CHARACTERIZATION OF CAL 27 ORAL SQUAMOUS CARCINOMA CELL LINE AS A
MODEL FOR CANCER STEM CELL STUDY
CHAI YUAN LIN
UNIVERSITI SAINS MALAYSIA
2016
CHARACTERIZATION OF CAL 27 ORAL SQUAMOUS CARCINOMA CELL LINE AS A
MODEL FOR CANCER STEM CELL STUDY
by
CHAI YUAN LIN
Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
SEPTEMBER 2016
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ACKNOWLEDGEMENTS
This study was conducted with funding from the Universiti Sains Malaysia- Postgraduate Research Grant Scheme (Grant number: 1001/CIPPM/846042).
Additionally, it was my honour to receive “MyBrain 15” Scholarship from the Ministry of Education (MOE), Malaysia. I am grateful to have these funding that support me financially from both Universti Sains Malaysia and MOE.
I would like to take this opportunity to express my gratitude to my supervisor Dr. Ong Ming Thong for his knowledge, patience and motivation while allowing me to work in my own way to grow into a research scientist. My sincere thanks also go to my co-supervisor Dr. Khairani Idah Mokhtar for her support and insightful comments. I could not have imagined any better advisors and mentors for my PhD study.
I would like to expand my appreciation to all other lecturers that encouraged me when I was lost in my study. I thank the administrative staffs, lab technologist, research assistant and research officer from INFORMM (both Penang and Kelantan) and also Dental School for assisting me in documentation and technical support. I thank my fellow lab mates OMT groupers: Veronica, Priscilla, Kok Lee, Darul, Tan Wei, Ron, Geethaa, Malini and Fini for the stimulating discussions and all the fun we had. Last but not least, a special thanks to my family that supported me whenever I need them the most.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SYMBOLS AND ABBREVIATIONS xiii
ABSTRAK xix
ABSTRACT xxi
CHAPTER 1: INTRODUCTION 1.1 Problem statement
1.2 Rationale of study 1.3 Objectives
CHAPTER 2: LITERATURE REVIEW 2.1 Cancer: The killer disease
2.2 Cancer progression 2.3 Types of cancer 2.4 Oral carcinoma
2.5 Properties of cancer cells
2.6 Stem cells at the top of hierarchy 2.7 Stem cells and cancer stem cells 2.8 History of concept development
1 3 4 5
6 6 7 9 10 11 16 19 20
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2.9 New era of cancer research-the cancer stem cell concept 2.10 Definition of cancer stem cells
2.11 Characteristics of cancer stem cells 2.12 CSC identification
2.12.1 Surface markers based approach 2.12.2 Function based approach
2.12.3 Transcription factors based approach 2.13 CSC isolation to enrichment
2.14 Cell adhesion
2.15 Epithelial-mesenchymal transition 2.16 Wnt signaling pathways
2.17 Issues in cell based assay and CAL 27 cell line
CHAPTER 3: MATERIALS AND METHODS 3.1 Flow chart overview
3.2 Preparation of reagents 3.2.1 Water
3.2.2 70% (v/v) of ethanol 3.2.3 75% (v/v) of ethanol
3.2.4 80% (v/v) and 95% (v/v) of ethanol 3.2.5 Transport medium
3.2.6 Dental pulp stem cells (DPSC) culture medium 3.2.7 Cell lines
3.2.8 Hs27 cell line culture medium 3.2.9 CAL 27 cell line culture medium
21 26 28 30 31 35 38 40 45 47 53 58
61 61 62 62 62 62 62 62 63 63 63 63
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3.2.10 Serum-free DMEM culture medium 3.2.11 Serum-free DMEM/F12 culture medium 3.2.12 Freezing medium
3.2.13 1X Phosphate buffered saline (PBS) 3.2.14 0.6% (w/v) and 1.0% (w/v) of agarose gel 3.2.15 MTT solution
3.2.16 Acidified isopropanol 3.2.17 Ice-cold methanol 3.2.18 Permeabilizing solution 3.2.19 Peroxidase blocking solution 3.2.20 Goat serum blocking solution 3.2.21 Protein extraction solution 3.2.22 5X protein sample buffer 3.2.23 30% of acrylamide 3.2.24 Resolving buffer 3.2.25 Stacking buffer
3.2.26 10% (w/v) of sodium dodecyl sulfate (SDS) 3.2.27 10% (w/v) of ammonium persulfate (APS) 3.2.28 10X running buffer
3.2.29 1X running buffer 3.2.30 Transfer buffer
3.2.31 0.5% (w/v) of Ponceau S staining solution 3.2.32 Tris buffered saline (TBS)
3.2.33 TBS-Tween® 20 buffer (TBST Washing buffer) 3.2.34 5% (w/v) of bovine serum albumin (BSA) blocking
solution
63 64 64 64 64 64 65 65 65 65 65 66 66 66 66 66 67 67 67 67 67 68 68 68 68
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3.2.35 5% (w/v) of skimmed milk blocking solution 3.3 Methods
3.3.1 Preparation of cells
3.3.1(a) Dissection of teeth
3.3.1(b) Isolation and culture of dental pulp stem cells 3.3.1(c) Culture of cell lines
3.3.1(d) Thawing of cells
3.3.1(e) Changing of culture media 3.3.1(f) Subculture of cells
3.3.1(g) Freezing of cells
3.3.1(h) Cell counting and plating
3.3.2 Identification and quantification of mesenchymal stem cell markers
3.3.2(a) Immunocytochemistry
3.3.2(b) Immunotyping by flow cytometry (CD105, CD34 and CD45)
3.3.3 Characterization of cancer stem cells properties 3.3.3(a) Cell proliferation assay
3.3.3(b) Soft agar assay 3.3.3(c) Matrigel assay 3.3.3(d) Cytotoxicity test
3.3.3(e) Flow cytometry (Pan-cytokeratin)
3.3.4 Determination of cell adhesive properties and regulated proteins
3.3.4(a) Serum-free DMEM/F12 culture 3.3.4(b) Flow cytometry (CD166)
68 69 69 69 69 70 71 71 72 72 73 74
74 77
78 78 79 80 81 82 83 83 83
vii 3.3.4(c) Immunofluorescence 3.3.5 Identification of signaling pathways
3.3.5(a) Cell lysis and sample preparation 3.3.5(b) SDS-PAGE
3.3.5(c) Western blot
3.3.5(d) RNA extraction and purification 3.3.5(e) cDNA synthesis
3.3.5(f) Real-time PCR
CHAPTER 4: RESULTS
4.1 Identification and quantification of mesenchymal stem cell markers 4.1.1 Morphological changes
4.1.2 Immunochemistry detection of CD105 expression
4.1.3 Immunophenotyping with mesenchymal stem cell markers 4.2 Characterization of CSC properties
4.2.1 Cell proliferation assay determination of cell doubling time 4.2.2 Soft agar assay evaluation of colony forming ability
4.2.3 MatrigelTM assay assessment of invasiveness and metastasis 4.2.4 MTT assay evaluation of treatment resistance
4.2.5 Flow cytometry determination of cytokeratin expression 4.3 Determination of cell adhesive properties and regulated proteins
4.3.1 Colony forming ability in serum-free culture 4.3.2 Immunophenotyping with CD166
4.3.3 Immunofluorescence with E-cadherin and vimentin 4.4 Identification of signaling pathways
84 85 85 86 87 91 92 93
97 97 97 99 101 105 105 108 112 115 117 119 119 121 123 126
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4.4.1 Expression level and phosphorylation of β-catenin 4.4.2 Non-simultaneous activation of Wnt/β-catenin targets 4.4.3 Downregulation of Wnt non-canonical upstream signals 4.4.4 Regulation of Wnt/planar cell polarity signaling
4.4.5 Non-regulated Wnt/Ca2+ signaling
4.4.6 Regulation of gene expression in quantitative real-time PCR
CHAPTER 5: DISCUSSION 5.1 Review
5.2 Identification and quantification of mesenchymal stem cell markers 5.2.1 Morphological changes
5.2.2 Expression of CD105
5.3 Characterization of cancer stem cell properties
5.3.1 Shortening of cell doubling time from cell proliferation assay 5.3.2 Colony forming ability in soft agar assay
5.3.3 Invasiveness and metastasis in Matrigel assay 5.3.4 Resistance to latex DCS fraction treatment
5.3.5 Reversible phenotype with cytokeratin expression
5.4 Determination of cell adhesive properties and regulated proteins 5.4.1 Development of colony forming ability in serum-free culture 5.4.2 Diminished population of CD166 expressing cells
5.4.3 E-cadherin and vimentin expression in immunofluorescence 5.5 Identification of signaling pathways
5.5.1 Canonical Wnt/β-catenin pathway 5.5.2 Noncanonical Wnt/PCP pathway
126 126 128 130 130 132
137 137 139 139 142 144 144 145 146 148 149 151 151 152 153 156 157 160
ix 5.5.3 Noncanonical Wnt/Ca2+ pathway 5.5.4 Regulation at transcription level
5.5.5 Integrated and convergent signaling pathways CHAPTER 6: CONCLUSION
CHAPTER 7: FUTURE STUDY REFERENCES
APPENDICES
163 165 168 172 174 177 195
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LIST OF TABLE
Page
Table 2.1 Cell surface phenotypes of CSCs 32
Table 3.1 Reagents from Immunoperoxidase Secondary Detection System Kit
75
Table 3.2 Preparation of 10% separating gel 87
Table 3.3 Preparation of 4% Stacking gel 88
Table 3.4 List of primary antibodies (Wnt canonical signaling) 90 Table 3.5 List of primary antibodies (Wnt/β-catenin activated
targets)
91
Table 3.6 List of primary antibodies (Wnt non-canonical signaling) 91
Table 3.7 Primary antibody (Endogenous control) 92
Table 3.8 List of secondary antibodies 92
Table 3.9 Template-primer mixture composition 93
Table 3.10 Master mix composition for cDNA synthesis 94
Table 3.11 List of primers for housekeeping genes 95
Table 3.12 List of primers for target genes 95
Table 3.13 Composition of master mix with primers (per reaction) 96
Table 3.14 Thermal profile for real-time PCR 97
Table 4.1 Mean of CAL 27 cell doubling time 109
Table 4.2 CFU count in soft agar assay 110
Table 4.3 Lethal concentration 50 (LD50) values for MTT assay 117
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LIST OF FIGURE
Page
Figure 2.1 Density-dependent inhibition 13
Figure 2.2 Hierarchy in tumours 23
Figure 2.3 Clonal evolution in stochastic and cancer stem cell model 24
Figure 2.4 Summarized characteristics of CSC 29
Figure 2.5 Functional assays to assess the renewal and differentiation potential of CSCs, committed progenitors and differentiated cells
42
Figure 2.6 Cell-cell and cell-matrix contacts in complexes that involve transmembrane proteins
49
Figure 2.7 The cycle of epithelial-cell plasticity 50
Figure 2.8 Carcinoma progression 53
Figure 2.9 Canonical Wnt signaling 56
Figure 2.10 Noncanonical Wnt signaling 57
Figure 2.11 Immunostaining of CAL 27 cells 61
Figure 4.1 Phase contrast images on different morphology of CAL 27 cells
98
Figure 4.2 Micrograph from immunocytochemistry for CD105 staining on CAL 27cells
101
Figure 4.3A Flow cytometry analysis for expression of CD105 on CAL 27cells
103
Figure 4.3B Flow cytometry analysis for expression of CD34 on CAL 27cells
104
Figure 4.3C Flow cytometry analysis for expression of CD45 on CAL 27cells
105
Figure 4.3D Forward scatter and side scatter of CAL 27 cells from flow cytometry analysis
107
Figure 4.4 Cell growth profile of CAL 27 cells 108
Figure 4.5A Images of CAL 27 cells in soft agar assay 111
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Figure 4.5B End-point quantification of CFU from soft agar assay 111 Figure 4.6A Images captured using phase contrast microscope in
Matrigel assay
113
Figure 4.6B Comparison of number of cells exhibited invasive and metastatic properties in Matrigel assay
114
Figure 4.7 Viability of CAL 27 cells with latex DCS fraction treatment
116
Figure 4.8 Flow cytometry analysis for Pan-cytokeratin expression in CAL 27 cells
118
Figure 4.9 Images of CAL 27-Y and CAL 27-O cells in serum-free culture
120
Figure 4.10 Flow cytometry analysis for CD166 expression in CAL 27 cells
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Figure 4.11A Immunofluorescence images of CAL 27 cells for E- cadherin expression
124
Figure 4.11B Immunofluorescence images of CAL 27 cells for vimentin expression
125
Figure 4.12 Western blot analysis of β-catenin and phospho-β-catenin expression
127
Figure 4.13 Western blot analysis of Wnt canonical targets expression 129 Figure 4.14 Western blot analysis of protein expression for upstream
of Wnt non-canonical signaling
129
Figure 4.15 Western blot analysis of protein expression for Wnt/PCP pathway
131
Figure 4.16 Western blot analysis of protein expression for Wnt/Ca2+
pathway
133
Figure 4.17 Amplification plot of real-time PCR with HPRT1 primers 134 Figure 4.18 Relative expression of the mRNA in real time PCR 136 Figure 5.1 Comparison of morphology of CAL 27-Y and CAL 27-O
cells with CAL 27 cells with EMT-like phenotype
141
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LIST OF SYMBOL AND ABBREVIATION
Abbreviation Description
AACR American Association for Cancer Research
AKT Protein kinase B
ALDH Aldehyde dehydrogenase
AMP Adenosine monophosphate
αMEM Alpha minimal essential medium
ABC ATP-binding cassette
ALCAM Activated leucocyte adhesion molecule AML Acute myeloid leukemia
APC Adenomatous polyposis coli
APS Ammonium persulfate
ATP Adenosine triphosphate
BCRP Breast cancer resistance protein BER base excision repair
BSA Bovine serum albumin
CAL 27 Human tongue squamous cell carcinoma cell (ATCC® CRL-2095TM) CAL 27-Y cells CAL 27 cells of 5th passage and below
CAL 27-O cells CAL 27 cells of 6th -20th passage CAM Cell adhesion molecules
CaMKII Calcium/calmodulin-dependent kinase II CCND1 Protein coding gene for cyclin D1
cDNA Complementary DNA
CD34 Hematopoietic progenitor cell antigen CD45 Lymphocyte common antigen
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CD105 Endoglin
CD166 Activated leucocyte cell adhesion molecule (ALCAM) CDK Cyclin-dependent kinase
CFU Colony forming unit
CK-1 Casein kinase-1
CO2 Carbon dioxide
CSC Cancer stem cell
DAB 3,3’-Diaminobenzidine
DCS Dialysed C-serum fraction DEPC Diethylpyrocarbonate
DMSO Dimethyl sulfoxide
DMEM Dulbecco’s Modified Eagle Medium
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide DPSC Dental pulp stem cell
Dvl Dishevelled
ECL substrate Enhanced chemiluminescent substrate ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor
EpCAM Epithelial cell adhesion molecule EMT Epithelial-mesenchymal transition ES cell Embryonic stem cell
FACS Fluorescence activated cell sorting
FBS Fetal bovine serum
xv FGF-2 Fibroblast growth factor 2
FGFR2 fibroblast-growth-factor receptor-2 FITC Fluorescein isothiocyanate
FSP1 Fibroblast-specific protein-1
Fzd Frizzled
GDP Guanosine diphosphate Gli1 GLI family zinc finger 1 GPCR G-protein coupled receptor GSK-3 Glycogen synthase kinase-3 GTP Guanosine triphosphate
HA Hyaluronan
HAS Heat-stable antigen
HBSS Hank’s Balance Salt Solution
HCl Hydrochloric acid
HGF Hepatocyte growth factor HIF-1 Hypoxia-inducible factor-1
HLA Human leucocyte antigen
HNSCC Head and neck squamous cell carcinoma
HPV Human papillomavirus
HSC Hematopoietic stem cell
Hs27 Human foreskin fibroblast cell (ATCC® CRL-1634TM) H2O2 Hydrogen peroxide
IARC International Agency for Research on Cancer
IgG Immunoglobulin G
IL-6 Interleukin-6
xvi iPS cell Induced pluripotent stem cell
ISCT International Society for Cellular Therapy
JAK-STAT Janus kinase–signal transducer and activator of transcription JNK c-Jun N-terminal kinases
LC50 Lethal concentration 50 LEF Lymphoid enhancer factor
LRP5/6 Low density lipoprotein related proteins 5 or 6 MACS magnetic activated cell sorting
MAP Mitogen activated protein MDR Multidrug resistance
MET Mesenchymal-epithelial transition
MF Microfilaments
MMP Matrix metalloproteinases
M-PER Mammalian protein extraction reagent MRP Multidrug resistance-associated protein
MSC Mesenchymal stem cell
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) mTOR Mammalian target of rapamycin
MYC Protein coding gene for c-Myc transcription factor NAD(P) Nicotinamide adenine dinucleotide phosphate NER Nucleotide excision repair
NFAT Nuclear factor of activated T cells
NFκB Nuclear factor κB
NLK Nemo-like kinase
NOD/SCID Non-obese diabetic /severe combined immunodeficiency
xvii OSCC Oral squamous cell carcinoma PBS Phosphate buffer saline PCP Planar cell polarity PCR Polymerase chain reaction
PE Phycoerythrin
PI3 kinase Phosphatidylinositol-3-kinase
PKA Protein kinase A
PKC Protein kinase C
POU Pit-Oct-Unc transcription factor pRb Retinoblastoma protein
PVDF Polyvinyl difluoride
RNA Ribonucleic acid
ROCK Rho-kinase
ROR Receptor tyrosine kinase-like orphan receptor rpm Revolutions per minute
RTK Receptor tyrosine kinase
RYK Atypical receptor tyrosine kinase; Receptor-like tyrosine kinase
SC Stem cell
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of the mean
Ser Serine
siRNA Small interfering RNA SP cell Side population cell TBS Tris buffered saline
TBST Tris buffered saline -Tween® 20 buffer
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TCF T-cell factor
TEMED Tetramethylethylenediamine TGF-β Transforming growth factor beta
Thr Threonine
TIC Tumour initiating cell
TLE Transducing-like enhancer proteins
Tm Melting temperature
TPC Tumour propagating cell
TP53 Protein coding gene for tumour protein p53 VEGF Vascular endothelial growth factor
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PENCIRIAN SEL TITIS SKUAMOUS KARSINOMA ORAL CAL 27 SEBAGAI MODEL UNTUK KAJIAN SEL STEM KANSER
ABSTRAK
Peratusan sel stem kanser (CSC) agak rendah di kalangan populasi sel barah.
Oleh itu, langkah-langkah pengasingan dan pengayaan CSC telah menjadi rutin bagi kerja-kerja penyelidikan yang melibatkan CSC. CAL 27 (ATCC® CRL-2095 TM) yang berasal daripada tisu lidah manusia, merupakan salah satu titisan sel epitelium skuamus karsinoma yang sering digunakan. Tujuan kajian ini dijalankan adalah untuk mewujudkan satu model titisan sel yang bersesuaian untuk mengkaji perkembangan CSC secara cekap, dengan mengurangkan masa bagi proses pengasingan dan juga langkah pengayaan sel. Kajian dijalankan untuk menentukan transformasi morfologi dan juga fisiologi sel titis CAL 27, terutamanya berkenaan dengan pengekspresian penanda sel stem mesenkima, tempoh masa penggandaan sel, rintangan terhadap rawatan, kebolehbalikan fenotaip, ciri invasif dan keupayaan metastatik. Selain itu, penglibatan lekatan sel dan peralihan epitelium-mesenkima (EMT), bersama dengan isyarat laluan Wnt juga diselidik. Hasilnya, pemanjangan sel CAL 27-O (sel CAL 27 dari generasi ke-6 dan seterusnya) dari sel CAL 27-Y (sel CAL 27 sebelum generasi ke-6) dikaitkan dengan peningkatan keupayaan invasif dan metastatik, rintangan rawatan yang lebih tinggi, pembalikan fenotaip yang mencirikan sel-sel epitelium, selain peningkatan kadar pembaharuan sel.
Tambahan pula, sel yang mengekspreskan penanda positif sel stem mesenkimia (CD105) telah meningkat lebih daripada 70%. Walaubagaimanapun, pengekspresian sel dengan protein lekatan sel (CD166) telah menyusut sekurang-
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kurangnya 90%, diiringi oleh peningkatan ekspresi vimentin dan penyusutan ekspresi E-cadherin untuk EMT. Selain itu, transformasi fenotaip didapati berkaitan dengan penyusutan aturan bagi laluan kekutuban sel satah (PCP) Wnt bukan kanonikal dalam sel CAL 27-O. Oleh itu, kajian ini boleh digunakan dalam kajian berkaitan CSC, untuk memahami perkembangan CSC dan juga untuk proses saringan dadah yang berpotensi untuk mensasarkan CSC.
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CHARACTERIZATION OF CAL 27 ORAL SQUAMOUS CARCINOMA CELL LINE AS A MODEL FOR CANCER STEM CELL STUDY
ABSTRACT
The percentage of cancer stem cells (CSCs) is relatively low in tumour bulk population. Therefore, isolation and enrichment steps for CSCs have become routine for research works involving CSC. CAL 27 (ATCC® CRL-2095 TM) is one of the commonly used squamous epithelial carcinoma cell lines derived from human tongue tissue. The present study was conducted with the objective to establish a suitable cell line model for studying CSC development efficiently, by reducing the time consuming isolation and enrichment steps. Experiments were carried out to determine the morphological as well as physiological transformations of CAL 27 cell line, particularly on the expression of mesenchymal stem cell markers, cell doubling time, treatment resistance, phenotype reversibility, invasiveness and metastatic ability. In addition to that, involvement of cell adhesion and epithelial-mesenchymal transition (EMT), together with Wnt signaling pathway were investigated. As for the results, elongation of CAL 27-O cells (CAL 27 cells from 6th passage onwards) from CAL 27-Y cells (CAL 27 cells below 6th passage) were found to be associated with enhanced invasive and metastatic capability, greater resistance to treatment, reversal of phenotype characterizing epithelial cells, besides enhanced proliferation. Moreover, cells expressing mesenchymal stem cell positive marker (CD105) was increased for more than 70% with the transition.
Contrasting to this, cells with cell-cell adhesion protein (CD166) was reduced by more than 90%, accompanied by overexpression of vimentin and downregulated expression of E-cadherin for EMT. Besides, the phenotypic transformations were
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associated with downregulation of Wnt noncanonical planar cell polarity (PCP) pathway in CAL 27-O cells. Hence, the study could be extended in CSC related study, to understand the development of CSCs and also for screening of potential drugs targeting CSCs.
1 CHAPTER 1 INTRODUCTION
Abnormal growth of cells generates tumours. Benign tumours are not harmful to body tissues. Hence, only malignant tumours are cancers. Malignant cancers refer to the abnormal growth of cells that damage or destroy normal body tissues.
Classification of cancer types is based on the types of cell that is initially affected.
The abnormal growth of cells harms the body as the uncontrollable cell division interferes with the digestive, nervous and circulatory systems, except in leukemia where cancer prohibits normal blood function.
Oral cancer is the most common site of malignancy in the head and neck region that affect lips, tongue, gums, cheeks, sinuses and pharynx. Most of the patients with oral cancer are diagnosed at advanced stage that causes therapeutic complications with high mortality rate. The factor accountable for the late diagnosis is that the early and curable lesions are usually manifested with some common symptoms such as swelling lumps, velvety patches, and persistent soreness.
The overall survival rate differs significantly between the carcinoma stages, decreases from 75-90% for Stage I to 10-22% for stage IV. High frequency of loco- regional recurrences is often associated with oral cancer, which is implicated for most of the post-surgery and radiotherapy treatment failures. Relapsed oral cancer undoubtedly denotes a tough challenge for clinical practitioners due to the aggressiveness and invasive behaviours (Da Silva et al., 2012).
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The time course of relapse manifestation and metastatic progression are unpredictable. Currently, chemotherapy remains as the only option of treatment for relapsed cancer whenever surgery or re-irradiation is not possible. However, development of drug resistance in cancer cells has limited the efficacy of the chemotherapy treatment (Da Silva et al., 2012). The high probability of oral cancer recurrence is frequently linked to ipsilateral or bilateral lymph node metastasis as the extensive lymphatic submucosal network enables the spreading of cancer cells to other site in head and neck region (Fan et al., 2011).
Cancer cell populations are heterogeneous in which the self-proliferative population is known as cancer stem cell (CSC). According to American Association for Cancer Research (AACR), CSCs are defined as a small subset of cancer cells that make up a pool of self-sustaining cells with the special ability to self-renew and generate heterogeneous lineages of cancer cells (Clarke et al., 2006). CSC population was first isolated by Bonnet and Dijk (1997) and Al Hajj (2003) in acute myeloid leukemia and solid tumours, respectively. The research obtained attention from many researchers ever since then.
Chemotherapy drugs that target the tumour bulk are only able to eliminate the chemotherapy sensitive non-CSC, while enriching the chemotherapy-resistant CSC and hence worsen the case by promoting recurrence (Frame and Maitland, 2011).
Characteristics such as high proliferative ability, invasiveness, migration capacity, treatment resistance and phenotype reversibility are known to associate with CSCs (Reya et al., 2001; Wicha et al., 2006; Sayed et al., 2011; Cabrera et al., 2015; Hong et al., 2015). These research findings indicated that CSCs are responsible for cancer
3
recurrence, treatment resistance and metastatic dissemination. The advancement of aggressiveness observed from relapsed oral cancer is parallel with the development of CSC.
Gioanni and colleagues established two new cell lines from squamous cell carcinomas of the tongue in 1982. One of the cell lines that was established was CAL 27 derived from a 56-year-old Caucasian male. The tumorigenicity of CAL 27 cells was confirmed in nude mice during cell line establishment. The cell line was established without any genetic manipulation (Gioanni et al., 1988).
Ever since then, CAL 27 cell line was widely used to build oral squamous cell carcinoma (OSCC) model for in vitro and in vivo studies, and thus considered as a representative cell line for OSCC study (Jiang et al., 2009). Based on CSC hypothesis, CSCs in CAL 27 cell line are responsible for the enhanced growth regulation, treatment resistance, phenotype plasticity, as well as invasiveness and metastatic ability of the cell line. Therefore, CAL 27 cell line was selected for this study.
1.1 Problem Statement
New findings involving CSC have been greatly discussed which brings about new ideas and directions for research. However, one of the challenges in CSC research is that the percentage of CSC population in tumour bulk is relatively low compared to non-CSC (Beck and Blanpain, 2013). Therefore, the process of identifying, isolating and enriching CSCs has always been a time-consuming process for the study. Currently, there is no such cellular model available to be used. In view
4
of this, a cellular model mimicking CSC development using currently available cell line could be a practical approach for screening study involving CSC.
The first part of this study focused on the morphological transformation of the cells, along with detecting and identifying CSC population from the bulk population of cells, using appropriate markers. Comparison on mesenchymal stem cell marker expression was performed against stem cells from the same region, dental pulp stem cells (DPSC). The second part of the study was directed towards characterization of the cancer-origin cell line according to CSC properties to recognize the presence and transformation of CSC in the cell line.
In the third part of the study, concurrent investigation on epithelial- mesenchymal transition (EMT) and cell adhesion proteins were done to provide an insight on the cellular regulation during CSC development. With these, investigation to reveal the missing puzzle on the signaling pathway(s) underlying CSC development, particularly related to the transformation of cell line, was performed in the last part of the study.
1.2 Rationale of study
Based on the understanding on CSC concept, identifying CSC from cancer- origin cell line is feasible. The CSC cellular model would be a more time-effective alternative for CSC research by reducing the time requirement for isolating and enriching CSCs for fundamental studies, as well as drugs screening. The model could be used to predict the development of CSC under defined circumstances created by researchers to understand carcinogenesis progression. Furthermore, the cellular
5
model can also be used to evaluate the efficiency of cancer treatment targeting CSC specifically.
1.3 Objectives
The general objective of this project is to establish a better-studied cellular model for CSC study by using CAL 27 cell line. In order to achieve the general objective, the following detailed objectives are defined:
1. To identify the morphological transformation of CAL 27 cells.
2. To identify and quantify the mesenchymal stem cell marker CD105 expressing population among CAL 27 cells.
3. To characterize cancer stem cell properties exhibited by CAL 27 cells.
4. To investigate on the protein regulation for cell adhesion and epithelial- mesenchymal transition (EMT) in CAL 27 cells.
5. To understand and propose a potential signaling pathway(s) involved in early cancer development.
6 CHAPTER 2 LITERATURE REVIEW 2.1 Cancer: The Killer Disease
Cancer has been so frequent that every one of us would have at least a relative or friend that was involved in fighting cancer. According to the last Health Facts 2013 from Ministry of Health (MoH) Malaysia, cancer is one of the top ten reasons of hospitalisation and one of the top five causes of death. The most frequently encountered cancer types among Malaysians are breast, colorectal and lung cancer.
Based on reports from the International Agency for Research on Cancer (IARC) Globocan of the World Health Organisation reported by Ferlay et al. (2013), one out of 19 Malaysians develops breast cancer, one in 33 develops colorectal cancer and one in 40 develops lung cancer. In Malaysia, the number of cancer incidence increased from 32,000 new cases in 2008 to about 37,000 in 2012. There were only 21,100 cancer mortality in 2008, but the figure has increased to 21,700 deaths in 2012.
In 2012, 14 million people were diagnosed with cancer worldwide, according to the World Cancer Report 2014, a new global cancer report compiled by IARC.
The striking fact reported in the same report is that cancer has overtaken heart disease as the number one killer in 2014. To reduce the number of cases, there is an accelerated demand for better understanding on cancer development for the most effective and efficient ways to control cancer.
7 2.2 Cancer Progression
Cancer can begin from any cell in the body. These cells start out as normal cells, but then changes with exposure to mutations and become neoplastic. The development of cancer is a multistep process in which cells gradually become malignant through a progressive series of alterations, indicated by development of cancer late in life. Mutations affect how cells grow, work, reproduce and die. They may cause the cell to continue growing and dividing uncontrollably instead of dying when it is the appropriate timing. Along the expansion of cancer cell population, random mutations occur in the individual cells, leading further to divergence in molecular identity (Korbel and Campbell, 2013).
The process of cell changes in which a cell loses its capacity to manage its rate of division, and thus becomes a cancer cell, is known as cell transformation.
Although there are many different types of cancer, they all start because of uncontrolled and abnormal growth of cells. The cancerous cell generally retains the structural and functional characteristics of the normal cell type from which it is derived. Almost 80% of all cancers were epithelial cancers from breast, colon, prostate and ovary tissues (Diaz-Cano, 2012).
The typical characteristics of cancer are often referred to the poorly differentiated phenotype, and the genetic and functional heterogeneity. Within tumours, functional heterogeneity was observed as cells exhibited variable proliferative and differentiation capacity. While environmental and other non-genetic factors are involved in tumorigenesis, it is commonly accepted that cancer occurs because of mutations in cancer-susceptible genes namely oncogenes and tumour
8
suppressor genes. Both oncogenes and tumour suppressor genes are gate-keepers in cancer initiation (Pizon et al., 2013).
Oncogenes are damaged version of normal genes, the proto-oncogene. These proto-oncogenes control various cell functions, particularly in cell growth and reproduction. When proto-oncogenes are mutated or activated to turn into oncogenes, cells grow out of control and the transformation promotes cancer cell growth.
Oncogenes have been discovered to take part in different stages of human cancers- tumour initiation, progression, angiogenesis and metastasis (Shortt and Johnstone, 2012).
On the other hand, tumour suppressor genes are the genes work to guard against cancer. These genes act as barriers that help to stop cell growth and regulate cell death such as apoptosis. However, if the tumour suppressor genes are damaged or missing, their implementations on cell proliferation and cell death are unchecked.
Thus, the alteration leads to cancer progression. For example, TP53; the tumour suppressor gene that prompts cell death, is often found to be damaged or missing in several types of cancer (Kitagishi et al., 2012).
Progressive changes of human cancer from pre-malignant lesions to fully malignant invasive tumours have been well recognized from the recorded cases.
Accumulation of genetic alteration converts pre-malignant cells into more aggressive malignant cells, producing primary tumour. At the early stage of primary tumour expansion, the cells are not invasive or metastatic. Due to the genetic instability in the cells, further genetic alterations occur. Thus, new clones with invasiveness and metastatic capacity are produced, which are the fully malignant cells. However, only
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a fraction of the cells in primary tumours acquire the invasive and metastatic ability, creating heterogeneity in the cell populations (Diaz-Cano, 2012).
2.3 Types of Cancer
Cancer can begin from abnormal proliferation of any of the cells in the body.
Therefore, there are more than a hundred distinctive cancer types, which can vary considerably in their aggressiveness and response to treatment.
The most noticeable issue in cancer pathology is the difference between benign and malignant tumours. A benign tumour remains restricted to its original location, but not invading the surrounding normal tissue or spreading to other body sites. In contrast, a malignant tumour is capable of both invading surrounding normal tissue and spreading throughout the body via the circulatory or lymphatic systems, a condition which is known as metastasis. Cancers, which are commonly referring to those malignant tumours, are dangerous due to their ability to invade and metastasize.
Benign tumours can usually be removed by surgery, but the spread of malignant tumours to distant body sites often renders effectiveness of localized treatment to these tumours (Valastyan and Weinberg, 2011).
Classifications of both benign and malignant tumours are based on the type of cell from which they arise. The three main categories for most of the cancer types are carcinomas, sarcomas, and leukaemias or lymphomas. Approximately 90% of human cancers are carcinomas, developing from epithelial cells. Sarcomas are solid tumours of connective tissues such as muscle, bone and cartilage. Leukemias and lymphomas arise from the blood-forming cells and from cells of the immune system respectively,
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occupy approximately 8% of human malignancies. Tumours are further classified according to their tissue of origin, for example lung or breast carcinomas (American Cancer Society, 2011)
2.4 Oral Carcinoma
95% of head and neck cancer cases are diagnosed as head and neck squamous cell carcinoma (HNSCC) that occur in over half a million people globally. HNSCC is the sixth most common malignancy in the world (Sayed et al., 2011). The prognosis for patients with recurrent or metastatic HNSCC is generally poor with low 5-year survival rates despite treatment advances over the past few decades, with surgery and radiation being the core treatment method (Suma et al., 2015).
Approximately half of those diagnosed cases of HNSCC occur specifically in the oral cavity, reported as oral squamous cell carcinomas, which might involve lip, tongue, gum, cheek, floor of mouth, sinuses and pharynx. The symptoms detected such as velvety patches, swelling, lumps or bumps in the oral region with persistent soreness can be easily mistaken as other infectious diseases (Tamashiro et al., 2013).
The factors leading to oral cancer include not only alcohol and tobacco that were known to be the traditional ones. However, there has been increasing number of incidence reported in young patients which are caused by betel quid chewing (Guha et al., 2014) and human papillomavirus (HPV). Oral cancer cases developed from HPV infection is a factor discovered recently, especially in the United States (Martin-Hernan et al., 2013).
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Despite recent advances in cancer detection and management, most of the patients present with late-stage disease because the clinical signs and symptoms of head and neck tumours are often nonspecific and tend to be mistaken for other common ailments. Consequently, it is essential to search for new biomarkers and effective therapy options to optimize cancer treatment (Martinez-Useros and Garcia- Foncillas, 2015).
2.5 Properties of Cancer Cells
Losing control over growth, cancerous cells are formed as a result of accumulated aberrations that interfere with many of the regulatory mechanisms. With the interference from these unregulated mechanisms, the reflected cell behaviour display abnormalities in cell proliferation, survival and differentiation that distinguish cancer cells from the normal counterparts. These properties serve as a characterizing description for cancer cells (Hanahan and Weinberg, 2011).
Cancer cells have reduced requirements for extracellular growth factors as compared to normal cells. Proliferation of most of the cells in our body is controlled, mostly by polypeptide growth factors. For certain cell types, the availability of serum growth factors is the key determinant for their proliferative capacity in culture. This property is particularly important in fibroblasts. The requirement for growth factors of cancerous cells is closely related to density-dependent inhibition, since the cell density is the key determinant for cells to enter quiescent stage (Yang and Xu, 2011).
Cancer cells are able to produce growth factors that stimulate their own proliferation in certain cases. Production of growth factors by a cancer cell can lead
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to continuous autocrine growth stimulation that stimulates cell division in the niches.
In this condition, cancer cells are less dependent on growth factors from other sources. Also, the reduced growth factor dependence of cancer cells is a result from aberrations in intracellular signaling systems, such as upregulation or hyperactivity of growth factor receptors or other proteins (Ras proteins or protein kinases) (Witsch et al., 2010).
Conversely, cancer cells secrete growth factors that promote the formation of new blood vessels in angiogenesis. Angiogenesis is needed to support the tumour outgrowth that grows beyond the size of normal cells. With increased requirement to oxygen and nutrients supply, new blood vessels are formed to support the need. In metastasis, the actively growing new capillaries formed are easily penetrated by the tumour cells, providing a ready opportunity for cancer cells to enter the circulatory system and begin the metastatic process (Witsch et al., 2010).
As illustrated in Figure 2.1, normal cells encounter density-dependant inhibition during cell growth. During proliferation, normal cells divide and multiply until they reach certain density. The multiplication stops proliferating and arrested in G0 stage of cell cycle to remain quiescent. The key distinction between cancer cells and normal cells in culture is that the cancer cells are unresponsive to density- dependent growth inhibition. Cancer cells proceed to continue growing towards high cell density in culture, mimicking their uncontrolled proliferation in vivo and in real case scenario (Leontieva et al., 2014; Reece et al., 2013).
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Figure 2.1: Density-dependant inhibition. Normal cells proliferate in culture until they reach a finite cell density coming into contact with other cells, at which point they become quiescent. Tumour cells, however, continue to proliferate independent of cell density. The concepts of ‘contact inhibition of cell proliferation’ and ‘contact inhibition of cell movement’ are often taken as two sides of the same coins.
(Takai et al., 2008)
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Cancer cells are not restricted by contact inhibition as opposed to normal cells. Normal fibroblasts migrate across the surface of a culture dish until they come into contact with a neighbouring cell. Further cell migration is inhibited that causes the normal fibroblast cells to adhere to each other, forming an organized arrangement of cells on the culture dish surface. Instead, cancer cells continue moving after contact with their neighbours, migrating over neighbouring cells. The growth is in a disordered, multi-layered pattern. Cancer cells are characteristically insensitive to such contact inhibition of growth and movement (Kim and Asthagiri, 2011; Batson et al., 2013).
Cancer cells are different from normal cells in their cell-cell and cell- matrix interactions. Most cancer cells are less adhesive than normal cells, due to reduced expression of cell surface adhesion molecules. For example, loss of E- cadherin in development of carcinomas (epithelial cancers), the key adhesion molecule has been observed. As a consequence of reduced expression of cell adhesion molecules, cancer cells are able to invade and metastasize to other tissues with less restricted interactions with other cells and tissues. The reduced adhesiveness of cancer cells also leads to morphological and cytoskeletal alterations (Jung et al., 2012).
In the microenvironment, cancer cells exhibit additional properties that empower them in invasion and metastasis. Malignant cells secrete proteases that digest extracellular matrix components, allowing the cancer cells to invade adjacent normal tissues. For instance, secretion of collagenase aids in digestion and
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penetration of malignant cells through basal laminae to invade underlying connective tissue in carcinoma development (Lu et al., 2011; Man et al., 2013).
Most cancer cells display defective differentiation, which is closely related to abnormal proliferation. Fully differentiated cells cease cell division or seldom divide.
Instead of normal differentiation, cancer cells are usually blocked at the stage of specialization, corresponding to their continued active proliferation. One of the examples to show the relationship between defective differentiation and malignancy can be observed in leukaemia. Leukemic cells fail to undergo terminal differentiation in generation of erythrocytes, lymphocytes, granulocytes, or macrophages. They remained to be at early stages of development at which they retain their ability for proliferation (Diaz-Cano, 2012).
In addition to that, many cancer cells fail to undergo apoptosis, the programmed cell death. As a result, the life spans of cancer cells are longer in comparison to normal cells. Failure of cancer cells to undergo apoptosis enables cancer cells to survive in condition deprived of growth factors from environmental signals, contrasting the survival of normal cells which rely on signal from growth factors or extracellular matrix that prevent apoptosis. Instead of undergoing apoptosis with DNA damage, cancer cells fail to correct the situation. Being non-responsive to apoptosis, cancer cells become resistant to radiation and chemotherapeutic drugs in treatment (Su et al., 2015).
16 2.6 Stem cells at the top of hierarchy
Stem cells have been a hot topic in biological science. The stem cells are unique due to their unlimited regenerative potential to produce more stem cells and tissue specific differentiation capacity to differentiate into diverse specialized cell types. With more research activities being involved, stem cell technology would be the trend for treatment for diseases (Duran and George, 2011).
Categorisation of stem cells can be based on the potency of the cells.
Totipotent stem cells differentiate into embryonic and extraembryonic cell types to give rise to a fully functional organism, as well as to every cell type of the body.
Pluripotent stem cells are derived from totipotent stem cells, can give rise to almost any cell types from any of the three germ layers or any cell types in adult organism.
Embryonic stem cells are in this category (Horie et al., 2011; Hima and Srilatha, 2011).
Multipotent stem cells are more differentiated cells, which mean that their possible lineages are less variable or more determined. Thus, multipotent stem cells are only capable to give rise to a limited number of cells from closely related family.
Adult hemapoietic stem cells and MSCs are in this category. For example, MSCs have been shown to produce bone, muscle, cartilage, fat, and other connective tissues.
These tissue specific stem cells are indispensable for maintaining tissue homeostasis and repair (Hima and Srilatha, 2011).
There are many prospective sources where stem cells can be obtained.
Embryonic stem (ES) cells are derivatives from the inner cell mass of a blastocyst,
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whereas fetal stem cells can be found in fetal organs. Cell therapy using stem cells introduce stem cells to targeted site of tissues to treat diseases with or without gene therapy. Pluripotent ES cells have been isolated from inner cell mass of early embryos for the almost limitless potency in biological research that is able to generate mostly all cell types. However, the promising research raised ethical issues, leading the development of induced pluripotent stem (iPS) cells. One key drawback of stem cell therapy using ES and iPS cells is on their potential for teratoma formation, compromising their application in regenerative medicine (Wei et al., 2013).
Cells have to be replaced even after complete maturation of an organism.
Adult stem cells have been isolated from various tissues, including central nervous system, bone marrow, retina, skeletal muscle and dental pulp (Hima and Srilatha, 2011; Abdullah et al., 2013). Stem cells play their role in internal repair system in most of the tissues, with their capability to divide and replenish damaged cells. The new cells after division can be channelled into different differentiation path to become more specialized cell types such as muscle cells, red blood cells and white blood cells. Hematopoietic stem cells (HSCs) have been widely used for allogeneic cell therapy, meaning that the donor is different from the recipient of the cells.
(Hima and Srilatha, 2011).
With the new hope generated from application of mesenchymal stem cells (MSCs) in biomedical field, MSCs catch the attention of scientists for more investigations. However, investigations using different isolation and expansion methods created ambiguities and inconsistency as it is difficult to compare and
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contrast study outcomes without a standard method to characterize MSC. To address the issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed to set the minimal criteria to define human MSC (Dominici et al., 2006).
The first criterion being set by ISCT is that the cells must exhibit adherence to plastic in standard culture conditions. Secondly, MSC must express CD105, CD73 and CD90 (≥ 95% of cell population), but not CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR (≤ 2% of cell population). Thirdly, these cells must be able to differentiate into osteoblasts, adipocytes and chondroblasts as demonstrated by staining of in vitro cell culture. This minimal set of criteria standardise a uniform characterization of MSC, so that exchange of data among researchers in the field becomes relevant. These criteria, particularly plastic adherence and marker expression, were applied in this study for characterizing stem cells (Dominici et al., 2006).
Stem cells isolated from adult dental pulp are termed as dental pulp stem cells (DPSCs). DPSCs can be collected from dental pulp by means of a non-invasive method that can be performed in adults using simple surgical wisdom teeth extraction, a routine surgical practice with less ethical issues. DPSCs have been studied for the ability to develop into active neurons, osteoblasts precursors, as well as chondrogenic and myogenic differentiation potential (Abdullah et al., 2013). In this study, DPSCs were used as a comparison control for stem cell characteristics as there is a close proximity between DPSCs and oral carcinoma concerning tissue of origin, and also concerning debates on ethical issues in stem cells research.
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On the other hand, stem cells have been used in replenishment of blood and immune system suffering from damage during cancer treatment by chemotherapy or radiotherapy. Other than immune-reconstitution, stem cells particularly MSCs have been reported to be used in cell-based bone reconstitution following chemotherapy and surgery in malignancies due to their regeneration and differentiation capacity (Suma et al., 2015).
2.7 Stem cells and CSCs
Scientists and oncologists have been working on a long-standing goal of understanding the development of cancer, so that a framework can be established for successful treatment. One of the frameworks that attracted much attention is to understand cancer as a perturbed mechanism arising from cells in normal tissues that retain the tissue specific developmental features (Diaz-Cano, 2012).
Furthermore, analysis on morphology and proliferation of stem cells leads to understanding of heterogeneity of stem cell population that not every cell in the population actively proliferates at the same time within a given tissue. With these observations in both normal and aberrant tissues, it has been hypothesized that the differentiated cells originate from the undifferentiated cells with regenerative ability (Beck and Blanpain, 2013).
Former research was led to focus on understanding the genetic changes that direct a cell towards aberration in development without considering which cells are affected by mutations. The most recent model for carcinogenesis is the “stem cell hypothesis”, in which CSCs are the sites of mutations that initiate malignancy. With
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this model, cancer has been presented in a hierarchy structure where CSCs are at the top of pyramid to perform self-renewal to support on-going tumour growth and tissue-specific differentiation (Mannelli and Gallo, 2012; Suma et al., 2015).
In fact, there are diverging opinions on the origin of CSCs. Some researchers supported the view that cancers are derived from naïve stem cells that retain self- renewal ability but accumulated mutations that lead to epigenetic and genetic changes for cancer development. On the other side, the opponents stated the possibility that CSCs can be derived from differentiated cells that undergo dedifferentiation and reactivate the self-propagating activity to cause malignancy (Clarke et al., 2006; Van de Stolpe, 2013).
2.8 History of concept development
The concept started when pathologist Virchow (1855) suggested that tumour could be derived from embryonic cells. Following this, Bruce and colleagues (1963) showed that only 1-4% of transplanted murine lymphoma cells can form colonies in vitro or can initiate a carcinoma in mouse spleen. The concept of cancer cells acquired basic features of normal stem-like cells, such as proliferation potential and differentiation capacity was proposed by Pierce and Speers (1988).
Previous research by Bonnet and Dick (1997), it was revealed that a single leukemic cell that was selected and isolated based on cluster of differentiation marker (CD markers) for CSCs, was able to transmit the disease in mouse after transplantation. The first solid tumour from which CSCs were identified and isolated was from breast cancers by Al-Hajj and colleagues (2003), which were characterized
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as a CD44high/CD24low subpopulation, and positive for epithelial cell surface antigen.
The 100 cells being selected using the specific phenotype were able to proliferate and form new tumours when transplanted in mice.
With these reports, more research works were initiated and reports on solid tumours were consecutively reported, as from Singh et al. (2003) on brain tumour, Collins et al. (2005) on prostate cancer, O’Brien et al. (2007) on colon cancer, Li et al. (2007) on pancreatic cancer, Quintana et al. (2008) on melanoma, Sell and Leffer (2008) on liver cancer. Nevertheless, there are still doubts and criticism over the existence of CSCs and their functional role in cancer progression (Maenhaut et al., 2010). On-going research on CSCs are still being carried out to answer the questions that seems to get more complicated with more data accumulating. The postulations and deductions on CSCs are reviewed from time to time to be in pace with the latest research reports.
2.9 New era of cancer research-the cancer stem cell concept
Most cancers remain not curable with the currently available therapeutic approaches, based on the knowledge that we have learnt about cancer biology in the last decades. Current treatments are based on the assumption that cancer is a proliferation-based disease. According to this assumption, the main treatments are anti-proliferative and they are non-specific and with serious side-effects. Although cytotoxic drugs and radiation reduce tumour burden, relapse of the disease occurs in the majority of the cases (Pisco and Huang, 2015).
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At least two models have been proposed to explain heterogeneity in cancer cells for the propagating properties and differentiation status: hierarchical cancer stem cell model as shown in Figure 2.2 and stochastic model. For each of the model, clonal evolution could happen as a result of interaction of microenvironment and cells, leading to accumulated epigenetic changes, as demonstrated in Figure 2.3 (Sayed et al., 2011; Beck and Blanpain, 2013).
Cancer stem cell concept has been derived from understanding on normal stem cells. The issue of stem cells has been raised corresponding to cancer development. It was found that stem cells and tumour cells exhibit common features on their unlimited proliferative and tissue-specific differentiation capacity (Trosko and Chang, 1989). The unique replication process in normal tissues allows a stem cell to receive signals from the microenvironment to divide and expand the stem cell population (Morrison and Kimble, 2006).
According to the cancer stem cell model, a small population of cancer cells known as CSCs make up the reservoir of self-sustaining cells with their unique properties to propagate and differentiate into the bulk of tumours. CSCs have the capacity to divide as well as to expand the cancer stem cell pool and to differentiate into the heterogeneous non-tumorigenic cancer cell types to form the bulk of the cancer cells within the tumour (Nguyen et al., 2012).
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Figure 2.2: Hierarchy in tumours. The cellular hierarchy in tumours implies that not all cells are equivalent and that only CSCs present long term self-renewal and differentiation potential. CSCs give rise to new CSCs, as well as more committed progenitors with more restricted renewal potential which eventually produce terminally differentiated cells.
(Beck and Blanpain, 2013)
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Figure 2.3: Clonal evolution in stochastic and cancer stem cell model
a. According to stochastic model, all tumour cells are equipotent and randomly self-renew to expand the population or differentiate to specific tissue.
b. With hierarchical CSC model, only a subset of tumour cells has the ability for long-term self-renewal and these cells give rise to progenitors with limited proliferative potential that eventually terminally differentiate.
c. Clonal evolution in stochastic model explains that tumour cells transform into more aggressive cell type with new somatic mutation.
d. Clonal evolution in CSC model describes the generation of clonal diversity with mutations, which further increase tumour heretogeneity.
(Beck and Blanpain, 2013)
