Stem cell-based therapies are promising in regenerative medicine. However, the success of cell therapy is greatly limited by the low engraftment rate to the target tissues. The present study demonstrated that human mesenchymal stem cells (hMSCs) were subjected to a self selection process via microencapsulation in collagen barrier when they were induced to migrate out from this barrier. While retaining the immuophenotype and self renewal capacity, the selected hMSCs showed a significantly better in vitro migratory response of than those cultured in traditional monolayer. The migratory response could be controlled by varying the fabrication parameters of the collagen barrier, including initial collagen concentration and cells seeding density. Affinity to adhere on endothelial cells layer is another engraftment related property. Significant difference was observed between these selected hMSCs and hMSCs in monolayer culture. In order to investigate the engraftment potential of the selected hMSCs, an animal model was performed. The selected hMSCs were transplanted intravenously into NOD/SCID mice under partial hepatectomy. Presence of human cells in the residual liver was determined by the presence of human HLA-ABC using flow cytometry after 48 hours, 1 week and 1 month. Engraftment of the selected hMSCs was significantly higher than that of monolayer cultured hMSCs in time point of 1 month. It demonstrated that the selected hMSCs favor the engraftment to the injured liver. Further investigation is required to determine the fate of the engrafted hMSCs in order to truly confirm their therapeutic potential. The current work demonstrated that collagen-hMSCs microsphere could act as a barrier to select hMSCs with enhanced in vitro migratory response and in vivo engraftment properties. These findings may contribute towards the development of better stem cell therapies. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). These TICs are believed to display unique survival mechanisms, and account for failure in therapeutic treatments. It is also believed that, effective treatments against the diseases can only be developed through targeting and eliminating these TICs. We previously identified TIC populations derived from hepatocellular carcinoma (HCC) that are characterized by membrane expression of CD133. As findings from our previous studies were mostly based on HCC cell lines, here, we first identified rare CD133+ subpopulations in freshly resected HCC specimens, but not their non-tumor counterparts. We also found increased CD133 expression to be associated with advanced disease stages, increased recurrence rate and poorer overall survival in HCC patients. Next, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133+ liver TICs isolated from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133+ cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133+ liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis and initiate tumors. In further support of these observations, IL-8 repression in CD133+ liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be also preferentially expressed in CD133+ liver TICs. Exogenous NTS treatment resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, key components of the MAPK signaling pathway. Enhanced IL-8 secretion by CD133+ TICs can in turn activate an IL-8 positive feedback loop through MAPK signaling. Subsequent studies from CD133 sorted cells found only CD133+ TICs, but not CD133- cells were able to response to exogenous NTS / IL-8 stimulations with concomitant up-regulation of CD133, suggested that the preferential expression of NTS / IL-8 signaling cascade was also important in CD133+ TICs self-renewal and maintenance. Further to its role as a liver TIC marker, CD133 also plays functional roles in conferring TICs properties via regulating NTS / IL-8 / CXCL1 / MAPK signaling. These results suggested that CD133+ liver TICs promote angiogenesis, tumorigenesis and selfrenewal through NTS-induced activation of the IL-8 signaling cascade. In conclusion, our findings had identified rare expressions of CD133 in clinical HCC specimens and hence its prognostic values. We also show for the first time the functional roles of CD133 in conferring tumorigenic potential to liver TICs. The characterization of underlying molecular signaling in CD133+ liver TICs in this study should provide not only a better understanding of the mechanisms regulating this specific population of cells but also novel insights that could allow the development of more effective therapeutic treatments of this disease. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Haematological stem cell transplantation (HSCT) is a revolutionary treatment for haematological malignancies. Although HSCT is potentially curative, patients usually develop stomatitis which is a common and debilitating complication after the transplantation. Furthermore, stomatitis may predispose patients to various complications which are associated with significantly increased morbidity and mortality. In some studies, ice water mouthwash has been shown to be an effective method for the prevention of stomatitis. However, a high-level evidence-based protocol on the prevention of stomatitis has not been fully developed and it is not commonly practiced in most HSCT centers at present. A well established protocol can help to minimize the patients’ suffering and avoid prolonged hospitalization. The nurses who are involved in patient education, assessment, care for, and coping with stomatitis, play an important role to bring these innovations into practice. In this regard, this translational research aims at developing an evidence-based protocol on using ice water mouthwash in the prevention of stomatitis for patients undergoing autologous HSCT. A systematic search for relevant literatures was performed with the use of five electronic databases. Six relevant studies were found. Critical appraisal on the relevant studies was conducted. The level of evidence extracted from the studies was graded according to the Scottish Intercollegiate Guidelines Network (SIGN) and were synthesized to establish the protocol for patients in the proposed setting. The implementation potential of the protocol was assessed in terms of the transferability, feasibility, and cost benefit ratio. An implementation and evaluation plan was established for comprehensive evidence-based protocol development. The successful implementation of the protocol will be beneficial for the patients undergoing HSCT as it may hasten their recovery, shorten their hospital stay, and minimize their distressing experience and suffering. / published_or_final_version / Nursing Studies / Master / Master of Nursing

The groundbreaking use of transcription factors (Oct4, Sox2, Klf4, c-Myc) in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides novel ways in regenerative medicine and disease modeling. The reprogramming process is a stepwise process involving global epigenetic remodeling. In recent years, small molecules like DNA methyltransferase inhibitor that alter the epigenetic status of cells were shown to enhance the reprogramming efficiency. It was postulated that chromatin modifying enzymes played an important role during the reprogramming process, and microRNAs (miRNAs) were the upstream regulators. The objectives of this study involve the identification of potential miRNAs regulating the expression of chromatin modifying enzymes and the study of their roles during reprogramming. Primary mouse embryonic fibroblasts (1o MEFs) were used for the establishment of a reprogramming system, where the delivery of transcription factors Oct4, Sox2, klf4 and cMyc was mediated by lentivirus. Another established secondary MEFs (2o MEFs) reprogramming system was also included in the study. Mouse iPSCs (miPSCs) derived from both systems were shown to express pluripotent markers. In-silico analysis predicted a set of miRNAs (miR-101, miR-135a, miR-148a and miR-148b) commonly targeted the chromatin modifying enzymes in mouse genome. Among them, miR-101 and miR-135a overexpression were found to inhibit the reprogramming efficiency significantly in both 1o and 2o MEFs. Conversely, the inhibition of miR-135a but not miR-101 expression significantly enhanced the reprogramming efficiency in both systems. In this study, it was postulated that miR-101 regulated enhancer of zeste homolog 2 (Ezh2) during reprogramming. Ezh2 was confirmed to be negatively regulated by miR-101 at protein level. The expression of Ezh2 was high in mouse embryonic stem cells (mESCs) but time dependently depressed during mESC differentiation, while its expression was increased during reprogramming of MEFs. Ezh2 expression was found to negatively correlate with miR-101 expression in these conditions. In addition, the knockdown of Ezh2 mimicked the inhibitory effect of miR-101 overexpression on reprogramming efficiency. The inhibitory role of miR-135a on reprogramming was linked to its potential target, Sirtuin 1 (Sirt1). Sirt1 was negatively regulated by miR-135a. The expression of miR-135a was upregulated upon mESC differentiation and decreased during reprogramming. Together with the previous finding in this laboratory, miR-135a expression was negatively correlated with Sirt1. Furthermore, miR-135a inhibition increased the proliferation rate of MEFs. More importantly, miPSCs reprogrammed from miR-135a knockdown MEFs maintained the pluripotent state. To further analyze the pluripotency of the miPSCs, the tetraploid complementation assay was established. Preliminary studies were performed to optimize the conditions for electrofusion. Although single electrofusion with a lower field strength (1000V/cm) resulted in lower fusion rate, the development of the mESC aggregated embryo was the best when compared to higher field strength and those with double electrofusion. Lastly, the mESCs aggregated into tetraploid embryo were mainly localize in the inner cell mass of the embryo. In conclusion, negative correlations were found between miR-101/Ezh2, and miR-135a/Sirt1 during somatic cell reprogramming. The identification of small molecules in reprogramming helps to understand the molecular mechanisms of reprogramming. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Hepatocellular carcinoma (HCC) is one of the most devastating malignancies worldwide with increasing incidences in both developed and developing countries. Survival rates have not been significantly improved over the past decades in spite of advances in detection and therapies for this disease, suggesting that current treatments may target the wrong cells, and miss the cancer stem cells (CSCs). The cancer stem cell hypothesis presents that tumor formation, proliferation and propagation are driven by a rare subpopulation of chemoresistant CSCs that are not killed by conventional therapies and go on to cause disease relapse. The objective of this study was to identify and characterize CSCs in HCC cell lines and human liver tumor specimens using CD90 as a potential marker. The number of CD90+ cells present in HCC cell lines was found to positively correlate with tumorigenicity potentials. Injection of as few as 2,000 sorted CD90+ cells from HCC cell lines resulted in the formation of tumor nodules in nude mice, whereas no tumors formed for CD90ˉcells in the same model. The tumor xenograft generated by injection of CD90+ cells sorted from previous xenograft in a serial xenotransplantation assay exhibited recapitulation of tumor heterogeneity to original primary tumor and consistent proportion of CD90+ and CD90ˉ cells which demonstrated self-renewal and differentiation capacities of CD90+CSCs. CD45ˉCD90+ cells were detected (0.03%–6.2%) in human liver tumor specimens, but were only present in minute quantities in normal, cirrhotic and non-tumorous tissues. More importantly, CD45ˉCD90+ cells sorted from primary HCC tumor also displayed tumorigenicity, self-renewal and lineage differentiation capacities. CD90+CSCs were found to be more resistant to therapeutic drugs compared to CD90- cells, as reflected by the results of enrichment of the CD90+ CSCs and longer survival rates after chemotherapeutic treatment. The high expression of genes, such as OCT4, MRP3, ABCG2, AKT1, BirC5, BCL2, HA and CD44, in CD90+CSCs may mediate chemoresistance. The majority of CD90+ cells co-expressed CD44, another stem cell marker. Blocking CD44 activities by anti-CD44 antibody increased apoptosis of CD90+ CSCs, sensitized CD90+CSCs to chemotherapeutic drugs in vitro, and decreased tumorigenic and metastatic potentials of CD90+CSCs in vivo, indicating that a therapeutic potential of targeting CD44. However side effects may be problematic due to the endogenous expression of CD44 in healthy tissues and normal lymphocytes. To identify novel gene targets specific to liver CSCs, a sensitive RNA-sequencing (RNA-Seq) technique was used to compare the gene expression profiles between CD90+CSCs sorted from HCC primary tumors and CD90+cells from adjacent non-tumorous tissue (CD90+NTSCs). The up-regulated genes in CD90+CSCs were associated with lipid metabolism, inflammation, and drug resistance. Among the differentially expressed genes, glypican-3 (GPC3) was specifically elevated in CD90+CSCs but not in CD90+NTSCs. Therefore, GPC3 could be a promising gene candidate for HCC therapy as targeting GPC3 should not induce damage to normal liver stem cells. In summary, CD90 is a liver CSCs marker. Identification of CD90+ CSCs in HCC provides new insight into cellular basis of hepatocarcinogenesis, recurrence and metastasis, which opens new avenues for the design of future CSC-targeted therapies. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate. The compounds melatonin and sPDZD2 have been suggested to act through the Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation. In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation. Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Despite major advances in pharmacological and surgical treatments of cardiovascular diseases (CVDs), clinical outcomes of patients with severe CVDs remain very poor. Most of medication and interventions currently available are only playing roles of preventing further damage to myocardium, declining the risk of on-going cardiovascular events, lifting the cardiac pumping efficiency and lower early mortality rates, none of these treatments can regenerate or repair damaged cardiac tissue or restore heart function. As a result, several new strategies have been explored to overcome limitations of current therapeutic approaches. One prospective is to replace dead cardiac vascular cells with young and green cells to repair or regenerate damaged heart myocardium. Several types of stem cells, including bone marrow hematopoietic stem cells, mesenchymal stem cells (MSCs), embryonic stem cell (ESCs)and induced pluripotent stem cells (iPSCs),have been tested as the candidates for treatment of CVDs. Among a myriad of types of stem cells, bone marrow derived MSCs(BM-MSCs) has received great attention based on several unique properties such as easy isolation and expansion, stable genetic background and low immunogenicity. However, the therapeutic efficacy of BM-MSCs derived from aging or diseased donors is impaired. The differentiation potential of BM-MSCs is gradually reduced with the increased culture time. Thus, it is urgent to identify some novel alternative sources for MSCs. Moreover, the potential mechanisms of MSCs therapy have not been understood totally. This thesis is designed to investigate the therapeutic efficacy and potential mechanisms of several novel types of MSCs, including hESC-MSCs and hiPSC-MSCs and Rap1-/--BM-MSCson several types of CVDs, including pulmonary arterial hypertension (PAH), dilated cardiomyopathy (DCM)and myocardial infarction (MI). In Chapter 4, it disclosed that hESC-MSCs have a better therapeutic efficacy than BM-MSCs in attenuation of PAH induced by monocrotaline in mice. The greater therapeutic potential of hESC-MSCs on PAH was not only attributed to the higher capacity of differentiation into de-novo vascular cells, but also attributed to higher cell survival rate and greater paracrine effects post-transplantation. In Chapter 5, it demonstrated that compared with BM-MSCs, iPSC-MSCs have a better therapeutic effect on doxorubicin-induced cardiomyopathy. Several potential mechanisms of action were involved in iPSC-MSCs-based therapy for cardiomyopathy. It demonstrated that iPSC-MSCs transplantation not only attenuated the generation of reactive oxygen species(ROS)and the level of inflammation, but also restored depletion of cardiac progenitor cells and promoted endogenous myocardial regeneration against doxorubicin induced cardiomyopathy. Moreover, mitochondrial transfer and paracrine actions of iPSC-MSCs played critical roles in the rescue for doxorubicin-induced cardiomyopathy. In Chapter 6, it uncovered that compared with wild type BM-MSCs,Rap1-/--BM-MSCs transplantation achieved a better benefit to MI induced by ligation of left anterior descending (LAD)coronary artery. Rap1-mediated NF-κB activity plays a key role in regulation MSCscytokine secretion profiles. The absence of Rap1 in MSCs leads to reduced pro-inflammatory cytokines secretion and enhanced MSCs survival capacity, thus yielding a better therapeutic efficacy. In conclusion, findings presented in this thesis provide important new insights regarding different novel types of MSCs, including those derived from ESC and iPSC. They have distinct mechanisms of action from BM-MSCs and provide superior therapeutic efficacy in various form of severe CVDs, including PAH and DCM. The safety and efficacy of these novel types of MSCs for treatment of CVDs deserve further investigations. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Pluripotent stem cells hold great promise in regenerative medicine. Theoretically, a variety of tissues can be generated from this progeny. The production of tailor-made stem cells for individualized patient treatment is the ultimate goal of stem cell based therapy. Human induced pluripotent stem cells (iPSCs) hold the precious key to success and promote the clinical application of stem cells. By reprogramming somatic cells, pluripotent stem cells can be generated in a patient-specific manner and subsequently differentiated into specific tissue for regeneration. Nonetheless exposure of hiPSCs to animal feeder cells and serum during generation and maintenance imposes a risk of transmitting animal pathogens to human subjects, thus hindering their potential therapeutic application. In addition, the efficacy of iPSC generation is < 1% of total somatic cells used. The first part of the study focused on the development of improved methods to produce a more efficient xenogen-free culture system to produce more clinically compatible iPSCs. Specific tissue or cells derived from stem cells may offer a solution and cell therapy using endothelial cells and their progenitors may be possible in treatment of severe cardiovascular diseases. In theory, endothelial cells can be generated from different sources of progenitor cells although no direct comparison of these various derived endothelial cells (ECs) has been reported. Thus in the second part of the study, the functional and physiological properties of BM, ESC and iPSC-ECs will be evaluated to determine their therapeutic potential in ischemic disease. A mouse hind limb ischemia model was used to assess and monitor neovascularization by the derived ECs. The results can provide further insight to evaluate the possibility of using iPSCEC as the cell source for patient-specific treatment. Use of pluripotent stem cells is a promising approach in therapeutic angiogenesis although numerous hurdles continue to hamper their widespread clinical use. Conditioned medium derived from progenitor cells may be another possible strategy in the treatment of ischemic diseases such that direct cell transplantation is avoided. Conditioned media produced from ex vivo culture of endothelial cells contain a combination of angiogenic factors that can be applied to promote neovascularization in ischemic tissue. Nonetheless the efficacy of this angiogenic application is unknown. The third part of the study focused on the potential application of EC-derived conditioned media in the treatment of ischemic disease using a mouse hind limb ischemia model. Some cardiovascular risk factors such as diabetes might affect endothelial cell function such that autologous application of ECs and their conditioned media is not feasible. A human embryonic stem cell line may offer and alternative means to obtain stable quality ECs and conditioned medium for therapeutic use. In summary, advances in stem cell technology hold great promise for the treatment of cardiovascular disease, further improved by the generation of patient-specific stem cells using iPSC technology. Vascular cells can be generated from different sources of stem cells with similar angiogenic properties and may be used in the treatment of ischemic diseases. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The fundamental principle of tissue engineering is to use appropriate cell source, combined with scaffolds and bioactive factors to develop tissue constructs which restore, maintain or improve tissue function. There is increasing data emphasizing the importance of mechanical signals and extracellular matrix (ECM) proteins presented by the scaffold in determining stem cell fate/functions which are critical to tissue construct maturation and success of stem cell-based therapies. Cell-matrix adhesions are one of the major mechanosensing machineries cells use to convert information provided by ECM ligands and mechanical signals presented by scaffolds into intracellular biochemical signaling cascades which lead to particular functional responses. Therefore, understanding how ECM ligands and mechanical signals regulate cell-matrix adhesion formation and activation of associated intracellular signaling proteins is fundamental to rational design of biomaterial and loading protocol for optimal cell functional responses in tissue constructs. In this study, we attempted to understand the regulatory effects of external mechanical signal and exogenous ECM protein on cell-matrix adhesion formation and associated intracellular signaling proteins of human mesenhymal stem cells, and in particular, to test the hypothesis that mechanical stimulation or exogenous ECM protein can lead to adhesion maturation into 3D-matrix adhesions in 3D collagen environment. We used microencapsulation technique to embed cells in 3D collagen environment, forming disc-shaped hMSC-collagen constructs. By immunofluorescent staining and confocal microscopy, we visualized changes in size, morphologies and molecular composition of the adhesions. First of all, 2D adhesions of hMSCs were characterized. We showed that hMSCs form well-organized αv integrin-based focal adhesions and fibrillar adhesions in 2D culture. To investigate the regulatory effects of mechanical signals on adhesion signaling and maturation, we used micromanipulator-based loading device to impose dynamic compression to hMSC-collagen constructs. We found that dynamic compression lead to enlargement of integrin αv adhesions which recruit focal adhesion kinase (FAK), vinculin and extracellular signal-regulated kinase (ERK). In addition, FAK was activated at enlarged integrin αv adhesions and translocated to peri-nuclear region after compression, suggesting that loading induces activation of FAK signaling pathways through increased integrin αv clustering. Moreover, we demonstrated that dynamic compression can induce 3D-matrix adhesion formation, indicating the role of external force in integrin α5-based adhesion maturation in 3D collagen environment. We explored the effect of exogenous ECM proteins on adhesion maturation of hMSCs by adding fibronectin into cell-collagen mixture during fabrication of collagen constructs. Our results demonstrated that the exogenous fibronectin can induce α5 integrin-based adhesion maturation into 3D-matrix adhesions in our collagen constructs in a dose-dependent manner. This study demonstrated that the effect of external mechanical signals and exogenous ECM ligands on adhesion signaling and maturation of hMSCs in 3D collagen environment. Our findings contribute towards mechanobiology of hMSCs in 3D context. In particular, our results showed that exogenous proteins or external loading can lead to 3D-matrix adhesion formation, which may serve as a potential way to enhance biological functions of hMSCs in collagen constructs, facilitating stem cell-based therapies. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Nasopharyngeal carcinoma (NPC), endemic in southern China and Southeast Asia, was ranked 7th as the most common new malignancy in Hong Kong. Metastatic and recurrent NPC have a poor prognosis despite recent advancement in medicine. Inactivation of tumor suppressor genes (TSGs) through the loss of chromosomal regions is frequently reported in NPC. With the recent discovery of cancer stem cells (CSCs), which are refractory to current therapies, a new paradigm shift in the perspective of cancer therapy development has emerged. For the first time, this study aims to unravel the complexity of NPC tumorigenicity for identifying more effective targets by studying the possible interplay between CSCs and TSGs. NPC cell lines had different expression profiles of CSC markers, confirming not all CSC markers are applicable to every tumor type. Although CD24/CD44 were expressed in NPC, however CD24+CD44+ NPC cells did not initiate tumor formation. By utilizing a cancer hybrid cell model with a transferred single copy of chromosome 3, physiological β-catenin up-regulated core stem cell markers through the activation of Wnt signaling pathway in NPC. Moreover, the down-regulation of β-catenin suppressed chemoresistance and inhibited cell proliferation, colony formation, angiogenesis, the epithelial-mesenchymal transition (EMT) process, and the tumor microenvironment factors. Amongst the tumor microenvironment factors, chemokine Rantes and matrix metalloproteinase were down-regulated when β-catenin was knocked down. Therefore, activation of Wnt signaling provide an alternative platform for identifying putative CSCs in NPC, leading to the identification of several prospective CSC markers in NPC. Down-regulation of IKBB, a NF-KB inhibitor, in the majority of NPC patients indicated that IKBB plays a prominent role as a TSG in NPC. In this study, IKBB was found to exert its tumor suppressive functions by abrogating tumor formation, cell migration, invasion and angiogenesis. Angiogenic factors, including Rantes, Upar, IL6 and IL8, were significantly down-regulated by IKBB. In addition, IKBB also suppressed the binding activity of NF-KB. The involvement of Akt/Gsk-3β pathways was also observed. Taken together, IKBB regulated NPC tumorigenesis through NF-KB/Akt/Gsk-3β and interaction with tumor microenvironment. Collectively, this study demonstrated that the progression of NPC is not simply initiated by a single signaling pathway, but a dynamic and complex interplay between multiple signaling networks and the tumor microenvironment. NPC tumorigenesis is hypothesized to be driven by orchestrated interaction between CSCs and TSGs through crosstalk with the tumor microenvironment. Amongst the major players in the tumor microenvironment, Rantes/CCL5, IL6, and the matrix metalloproteinase are envisaged to induce angiogenesis, EMT, and metastasis in NPC. This dynamic intercommunication between CSCs and tumor suppressor IKBB signaling networks may shed better insights on modulation of the major hallmarks of cancer in orchestrating NPC development. The modulation of the major hallmarks of cancer by CSCs and IKBB, a TSG, involves promotion of aberrant proliferation, enhancement of invasion and metastasis, induction of angiogenesis, circumvention of tumor suppressors, and prevention of cell death. Taken together, selective and synergistic co-targeting these signaling networks and the tumor microenvironment will provide a more effective new modality of treatments for NPC. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy