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Cancer Stem Cells and Epithelial Ovarian Cancer

Cancer Stem Cells and Epithelial Ovarian Cancer Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 105269, 9 pages doi:10.1155/2010/105269 Review Article 1 1 1, 2 Sheetal Dyall, Simon A. Gayther, and Dimitra Dafou Gynaecological Cancer Research Laboratory, EGA UCL Institute for Women’s Health, London WC1E 6DD, UK King’s College School of Medicine, Guy’s Hospital, London SE1 9RT, UK Correspondence should be addressed to Sheetal Dyall, s.dyall@ucl.ac.uk Received 15 June 2010; Accepted 9 November 2010 Academic Editor: Peter E. Schwartz Copyright © 2010 Sheetal Dyall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The cancer stem cell hypothesis is becoming more widely accepted as a model for carcinogenesis. Tumours are heterogeneous both at the molecular and cellular level, containing a small population of cells that possess highly tumourigenic “stem-cell” properties. Cancer stem cells (CSCs), or tumour-initiating cells, have the ability to self-renew, generate xenografts reminiscent of the primary tumour that they were derived from, and are chemoresistant. The characterisation of the CSC population within a tumour that drives its growth could provide novel target therapeutics against these cells specifically, eradicating the cancer completely. There have been several reports describing the isolation of putative cancer stem cell populations in several cancers; however, no defined set of markers has been identified that conclusively characterises “stem-like” cancer cells. This paper highlights the current experimental approaches that have been used in the field and discusses their limitations, with specific emphasis on the identification and characterisation of the CSC population in epithelial ovarian cancer. 1. Introduction represent suitable targets for new therapeutic approaches for epithelial ovarian cancers (EOC). Ovarian cancer (OC) is the sixth most lethal malignancy The mechanisms underlying chemoresistance in cancer in women in the western world. Over 90% of malignant are not clear. One hypothesis is that cancers are driven by tumours are epithelial. It has been hypothesised that tumours a subset of highly tumourigenic cells with stem cell properties can arise either from a single layer of cells covering the ovary within the tumour, cancer stem cells (CSCs). The term CSC (the ovarian surface epithelium or OSE) or from the epithe- is not meant to suggest that these cells have any association lial lining of the fimbrial end of the fallopian tube [1]. The with adult tissue stem cells; more recently, the term cancer aetiology of OC remains poorly understood. One proposed or tumour initiating cells (CIC or TIC) has come to be model is the incessant ovulation hypothesis, which postulates thought of as more appropriate. As the term CSC is most that continuous rupture of the OSE during ovulation and commonly used in the literature to describe these cells, for subsequent cell proliferation leading to repair make OSE cells the purpose of this paper, tumourigenic cancer cells with more susceptible to malignant transformation. stem cell properties will be referred to as CSC. According to this model, only the CSCs, but not the remaining cells in Approximately 70% of patients diagnosed with ovarian the tumour, can propagate tumourigenesis. CSCs have been cancer have advanced stage disease, partly because symp- implicated in tumour initiation, progression, metastasis, and toms are vague and can be confused with gastrointestinal drug resistance. complaints (e.g., bloating, constipation and mild abdominal pain) [2, 3]. Despite improvements in debulking surgery and initial good responses to platinum-based chemotherapies, 2. Cancer Stem Cells survival rates for the disease remain poor due to the devel- opment of chemoresistant disease, and less than 60% of A recent AACR workshop defined the CSC as a malignant cases survive more than 5 years. Thus, the identification cancer cell with a stem cell phenotype [4]. Whilst the CSC of molecular markers that target chemoresistance may hypothesis does not specifically address the mechanisms of 2 Journal of Oncology + − malignant transformation, it has been suggested that CSCs leukemia [13], in which the population of CD34 CD38 are the malignant counterparts of normal adult tissue stem cells isolated and transplanted into SCID mice formed cells, which, due to dysregulated signalling pathways, are tumours that had similar features to human leukemia, unable to maintain stem cell homeostasis. As is the case with whilst populations containing cells with different cell surface tissue stem cells, CSCs are thought to reside at the top of the marker profiles did not. Since then, several studies have lineage hierarchy and give rise to differentiated cells, which characterised the CSC in other types of tumours (Table 1). themselves have no potential for self-renewal, and therefore Classically, stem cells are defined by their two main char- do not contribute significantly to tumour growth. acteristics: self-renewal and pluripotency. And so by infer- The idea that tissue stem cells are the underlying cells for ence, to be classified as a CSC, a cell must meet the following carcinogenesis is attractive. Due to their long lifespan, stem criteria: (i) the ability of self-renewal and differentiation into cellsremaininatissue forlongerperiods of time compared different cell types, (ii) the ability to propagate tumours to their differentiated progeny, thereby making them more in vivo, (iii) the expression of distinct markers that permit likely to acquire transforming mutations. Additionally, it is consistent isolation, and (iv) restriction to a minority of the generally accepted that stem cells are more resistant to cell population (although this latter is now debatable, due to apoptosis and DNA damage and are therefore more likely to recent evidence showing that the microenvironment of mice survive any insults [5, 6]. Whilst being quiescent in normal alters the tumour-forming efficiency of engrafted cells) [14]. tissue, stem cells are able to maintain the stem cell pool by Table 1 summarises the various techniques and experi- undergoing asymmetric cell division during processes such mental approaches that have been used to isolate the putative as tissue damage. During this process, a stem cell divides CSC component from primary tumours. The approaches asymmetrically to generate an identical daughter cell (i.e., used rely on the intrinsic and functional properties of stem another stem cell) that is committed to differentiation. Adult cells. The most commonly used approach is the tumour stem cell cangiverisetoawide rangeofdifferentiated cells sphere assay, which evaluates the ability of cells to grow and it has been suggested that CSCs undergo asymmetric cell as nonadherent spheroids under non-differentiating con- division to generate the different cell types within a tumour, ditions, and cell surface antigen profile of subpopulations thereby contributing to tumour heterogeneity often seen in found within tumours. The dye exclusion assay enables the many cancers. The stem cell pool is also tightly regulated by isolation of a side population of cells that extrude lipophilic signalling pathways from the microenvironment of the stem dyes due to the overexpression of drug efflux pumps; the cell niche, and several of these pathways, including Hedgehog enzyme aldehyde dehydrogenase 1 is thought to be prefer- [7], and Wnt [8], have been implicated in carcinogenesis. entially active in stem-like cells. The tumourigenicity of the The CSC hypothesis suggests a paradigm shift in the putative CSC component is then verified by engraftment into understanding of carcinogenesis and tumour cell biology. immunosuppressed mice. The aim is to show that a minimal This may have fundamental implications in therapeutic number of stem-like cells can give rise to tumours of the interventions, including an explanation for the development same histopathology as the original primary tumours that of chemoresistance. A role for CSCs in propagating and they have been isolated from. maintaining metastasis has been proposed [9, 10]. Many features of tumours can be explained by the inherent char- acteristics of stem cells; for example, tumours selfrenew and 2.1. Assaying Cancer Stem Cells heterogeneity can be attributed to multilineage differentia- 2.1.1. The Tumour Sphere Assay. Whilst there are no estab- tion. Dontu et al. [11] have proposed that breast cancers lished protocols for isolating pure populations of CSCs, the may arise from mammary stem cells, or early progenitor general aim of many studies has been to enrich for cell cells. It has also been shown that hypermethylation at populations that show features that are characteristic of Polycomb promoter regions lock stem cells in a state of normal stem cells, primarily the ability to self-renew and self-renewal, which can lead to aberrant clonal expansion, differentiate into different lineages. The tumourigenicity of thus leading to cancer predisposition [12]. Even so, the the isolated cell populations is then confirmed by assessing contrasting hypothesis that the dedifferentiation of mature the tumour-forming potential of these cells in appropriate cells to a more pluripotent state as a potential mechanism mice models. A commonly used approach for CSC enrich- for the development of stem cell-like features by cancer cells ment is the tumour sphere assay, which evaluates the ability cannot be dismissed. of cancer cells to grow as multicellular spheroids under non- Cancer cells with stem cell properties have been identified differentiating and non-adherent conditions, a characteristic in acute myeloid leukemia and several solid malignancies believed to be indicative of self-renewal. (Table 1). Whilst the fact that tissue stem cells are the target of transformation may be true for certain malignancies, this has In a typical tumour sphere assay, cells from a primary yet to be proven as a universal mechanism of tumorigenesis. tumour or cancer cell line are dissociated into a single cell Consequently, the terms cancer and tumour-initiating cells suspension and cultured in a serum-free growth factor- are thought to be more appropriate as they define cells with rich medium containing primarily epidermal growth factor tumour-initiating potential, without any reference to an (EGF) and fibroblast growth factor (FGF). Multicellular origin from tissue stem cells. spheroids of enriched CSCs that form can undergo serial The first strong evidence for the existence of cancer stem dissociation and passaging to further enrich for stem cell-like cells in human tumours came from a study in acute myeloid cells. Journal of Oncology 3 Table 1: Studies that have attempted to characterise the cancer stem cell (CSC) component in the cancers are listed. Only the most relevant studies are referenced. The different experimental techniques used in the referenced studies are reviewed; this highlights the lack of consensus in the approach that can be best used to identify stem-like cells in cancers. In vitro characterization Cell surface In vivo Cancer type References b c d e antigen profile characterisation Dye exclusion assay Tumour sphere assay Clonality assays ALDH assay H 33342 (5 μg/mL) 2 Colony forming unit assay: SFM hours, 37 C; number of colonies from + − Leukaemia CD34 /CD38 Not done SCID [15, 16] 20 ng/mL Interleukin-3 Inhibitor KO143 leukemic SP and haematopoetic (200 nM) 100 ng/mL stem cell factor SP compared per 10 cells plated SFM H 33342 (5 μg/mL) 90 20 ng/mL EGF, minutes at 37 C Primary spheres dissociated and Brain CD133 Inhibitors plated at cell dilutions from Not done NOD-SCID [17, 18] 20 ng/mL bFGF Verapamil (50 μM) or 200 cells/well to 1 cell. 10 ng/mL LIF reserpine (100 μM). 60 μg/mL N-acetylcysteine SFM + [−]/low CD44 CD24 H33342 (5 μg/mL) for 10 ng/mL bFGF Single cells (1 cells per well in ALDEFLUOR- + ◦ Breast OCT4 90 min, 37 C NOD-SCID [19, 20] 20 ng/mL EGF 96-well plate) positive CD133 Inhibitor FTC 10 μM 5 μg/mL insulin 0.4% BSA SFM H33342 (5 μg/mL) for 0.6% glucose, 1.5–2 hours, 37 C, Single-cell derived tumour 9.6 μg/mL putrescine, Inhibitor verapamil at spheres (unsorted and CD133 , Colon CD133 Not done NOD-SCID [21, 22] 6.3 ng/mL progesterone 50 μMfor 10 minutesat either spheroid-derived or room temperature primary) ITS before adding H33342 20 ng/mL EGF 10 ng/mL bFGF H33342 (5 μg/mL) for 90 minutes, 37 C NOD-SCID Endometrial —— Notdone [23] Inhibitor verapamil (50 μM) 4 Journal of Oncology Table 1: Continued. Cell surface In vitro characterization In vivo Cancer type References antigen profile characterisation b c d e Dye exclusion assay Tumour sphere assay Clonality assays ALDH assay H33342 5 μg/mL at varying time points, CD44 NOD-SCID Head and ◦ + − 37 C; SCM Single cells GFP and GFP Not done [24–26] neck Inhibitor reserpine (5 μg/mL) SFM 10 ng/mL FGF 1 cell/well in SFM by limiting + 20 ng/mL EGF Pancreas CD133 — Not done Nude mice [27] dilution 5 μg/mL insulin ITS 0.4% BSA SFM H33342 (5 μM) for 90 Single cells were plated at + hi minutes, 37 C; CD44 , α β , B27 Prostate 1,000/mL on a plate coated with Not done SCID [9, 28, 29] Inhibitor verapamil CD133 10 ng/mL EGF 0.5% agar (50 μM) 10 ng/mL bFGF SFM H33342 (5 μM) for 90 5 μg/mL insulin + ◦ CD133 minutes, 37 C; Ovary — Not done BALB/c-nu/nu [30–37] + + 20 ng/mL EGF CD44 CD117 Inhibitor verapamil (50 μM) 10 ng/mL bFGF 0.4% BSA Cell surface antigen profile expression by flow cytometry and immunohistochemistry techniques Dye Exclusion flow cytometry assays following treatment with Hoechst, coupled with ABC multidrug transporter inhibitors to isolate side population cells Sphere formation assays for multicellular spheroid forming ability under nonadherent conditions, which is characteristic of stem cells Clonality assays measure the ability of single cells to generate spheroids/clones Aldehyde Dehydrogenase assays measure the levels of ALDH1 in cells Soft agar assay measures anchorage-independent growth due to loss of contact-inhibition and is an indicator of tumourigenicity. EGF: Epidermal growth factor bFGF: Basic fibroblast growth factor LIF: Leukemia inhibitory factor NSF: Neuronal survival factor BPE: Bovine pituitary extract KO143: Inhibitor of breast cancer resistant protein (BRCP) SFM: Serum-free media SCM: Serum-containing media. Journal of Oncology 5 Although this is one of the most commonly used tech- overexpress ATP-binding cassette (ABC) transporter proteins niques to evaluate self-renewal, the results from such assays on their cell surface, thus contributing to the intrinsic resis- can be misleading. The characteristic of self-renewal can only tance of stem cells to chemotherapy and radiotherapy. This be inferred from the presence of tumour spheres in culture characteristic has been implicated in multidrug resistance in if it can be shown that each tumour sphere has arisen from cancers, as overexpression of multi-drug resistant proteins a single cell under proliferative nonadherent conditions; yet, enables cancer cells to remove chemotherapeutic drugs from there are few published studies that have actually demon- their cytoplasm. strated this [38]. A detailed analysis of the methodologies When stained with the fluorescent dyes, cells overexpress- used in many studies shows that starting cell densities in ing the ABC transporter proteins will eliminate the dye from the tumour sphere assay are fairly high (e.g., 1000 cells/mL). their cytoplasm, thus producing a lower signal than cells These studies aim to utilise single cell suspensions, but the not overexpressing ABC proteins. By combining fluorescent high cell densities do not preclude the possibility that the staining with fluorescent-activated cell sorting (FACS) in spheroids that form may have arisen from the aggregation a cell population, a side population of cells (SP) can be of mixed cell population and therefore would not be clonal. isolated that is enriched for stem “like” cells. As a control for For neurospheres, it has been shown that spheroids are clonal the SP phenotype, cells are stained in the presence of ABC only when cells are plated at low density [39]. transporter inhibitors, which reverse the SP phenotype. The most commonly used inhibitor is verapamil, which targets P-glycoprotein encoded by the gene MDR1. A less frequently 2.1.2. Cell Surface Specific Antigen Profile. Several studies used inhibitor is fumitremorgin C, which specifically targets have prospectively isolated CSCs by looking for the presence the BCRP transporter (encoded by ABCG2). Cells that retain of extracellular markers that are thought to be stem cell- Hoechst are thought to contain terminally-differentiated specific. The markers most commonly used are CD133 and cells that are unable to self-renew; thus this is a marker CD44. CD133, a penta-membrane glycoprotein, has been commonly used for identifying the non-SP fraction of a cell found to interact with cholesterol and may be involved population. in maintaining membrane topology and membrane lipid SP analysis, which was originally used to identify composition. Its expression has been shown to be restricted haematopoietic stem cells from murine bone marrow [43], to plasma membrane protrusions in epithelial cells [40]. It is has been used to isolate putative CSCs in several studies also thought to be a marker for neuroepithelial progenitor in primary tumours and cell lines [19, 28, 29, 36, 44–50]. cells and in haematopoietic progenitor cells. CD133 was Reviewing the published literatures suggests that the per- originally identified as a neural stem cell marker. It has been centage of SP cells that can be isolated from whole cell used to characterise CSCs in brain tumours [17]aswell line populations can range from 0%–20%. However, in the as several other cancers, including, prostate, colon, ovary, majority of studies, the SP cells form a minority (<5%) of and pancreas (Table 1). In breast cancer, CSCs have been + − the bulk cell population, supporting the idea that CSCs, like characterised as CD44 and CD24 [41]. The limitation of stem cells, constitute a small proportion of cell cultures. using cell surface marker expression to characterise CSCs is that the approach requires prior knowledge of cell surface SP analysis provides a rapid method for isolating stem markers that are expressed by the putative CSCs in the tissue cell-like cells; it relies on the functional characteristic of of interest, and often the choice of markers is inferred from stem cells and requires no prior knowledge of extracellular known expression of markers in normal adult stem cells. markers. Nevertheless, the use of Hoechst as a method of Whilst CD133 and CD44 are thought to be indicative enriching for CSC has received criticism. It has been reported of a CSC phenotype, it is not clear if they are universal that Hoechst treatment reduces the clonogenicity of the markers for characterising CSCs derived from all types of breast cancer cell line MCF7 and the ovarian cancer cell line tumours. Furthermore, the expression of CD133 and CD44 SKOV3 due to a loss in viability [51]. In this study, single may not be restricted to the CSC population and may be cells from unsorted cell populations, when grown in serum- present in early progenitor cells. Their functions are largely containing media under adherent conditions, generated unknown, although there are reports implicating CD44 in a clone that would satisfy the criteria for self-renewal. metastasis and chemoresistance. Recent evidence suggests Additionally, the same group reported that both SP and non- that CD133 is expressed in epithelial cells and may be SP cells generated clones in vitro and non-SP cells were able involved in maintaining pluripotency [40]. to form both SP and non-SP cells, suggesting that non-SP Epithelial-to-mesenchymal transition (EMT) is also cells can self-renew as well. a feature of carcinogenesis, as cells acquire a more mesenchy- mal phenotype during neoplastic transformation. A link 2.1.4. Aldehyde Dehydrogenase Expression. More recently, between CSC and EMT has been suggested, whereby trans- it has been suggested that an elevated level of aldehyde formed human mammary epithelial cells that have under- dehydrogenase 1 (ALDH1) activity can select for stem- gone EMT show a gain of the CSC phenotype [42]. like cells in both normal and malignant tissues. ALDH1 is a detoxifying enzyme that is involved in the metabolism 2.1.3. Hoechst 33342 Dye Exclusion Assay. Stem cells actively of vitamin A to retinoic acid in the liver. It has been efflux lipophilic compounds such as the fluorescence dyes suggested that retinoic acid signalling is a probable protective Hoechst 33342 and rhodamine from their cytoplasm, as they mechanism against oxidative damage in stem cells; recently, 6 Journal of Oncology levels of ALDH1 have been used as CSC markers in the described for CSCs by these studies, both studies reported isolation of putative CSC, and are thought to be more specific that the CSC phenotype was more resistant to platinum- to the stem cell phenotype [10, 52–55]. In the colon, it based therapy, which again, supports the idea that CSCs may has been shown that the expressions of CD44 and CD133, be responsible for chemoresistance. which have previously been used to isolate CSC populations, Generally, these studies highlight the lack of consensus are not restricted to the stem cell populations, which are about the molecular characteristics of ovarian CSCs. It is located at the base of colonic crypt, but also to populations of likely that the expression of markers overlaps, and both rapidly proliferating cells located further up along the crypt. CD133 and CD44 characterise the ovarian CSC. Alterna- The expression of ALDH1, however, is more limited to the tively, there may be more than one population of cells base of the crypt. with stem cell properties in ovarian cancers. The study by Bapat et al. [32] postulated that stem cells are the target of transformation in ovarian cancer, due to the fact that 2.1.5. In Vivo Tumourigenicity Assays. Following the identi- a few of the clones isolated from ovarian cancer ascites fication of the putative CSC population, cells are engrafted spontaneously immortalised in culture, suggesting a model into appropriate mice models to show that they are more for disease development. tumourigenic than their non-CSC counterparts. This is cur- rently believed to be the gold-standard in the field. Generally, a small number of CSCs and non-CSCs are transplanted 4. Issues with the Current into mice, and it is shown that CSCs have a higher tumour- Experimental Approaches forming capacity than non-CSCs. As described above, the experimental approaches used to identify and characterise populations of putative CSC can be 3. Cancer Stem Cells and Ovarian Cancer classed in three major categories: (1) their isolation based on Several characteristics of CSCs have been implicated in the expression of extracellular markers, (2) their growth in chemoresistance and radiotherapy. Due to the overexpres- tumour sphere assays under non-differentiating conditions, sion of extracellular multi-drug transport proteins, CSCs are and (3) dye exclusion due to the overexpression of drug efflux able to efflux chemotherapeutic drugs from their cytoplasm. pumps. Additionally, chemotherapeutic treatment is thought Stem cells, and possibly CSCs, are inherently quiescent; to enrich for CSCs; thus, when exposed to chemotherapy however, current drugs target rapidly cycling cells. Thus, the drugs, CSCs are selected for [56]. It is not clear whether hypothesis is that whilst the majority of cells in the bulk of chemoresistance is an inherent feature of CSCs or whether the tumour are eliminated, CSCs evade chemotherapy and they acquire this property throughout tumorigenesis. are able to reinitiate tumour development. The most common approach that has been used to isolate There is still uncertainty relating to the identification CSCs is to select for cells by the expression of extracellular of CSCs in ovarian cancer. Several papers have described markers that may be associated with stemness. However, the isolation of ovarian CSCs using different approaches. the rationale underlying the selection of markers used for The first study to be published identified clones established this purpose is not always apparent. Although CD44 and from tumour ascites that were able to form anchorage- CD133 are the two most commonly used markers, it is independent spheroids and were shown to express the stem unclear whether or not they are ubiquitous. CD133 has cell markers Oct 3/4 and Nanog and the progenitor marker been extensively used to isolate CSCs in cancers of the brain Nestin [32]. Using flow cytometry, Ferrandina et al. [34] [18], prostate [57], pancreas [58], and ovary [31, 34]; but reported that two isoforms of CD133 (isoforms 1 and 2) whilst it has been successfully used to isolate brain tumour were both expressed in human ovarian tumours at a higher stem cells, the use of CD133 in defining CSCs in colon and frequency than in normal ovaries and metastatic omental ovarian cancer has been more controversial. In the study by lesions [34]. Szotek et al. [50] used flow cytometry to Ferrandina et al., CD133 was suggested to be a marker for isolate a side population of cells from genetically engineered ovarian cancer and was also used as an ovarian CSC marker mouse ovarian cancer cell lines that expressed the multi- in the study by Baba et al. [31]. Zhang et al. on the other hand + + drug transporter protein BCRP1 and were insensitive to have suggested that CD44 CD117 phenotype is indicative doxorubicin, suggesting a possible link between CSCs and of the ovarian CSC phenotype. In all of these studies, the chemoresistance. They also isolated a similar, although much characteristics of the stem cell phenotype were confirmed smaller, side population of cells from the human ovarian by in vivo models and spheroid forming assays. It is likely cancer cell lines IGROV-1, OVCAR3, and SKOV3, but these that both CD133 and CD44 are markers for stem cell-like SP cells were not further characterised. cells in epithelial OCs but there are no existing studies that Two other studies have independently defined the ovar- have compared the expression of CD44 and CD133 in EOCs. + + ian cancer stem cell by evaluating the CD44 CD117 [37] It is possible that both markers are expressed by the same and CD133 [31] phenotypes. The latter suggests an epige- population of ovarian CSC; alternatively, they may be specific netic regulation of the CD133 promoter. Additionally, using to different populations of CSCs, or there may be other as yet CD44, stem-like cells were enriched from patients’ samples unidentified markers that are ubiquitous markers for ovarian and were characterised by Myd88 expression and chemokine CSCs. Several studies have shown that ovarian cancers are and cytokine production [30]. Despite the different profiles extremely heterogeneous, and this may suggest that different Journal of Oncology 7 populations of CSC can be responsible for tumourigenicity [6] S. Bao, Q. Wu, R. E. McLendon et al., “Glioma stem cells promote radioresistance by preferential activation of the DNA in different histological subtypes. damage response,” Nature, vol. 444, no. 7120, pp. 756–760, Another issue raised by the published literature is that caution needs to be applied when interpreting the results [7] S. Liu, G. Dontu, I. D. Mantle et al., “Hedgehog signaling and from tumour sphere assays. It is only possible to infer self- Bmi-1 regulate self-renewal of normal and malignant human renewal if it has conclusively been shown that multicellular mammary stem cells,” Cancer Research, vol. 66, no. 12, pp. spheroids arise from single cells. Furthermore, it is important 6063–6071, 2006. to show that single cells isolated from such spheroids can [8] H. Korkaya, A. Paulson, E. Charafe-Jauffret et al., “Regulation themselves generate spheroids through serial passaging. of mammary stem/progenitor cells by PTEN/Akt/β-catenin Despite the amount of literature on CSCs, it is still not signaling,” PLoS Biology, vol. 7, no. 6, Article ID e1000121, clear as to what constitutes a universal CSC-specific profile. The definition of the CSC hypothesis is constantly evolving [9] J. Miki, B. Furusato, H. Li et al., “Identification of puta- to specifically address the mechanism of neoplastic transfor- tive stem cell markers, CD133 and CXCR4, in hTERT- immortalized primary nonmalignant and malignant tumor- mation in different tumour types. It is becoming increasingly derived human prostate epithelial cell lines and in prostate evident that there may be more than one population of CSC cancer specimens,” Cancer Research, vol. 67, no. 7, pp. 3153– within a tumour. In addition, the cell of origin of CSCs 3161, 2007. is not evident, as CSCs can arise from either stem cells or [10] E. Charafe-Jauffret, C. Ginestier, F. Iovino et al., “Breast cancer progenitor cells. To be a true CSC, all the established criteria cell lines contain functional cancer stem sells with metastatic that characterise the stem cell properties of a cancer cell need capacity and a distinct molecular signature,” Cancer Research, to be satisfied. vol. 69, no. 4, pp. 1302–1313, 2009. [11] G. Dontu, W. M. Abdallah, J. M. Foley et al., “In vitro prop- 5. Concluding Remarks agation and transcriptional profiling of human mammary stem/progenitor cells,” Genes and Development, vol. 17, no. 10, Ovarian cancer is a heterogeneous disease with histologically pp. 1253–1270, 2003. defined subtypes, and it is highly probable that CSCs [12] M. Widschwendter, H. Fiegl, D. Egle et al., “Epigenetic stem cell signature in cancer,” Nature Genetics,vol. 39, no.2,pp. are involved in tumour development. Despite the number 157–158, 2007. of studies attempting to isolate ovarian CSCs, a well- [13] J. E. Dick, M. Bhatia, O. Gan, and U. Kapp, “Assay of human characterised profile has not been established. It is essential stem cells by repopulation of NOD/SCID mice,” Stem Cells, that further studies are carefully designed to address all vol. 15, supplement 1, pp. 199–207, 1997. the functional characteristics of stem cells covering: (i) self- [14] E. Quintana, M. Shackleton, M. S. Sabel, D. R. Fullen, T. M. renewal, where a single CSC is shown to form multicellular Johnson, and S. J. Morrison, “Efficient tumour formation by spheroid in vitro and (ii) multilineage differentiation such single human melanoma cells,” Nature, vol. 456, no. 7222, pp. that only the putative CSC would divide asymmetrically to 593–598, 2008. generate several cell types. The tumourigenicity of these cells [15] D. Bonnet and J. E. Dick, “Human acute myeloid leukemia thenneedstobevalidatedinanappropriate in vivo model is organized as a hierarchy that originates from a primitive that is permissible to tumour formation. The development hematopoietic cell,” Nature Medicine, vol. 3, no. 7, pp. 730– of chemoresistant disease represents a major hindrance to 737, 1997. successful ovarian cancer treatment, and the identification [16] B. Moshaver, A. Van Rhenen, A. Kelder et al., “Identification of of a molecular profile of ovarian CSC may aid to the a small subpopulation of candidate leukemia-initiating cells in development of more effective targeted therapy. the side population of patients with acute myeloid leukemia,” Stem Cells, vol. 26, no. 12, pp. 3059–3067, 2008. [17] S. K. Singh, I. D. Clarke, M. Terasaki et al., “Identification of References a cancer stem cell in human brain tumors,” Cancer Research, vol. 63, no. 18, pp. 5821–5828, 2003. [1] L. Dubeau, “The cell of origin of ovarian epithelial tumours,” [18] S. K. Singh, C. Hawkins, I. D. Clarke et al., “Identification The Lancet Oncology, vol. 9, no. 12, pp. 1191–1197, 2008. of human brain tumour initiating cells,” Nature, vol. 432, no. [2] C.Wikborn,F.Pettersson, andP.J.Moberg, “Delay in 7015, pp. 396–401, 2004. diagnosis of epithelial ovarian cancer,” International Journal of [19] K. Engelmann, H. Shen, and O. J. Finn, “MCF7 side popula- Gynecology and Obstetrics, vol. 52, no. 3, pp. 263–267, 1996. tion cells with characteristics of cancer stem/progenitor cells [3] B. D. Rufford, I. J. Jacobs, and U. Menon, “Feasibility of express the tumor antigen MUC1,” Cancer Research, vol. 68, screening for ovarian cancer using symptoms as selection no. 7, pp. 2419–2426, 2008. criteria,” British Journal of Obstetrics and Gynaecology, vol. 114, no. 1, pp. 59–64, 2007. [20] D. Ponti, A. Costa, N. Zaffaroni et al., “Isolation and in vitro propagation of tumorigenic breast cancer cells with [4] M.F.Clarke, J. E. Dick,P.B.Dirks et al., “Cancerstemcells— stem/progenitor cell properties,” Cancer Research, vol. 65, no. perspectives on current status and future directions: AACR 13, pp. 5506–5511, 2005. workshop on cancer stem cells,” Cancer Research, vol. 66, no. 19, pp. 9339–9344, 2006. [21] R. T. Sussman,M.S.Ricci,L.S.Hart, Y. S. Shi, andW.S. [5] A. K. Croker and A. L. Allan, “Cancer stem cells: implications El-Deiry, “Chemotherapy-resistant side-population of colon for the progression and treatment of metastatic disease,” cancer cells has a higher sensitivity to TRAIL than the non- Journal of Cellular and Molecular Medicine, vol. 12, no. 2, pp. SP, a higher expression of c-Myc and TRAIL-receptor DR4,” 374–390, 2008. Cancer Biology and Therapy, vol. 6, no. 9, pp. 1490–1495, 2007. 8 Journal of Oncology [22] L. Ricci-Vitiani, D. G. Lombardi, E. Pilozzi et al., “Identifi- [38] L. Vermeulen, M. Todaro, F. De Sousa Mello et al., “Single- cation and expansion of human colon-cancer-initiating cells,” cell cloning of colon cancer stem cells reveals a multi-lineage Nature, vol. 445, no. 7123, pp. 111–115, 2007. differentiation capacity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 36, pp. [23] A. M. Friel, P. A. Sergent, C. Patnaude et al., “Functional 13427–13432, 2008. analyses of the cancer stem cell-like properties of human [39] B. L. K. Coles-Takabe, I. Brain, K. A. Purpura et al., endometrial tumor initiating cells,” Cell Cycle,vol. 7, no.2,pp. “Don’t look: growing clonal versus nonclonal neural stem cell 242–249, 2008. colonies,” Stem Cells, vol. 26, no. 11, pp. 2938–2944, 2008. [24] L. J. Harper, K. Piper, J. Common, F. Fortune, and I. C. [40] D. Corbeil, K. Rop ¨ er, A. Hellwig et al., “The human AC133 Mackenzie, “Stem cell patterns in cell lines derived from head hematopoietic stem cell antigen is also expressed in epithelial and neck squamous cell carcinoma,” Journal of Oral Pathology cells and targeted to plasma membrane protrusions,” Journal and Medicine, vol. 36, no. 10, pp. 594–603, 2007. of Biological Chemistry, vol. 275, no. 8, pp. 5512–5520, 2000. [25] M. Locke, M. Heywood, S. Fawell, and I. C. Mackenzie, [41] M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, “Retention of intrinsic stem cell hierarchies in carcinoma- and M. F. Clarke, “Prospective identification of tumorigenic derivedcelllines,” Cancer Research, vol. 65, no. 19, pp. 8944– breast cancer cells,” Proceedings of the National Academy of 8950, 2005. Sciences of the United States of America, vol. 100, no. 7, pp. [26] M. E. Prince, R. Sivanandan, A. Kaczorowski et al., “Iden- 3983–3988, 2003. tification of a subpopulation of cells with cancer stem cell [42] S. A. Mani, W. Guo, M. J. Liao et al., “The epithelial- properties in head and neck squamous cell carcinoma,” mesenchymal transition generates cells with properties of stem Proceedings of the National Academy of Sciences of the United cells,” Cell, vol. 133, no. 4, pp. 704–715, 2008. States of America, vol. 104, no. 3, pp. 973–978, 2007. [43] M. A. Goodell, K. Brose, G. Paradis, A. S. Conner, and R. [27] S. Gou, T. Liu, C. Wang et al., “Establishment of clonal colony- C. Mulligan, “Isolation and functional properties of murine forming assay for propagation of pancreatic cancer cells with hematopoietic stem cells that are replicating in vivo,” Journal stem cell properties,” Pancreas, vol. 34, no. 4, pp. 429–435, of Experimental Medicine, vol. 183, no. 4, pp. 1797–1806, 1996. [44] S. K. Addla, M. D. Brown, C. A. Hart,V.A.C.Ramani, [28] M. D. Brown, P. E. Gilmore, C. A. Hart et al., “Characterization and N. W. Clarke, “Characterization of the Hoechst 33342 of benign and malignant prostate epithelial Hoechst 33342 side population from normal and malignant human renal side populations,” Prostate, vol. 67, no. 13, pp. 1384–1396, epithelial cells,” American Journal of Physiology, vol. 295, no. 3, pp. F680–F687, 2008. [29] L. Patrawala, T. Calhoun, R. Schneider-Broussard, J. Zhou, [45] M. Christgen, M. Ballmaier, H. Bruchhardt, R. Wasielewski, K. Claypool, and D. G. Tang, “Side population is enriched H. Kreipe, and U. Lehmann, “Identification of a distinct in tumorigenic, stem-like cancer cells, whereas ABCG2 side population of cancer cells in the Cal-51 human breast and ABCG2 cancer cells are similarly tumorigenic,” Cancer carcinoma cell line,” Molecular and Cellular Biochemistry, vol. Research, vol. 65, no. 14, pp. 6207–6219, 2005. 306, no. 1-2, pp. 201–212, 2007. [30] A. B. Alvero, R. Chen, H. H. Fu et al., “Molecular phenotyping [46] M. A. Harris, H. Yang, B. E. Low et al., “Cancer stem cells of human ovarian cancer stem cells unravel the mechanisms are enriched in the side population cells in a mouse model forrepairand chemo-resistance,” Cell Cycle,vol. 8, no.1,pp. of glioma,” Cancer Research, vol. 68, no. 24, pp. 10051–10059, 158–166, 2009. [31] T. Baba,P.A.Convery,N.Matsumura et al., “Epigenetic [47] P. C. Hermann, S. L. Huber, T. Herrler et al., “Distinct regulation of CD133 and tumorigenicity of CD133+ ovarian populations of cancer stem cells determine tumor growth and cancer cells,” Oncogene, vol. 28, no. 2, pp. 209–218, 2009. metastatic activity in human pancreatic cancer,” Cell Stem Cell, [32] S. A. Bapat, A. M. Mali, C. B. Koppikar, and N. K. Kurrey, vol. 1, no. 3, pp. 313–323, 2007. “Stem and progenitor-like cells contribute to the aggressive [48] M. Kim, H. Turnquist, J. Jackson et al., “The multidrug resis- behavior of human epithelial ovarian cancer,” Cancer Research, tance transporter ABCG2 (breast cancer resistance protein 1) vol. 65, no. 8, pp. 3025–3029, 2005. effluxes Hoechst 33342 and is overexpressed in hematopoietic [33] M. D. Curley,V.A.Therrien,C.L.Cummingsetal., “CD133 stem cells,” Clinical Cancer Research, vol. 8, no. 1, pp. 22–28, expression defines a tumor initiating cell population in primary human ovarian cancer,” Stem Cells, vol. 27, no. 12, [49] M. D. Brown, P. E. Gilmore, C. A. Hart et al., “Characterization pp. 2875–2883, 2009. of benign and malignant prostate epithelial Hoechst 33342 [34] G. Ferrandina, G. Bonanno, L. Pierelli et al., “Expression side populations,” Prostate, vol. 67, no. 13, pp. 1384–1396, of CD133-1 and CD133-2 in ovarian cancer,” International Journal of Gynecological Cancer, vol. 18, no. 3, pp. 506–514, [50] P. P. Szotek, R. Pieretti-Vanmarcke, P. T. Masiakos et al., “Ovarian cancer side population defines cells with stem cell- [35] M. D. Curley,V.A.Therrien,C.L.Cummingsetal., “CD133 like characteristics and Mullerian inhibiting substance respon- expression defines a tumor initiating cell population in siveness,” Proceedings of the National Academy of Sciences of the primary human ovarian cancer,” Stem Cells, vol. 27, no. 12, United States of America, vol. 103, no. 30, pp. 11154–11159, pp. 2875–2883, 2009. [36] L. Moserle, S. Indraccolo, M. Ghisi et al., “The side population [51] Y. Zhong, C. Zhou, W. Ma et al., “Most MCF7 and SK-OV3 of ovarian cancer cells is a primary target of IFN-α antitumor cells were deprived of their stem nature by Hoechst 33342,” effects,” Cancer Research, vol. 68, no. 14, pp. 5658–5668, 2008. Biochemical and Biophysical Research Communications, vol. 364, no. 2, pp. 338–343, 2007. [37] S. Zhang, C. Balch, M. W. Chan et al., “Identification and characterization of ovarian cancer-initiating cells from [52] E. H. Huang, M. J. Hynes, T. Zhang et al., “Aldehyde dehydrogenase 1 is a marker for normal and malignant human primary human tumors,” Cancer Research, vol. 68, no. 11, pp. 4311–4320, 2008. colonic stem cells (SC) and tracks SC overpopulation during Journal of Oncology 9 colon tumorigenesis,” Cancer Research, vol. 69, no. 8, pp. 3382–3389, 2009. [53] D. A. Hess, L. Wirthlin, T. P. Craft et al., “Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells,” Blood, vol. 107, no. 5, pp. 2162–2169, 2006. [54] C. Ginestier, M. H. Hur, E. Charafe-Jauffret et al., “ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome,” Cell Stem Cell, vol. 1, no. 5, pp. 555–567, 2007. [55] J. E. Carpentino,M.J.Hynes,H.D.Appelmanetal., “Alde- hyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer,” Cancer Research, vol. 69, no. 20, pp. 8208–8215, 2009. [56] V. Levina, A. M. Marrangoni, R. DeMarco, E. Gorelik, and A. E. Lokshin, “Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties,” PLoS One, vol. 3, no. 8, Article ID e3077, 2008. [57] G. D. Richardson, C. N. Robson, S. H. Lang, D. E. Neal, N. J. Maitland, and A. T. Collins, “CD133, a novel marker for human prostatic epithelial stem cells,” Journal of Cell Science, vol. 117, no. 16, pp. 3539–3545, 2004. [58] H. Immervoll, D. Hoem, P. Sakariassen, O. J. Steffensen, and A. Molven, “Expression of the ”stem cell marker” CD133 in pancreas and pancreatic ductal adenocarcinomas,” BMC Cancer, vol. 8, article 48, 2008. 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Cancer Stem Cells and Epithelial Ovarian Cancer

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Copyright © 2010 Sheetal Dyall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2010/105269
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Abstract

Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 105269, 9 pages doi:10.1155/2010/105269 Review Article 1 1 1, 2 Sheetal Dyall, Simon A. Gayther, and Dimitra Dafou Gynaecological Cancer Research Laboratory, EGA UCL Institute for Women’s Health, London WC1E 6DD, UK King’s College School of Medicine, Guy’s Hospital, London SE1 9RT, UK Correspondence should be addressed to Sheetal Dyall, s.dyall@ucl.ac.uk Received 15 June 2010; Accepted 9 November 2010 Academic Editor: Peter E. Schwartz Copyright © 2010 Sheetal Dyall et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The cancer stem cell hypothesis is becoming more widely accepted as a model for carcinogenesis. Tumours are heterogeneous both at the molecular and cellular level, containing a small population of cells that possess highly tumourigenic “stem-cell” properties. Cancer stem cells (CSCs), or tumour-initiating cells, have the ability to self-renew, generate xenografts reminiscent of the primary tumour that they were derived from, and are chemoresistant. The characterisation of the CSC population within a tumour that drives its growth could provide novel target therapeutics against these cells specifically, eradicating the cancer completely. There have been several reports describing the isolation of putative cancer stem cell populations in several cancers; however, no defined set of markers has been identified that conclusively characterises “stem-like” cancer cells. This paper highlights the current experimental approaches that have been used in the field and discusses their limitations, with specific emphasis on the identification and characterisation of the CSC population in epithelial ovarian cancer. 1. Introduction represent suitable targets for new therapeutic approaches for epithelial ovarian cancers (EOC). Ovarian cancer (OC) is the sixth most lethal malignancy The mechanisms underlying chemoresistance in cancer in women in the western world. Over 90% of malignant are not clear. One hypothesis is that cancers are driven by tumours are epithelial. It has been hypothesised that tumours a subset of highly tumourigenic cells with stem cell properties can arise either from a single layer of cells covering the ovary within the tumour, cancer stem cells (CSCs). The term CSC (the ovarian surface epithelium or OSE) or from the epithe- is not meant to suggest that these cells have any association lial lining of the fimbrial end of the fallopian tube [1]. The with adult tissue stem cells; more recently, the term cancer aetiology of OC remains poorly understood. One proposed or tumour initiating cells (CIC or TIC) has come to be model is the incessant ovulation hypothesis, which postulates thought of as more appropriate. As the term CSC is most that continuous rupture of the OSE during ovulation and commonly used in the literature to describe these cells, for subsequent cell proliferation leading to repair make OSE cells the purpose of this paper, tumourigenic cancer cells with more susceptible to malignant transformation. stem cell properties will be referred to as CSC. According to this model, only the CSCs, but not the remaining cells in Approximately 70% of patients diagnosed with ovarian the tumour, can propagate tumourigenesis. CSCs have been cancer have advanced stage disease, partly because symp- implicated in tumour initiation, progression, metastasis, and toms are vague and can be confused with gastrointestinal drug resistance. complaints (e.g., bloating, constipation and mild abdominal pain) [2, 3]. Despite improvements in debulking surgery and initial good responses to platinum-based chemotherapies, 2. Cancer Stem Cells survival rates for the disease remain poor due to the devel- opment of chemoresistant disease, and less than 60% of A recent AACR workshop defined the CSC as a malignant cases survive more than 5 years. Thus, the identification cancer cell with a stem cell phenotype [4]. Whilst the CSC of molecular markers that target chemoresistance may hypothesis does not specifically address the mechanisms of 2 Journal of Oncology + − malignant transformation, it has been suggested that CSCs leukemia [13], in which the population of CD34 CD38 are the malignant counterparts of normal adult tissue stem cells isolated and transplanted into SCID mice formed cells, which, due to dysregulated signalling pathways, are tumours that had similar features to human leukemia, unable to maintain stem cell homeostasis. As is the case with whilst populations containing cells with different cell surface tissue stem cells, CSCs are thought to reside at the top of the marker profiles did not. Since then, several studies have lineage hierarchy and give rise to differentiated cells, which characterised the CSC in other types of tumours (Table 1). themselves have no potential for self-renewal, and therefore Classically, stem cells are defined by their two main char- do not contribute significantly to tumour growth. acteristics: self-renewal and pluripotency. And so by infer- The idea that tissue stem cells are the underlying cells for ence, to be classified as a CSC, a cell must meet the following carcinogenesis is attractive. Due to their long lifespan, stem criteria: (i) the ability of self-renewal and differentiation into cellsremaininatissue forlongerperiods of time compared different cell types, (ii) the ability to propagate tumours to their differentiated progeny, thereby making them more in vivo, (iii) the expression of distinct markers that permit likely to acquire transforming mutations. Additionally, it is consistent isolation, and (iv) restriction to a minority of the generally accepted that stem cells are more resistant to cell population (although this latter is now debatable, due to apoptosis and DNA damage and are therefore more likely to recent evidence showing that the microenvironment of mice survive any insults [5, 6]. Whilst being quiescent in normal alters the tumour-forming efficiency of engrafted cells) [14]. tissue, stem cells are able to maintain the stem cell pool by Table 1 summarises the various techniques and experi- undergoing asymmetric cell division during processes such mental approaches that have been used to isolate the putative as tissue damage. During this process, a stem cell divides CSC component from primary tumours. The approaches asymmetrically to generate an identical daughter cell (i.e., used rely on the intrinsic and functional properties of stem another stem cell) that is committed to differentiation. Adult cells. The most commonly used approach is the tumour stem cell cangiverisetoawide rangeofdifferentiated cells sphere assay, which evaluates the ability of cells to grow and it has been suggested that CSCs undergo asymmetric cell as nonadherent spheroids under non-differentiating con- division to generate the different cell types within a tumour, ditions, and cell surface antigen profile of subpopulations thereby contributing to tumour heterogeneity often seen in found within tumours. The dye exclusion assay enables the many cancers. The stem cell pool is also tightly regulated by isolation of a side population of cells that extrude lipophilic signalling pathways from the microenvironment of the stem dyes due to the overexpression of drug efflux pumps; the cell niche, and several of these pathways, including Hedgehog enzyme aldehyde dehydrogenase 1 is thought to be prefer- [7], and Wnt [8], have been implicated in carcinogenesis. entially active in stem-like cells. The tumourigenicity of the The CSC hypothesis suggests a paradigm shift in the putative CSC component is then verified by engraftment into understanding of carcinogenesis and tumour cell biology. immunosuppressed mice. The aim is to show that a minimal This may have fundamental implications in therapeutic number of stem-like cells can give rise to tumours of the interventions, including an explanation for the development same histopathology as the original primary tumours that of chemoresistance. A role for CSCs in propagating and they have been isolated from. maintaining metastasis has been proposed [9, 10]. Many features of tumours can be explained by the inherent char- acteristics of stem cells; for example, tumours selfrenew and 2.1. Assaying Cancer Stem Cells heterogeneity can be attributed to multilineage differentia- 2.1.1. The Tumour Sphere Assay. Whilst there are no estab- tion. Dontu et al. [11] have proposed that breast cancers lished protocols for isolating pure populations of CSCs, the may arise from mammary stem cells, or early progenitor general aim of many studies has been to enrich for cell cells. It has also been shown that hypermethylation at populations that show features that are characteristic of Polycomb promoter regions lock stem cells in a state of normal stem cells, primarily the ability to self-renew and self-renewal, which can lead to aberrant clonal expansion, differentiate into different lineages. The tumourigenicity of thus leading to cancer predisposition [12]. Even so, the the isolated cell populations is then confirmed by assessing contrasting hypothesis that the dedifferentiation of mature the tumour-forming potential of these cells in appropriate cells to a more pluripotent state as a potential mechanism mice models. A commonly used approach for CSC enrich- for the development of stem cell-like features by cancer cells ment is the tumour sphere assay, which evaluates the ability cannot be dismissed. of cancer cells to grow as multicellular spheroids under non- Cancer cells with stem cell properties have been identified differentiating and non-adherent conditions, a characteristic in acute myeloid leukemia and several solid malignancies believed to be indicative of self-renewal. (Table 1). Whilst the fact that tissue stem cells are the target of transformation may be true for certain malignancies, this has In a typical tumour sphere assay, cells from a primary yet to be proven as a universal mechanism of tumorigenesis. tumour or cancer cell line are dissociated into a single cell Consequently, the terms cancer and tumour-initiating cells suspension and cultured in a serum-free growth factor- are thought to be more appropriate as they define cells with rich medium containing primarily epidermal growth factor tumour-initiating potential, without any reference to an (EGF) and fibroblast growth factor (FGF). Multicellular origin from tissue stem cells. spheroids of enriched CSCs that form can undergo serial The first strong evidence for the existence of cancer stem dissociation and passaging to further enrich for stem cell-like cells in human tumours came from a study in acute myeloid cells. Journal of Oncology 3 Table 1: Studies that have attempted to characterise the cancer stem cell (CSC) component in the cancers are listed. Only the most relevant studies are referenced. The different experimental techniques used in the referenced studies are reviewed; this highlights the lack of consensus in the approach that can be best used to identify stem-like cells in cancers. In vitro characterization Cell surface In vivo Cancer type References b c d e antigen profile characterisation Dye exclusion assay Tumour sphere assay Clonality assays ALDH assay H 33342 (5 μg/mL) 2 Colony forming unit assay: SFM hours, 37 C; number of colonies from + − Leukaemia CD34 /CD38 Not done SCID [15, 16] 20 ng/mL Interleukin-3 Inhibitor KO143 leukemic SP and haematopoetic (200 nM) 100 ng/mL stem cell factor SP compared per 10 cells plated SFM H 33342 (5 μg/mL) 90 20 ng/mL EGF, minutes at 37 C Primary spheres dissociated and Brain CD133 Inhibitors plated at cell dilutions from Not done NOD-SCID [17, 18] 20 ng/mL bFGF Verapamil (50 μM) or 200 cells/well to 1 cell. 10 ng/mL LIF reserpine (100 μM). 60 μg/mL N-acetylcysteine SFM + [−]/low CD44 CD24 H33342 (5 μg/mL) for 10 ng/mL bFGF Single cells (1 cells per well in ALDEFLUOR- + ◦ Breast OCT4 90 min, 37 C NOD-SCID [19, 20] 20 ng/mL EGF 96-well plate) positive CD133 Inhibitor FTC 10 μM 5 μg/mL insulin 0.4% BSA SFM H33342 (5 μg/mL) for 0.6% glucose, 1.5–2 hours, 37 C, Single-cell derived tumour 9.6 μg/mL putrescine, Inhibitor verapamil at spheres (unsorted and CD133 , Colon CD133 Not done NOD-SCID [21, 22] 6.3 ng/mL progesterone 50 μMfor 10 minutesat either spheroid-derived or room temperature primary) ITS before adding H33342 20 ng/mL EGF 10 ng/mL bFGF H33342 (5 μg/mL) for 90 minutes, 37 C NOD-SCID Endometrial —— Notdone [23] Inhibitor verapamil (50 μM) 4 Journal of Oncology Table 1: Continued. Cell surface In vitro characterization In vivo Cancer type References antigen profile characterisation b c d e Dye exclusion assay Tumour sphere assay Clonality assays ALDH assay H33342 5 μg/mL at varying time points, CD44 NOD-SCID Head and ◦ + − 37 C; SCM Single cells GFP and GFP Not done [24–26] neck Inhibitor reserpine (5 μg/mL) SFM 10 ng/mL FGF 1 cell/well in SFM by limiting + 20 ng/mL EGF Pancreas CD133 — Not done Nude mice [27] dilution 5 μg/mL insulin ITS 0.4% BSA SFM H33342 (5 μM) for 90 Single cells were plated at + hi minutes, 37 C; CD44 , α β , B27 Prostate 1,000/mL on a plate coated with Not done SCID [9, 28, 29] Inhibitor verapamil CD133 10 ng/mL EGF 0.5% agar (50 μM) 10 ng/mL bFGF SFM H33342 (5 μM) for 90 5 μg/mL insulin + ◦ CD133 minutes, 37 C; Ovary — Not done BALB/c-nu/nu [30–37] + + 20 ng/mL EGF CD44 CD117 Inhibitor verapamil (50 μM) 10 ng/mL bFGF 0.4% BSA Cell surface antigen profile expression by flow cytometry and immunohistochemistry techniques Dye Exclusion flow cytometry assays following treatment with Hoechst, coupled with ABC multidrug transporter inhibitors to isolate side population cells Sphere formation assays for multicellular spheroid forming ability under nonadherent conditions, which is characteristic of stem cells Clonality assays measure the ability of single cells to generate spheroids/clones Aldehyde Dehydrogenase assays measure the levels of ALDH1 in cells Soft agar assay measures anchorage-independent growth due to loss of contact-inhibition and is an indicator of tumourigenicity. EGF: Epidermal growth factor bFGF: Basic fibroblast growth factor LIF: Leukemia inhibitory factor NSF: Neuronal survival factor BPE: Bovine pituitary extract KO143: Inhibitor of breast cancer resistant protein (BRCP) SFM: Serum-free media SCM: Serum-containing media. Journal of Oncology 5 Although this is one of the most commonly used tech- overexpress ATP-binding cassette (ABC) transporter proteins niques to evaluate self-renewal, the results from such assays on their cell surface, thus contributing to the intrinsic resis- can be misleading. The characteristic of self-renewal can only tance of stem cells to chemotherapy and radiotherapy. This be inferred from the presence of tumour spheres in culture characteristic has been implicated in multidrug resistance in if it can be shown that each tumour sphere has arisen from cancers, as overexpression of multi-drug resistant proteins a single cell under proliferative nonadherent conditions; yet, enables cancer cells to remove chemotherapeutic drugs from there are few published studies that have actually demon- their cytoplasm. strated this [38]. A detailed analysis of the methodologies When stained with the fluorescent dyes, cells overexpress- used in many studies shows that starting cell densities in ing the ABC transporter proteins will eliminate the dye from the tumour sphere assay are fairly high (e.g., 1000 cells/mL). their cytoplasm, thus producing a lower signal than cells These studies aim to utilise single cell suspensions, but the not overexpressing ABC proteins. By combining fluorescent high cell densities do not preclude the possibility that the staining with fluorescent-activated cell sorting (FACS) in spheroids that form may have arisen from the aggregation a cell population, a side population of cells (SP) can be of mixed cell population and therefore would not be clonal. isolated that is enriched for stem “like” cells. As a control for For neurospheres, it has been shown that spheroids are clonal the SP phenotype, cells are stained in the presence of ABC only when cells are plated at low density [39]. transporter inhibitors, which reverse the SP phenotype. The most commonly used inhibitor is verapamil, which targets P-glycoprotein encoded by the gene MDR1. A less frequently 2.1.2. Cell Surface Specific Antigen Profile. Several studies used inhibitor is fumitremorgin C, which specifically targets have prospectively isolated CSCs by looking for the presence the BCRP transporter (encoded by ABCG2). Cells that retain of extracellular markers that are thought to be stem cell- Hoechst are thought to contain terminally-differentiated specific. The markers most commonly used are CD133 and cells that are unable to self-renew; thus this is a marker CD44. CD133, a penta-membrane glycoprotein, has been commonly used for identifying the non-SP fraction of a cell found to interact with cholesterol and may be involved population. in maintaining membrane topology and membrane lipid SP analysis, which was originally used to identify composition. Its expression has been shown to be restricted haematopoietic stem cells from murine bone marrow [43], to plasma membrane protrusions in epithelial cells [40]. It is has been used to isolate putative CSCs in several studies also thought to be a marker for neuroepithelial progenitor in primary tumours and cell lines [19, 28, 29, 36, 44–50]. cells and in haematopoietic progenitor cells. CD133 was Reviewing the published literatures suggests that the per- originally identified as a neural stem cell marker. It has been centage of SP cells that can be isolated from whole cell used to characterise CSCs in brain tumours [17]aswell line populations can range from 0%–20%. However, in the as several other cancers, including, prostate, colon, ovary, majority of studies, the SP cells form a minority (<5%) of and pancreas (Table 1). In breast cancer, CSCs have been + − the bulk cell population, supporting the idea that CSCs, like characterised as CD44 and CD24 [41]. The limitation of stem cells, constitute a small proportion of cell cultures. using cell surface marker expression to characterise CSCs is that the approach requires prior knowledge of cell surface SP analysis provides a rapid method for isolating stem markers that are expressed by the putative CSCs in the tissue cell-like cells; it relies on the functional characteristic of of interest, and often the choice of markers is inferred from stem cells and requires no prior knowledge of extracellular known expression of markers in normal adult stem cells. markers. Nevertheless, the use of Hoechst as a method of Whilst CD133 and CD44 are thought to be indicative enriching for CSC has received criticism. It has been reported of a CSC phenotype, it is not clear if they are universal that Hoechst treatment reduces the clonogenicity of the markers for characterising CSCs derived from all types of breast cancer cell line MCF7 and the ovarian cancer cell line tumours. Furthermore, the expression of CD133 and CD44 SKOV3 due to a loss in viability [51]. In this study, single may not be restricted to the CSC population and may be cells from unsorted cell populations, when grown in serum- present in early progenitor cells. Their functions are largely containing media under adherent conditions, generated unknown, although there are reports implicating CD44 in a clone that would satisfy the criteria for self-renewal. metastasis and chemoresistance. Recent evidence suggests Additionally, the same group reported that both SP and non- that CD133 is expressed in epithelial cells and may be SP cells generated clones in vitro and non-SP cells were able involved in maintaining pluripotency [40]. to form both SP and non-SP cells, suggesting that non-SP Epithelial-to-mesenchymal transition (EMT) is also cells can self-renew as well. a feature of carcinogenesis, as cells acquire a more mesenchy- mal phenotype during neoplastic transformation. A link 2.1.4. Aldehyde Dehydrogenase Expression. More recently, between CSC and EMT has been suggested, whereby trans- it has been suggested that an elevated level of aldehyde formed human mammary epithelial cells that have under- dehydrogenase 1 (ALDH1) activity can select for stem- gone EMT show a gain of the CSC phenotype [42]. like cells in both normal and malignant tissues. ALDH1 is a detoxifying enzyme that is involved in the metabolism 2.1.3. Hoechst 33342 Dye Exclusion Assay. Stem cells actively of vitamin A to retinoic acid in the liver. It has been efflux lipophilic compounds such as the fluorescence dyes suggested that retinoic acid signalling is a probable protective Hoechst 33342 and rhodamine from their cytoplasm, as they mechanism against oxidative damage in stem cells; recently, 6 Journal of Oncology levels of ALDH1 have been used as CSC markers in the described for CSCs by these studies, both studies reported isolation of putative CSC, and are thought to be more specific that the CSC phenotype was more resistant to platinum- to the stem cell phenotype [10, 52–55]. In the colon, it based therapy, which again, supports the idea that CSCs may has been shown that the expressions of CD44 and CD133, be responsible for chemoresistance. which have previously been used to isolate CSC populations, Generally, these studies highlight the lack of consensus are not restricted to the stem cell populations, which are about the molecular characteristics of ovarian CSCs. It is located at the base of colonic crypt, but also to populations of likely that the expression of markers overlaps, and both rapidly proliferating cells located further up along the crypt. CD133 and CD44 characterise the ovarian CSC. Alterna- The expression of ALDH1, however, is more limited to the tively, there may be more than one population of cells base of the crypt. with stem cell properties in ovarian cancers. The study by Bapat et al. [32] postulated that stem cells are the target of transformation in ovarian cancer, due to the fact that 2.1.5. In Vivo Tumourigenicity Assays. Following the identi- a few of the clones isolated from ovarian cancer ascites fication of the putative CSC population, cells are engrafted spontaneously immortalised in culture, suggesting a model into appropriate mice models to show that they are more for disease development. tumourigenic than their non-CSC counterparts. This is cur- rently believed to be the gold-standard in the field. Generally, a small number of CSCs and non-CSCs are transplanted 4. Issues with the Current into mice, and it is shown that CSCs have a higher tumour- Experimental Approaches forming capacity than non-CSCs. As described above, the experimental approaches used to identify and characterise populations of putative CSC can be 3. Cancer Stem Cells and Ovarian Cancer classed in three major categories: (1) their isolation based on Several characteristics of CSCs have been implicated in the expression of extracellular markers, (2) their growth in chemoresistance and radiotherapy. Due to the overexpres- tumour sphere assays under non-differentiating conditions, sion of extracellular multi-drug transport proteins, CSCs are and (3) dye exclusion due to the overexpression of drug efflux able to efflux chemotherapeutic drugs from their cytoplasm. pumps. Additionally, chemotherapeutic treatment is thought Stem cells, and possibly CSCs, are inherently quiescent; to enrich for CSCs; thus, when exposed to chemotherapy however, current drugs target rapidly cycling cells. Thus, the drugs, CSCs are selected for [56]. It is not clear whether hypothesis is that whilst the majority of cells in the bulk of chemoresistance is an inherent feature of CSCs or whether the tumour are eliminated, CSCs evade chemotherapy and they acquire this property throughout tumorigenesis. are able to reinitiate tumour development. The most common approach that has been used to isolate There is still uncertainty relating to the identification CSCs is to select for cells by the expression of extracellular of CSCs in ovarian cancer. Several papers have described markers that may be associated with stemness. However, the isolation of ovarian CSCs using different approaches. the rationale underlying the selection of markers used for The first study to be published identified clones established this purpose is not always apparent. Although CD44 and from tumour ascites that were able to form anchorage- CD133 are the two most commonly used markers, it is independent spheroids and were shown to express the stem unclear whether or not they are ubiquitous. CD133 has cell markers Oct 3/4 and Nanog and the progenitor marker been extensively used to isolate CSCs in cancers of the brain Nestin [32]. Using flow cytometry, Ferrandina et al. [34] [18], prostate [57], pancreas [58], and ovary [31, 34]; but reported that two isoforms of CD133 (isoforms 1 and 2) whilst it has been successfully used to isolate brain tumour were both expressed in human ovarian tumours at a higher stem cells, the use of CD133 in defining CSCs in colon and frequency than in normal ovaries and metastatic omental ovarian cancer has been more controversial. In the study by lesions [34]. Szotek et al. [50] used flow cytometry to Ferrandina et al., CD133 was suggested to be a marker for isolate a side population of cells from genetically engineered ovarian cancer and was also used as an ovarian CSC marker mouse ovarian cancer cell lines that expressed the multi- in the study by Baba et al. [31]. Zhang et al. on the other hand + + drug transporter protein BCRP1 and were insensitive to have suggested that CD44 CD117 phenotype is indicative doxorubicin, suggesting a possible link between CSCs and of the ovarian CSC phenotype. In all of these studies, the chemoresistance. They also isolated a similar, although much characteristics of the stem cell phenotype were confirmed smaller, side population of cells from the human ovarian by in vivo models and spheroid forming assays. It is likely cancer cell lines IGROV-1, OVCAR3, and SKOV3, but these that both CD133 and CD44 are markers for stem cell-like SP cells were not further characterised. cells in epithelial OCs but there are no existing studies that Two other studies have independently defined the ovar- have compared the expression of CD44 and CD133 in EOCs. + + ian cancer stem cell by evaluating the CD44 CD117 [37] It is possible that both markers are expressed by the same and CD133 [31] phenotypes. The latter suggests an epige- population of ovarian CSC; alternatively, they may be specific netic regulation of the CD133 promoter. Additionally, using to different populations of CSCs, or there may be other as yet CD44, stem-like cells were enriched from patients’ samples unidentified markers that are ubiquitous markers for ovarian and were characterised by Myd88 expression and chemokine CSCs. Several studies have shown that ovarian cancers are and cytokine production [30]. Despite the different profiles extremely heterogeneous, and this may suggest that different Journal of Oncology 7 populations of CSC can be responsible for tumourigenicity [6] S. Bao, Q. Wu, R. E. McLendon et al., “Glioma stem cells promote radioresistance by preferential activation of the DNA in different histological subtypes. damage response,” Nature, vol. 444, no. 7120, pp. 756–760, Another issue raised by the published literature is that caution needs to be applied when interpreting the results [7] S. Liu, G. Dontu, I. D. Mantle et al., “Hedgehog signaling and from tumour sphere assays. It is only possible to infer self- Bmi-1 regulate self-renewal of normal and malignant human renewal if it has conclusively been shown that multicellular mammary stem cells,” Cancer Research, vol. 66, no. 12, pp. spheroids arise from single cells. Furthermore, it is important 6063–6071, 2006. to show that single cells isolated from such spheroids can [8] H. Korkaya, A. Paulson, E. Charafe-Jauffret et al., “Regulation themselves generate spheroids through serial passaging. of mammary stem/progenitor cells by PTEN/Akt/β-catenin Despite the amount of literature on CSCs, it is still not signaling,” PLoS Biology, vol. 7, no. 6, Article ID e1000121, clear as to what constitutes a universal CSC-specific profile. The definition of the CSC hypothesis is constantly evolving [9] J. Miki, B. Furusato, H. Li et al., “Identification of puta- to specifically address the mechanism of neoplastic transfor- tive stem cell markers, CD133 and CXCR4, in hTERT- immortalized primary nonmalignant and malignant tumor- mation in different tumour types. It is becoming increasingly derived human prostate epithelial cell lines and in prostate evident that there may be more than one population of CSC cancer specimens,” Cancer Research, vol. 67, no. 7, pp. 3153– within a tumour. In addition, the cell of origin of CSCs 3161, 2007. is not evident, as CSCs can arise from either stem cells or [10] E. Charafe-Jauffret, C. Ginestier, F. Iovino et al., “Breast cancer progenitor cells. To be a true CSC, all the established criteria cell lines contain functional cancer stem sells with metastatic that characterise the stem cell properties of a cancer cell need capacity and a distinct molecular signature,” Cancer Research, to be satisfied. vol. 69, no. 4, pp. 1302–1313, 2009. [11] G. Dontu, W. M. Abdallah, J. M. Foley et al., “In vitro prop- 5. Concluding Remarks agation and transcriptional profiling of human mammary stem/progenitor cells,” Genes and Development, vol. 17, no. 10, Ovarian cancer is a heterogeneous disease with histologically pp. 1253–1270, 2003. defined subtypes, and it is highly probable that CSCs [12] M. Widschwendter, H. Fiegl, D. Egle et al., “Epigenetic stem cell signature in cancer,” Nature Genetics,vol. 39, no.2,pp. are involved in tumour development. Despite the number 157–158, 2007. of studies attempting to isolate ovarian CSCs, a well- [13] J. E. Dick, M. Bhatia, O. Gan, and U. Kapp, “Assay of human characterised profile has not been established. It is essential stem cells by repopulation of NOD/SCID mice,” Stem Cells, that further studies are carefully designed to address all vol. 15, supplement 1, pp. 199–207, 1997. the functional characteristics of stem cells covering: (i) self- [14] E. Quintana, M. Shackleton, M. S. Sabel, D. R. Fullen, T. M. renewal, where a single CSC is shown to form multicellular Johnson, and S. J. Morrison, “Efficient tumour formation by spheroid in vitro and (ii) multilineage differentiation such single human melanoma cells,” Nature, vol. 456, no. 7222, pp. that only the putative CSC would divide asymmetrically to 593–598, 2008. generate several cell types. The tumourigenicity of these cells [15] D. Bonnet and J. E. Dick, “Human acute myeloid leukemia thenneedstobevalidatedinanappropriate in vivo model is organized as a hierarchy that originates from a primitive that is permissible to tumour formation. The development hematopoietic cell,” Nature Medicine, vol. 3, no. 7, pp. 730– of chemoresistant disease represents a major hindrance to 737, 1997. successful ovarian cancer treatment, and the identification [16] B. Moshaver, A. Van Rhenen, A. Kelder et al., “Identification of of a molecular profile of ovarian CSC may aid to the a small subpopulation of candidate leukemia-initiating cells in development of more effective targeted therapy. the side population of patients with acute myeloid leukemia,” Stem Cells, vol. 26, no. 12, pp. 3059–3067, 2008. [17] S. K. Singh, I. D. Clarke, M. Terasaki et al., “Identification of References a cancer stem cell in human brain tumors,” Cancer Research, vol. 63, no. 18, pp. 5821–5828, 2003. [1] L. Dubeau, “The cell of origin of ovarian epithelial tumours,” [18] S. K. Singh, C. Hawkins, I. D. Clarke et al., “Identification The Lancet Oncology, vol. 9, no. 12, pp. 1191–1197, 2008. of human brain tumour initiating cells,” Nature, vol. 432, no. [2] C.Wikborn,F.Pettersson, andP.J.Moberg, “Delay in 7015, pp. 396–401, 2004. diagnosis of epithelial ovarian cancer,” International Journal of [19] K. Engelmann, H. Shen, and O. J. Finn, “MCF7 side popula- Gynecology and Obstetrics, vol. 52, no. 3, pp. 263–267, 1996. tion cells with characteristics of cancer stem/progenitor cells [3] B. D. Rufford, I. J. Jacobs, and U. Menon, “Feasibility of express the tumor antigen MUC1,” Cancer Research, vol. 68, screening for ovarian cancer using symptoms as selection no. 7, pp. 2419–2426, 2008. criteria,” British Journal of Obstetrics and Gynaecology, vol. 114, no. 1, pp. 59–64, 2007. [20] D. Ponti, A. Costa, N. Zaffaroni et al., “Isolation and in vitro propagation of tumorigenic breast cancer cells with [4] M.F.Clarke, J. E. Dick,P.B.Dirks et al., “Cancerstemcells— stem/progenitor cell properties,” Cancer Research, vol. 65, no. perspectives on current status and future directions: AACR 13, pp. 5506–5511, 2005. workshop on cancer stem cells,” Cancer Research, vol. 66, no. 19, pp. 9339–9344, 2006. [21] R. T. Sussman,M.S.Ricci,L.S.Hart, Y. S. Shi, andW.S. [5] A. K. Croker and A. L. Allan, “Cancer stem cells: implications El-Deiry, “Chemotherapy-resistant side-population of colon for the progression and treatment of metastatic disease,” cancer cells has a higher sensitivity to TRAIL than the non- Journal of Cellular and Molecular Medicine, vol. 12, no. 2, pp. SP, a higher expression of c-Myc and TRAIL-receptor DR4,” 374–390, 2008. Cancer Biology and Therapy, vol. 6, no. 9, pp. 1490–1495, 2007. 8 Journal of Oncology [22] L. Ricci-Vitiani, D. G. Lombardi, E. Pilozzi et al., “Identifi- [38] L. Vermeulen, M. Todaro, F. De Sousa Mello et al., “Single- cation and expansion of human colon-cancer-initiating cells,” cell cloning of colon cancer stem cells reveals a multi-lineage Nature, vol. 445, no. 7123, pp. 111–115, 2007. differentiation capacity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 36, pp. [23] A. M. Friel, P. A. Sergent, C. Patnaude et al., “Functional 13427–13432, 2008. analyses of the cancer stem cell-like properties of human [39] B. L. K. Coles-Takabe, I. Brain, K. A. Purpura et al., endometrial tumor initiating cells,” Cell Cycle,vol. 7, no.2,pp. “Don’t look: growing clonal versus nonclonal neural stem cell 242–249, 2008. colonies,” Stem Cells, vol. 26, no. 11, pp. 2938–2944, 2008. [24] L. J. Harper, K. Piper, J. Common, F. Fortune, and I. C. [40] D. Corbeil, K. Rop ¨ er, A. Hellwig et al., “The human AC133 Mackenzie, “Stem cell patterns in cell lines derived from head hematopoietic stem cell antigen is also expressed in epithelial and neck squamous cell carcinoma,” Journal of Oral Pathology cells and targeted to plasma membrane protrusions,” Journal and Medicine, vol. 36, no. 10, pp. 594–603, 2007. of Biological Chemistry, vol. 275, no. 8, pp. 5512–5520, 2000. [25] M. Locke, M. Heywood, S. Fawell, and I. C. Mackenzie, [41] M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, “Retention of intrinsic stem cell hierarchies in carcinoma- and M. F. Clarke, “Prospective identification of tumorigenic derivedcelllines,” Cancer Research, vol. 65, no. 19, pp. 8944– breast cancer cells,” Proceedings of the National Academy of 8950, 2005. Sciences of the United States of America, vol. 100, no. 7, pp. [26] M. E. Prince, R. Sivanandan, A. Kaczorowski et al., “Iden- 3983–3988, 2003. tification of a subpopulation of cells with cancer stem cell [42] S. A. Mani, W. Guo, M. J. Liao et al., “The epithelial- properties in head and neck squamous cell carcinoma,” mesenchymal transition generates cells with properties of stem Proceedings of the National Academy of Sciences of the United cells,” Cell, vol. 133, no. 4, pp. 704–715, 2008. States of America, vol. 104, no. 3, pp. 973–978, 2007. [43] M. A. Goodell, K. Brose, G. Paradis, A. S. Conner, and R. [27] S. Gou, T. Liu, C. Wang et al., “Establishment of clonal colony- C. Mulligan, “Isolation and functional properties of murine forming assay for propagation of pancreatic cancer cells with hematopoietic stem cells that are replicating in vivo,” Journal stem cell properties,” Pancreas, vol. 34, no. 4, pp. 429–435, of Experimental Medicine, vol. 183, no. 4, pp. 1797–1806, 1996. [44] S. K. Addla, M. D. Brown, C. A. Hart,V.A.C.Ramani, [28] M. D. Brown, P. E. Gilmore, C. A. Hart et al., “Characterization and N. W. Clarke, “Characterization of the Hoechst 33342 of benign and malignant prostate epithelial Hoechst 33342 side population from normal and malignant human renal side populations,” Prostate, vol. 67, no. 13, pp. 1384–1396, epithelial cells,” American Journal of Physiology, vol. 295, no. 3, pp. F680–F687, 2008. [29] L. Patrawala, T. Calhoun, R. Schneider-Broussard, J. Zhou, [45] M. Christgen, M. Ballmaier, H. Bruchhardt, R. Wasielewski, K. Claypool, and D. G. Tang, “Side population is enriched H. Kreipe, and U. Lehmann, “Identification of a distinct in tumorigenic, stem-like cancer cells, whereas ABCG2 side population of cancer cells in the Cal-51 human breast and ABCG2 cancer cells are similarly tumorigenic,” Cancer carcinoma cell line,” Molecular and Cellular Biochemistry, vol. Research, vol. 65, no. 14, pp. 6207–6219, 2005. 306, no. 1-2, pp. 201–212, 2007. [30] A. B. Alvero, R. Chen, H. H. Fu et al., “Molecular phenotyping [46] M. A. Harris, H. Yang, B. E. Low et al., “Cancer stem cells of human ovarian cancer stem cells unravel the mechanisms are enriched in the side population cells in a mouse model forrepairand chemo-resistance,” Cell Cycle,vol. 8, no.1,pp. of glioma,” Cancer Research, vol. 68, no. 24, pp. 10051–10059, 158–166, 2009. [31] T. Baba,P.A.Convery,N.Matsumura et al., “Epigenetic [47] P. C. Hermann, S. L. Huber, T. Herrler et al., “Distinct regulation of CD133 and tumorigenicity of CD133+ ovarian populations of cancer stem cells determine tumor growth and cancer cells,” Oncogene, vol. 28, no. 2, pp. 209–218, 2009. metastatic activity in human pancreatic cancer,” Cell Stem Cell, [32] S. A. Bapat, A. M. Mali, C. B. Koppikar, and N. K. Kurrey, vol. 1, no. 3, pp. 313–323, 2007. “Stem and progenitor-like cells contribute to the aggressive [48] M. Kim, H. Turnquist, J. Jackson et al., “The multidrug resis- behavior of human epithelial ovarian cancer,” Cancer Research, tance transporter ABCG2 (breast cancer resistance protein 1) vol. 65, no. 8, pp. 3025–3029, 2005. effluxes Hoechst 33342 and is overexpressed in hematopoietic [33] M. D. Curley,V.A.Therrien,C.L.Cummingsetal., “CD133 stem cells,” Clinical Cancer Research, vol. 8, no. 1, pp. 22–28, expression defines a tumor initiating cell population in primary human ovarian cancer,” Stem Cells, vol. 27, no. 12, [49] M. D. Brown, P. E. Gilmore, C. A. Hart et al., “Characterization pp. 2875–2883, 2009. of benign and malignant prostate epithelial Hoechst 33342 [34] G. Ferrandina, G. Bonanno, L. Pierelli et al., “Expression side populations,” Prostate, vol. 67, no. 13, pp. 1384–1396, of CD133-1 and CD133-2 in ovarian cancer,” International Journal of Gynecological Cancer, vol. 18, no. 3, pp. 506–514, [50] P. P. Szotek, R. Pieretti-Vanmarcke, P. T. Masiakos et al., “Ovarian cancer side population defines cells with stem cell- [35] M. D. Curley,V.A.Therrien,C.L.Cummingsetal., “CD133 like characteristics and Mullerian inhibiting substance respon- expression defines a tumor initiating cell population in siveness,” Proceedings of the National Academy of Sciences of the primary human ovarian cancer,” Stem Cells, vol. 27, no. 12, United States of America, vol. 103, no. 30, pp. 11154–11159, pp. 2875–2883, 2009. [36] L. Moserle, S. Indraccolo, M. Ghisi et al., “The side population [51] Y. Zhong, C. Zhou, W. Ma et al., “Most MCF7 and SK-OV3 of ovarian cancer cells is a primary target of IFN-α antitumor cells were deprived of their stem nature by Hoechst 33342,” effects,” Cancer Research, vol. 68, no. 14, pp. 5658–5668, 2008. Biochemical and Biophysical Research Communications, vol. 364, no. 2, pp. 338–343, 2007. [37] S. Zhang, C. Balch, M. W. Chan et al., “Identification and characterization of ovarian cancer-initiating cells from [52] E. H. Huang, M. J. Hynes, T. Zhang et al., “Aldehyde dehydrogenase 1 is a marker for normal and malignant human primary human tumors,” Cancer Research, vol. 68, no. 11, pp. 4311–4320, 2008. colonic stem cells (SC) and tracks SC overpopulation during Journal of Oncology 9 colon tumorigenesis,” Cancer Research, vol. 69, no. 8, pp. 3382–3389, 2009. [53] D. A. Hess, L. Wirthlin, T. P. Craft et al., “Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells,” Blood, vol. 107, no. 5, pp. 2162–2169, 2006. [54] C. Ginestier, M. H. Hur, E. Charafe-Jauffret et al., “ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome,” Cell Stem Cell, vol. 1, no. 5, pp. 555–567, 2007. [55] J. E. Carpentino,M.J.Hynes,H.D.Appelmanetal., “Alde- hyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer,” Cancer Research, vol. 69, no. 20, pp. 8208–8215, 2009. [56] V. Levina, A. M. Marrangoni, R. DeMarco, E. Gorelik, and A. E. Lokshin, “Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties,” PLoS One, vol. 3, no. 8, Article ID e3077, 2008. [57] G. D. Richardson, C. N. Robson, S. H. Lang, D. E. Neal, N. J. Maitland, and A. T. Collins, “CD133, a novel marker for human prostatic epithelial stem cells,” Journal of Cell Science, vol. 117, no. 16, pp. 3539–3545, 2004. [58] H. Immervoll, D. Hoem, P. Sakariassen, O. J. Steffensen, and A. Molven, “Expression of the ”stem cell marker” CD133 in pancreas and pancreatic ductal adenocarcinomas,” BMC Cancer, vol. 8, article 48, 2008. 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