2
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
Reviewer 3
Huynh Nghia, 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.

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
6

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.


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

ii
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

2.2.13. 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

iv
3.2.2.1. Expression of BCSC markers CD44
+
CD24
-
59
3.2.2.2. In vitro self renewal 60

v
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

BM
Bone marrow
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
DC
Dendritic cell
DMEM
Dulbecco's Modified Eagle Medium
DMSO
Dimethyl sulfoxide
EDTA
Ethylenediaminetetraacetic acid

HLA
Human leukocyte antigen
HSC
Hematopoietic stem cell
IAP1
Inhibitor of Apoptosis Protein 1
IFN
Interferon
IL
Interleukin
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
MUC-1
Mucin 1
NAIP

RNAi
RNA interference
ROS
Reactive oxygen species
SC
Stem cell
SCID
Severe combined immunodeficency
shRNA
Small hairpin RNA
siRNA
Small interfering RNA
SP
Side population
SSC
Side scatter
TAC
Transit-amplifying cells
TGF
Transforming growth factor
TLR
Toll-like receptor
TNF-α
Tumor necrosis factor alpha
UCB
Umbilical cord blood
VEGF
Vascular endothelial growth factor
VNBRC
Vietnamese breast cancer cell

x
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

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

xii
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

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

2
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.

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) self-
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
SC population. There are asymmetric division that a 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).

4

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

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].

6
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


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

-
(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
-/dim
lin
-
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.

10

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

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