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 Tran Linh Thuoc, Professor, PhD Reviewer Nguyen Sao Trung, Professor, PhD Reviewer Huynh Nghia, PhD Independent reviewer Tran Cat Dong, Associate Professor, PhD Independent reviewer Nguyen Dang Quan, PhD SUPERVISORS: Truong Dinh Kiet, Professor, PhD Le Van Dong, PhD., MD Ho Chi Minh City – 2012 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   THỰC  NGHIỆM Chuyên  ngành:  Sinh  lý  Người  và  Động  vật Mã  số: 62 42 30 01 Phản  biện GS.TS  Trần  Linh  Thước Phản  biện  2 GS.TS  Nguyễn  Sào  Trung Phản  biện TS  Huỳnh  Nghĩa Phản  biện  độc  lập PGS.TS  Trần  Cát  Đông Phản  biện  độc  lập TS  Nguyễn  Đăng  Quân Cán  bộ  hướng  dẫn: GS.TS  Trương  Đình  Kiệt   TS.BS  Lê  Văn  Đơng   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 Chapter 1: LITERATURE REVIEW 1.1 STEM CELLS AND CANCER STEM CELLS 1.1.1 Stem cells 1.1.2 Cancer stem cells 1.1.2.1 Tumor contains cancer cells with SC properties 1.1.2.2 Cancer stem cell hypothesis 1.2 BREAST CANCER AND BREAST CANCER STEM CELLS 1.2.1 Breast cancer 1.2.2 Breast cancer stem cells 1.2.2.1 Markers, identification and isolation 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 i 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 ii 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 iii 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 iv 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-Agloaded 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 v LIST OF ABBREVIATIONS Ab Antibody ABC ATP-binding cassette ABCG2 ATP-binding cassette sub-family G member Ag Antigen ALDH Aldehyde dehydrogenase AML Acute myeloid leukaemia APC Antigen presenting cell ATM signaling Ataxia Telangiectasia Mutated signaling BCL-2 B-cell lymphoma BCL-XL B-cell lymphoma-extra large BCSC Breast cancer stem cell bFGF Basic fibroblast growth factor BM Bone marrow BRCA Breast cancer protein BRUCE Baculoviral IAP repeat-containing protein 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 EGF Epidermal growth factor vi ELISA Enzyme-Linked ImmunoSorbent Assay ER Endoplasmic reticulum ESA Epithelial surface antigen FACS Fluorescent activated cell sorting FBS Fetal bovine serum FITC Fluorescein isothiocyanate FTC Fumitremorgin C 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 HLA Human leukocyte antigen HSC Hematopoietic stem cell IAP1 Inhibitor of Apoptosis Protein IFN Interferon IL Interleukin LAK Lymphokine-activated killer cell MACS Magnetic activated cell sorting MCF-7 Michigan Cancer Foundation - MCM Monocyte conditioned medium MEGS Mammary Epithelial Growth Supplement MHC Major histocompatibility complex MLV Murine leukemia virus MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide MUC-1 Mucin NAIP NLR family, apoptosis inhibitory protein NK Natural killer cell NOD Non-obese diabetic vii 10 colonies would be exceptionally unlikely to contain similar insertion sites to disrupt the function of the genes Down-regulation of CD44 caused changes in the phenotype of BCSCs CD44 knockdown BCSCs lost the BCSC phenotype and showed reduced expression of genes related to stemness, metastasis and tumorigenesis, especially Muc-1 and Bcl-2 In addition, the cell cycle changed to resemble that seen in differentiated cells (non-BCSCs), and anti-tumor drug resistance and tumorigenic potential in NOD/SCID mice were both reduced These data indicate that BCSCs were differentiated into non-BCSCs by CD44 knockdown These results suggest that a combination of differentiation therapy aimed at down-regulation of CD44 in BCSCs, together with chemical or radiation therapies, represents a promising therapeutic strategy for the treatment of breast cancer, and also suggest that RNAi gene therapy could provide a novel differentiation strategy 4.3.3 CD44 gene therapy suppressed the breast tumors on NOD/SCID mice CSCs are considered the origin of malignant tissues The existence of CSCs has been recently confirmed in solid tumors of the brain, prostate, pancreas, liver, colon, head and neck, lung and skin [34];[96];[116];[187];[260];[285] Moreover, CD44+CD24- cells have been identified as BCSCs [33] Since CSC discovery, the study of cancer treatment in general and particularly breast cancer has gradually focused on targeting CSCs Thus far, targeting of BCSCs has been performed using various approaches, but mainly targets two aspects: self-renewal and differentiation of BCSCs To influence selfrenewal and differentiation, signaling pathways that are important in BCSCs such as Wnt, Notch, and Hedgehog [147];[297];[320] can be targeted There are numerous methods to target signaling pathways including gene therapy, immunotherapy and selective chemotherapy In our previous studies, we found that CD44 downregulation reduces the drug resistance of BCSCs to doxorubicin In this study, we used an experimental treatment to target BCSCs by combining gene therapy targeting CD44 and doxorubicin treatment First, we established a BCSC line that stably expressed GFP to monitor the xenografted breast cancer tumor in mice BCSCs were transduced with a lentiviral 98 vector carrying copGFP and a puromycin resistance gene for selection Because random insertion of lentiviral DNA into the genome can cause detrimental mutations, we isolated CD44+CD24- cells from GFP-BCSCs using s magnetic cell separation method and re-analyzed with flow cytometry Indeed a study showed that lentiviral vectors demonstrate a low tendency to integrate into genes that cause cancer [64] Another study also found that there is no increase in tumor incidence and no earlier onset of tumors in a mouse strain following the use of lentiviral vectors [221] GFP expressing BCSC1 maintained a tumorigenic capacity and formed malignant tumors in NOD/SCID mice with numerous poorly differentiated and abnormal cells Next, we determined the appropriate dose of virus particles to infect tumors, which was considered as the IFUs that down-regulated CD44 at the highest rate To determine the appropriate dose, we conducted serial assays with ratios between cells and IFUs at 2:1, 1:1 and 1:2, respectively CD44 down-regulation was highest using double the IFUs compared with that of the cell number To determine the number of cells in a tumor, we measured the tumor size at the time of treatment The number of tumor cells is calculated as cm3 tumor contains ~1×109 cells [88] Although recent studies have supported this claim [49];[100];[236] Experiments using the same mouse breed under the same condition are considered to possess similar tumor volumes among the mice Lentiviral vector-injected mice were treated with doxorubicin after 48 h This period of time was chosen because previous studies show that viruses infect target cells and inhibit CD44 expression after 24 h The doxorubicin dose used was mg/kg body weight and was chosen based on a previous study [239] The results showed significant differences about size and weight of tumors between treated mice compared with that of the controls Doxorubicin treatment and CD44 siRNA therapy alone or in combination inhibited tumor growth Tumor inhibition with doxorubicin treatment and CD44 shRNA therapy alone was identical, while a significant difference (P