chemotherapy as well as reduce the invasion In theory, CSC targeted therapy can attack totally CSCs so that the tumor will be degenerate While traditional cancer therapy only attack and kill the tumor

self-cells but not CSCs, so tumor can shrink but grows back (Fig 1.7)

Chemo- or radioresistance markedly impairs the efficacy of cancer therapy and involves anti-apoptotic signal transduction pathways that prevent cell death [32];[56];[182] Up to date, there are some molecular mechanisms that may account for the resistance of BCSCs to cytotoxic agents [38]

Figure 1 7 Differences in CSCs targeting therapy and traditional cancer therapy in breast cancer treatment

Over-expression of some proteins relating to multidrug resistance was recognized in BCSCs [95] BCSCs may also express high levels of anti-apoptotic proteins, such as survivin and BCL-XL [325] Moreover, efficiency of DNA repair increased in BCSCs, which may help them resistant to traditional agents [110]

Figure 1 8 Targeting signal transduction pathways in BCSCs

Schematic illustration of key signal transduction pathways, therapeutic targets, and targetingagents (shown in red) These pathways include Notch; Hedgehog; Wnt; human epidermal growth factor receptor 2 (HER2)–Akt; and cytokine loops, including interleukin (IL)-6 and IL-8 [331]

Self-renewal pathways hold vital roles in SCs as well as CSCs There are some signaling pathways that express strongly in SCs such as Wnt, Notch, and Hedgehog (Fig 1.8) These pathways can help BCSCs maintaining the phenotype during the tumor growth (so-called self-renewal) In fact, there are some experiments showed that if they were dys-regulated in mammary gland, mice can get breast cancer [147];[165];[297];[320] And in almost of human BCSC line, these pathways are also dys-regulated [161];[196];[245]

One of the most pathways relating the self-renewal is Her2-signalling pathway Up to date, there are many therapies targeting to Her-2 Almost they were developed to suppress the Her2-overexpressing breast cancer Some agents were

invented such as trastuzumab and lapatinib (Fig 1.8) In vivo trastuzumab and

lapatinib can improve progression-free survival and overall survival of patients with

advanced disease [173] And in vitro trastuzumab can reduce the BCSC population

[174] However, these agents also met some limitations, especially nearly 50% of patients who respond to HER2-targeted agents relapse within a year [228]

1.3.1.2 Indirectly targeting on BCSC microenvironment

Microenvironment is space surrounding SCs and termed the SC niche In tumors, this niche contains a variety of cellular elements that include inflammatory cells, fibroblasts, endothelial cells, and mesenchymal stem cells

Interleukin IL-6 and IL-8 are proved as regulators of BSCS self-renewal in in

vitro and xenograft models [173];[279] Korkaya et al (2008) showed that

chemotherapy in breast cancer could increase locally IL-8 and result the increase in CSC populations [174] In some other studies, the relations of IL-6 and IL-8 with development of metastasis and poor outcome also recorded [150];[279]

These studies suggest that cytokines such as IL6 and IL8 play a vital role in regulating CSCs inside the niche So micro-environment targeting therapy maybe is

an effective therapy in cancer treatment In fact, when statin – an anti-inflammatory was used in breast cancer patient the breast cancer risk was decreased (Kochhar RKV, 2005) Some antibodies that bind to IL-8 receptor CXCR1 or the small molecule CXCR1/CXCR2 such as repertaxin inhibit the tumor growth and

metastasis [173] (Fig 1.8)

1.3.2 Killing BCSCs by specific markers

1.3.2.1 Chemotherapy causes differentiation or apoptosis of BCSCs

In the recent study, stealth liposomal daunorubicin plus tamoxifen was developed for eradicating breast cancer cells together with CSCs The particles contained daunorubicin and tamoxifen with 95% and 90%, respectively Stealth liposomal daunorubicin plus tamoxifen could eliminate both breast cancer cells and BCSCs [126] In other research, all-trans retinoic acid stealth liposomes in combination with a cytotoxic agent, vinorelbine stealth liposomes were also developed for preventing the relapse of breast cancer The research also showed that all-trans retinoic acid stealth liposomes with vinorelbine stealth liposomes strongly

inhibited the recurrence of tumor in mice model [190];[193] Metformin, a standard

drug for diabetes, also inhibits cellular transformation and selectively kills BCSCs Metfromin and doxorubicin based therapy reduced tumor mass and prevented

relapse in a xenograft mouse model [140] Some chemicals such as all-trans retinoic acid,  metformin…  are  confirmed  as  SC  differentiating  agents [111] A combination

of the differentiation agents and anti-tumor drugs is considered as an effective way

to eliminate the CSCs as well as preventing recurrence

1.3.2.2 Immune cell based immunotherapy

There are some kinds of immune cells applied in immunotherapy However, dendritic cells were used most popular Killed breast cancer cell derived antigen loaded DCs were considered a novel approach to breast cancer immunotherapy Killed breast cancer cells could be captured by immature DCs After induced maturation, they can efficiently present MHC class I and class II peptides to CD8+and CD4+ T lymphocytes [232] DCs loaded with killed breast cancer cells can prime nạve CD8+ T cells to differentiate into effector CTLs Importantly, among

the tumor CTLs, Saito et al found that CTLs specific for HLA-A2 restricted

peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and surviving [276] Some researches fused tumor cells with DCs to aim improvement the efficacy of tumor antigen presentation DCs pulsed

with apoptotic breast tumor cells could elicit effective antitumor T cell responses in

vitro [86] Animal models have demonstrated that vaccination with DC/tumor

fusions can protect a lethal challenge with tumor cells and regressing disease Preclinical studies have also shown that fusion cells stimulate cellular immune responses that are capable of lysing autologous tumor cells [39]

In Phase I study, 32 patients were vaccinated with 105 to 4.106 fusion cells There was no significant treatment-related toxicity and no clinical evidence of autoimmunity In a subset of patients, there is an increase about the percentage of CD4+ and CD8+ T cells expressing intracellular IFN-gamma in response to in vitro

exposure to tumor lysate with two patients exhibited disease regressions, five patients with renal carcinoma and one patient had disease stabilization [40]

1.3.2.3 Oncolytic virus

At year 1977, some transformed cell lines sensitized to the human reovirus were regconized [130] Reovirus is an oncolytic virus that is not associated with significant disease in humans, but is selectively able to replicate in cancer cells

After that some experiments showed that reovirus was able to replicate efficiently in many human cancer cell lines (brain, breast, lymphoma, ovarian, bladder, spinal, and colon) [30];[139];[166];[235];[332];[341] Therefore reovirus was considered

as an anti-cancer therapeutic agent Intratumoral injection of reovirus induced tumor regression in immune-compromised mice bearing the human tumors [30];[139];[166];[235];[332];[341] Many promising anti-cancer agents based on oncolytic viruses have recently entered into clinical trials and demonstrated encouraging safety and efficacy

In breast cancer, reovirus can infect all the 6 examined breast cancer cell lines, after infection there is 50% or greater cytolysis was demonstrated at day 7 after infection [131] In the recent research, oncolytic reovirus has the potential to induce tumor regression in breast cancer patients More important, the BCSC population was equally reduced and was as susceptible to reovirus treatment as the non-BCSC population [209] In the present there is not clinical trial using reovirus therapy; however reovirus therapy is significant potential therapy for breast cancer treatment in the future

1.4 KNOCK DOWN GENE THERAPY AND IMMUNOTHERAPY

1.4.1 Knock down gene therapy for cancer

1.4.1.1 General introduction

RNA interference (RNAi) induces gene silencing at a level of transcription mediated by double stranded RNA This is a popular method used to

post-knock-down expression of interest gene (Fig 1.9) There are two ways to delivery

siRNA (double-stranded interference) to the target cells, including non-viral and viral vectors In comparison to non-viral vector, the delivery efficacy of viral vector gets higher efficacy Non-viral vectors are direct transfection of siRNA into target cells while viral vector brings a sequence that will express to short hairpin RNA (shRNA) and become 21 bp siRNA after cut by Dicer enzyme

Figure 1 9 Some gene knock-down strategies

Knock down of a gene using siRNA is used with a variety of strategies such

as inhibition of over-expressed oncogenes, promoting apoptosis, regulating cell cycle, anti-angiogenesis and enhancing the efficacy of chemotherapy and radio-therapy RNAi technology has become an excellent strategy for cancer gene therapy from it was discovered The phenomenon was first recognized in transgenic plants

and RNAi was named in the genome research of the worm Caenorhabditis elegans

by Fire et al in 1998 However, it was Elbashir et al who first discovered the RNAi

phenomenon in cultured mammalian cells, in 2001 [94]

In mammalian cells, chemically synthesized siRNA can activate the interferon system, which leads to a shutdown of the synthesis in some proteins and

non-specific degradation of cellular mRNA To improve the efficacy, Kim et al synthesized siRNA in vitro using   T7   RNA   polymerase   which   lacked   the   5′-

triphosphate to avoid the interferon response [168] However, a high dose should be avoided, because high dose of siRNA still can induce interferon To overcome these limitations, other viral vectors are used to express shRNAs by RNA polymerase-III promoter The expressed shRNA can be cut into ~21 bp siRNA by a

RNAse III family nuclease called Dicer enzyme (Fig 1.10)

Figure 1 10 Diagrams of three general ways of encoding siRNA in a plasmid or viral vector [70]

1.4.1.2 Non-viral vector vs viral vector

Non-viral vector RNAi usually adopted chemically synthesized And they were delivered into target cells by non-viral vehicles, such as lipophilic molecules,

polymer vectors, nanoparticles To improve the efficacy of transfection, Elbashir et

al (2001) investigated the use of liposome to deliver siRNA into cultured

mammalian cells [94]; and Bologna et al (2003) used polyethylenimine (PEI) as

transfection factor to deliver a broad panel of nucleic acids such as siRNAs into

cultured cells [51]; Schiffelers et al (2004) achieved target sterically stabilized

nanoparticles delivering the siRNA oligonucleotide into tumor cells via ligand targeted [282] In common, non-viral vectors are much safer and easier to produce than viral vectors However, clinical applications are still limited by low transfer efficiency Recently, many types of viral vectors have been used as siRNA carriers Mouse Moloney leukemia virus has been developed as siRNA retroviral delivery systems, with a high efficacy of entering a wide range of hosts and specificity of

targeting neoplastic cells (Fig 1.11)

Abbas-Terki et al (2002) designed lentiviral vectors to encode siRNA

against enhanced green fluorescent protein (EGFP) and silencing immediately occurs after 72 h post-infection and stables for at least 25 d [22] Adenovirus can be considered as the main alternative system Adenovirus is superior to other virus vectors in introducing genetic material into host cells (1) It may be grown at high titer (1012 Pfu/mL) (2) It can reasonably accommodate a large gene fragment (up to

8 kb) (3) It can infect most mammalian cell types (both replicative and

non-replicative) (4) It is easy to handle However, this system also holds many limitations In one cancer gene therapy study, scientists use an adenoviral vector containing wild-type p53 complementary DNA (Ad-p53) was administrated to 25 patients with non-small-cell lung cancer (NSCLC) The results showed that only two patients had partial responses (8%) One reasonable explanation is that the body can be tolerant to repeatedly intra-tumoral injection of Ad-p53 as well as it is difficult to reach a 100% of infection rate [306]

Figure 1 11 siRNA and shRNA activity [127]

1.4.1.3 siRNA strategies in cancer treatment

a Silencing over-expressed oncogenes

It is said that overexpression of oncogene such ras and myc plays an essential role in oncogenesis In fact, oncogenic mutations in the ras gene are present in

approximately 30% of all cancers Silencing these genes promised many excellent

results in cancer treatment Brummelkamp et al (2002) designed a set of K-ras

siRNAs in viral vectors [58] The results showed that after infect with siRNA viral

vector, K-ras expression had strongly decreased in tumor cells and tumors was

inhibited In another study, Fleming et al (2005) used siRNA to silence mutant

K-ras in of pancreatic adenocarcinoma The results also showed that siRNA inhibited

K-ras and resulted in reducing malignant tumors formation, migration and

angiogenesis in mice charged with tumor cells [108]

About c-Myc, this protein is over-expressed in a wide variety of human cancers with 80% of breast cancers, 70% of colon cancers, 90% of gynecological cancers and 50% of hepatocellular carcinomas Inhibition of this gene by siRNA also reduced tumor growth in nude mice [324]

b Promoting apoptosis

Apoptosis, the process of programmed cell death, hold extremely important

in cancer A cell goes to the apoptosis depending on a balance between apoptotic and anti-apoptotic members Bcl-2 family proteins are anti-apoptotic members in mitochondria They play a crucial role in apoptosis regulation Some inhibitor of apoptosis such as NAIP, c-IAP1, c-IAP2, XIAP, Survivin and BRUCE also hold important in maintaining anti-apoptosis of cells So that knocking down the anti-apoptosis protein will push cells go to apoptosis For example, knock down

pro-of three anti-apoptotic genes such as XIAP, Survivin and Bcl-2 in two pancreatic cancer cell lines cause induction of apoptosis in pancreatic cancer cells [273] In another study, inhibition of survivin reduces cell proliferation and induces apoptosis

in human endometrial cancer [27]

c Regulating cell cycle

The dys-regulation of the cell cycle mechanism is considered as one of the main reason of carcinogenesis Cell cycle is regulated by a complex interaction of specific regulatory proteins, such as cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors (CKIs) Cyclin E is believed to control G1/S phase progression Cyclin E over-expression can trigger G1 progression, causing genome instability and carcinogenesis So, some regulatory proteins especially cyclin E by knocking down it can be a promising therapy to inhibit cancer cell growth In

hepatocarcinoma cell model, Li et al (2003) showed that siRNA oligos can induce

apoptosis and suppress tumor formation in nude mice [185] In recently research,

He et al (2009) showed that inhibition of cyclin E expression in breast cancer cells

can block their cell cycle at G1 phase, reduce their cell growth, differentiation and proliferation, and increase their sensitivity to chemotherapy [134] In similar to inhibition of cyclin E, knocking down of cyclin D1 by siRNA using oligofectamine

in MCF-7 also reduced the proliferation of MCF-7 cells, caused cell cycle arrest at G1 phase and lost colony forming ability [155]

d Inhibiting angiogenesis

Angiogenesis exerts a critical role in tumor growth and metastasis Up to now, more than 20 angiogenic factors have been identified, including VEGF, TGF, bFGF, Angiogenin and PDGF, while Angiostatin, Endostatin, Vasostain, Tumstatin and IL-12 are negative factors Inhibition or reduction of angiogenic stimulators by

RNAi can be an effective anti-angiogenic strategy in cancer therapy Chen et al

(2005) inhibited VEGF-C expression by siRNA and the resulting in reducing

growth of lymph node and lung metastasis [70] Shen et al (2007) used siRNA to

knock down vascular endothelial growth factor receptor 1 (VEGF- R1) This study also showed that VEGF-R1 also had therapeutic potential in antitumor [289]

1.4.2 Immunotherapy for cancer by dendritic cells

1.4.2.1 Immunotherapy

Immunotherapy for cancer is cancer treating therapy stimulating the immune system by a variety of reagents such as vaccines, infusion of immune cells, or cytokines These reagents will stimulate the immune systems through one of several mechanisms: 1) by stimulating the anti-tumor response, either by increasing the number of effector cells or by producing one or more soluble mediators such as lymphokine; 2) by decreasing suppressor mechanisms; 3) by altering tumor cells to increase their immunogenicity and make them more susceptible to immunologic defenses; and 4) by improving tolerance to cytotoxic drugs or radiotherapy, such as stimulating BM function with GM-CSF There are some immunotherapeutic strategies: (1) Immunotherapy for cancer; (2) Vaccination; (3) Increase immune system activity; (4) Disease burden and immunotherapy

1.4.2.2 Immunotherapy for cancer

Cancer immunotherapy is immunotherapy that is specific for cancer to reject cancer Its main target is acting the patient's immune system to attack the malignant

tumor cells causing the disease This therapy can perform by injecting a cancer vaccine such as Dendreon's Provenge, in which case the patient can be trained to recognized tumor cells as future target or administering antibodies as drugs that they can trigger the immune system to destroy the tumor cells Another approach in immunotherapy for cancer is cell based immunotherapy That means we can use the

immune cells such as NK cells, LAK, CTLs, DC, etc that are either activated in

vivo or in vitro and transfused back to the patient after that This therapy can get the

best results because the immune system can target the targets However, this therapy also gets some problems such as tumor cells display unusual antigens or non-specific antigen as well as other kinds of tumor cells display cell surface receptors that are rare or absent

1.4.2.3 Dendritic cells based immunotherapy

DCs were firstly discovered by Steinman and Cohn (1973) [301] Until now, many studies had been performed to identify the origin, phenotypes, and functions

of DCs, as well as their subtypes DCs are one of antigen-presenting cells They act

as messengers between the innate and adaptive immunity In the body, DCs originated from HSCs In the differentiation process, these progenitor cells initially transform into immature dendritic cells (imDCs) These cells are characterized by high endocytic activity and low T-cell activation They can engulf viruses as well as bacteria in the surrounding environment through pattern recognition receptors, toll-like receptors (TLRs) ImDCs probably also originated from monocytes, which circulate throughout the human body When recognizing the suitable signals,

monocytes will turn into either DCs or macrophages In vitro DCs can be

successfully generated from monocytes or CD34-positive HSCs from BM,

peripheral blood, and UCB [47];[203];[224];[268];[270];[303];[311] (Fig 1.12)

A widely used procedure is to induce monocytes or HSCs into imDCs by platting them in a tissue culture flask and treating with IL-4 and GM-CSF for 1 week Further treatment with α-TNF helps imDCs differentiate into mature DCs

DC therapy for cancer treatment is based on the use of DCs to present tumor antigens to nạve T cells Subsequently, T cells trigger tumor-specific immune response In this strategy, monocytes or HSCs are firstly harvested from UCB, BM