Cancer stem cell: Fundamental experimental pathological concepts and updates

Experimental and Molecular Pathology 98 (2015) 184–191

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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp

Review

Cancer stem cell: Fundamental experimental pathological concepts and updates Farhadul Islam a,1, Bin Qiao b,1, Robert A. Smith a,c, Vinod Gopalan a, Alfred K.-Y. Lam a,d,⁎ a

Cancer Molecular Pathology, School of Medicine and Griffith Health Institute, Griffith University, Gold Coast, Queensland, Australia Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Genomics Research Centre, Institute of Health and Biomedical Innovation, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia d Department of Pathology, Gold Coast University Hospital, Gold Coast, Queensland, Australia b c

a r t i c l e

i n f o

Article history: Received 29 January 2015 Accepted 4 February 2015 Available online 7 February 2015 Keywords: Cancer stem cell Origin Plasticity Microenvironment Heterogeneity

a b s t r a c t Cancer stem cells (CSCs) are a subset of cancer cells which play a key role in predicting the biological aggressiveness of cancer due to its ability of self-renewal and multi-lineage differentiation (stemness). The CSC model is a dynamic one with a functional subpopulation of cancer cells rather than a stable cell population responsible for tumour regeneration. Hypotheses regarding the origins of CSCs include (1) malignant transformation of normal stem cells; (2) mature cancer cell de-differentiation with epithelial–mesenchymal transition and (3) induced pluripotent cancer cells. Surprisingly, the cancer stem cell hypothesis originated in the late nineteenth century and the existence of haematopoietic stem cells was demonstrated a century later, demonstrating that the concept was possible. In the last decade, CSCs have been identified and isolated in different cancers. The hallmark traits of CSCs include their heterogeneity, interaction with microenvironments and plasticity. Understanding these basic concepts of CSCs is important for translational applications using CSCs in the management of patients with cancer. © 2015 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Cancer stem cell model . . . . . . . . . . . . . 3. Evolution of the CSC model . . . . . . . . . . . 4. Origin of CSCs (Fig. 1) . . . . . . . . . . . . . . 5. Milestones in CSC hypothesis development (Fig. 3) . 6. Proportion of CSC in cancer . . . . . . . . . . . 7. Heterogeneity . . . . . . . . . . . . . . . . . 8. CSC niche . . . . . . . . . . . . . . . . . . . 9. CSC plasticity . . . . . . . . . . . . . . . . . 10. Conclusion . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Tissue renewal is maintained by normal stem cells through a tightly regulated process of self-renewal and cell death (Pierce and Speers,

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1988; Pardal et al., 2005). Dysregulation of this process is a key event in cancer pathogenesis (Al-Hajj et al., 2004). Cancer might be considered as an abnormal organ/tissue, in which minor subpopulations of tumorigenic cells having aberrant self-renewal capacities give rise to

⁎ Corresponding author at: Griffith Medical School, Gold Coast Campus, Gold Coast QLD 4222, Australia. E-mail address: a.lam@griffith.edu.au (A.K.-Y. Lam). 1 Both authors contribute equally to the manuscript.

http://dx.doi.org/10.1016/j.yexmp.2015.02.002 0014-4800/© 2015 Elsevier Inc. All rights reserved.

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different lineages of cancer cells. Recent studies have indicated that these self-renewing tumorigenic cells called cancer stem cells (CSCs) are mainly responsible for resistance to chemo-radiation therapy and cancer relapses (Vermeulen et al., 2012; Aulmann et al., 2010; Fillmore and Kuperwasser, 2008; Francipane et al., 2013; Ghods et al., 2007; Resetkova et al., 2010; Williams et al., 2013). Research is needed to target the stem cell populations which attribute the tumour growth and progression. In this review, we presented the recent information of the basic concepts, development milestones and the characteristics of CSCs. 2. Cancer stem cell model Cancer is constituted of a heterogeneous population of cells differing in morphology, gene expression, proliferative capacity and invasiveness. This heterogeneity may occur as a result of cancer being hierarchically organized with a subset of cancer cells, called cancer stem cells (CSCs) or cancer initiating cells, at their apex, which have the capacity of stemness (Clarke et al., 2006; Vermeulen et al., 2008). CSCs potentially explain several phenomena of cancer such as minimal residual disease, resistance to chemo-radiation therapy, cancer recurrence and metastases (Vermeulen et al., 2012). Also, CSCs are believed to play a key role in predicting the biological aggressiveness of cancer, due to their ability of self-renewal and multi-lineage differentiation (stemness) through either asymmetric or symmetric division (Lee et al., 2011). In contrast, offspring of offspring, the progenitor cells and differentiated cancer cells, lose the capacity for stemness, thus no longer contributing to biological aggressiveness, though they cause much of the overall damage (Lee et al., 2011). Although these scientific hypotheses are appealing, they lack conclusive experimental evidences and they have stimulated controversies among cancer scientists. The current gold standard method to identify CSCs is via serial transplantation of cancer cells in animal models. As this xeno-transplantation model is an operational definition, it probably underestimates the total CSC number and is biased to the detection of those CSCs with more aggressive biological behaviour (Quintana et al., 2008; Lapidot, 2001). 3. Evolution of the CSC model Cancer is known to arise from a single cell in specific tissues through a series of genetic and/or epigenetic events that cause ectopic production of growth related genes. These alterations in turn interfere with the mechanisms that normally control a stable physiological cell output in that tissue or initiate genomic instability state (Nowell, 1976; Baylin and Jones, 2011; Greaves and Maley, 2012; Stratton, 2011). Thus cancer ideally comes from abnormal clones that, at least initially, preserve many properties of the hierarchical structure of the normal tissue in which the cancer has arisen (Valent et al., 2012). According to the CSC model, the ultimate result of this accumulation of genetic and epigenetic “hits” is the development of at least one cell with CSC traits that can produce more CSCs and more differentiated offspring. In the past, the CSC model was a static one. However, in recent times, it has been revised to a dynamic one, where CSCs were believed to be converted into more transient cell types. Accordingly, progeny of mutated cells may acquire the capacity for self-renewal through de-differentiation of progenitor cells as well as reversal of differentiation in fully differentiated cells (Scheel et al., 2011). Thus, CSCs are a functional subpopulation of cancer cells rather than a stable cell population. 4. Origin of CSCs (Fig. 1) The precise origin of CSCs is an ambiguous issue at present. There are three main hypotheses for the acquisition of the properties of a stem cell for cancer cells in the mainstream of the scientific world. These include the hypotheses of (1) malignant transformation of normal stem cells;

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(2) mature cancer cell dedifferentiation with epithelial–mesenchymal transition (EMT) and (3) induced pluripotent cancer cells. The first hypothesis of the origin of CSCs proposed that CSCs are the product of malignant transformation of adult stem cells (Fillmore and Kuperwasser, 2008; Swords et al., 2005; Todaro et al., 2007). Smalley and Ashworth first suggested that CSCs may derive from normal stem cells that have acquired mutations and have lost their ability to selfregulate cell proliferation (Smalley and Ashworth, 2003). Cells with different malignant potentials would be present in any cancers with a defined set of genetic and epigenetic changes. Also, differentiated cells that have lost their tumour propagation capacity and cells that possess a clonogenic potential may exist in an established cancer (Vermeulen et al., 2008). These findings indicate that cells having the same genotypic makeup can exhibit a completely different potential to initiate a cancer. There are normal stem cells in human bodies which are responsible for tissue repair, termed adult stem cells or somatic stem cells (Igarashi et al., 2008). Nowadays, more scholars support the hypothesis that CSCs originate from adult stem cells that have undergone accumulation of different degrees of epigenetic and genetic alterations and propose the following two reasons. First, for a normal somatic cell to transform into a malignant cell, it must accumulate many mutations. However as mutations are events of low frequency, the accumulation may take several years, even decades. In this process, no cell would survive so long, other than adult stem cells, with their properties of self-renewal and differentiation. Second, CSCs share several properties with normal stem cells, such as the capacity for self-renewal and the ability to differentiate, which would require still further mutation for a somatic cell to acquire. On the other hand, studies have found that progenitor cells of CSCs can reacquire the self-renewal capacity through further genetic mutations and epigenetic modifications (Cozzio et al., 2003; Jamieson et al., 2004; Krivtsov et al., 2006). Thus, the second hypothesis for the origin of CSCs is that CSCs are acquired from tumour cells themselves via cellular dedifferentiation. Since normal breast stem cells and CSCs of breast cancer seem to have a mesenchymal phenotype and display gene expression characteristic of epithelial–mesenchymal transition, Weinberg's group demonstrated in 2008 that CSCs can be enriched within an existing malignancy, by “dedifferentiation” of mature tumour cells through an EMT pathway (Mani et al., 2008). EMT gives differentiated tumour cells the ability to self-renew, thus allowing the formation of distant sites of metastasis (Qiao et al., 2012, 2013). A similar study was made by Brabletz's group in colorectal cancer. In that study, it was revealed that EMT-associated genes and stemnessassociated genes were both expressed at the invasive front of cancer cells (Spaderna et al., 2007). More importantly, a study supported the above concept by detecting co-expression of EMT markers and stem cell markers in spindle tumour cells inside the blood vessels of patients with metastasis (Aktas et al., 2009). Due to the fact that invasion into the blood is the second step of cancer cell metastasis, the concept of migrating cancer stem cells should be an area for future study. Evidence of a potential link between cancer stem cells and EMT was reported in 2007. Before the milestone study of Weinberg's group in 2008, Zeng's group in 2007 isolated and identified a cancer stem cell–like side population in human nasopharyngeal carcinoma cell lines by flow cytometry (Mani et al., 2008; Wang et al., 2007). A total of 10,000 freshly sorted cells from this side population formed tumours in NOD/SCID mice. Cells formed outside this population, however, could not form tumours until the number increased to 200,000. More interestingly, growth of the side population cells in complete medium in vitro leads to morphology alteration into many long filaments, causing the cells to resemble fibroblasts, not polygonal squamous epithelial cells. It was implied that epithelial cancer stem cells were akin to fibroblasts, the prototype of migrating cancer stem cells. The third hypothesis of the origin of CSCs is related to the recent development of induced pluripotent stem cells (iPS). Induced pluripotent stem cells (iPS) are artificially derived from a non-pluripotent cell

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Fig. 1. Origin of cancer stem cells: (A) Top panel explains the regulated hierarchy of normal tissue populations in which cell turnover is a feature of tissue maintenance, where stem cells produce progenitor/transit-amplifying cells to generate mature differentiated cells. (Ia) During multi-step tumour pathogenesis, the genetic and/or epigenetic alterations in stem or progenitor cells leads to deregulated cell production with various degrees of aberrations and loss of many traits of normal cells. The ultimate phenotype is a premalignant lesion. (Ib) With further accumulation of mutations, premalignant stem cells can be converted into cancer stem cells (hypothesis I). (II) Cancer stem cells can produce cancer progenitor cells, which in turn generate different subpopulations of cancer cells. In the presence of a malignant microenvironment, some differentiated cancer cells can also gain self-renewal as well as cancer stem cell properties by de-differentiation (hypothesis 2). (III) Conversion of normal somatic cells to cancer stem cells by cellular reprogramming: induced expression of transcription factors (Yamanaka factors) in normal somatic cells can produce the cancer stem cell (hypothesis 3).

in vitro, typically an adult somatic cell, by inducing the expression of specific genes, causing direct reprogramming of somatic cells through the transduction of four transcriptional factors, namely OCT3/4 (also known as POU5F1), Sox2, c-Myc and Klf4 (Takahashi and Yamanaka, 2006). These four transcriptional factors are also known as “Yamanaka factors” as the development of mouse iPS cells was first reported by Takahashi and Yamanaka (2006). Seeding mouse iPS cells in a mouse womb can produce new live mice though with an increased risk of teratoma (Okita et al., 2008). Hence, it has been proposed that iPS formation is a process of reprogramming that resembles the transforming of normal cells into tumour cells. This hypothesis was studied by two independent groups most recently in vitro and in vivo. Nishi and his colleagues acquired cells with cancer stem cell properties but not iPS properties from MCF-10A (non-tumorigenic immortalized mammary epithelial cell) cells by “Yamanaka factors” transduction in vitro (Nishi et al., 2014). On the other hand, a group of scientists using a transgenic mouse model found that a tumour mass which resembles Wilms' tumour could be initiated in the kidney of mice in the middle stage of reprogramming suggesting that transient expression of reprogramming factors in vivo should result in tumour development (Ohnishi et al., 2014). Our group acquired the preliminary data by immunofluorescence assay that OCT4 and Sox2 double positive cells (OCT4+Sox2+) were detected in precancerous lesions (oral lichen planus and oral leukoplakia) and oral squamous cell carcinoma, but not in normal oral epithelia (Fig. 2) (unpublished data). This finding is in line with Ohnishi's report that OCT4+Sox2 + cells might be premature termination of reprogramming of cells and these cells would initiate neoplasm in vivo. Thus, the third origin of CSCs is hypothesized that endogenous reprogramming or residues of the embryo are the source of CSCs. Meanwhile, induced pluripotency and oncogenic transformation could be acknowledged as a related process (Fig. 1).

Overall, these findings suggest that CSC models are not mutually exclusive. In a cancer, different subsets of cancer cells can act differently, as well as being influenced by their micro-environments (Malanchi et al., 2012; Odoux et al., 2008). 5. Milestones in CSC hypothesis development (Fig. 3) The cancer stem cell hypothesis originated in some degree from the old concept, ‘embryonal-rest hypothesis’ of tumour formation, described by two pathologists, Rudolph Virchow and Julius Cohnheim in the late nineteenth century (Virchow, 1855; Cohnheim, 1867). More than 100 years later in 1961, James Till and Ernest McCulloch suggested the existence of the haematopoietic stem cells, based on experiments demonstrating the ability of bone marrow to provide protection to mice from a lethal dose of irradiation (Till and McCulloch, 1961). In the same year, Chester Southam and Alexander Brunschwing showed that tumour cells which were harvested from a patient with disseminated malignancy can be injected subcutaneously into the same patient and reduce the frequency of tumour formation. This study has also noted that tumours were only initiated when over 1,000,000 cells were injected (Southam and Brunschwig, 1961). Similarly, other studies demonstrated that only 1–4% of transplanted murine lymphoma cells formed colonies in the spleens of recipient animals (Park et al., 1971; Bruce and Van Der Gaag, 1963). In the 1970s, researchers showed that solid tumour cells vary in their ability to proliferate in a similar way to haematopoietic cancer. Anne Hamburger and Sydney Salmon found that only 1 in 1000 to 1 in 5000 cells isolated from solid tumours such as lung, ovarian and brain tumours were capable of forming colonies (Hamburger and Salmon, 1977). These results indicated that only these few cells have the potential to form secondary cancer. These observations led the researchers to

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Fig. 2. Immunofluorescence assay showing the Sox-2 and Oct-4 expression in oral tissues. OCT4 and Sox2 double positive cells (OCT4+Sox2+) were detected in precancerous lesions (oral lichen planus [OLP] and oral leukoplakia [OLK]) and oral squamous cell carcinoma [OSCC], but not in normal oral epithelia. DAPI (4′,6-diamidino-2-phenylindole), a nuclear fluorescent stain, was used as controls.

speculate that the entire population of a tumour's cells might arise from a few so-called ‘cancer stem cells’ (Hamburger and Salmon, 1977). Also, some groups in 1970s and 1980s updated this concept and hypothesized that cancer may arise from the maturation arrest in tissuespecific stem cells (Pierce and Johnson, 1791; Potter, 1978; Sell and Pierce, 1994). In 1994, Lapidot et al. first reported the identification of cancer initiating/cancer stem cells in acute myeloid leukaemia by transplantation of selective subpopulation of cells into severe combined immune-deficient (SCID) mice (Lapidot et al., 1994). They also reported that the proportion of leukaemia initiating cells in the peripheral blood of patients with acute myeloid leukaemia was 1 in 250,000 cells (Lapidot et al., 1994).

The most remarkable and definitive evidence regarding the existence of CSCs was demonstrated by Bonnet and Dick in 1997 on leukemic stem cells (Bonnet and Dick, 1997). They showed that leukemic stem cells possess characteristic functional properties of stem cells by isolating sub-populations of cells expressing CD34+CD38− and then transplanting them into non-obese, non-diabetic and immunodeficient mice (Bonnet and Dick, 1997). This study also noted that these leukemic stem cells could reconstitute the full spectrum of phenotypes from patients with acute myeloid leukaemia (Bonnet and Dick, 1997). These results highlighted the importance of defining of CSC by a functional assay such as xeno-transplantation. Six years later, Al-Hajj et al. demonstrated for the first time the presence of CSCs in a solid

Fig. 3. The milestones for the development of concepts of cancer stem cell. CSC: cancer stem cell.

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cancer (breast cancer), while Singh and colleagues reported selfrenewal properties in brain cancer using in vitro tumour sphere assays (Al-Hajj et al., 2003, 2004). In the last decade, CSCs have also been identified and isolated in other solid tumours, including lung, colonic, prostatic and pancreatic cancers. The results of all these studies suggested that cancer derives from a subpopulation of CSCs capable of selfrenewal to initiate and sustain cancer growth (Singh et al., 2003; Collins et al., 2005; O'Brien et al., 2007). In recent years, three individual research groups demonstrated the acquisition of self-renewal capabilities in non-CSC populations within a tumour using cancer cell lines from primary cancers (Chaffer et al., 2011; Iliopoulos et al., 2011; Chaffer et al., 2013). These studies have brought the clonal evolution and CSC hypotheses more closely together (Chaffer et al., 2011; Iliopoulos et al., 2011; Chaffer et al., 2013). Among them, Weinberg et al. examined the ability of non-CSCs reacquiring the self-renewal (stemness) potential and reported that this interconversion was stimulated through oncogenic transformation (Chaffer et al., 2011). Iliopoulos and colleagues revealed a dynamic equilibrium of CSC and non-CSC populations in breast cancer, which was mediated by interleukin-6 (Iliopoulos et al., 2011). Also, Chaffer et al. found that non-CSCs in basal type breast carcinomas were able to switch to a CSC-like state, dependent on zinc finger E-box-binding homeobox 1 (ZEB1), a regulator that has been found to be involved in EMT (Chaffer et al., 2013). These studies thus confirm the conversion of non-stem cell to stem cell-like population. In 2012, Parada et al. identified a subpopulation of cells within mouse gliomas using a nestin-ΔTK-IRES GFP transgene and these cells were found to be the source of entirely new colonies of tumour growth after being treated with temozolomide (TMZ) (Chen et al., 2012). In the same year, Clevers and co-workers demonstrated that single intestinal Lrg5 (+) stem cells have the ability to initiate and maintain intestinal adenoma. They observed these phenomena by introducing a gene that would allow cells and their progeny to emit four different colours upon activation with the treatment of tamoxifen (a chemo-drug that allowed single stem cells to adopt one of the four colours) (Schepers et al., 2012). Also, Driessens et al. showed the generation of cellular hierarchy dependent on the stage of cancer growth and progression from benign skin tumour to cancer, accompanied by an expansion of the CSCs and a reduction of the population of non-CSCs (Driessens et al., 2012). All the three studies indicated the identification of a subpopulation of cells that could allow cancer cells to self-renew, differentiate and regenerate. These studies also provided compelling evidence of the existence of CSCs in a model of carcinogenesis. 6. Proportion of CSC in cancer The actual frequency of CSCs in cancers is still unclear. Studies on different cancers demonstrated that these tumorigenic cells in cancer are very rare (0–2%) (Todaro et al., 2007; Singh et al., 2003; O'Brien et al., 2007; Eramo et al., 2007; Ricci-Vitiani et al., 2007). O'Brien et al. noted that only one cell (CD133+) in 5.7 × 104 unsorted human colon cancer cells was able to generate tumour after xenotransplantation in NOD/ SCID mice (O'Brien et al., 2007). Another study by Todaro et al. revealed that only 2% of cells (CD133+) are tumorigenic in human colon cancers and these cells have the potential of tumour formation and therapy resistance (Todaro et al., 2007). Similarly, a study on human acute myeloid leukaemia showed that only (~ 1/106) CSCs had self-renewal and tumour forming capacity in immunosuppressed mice (Hope et al., 2004). On the other hand, several studies reported that tumour initiating cells are not so rare but present in human cancer in relatively large numbers (Kelly et al., 2007; Civenni et al., 2011; Shmelkov et al., 2008; Boiko et al., 2010). Kelly et al. demonstrated in a mice model that in leukaemia and lymphoma, at least one in ten (N10%) cancer cells have the potential of tumour formation in histocompatible recipients (Kelly et al., 2007). Also, Boiko and co-workers identified CD271+ cells as melanoma stem cells and observed the frequency of

these cells in different patients between 2.5–41% (Boiko et al., 2010). Thus, the frequency of CSCs present in cancers is highly debatable and its numbers appear to vary according to the types and stages of cancers. Since the isolation and transplantation of CSCs are functional assays for their detection, it has potential to be biased in different experimental conditions, especially by not detecting cells which may be capable of initiating new tumours in the original host, but are unable to do so in a xenotransplantation. Further research, especially single cell tracking systems with innovative technologies is needed for more conclusive information. 7. Heterogeneity There are several forms of intra-tumour heterogeneity that exist in different haematological and solid cancers (Marusyk et al., 2012). The presence of such diversity occurs as a result of large numbers of genetic/epigenetic abnormalities, which in turn, could lead to the occurrence of different sub-populations of cells within the cancer. CSCs have different combinations of genetically derived cells with particular predispositions for growth, survival and dominance in the tumour micro-environment (Pattabiraman and Weinberg, 2014). The CSC model hypothesizes that difference between functionally important properties of individual tumour cells arise from variations in their differentiation status. Thus, non-genetic factors may contribute most to tumour heterogeneity. Intra-tumoural phenotypic heterogeneity has been linked to different clinical outcomes such as differences in ability to metastasise, resistance to therapy and prognosis of the patients with cancer (Clevers, 2011). In principle, higher frequency of CSCs in a cancer is associated with aggressive clinical parameters, leading to the concept that CSCs are more treatment resistant and metastatic than non-CSC cancer cells. The existence of CSCs also represents an entirely distinct dimension of intra-tumoural heterogeneity. Thus, the subpopulations of cancer cells within a cancer may carry their own group of CSCs, and these CSCs and non-CSCs will share heritable genetic and epigenetic alterations (Pattabiraman and Weinberg, 2014). Similar to intra-tumour heterogeneity, the inter-tumour heterogeneity of CSCs has been identified (Lapouge et al., 2012). As cancer progresses, the properties and numbers of CSCs change among different stages of cancer. For example, the proportion of CSCs in squamous cell carcinoma changes as the cancer progresses. The carcinoma also has different levels of invasiveness as the number of CSCs increases (Lapouge et al., 2012). With the development of advanced technologies in molecular diagnosis, these forms of heterogeneity are now well known to the scientific community. However, the actual cause and real-time effects of such non-genetic heterogeneity are still poorly understood. 8. CSC niche Stem cells in normal tissues require crosstalk with their microenvironment to achieve an optimal balance among self-renewal, activation and differentiation (Medema and Vermeulen, 2011; Shestopalov and Zon, 2012). A similar concept could hold for CSC, as most CSCs originate from cells in normal tissues, in which local cues have an important regulatory function and have not yet acquired completely autonomous growth ability. The regulation of stemness properties in CSCs is still unclear as majority of the studies in this area are either done with isolated or cultured CSCs (Todaro et al., 2007; Singh et al., 2003; O'Brien et al., 2007; Lapouge et al., 2012). Several studies suggested that the tumour microenvironment, tentatively called the CSC niche is essential for the production and development of CSCs (Medema and Vermeulen, 2011; Borovski et al., 2009; Ricci-Vitiani et al., 2010). For example, in glioma, CSCs reside near endothelial cells, which are supposed to stimulate stemness through factors like Notch, diffusible factors/growth related factors and the presence of some supportive endothelial-like cells which are tumour derived. This suggests that regulation of CSCs' niche

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in glioma is intrinsic (Borovski et al., 2009; Ricci-Vitiani et al., 2010; Calabrese et al., 2007; Wang et al., 2010). Similarly, secretory proteins such as activin and nodal are not only produced by pancreatic CSCs, but also secreted by stellate cells, stimulating CSCs in a paracrine fashion (Lonardo et al., 2012). These two proteins promote pancreatic cancer stem cell sphere formation and invasiveness (Lonardo et al., 2012). It was reported that mesenchymal cells in breast cancer support CSCs through a signalling loop dependent on Interleukin-6 and chemokine C-X-C motif ligand 7 (CXCL7) (Liu et al., 2011). Also, in colon cancer, myofibroblasts support the CSCs through hepatocyte growth factor (Vermeulen et al., 2010). Recent studies indicated that CSCs respond to anti-tumour agents differently both in vivo and in vitro, reinforcing the concept that the niche in which a CSC is located may be an important determinant of how it responds to a given therapy (Junttila and de Sauvage, 2013). Such a CSC niche model also implies that less malignant tumours may depend more on their (cancer micro-environment) niche. These less advanced pathological stages cancers may fail to form a new tumour following implants into mice as they cannot reform their supporting microenvironment (Valent et al., 2012). In biologically advanced cancers, such as metastatic melanoma, this dependency could be lost by continued mutations, eventually resulting in a distant neoplasm, where all cells have CSC qualities solely determined by tumour-intrinsic mechanisms. This concept may also affect CSC marker selection and the success rate of xenotransplantation assays, as niche cells or niche-derived factors would be lost when isolating tumour cells for experimental designs (Medema, 2013). Thus, the tumour microenvironment as well as CSC niche could be crucial components in achieving CSC qualities. 9. CSC plasticity The term “plasticity” refers to the capacity of tissue-derived stem cells to exhibit a phenotypic potential that extends beyond the differentiated cell phenotypes of their resident tissue (Eisenberg and Eisenberg, 2003). Plasticity is an important feature of adult stem cells and it plays an important role in the regulation and differentiation of stem cells (Petersen et al., 1999; Bjornson et al., 1999). Bone marrow-derived stem cells which give rise to hepatocyte and neuronal stem cells have been shown to trans-differentiated into haematopoietic stem cells (Petersen et al., 1999; Bjornson et al., 1999). This inter-conversion of cell types in stem cells raises debate about whether, a stem cell is a concrete cellular entity, or possibly a more functional entity. This means any cells can be converted into a stem cell depending on the physiological demands of the cellular context (Blau et al., 2001; Zipori, 2004). In CSCs, the inherent trait of stemness may reflect a transient state that is regulated by tumour micro-environment parameters rather than by intrinsic features of the cells (Vermeulen et al., 2008). In the context of CSCs, the term ‘plasticity’ generally refers to the properties that a phenotypically differentiated cell has which can de-differentiate and allow acquisition of stem cell characteristics. In the static CSC model, this plasticity of the CSC compartment is often debatable, but in the case of fluidic CSC models, the de-differentiation of differentiated tumour cells into CSC has been demonstrated and many studies support this hypothesis (Petersen et al., 1999; Bjornson et al., 1999; Blau et al., 2001; Zipori, 2004). This implies that the CSC compartment is not fixed or stable over time. It was demonstrated that CSCs in culture are able to induce dedifferentiation in the presence of fibroblast growth factor and epidermal growth factor in brain cancer cells (Grinspan et al., 1996; Doetsch et al., 2002). Thus programming of CSC differentiation can be reversible and extrapolating this point in cancer would mean that the CSC population in a malignancy is unstable over time. 10. Conclusion The hypotheses of CSC models continue to evolve. Experimental evidence suggested that the conventional clonal evolution model and the

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modern CSC model are not mutually exclusive but rather co-exist and complement each other in cancer pathogenesis. In cancer treatment as it currently exists, the tumour bulk is eliminated as completely as possible in order to prevent recurrence and metastasis, but CSCs often remain to cause just these results. Future clarification of the molecular and cellular behaviour underlying CSC biology and cancer heterogeneity may lead to the development of effective and novel treatment modalities that can eliminate the bulk of the tumour cells by killing of the CSCs, providing a more effective way to prevent metastasis and recurrence. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgment The authors would like to thank Griffith University (Visiting Fellowship and Higher Degree Research scholarship) and the National Natural Science Foundation of China (Grant No. 81200796) for funding support for this manuscript. References Aktas, B., Tewes, M., Fehm, T., Hauch, S., Kimmig, R., Kasimir-Bauer, S., 2009. Stem cell and epithelial–mesenchymal transition markers are frequently overexpressed in circulating tumour cells of metastatic breast cancer patients. Breast Cancer Res. 11, R46. Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J., Clarke, M.F., 2003. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 100, 3983–3988. Al-Hajj, M., Becker, M.W., Wicha, M., Weissman, I., Clarke, M.F., 2004. Therapeutic implications of cancer stem cells. Curr. Opin. Genet. Dev. 14, 43–47. Aulmann, S., Waldburger, N., Penzel, R., Andrulis, M., Schirmacher, P., Sinn, H.P., 2010. Reduction of CD44(+)/CD24(−) breast cancer cells by conventional cytotoxic chemotherapy. Hum. Pathol. 41, 574–581. Baylin, S.B., Jones, P.A., 2011. A decade of exploring the cancer epigenome—biological and translational implications. Nat. Rev. Cancer 11, 726–734. Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Magli, M.C., Vescovi, A.L., 1999. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 283, 534–537. Blau, H.M., Brazelton, T.R., Weimann, J.M., 2001. The evolving concept of a stem cell: entity or function? Cell 105, 829–841. Boiko, A.D., Razorenova, O.V., van de Rijn, M., Swetter, S.M., Johnson, D.L., Ly, D.P., Butler, P.D., Yang, G.P., Joshua, B., Kaplan, M.J., Longaker, M.T., Weissman, I.L., 2010. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466, 133–137. Bonnet, D., Dick, J.E., 1997. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3, 730–737. Borovski, T., Verhoeff, J.J., ten Cate, R., Cameron, K., de Vries, N.A., van Tellingen, O., Richel, D.J., van Furth, W.R., Medema, J.P., Sprick, M.R., 2009. Tumor microvasculature supports proliferation and expansion of glioma-propagating cells. Int. J. Cancer 125, 1222–1230. Bruce, W.R., Van Der Gaag, H., 1963. A quantitative assay for the number of murine lymphoma cells capable of proliferation in vivo. Nature 199, 79–80. Calabrese, C., Poppleton, H., Kocak, M., Hogg, T.L., Fuller, C., Hamner, B., Oh, E.Y., Gaber, M.W., Finklestein, D., Allen, M., Frank, A., Bayazitov, I.T., Zakharenko, S.S., Gajjar, A., Davidoff, A., Gilbertson, R.J., 2007. A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69–82. Chaffer, C.L., Brueckmann, I., Scheel, C., Kaestli, A.J., Wiggins, P.A., Rodrigues, L.O., Brooks, M., Reinhardt, F., Su, Y., Polyak, K., Arendt, L.M., Kuperwasser, C., Bierie, B., Weinberg, R.A., 2011. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc. Natl. Acad. Sci. U. S. A. 108, 7950–7955. Chaffer, C.L., Marjanovic, N.D., Lee, T., Bell, G., Kleer, C.G., Reinhardt, F., D'Alessio, A.C., Young, R.A., Weinberg, R.A., 2013. Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity. Cell 154, 61–74. Chen, J., Li, Y., Yu, T.S., McKay, R.M., Burns, D.K., Kernie, S.G., Parada, L.F., 2012. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522–526. Civenni, G., Walter, A., Kobert, N., Mihic-Probst, D., Zipser, M., Belloni, B., Seifert, B., Moch, H., Dummer, R., van den Broek, M., Sommer, L., 2011. Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth. Cancer Res. 71, 3098–3109. Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., Visvader, J., Weissman, I.L., Wahl, G.M., 2006. Cancer stem cells—perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 66, 9339–9344. Clevers, H., 2011. The cancer stem cell: premises, promises and challenges. Nat. Med. 17, 313–319. Cohnheim, J., 1867. Ueber entzundung und eiterung. Pathol. Anat. Physiol. Klin. Med. 40, 1–79.

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