Journal of Pathology J Pathol 2010; 222: 335344 Published online 17 September 2010 in Wiley Online Library (wileyonlinelibrary.

com) DOI: 10.1002/path.2772

INVITED REVIEW

Finding cancer stem cells: are aldehyde dehydrogenases t for purpose?

Malcolm R Alison,1 * Naomi J Guppy,1 Susan ML Lim2 and Linda J Nicholson3
1 2 3

Blizard Institute of Cell and Molecular Science, Barts and London School of Medicine, London, UK Department of Orthopaedic Surgery, National University of Singapore, Singapore Division of Cancer Studies, Kings College London, London, UK

*Correspondence to: Professor Malcolm R Alison, Centre for Diabetes, Blizard Institute of Cell and Molecular Science, Queen Mary University of London, 4 Newark Street, London E1 4AT, UK e-mail: m.alison@qmul.ac.uk This review is largely based on a search of PubMed in the years 20092010 using the terms ALDH or ALDEFLUOR combined with cancer stem cells.

Abstract
Despite many years of intensive effort, there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison, the study of cancer stem cells is still in its infancy; so, unsurprisingly, there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal, clonogenicity, multipotentiality, adherence to the niche, and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs), probably signicant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules, and contributing to drug resistance. Antibodies are available against the ALDH enzyme family, but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identied by the ALDEFLUOR kit and sorted by uorescence activated cell sorting (FACS). For many human tumours, but notably breast cancer, cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodecient mice, and indeed the frequency of so-called ALDHbri cells in many tumours can be an independent prognostic indicator.
Copyright 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: ALDEFLUOR; aldehyde dehydrogenases; cancer stem cells; drug resistance; immunodecient mice; prognostic markers

Received 4 August 2010; Revised 20 August 2010; Accepted 24 August 2010

No conicts of interest were declared.

Introduction
We now believe that most tumours contain a subpopulation of malignant cells with stem cell properties; these stem-like cells are identied by their ability to give rise to new tumours when xenografted, usually in small numbers, into immunodecient micehence are often referred to as tumour-initiating cells (TICs) rather than cancer stem cells [1]. While the founder cell of many malignancies is likely to be a normal stem cell or a more lineage-committed progenitor cell (particularly for some leukaemias, reviewed in ref 1), it is currently unclear whether TICs are the direct descendants of transformed normal stem cells or whether they are derived from aberrant de-differentiation of more mature neoplastic cells at a later stage of cancer progression. In an expanding tumour, TICs are believed to self-renew and to give rise to a hierarchy of progenitor and differentiated cells (one cause of tumour heterogeneity), albeit in an unorthodox manner that gives rise to more TICs through symmetric divisions [2,3].
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Operationally, TICs are dened as a population of cells that can be prospectively isolated based on the expression of a specic molecule or combination of molecules (eg CD133, CD44, side population, ALDH) and that are more tumourigenic than the bulk unsorted tumour population. The gold standard tumourigenicity assay for human TICs is the transplantation of human cells into immunodecient mice that lack major elements of the immune system and therefore do not reject the human cells. Recipient mice that are used have mainly been nude and non-obese diabetic/severe combined immunodecient (NOD/SCID) mice, but more recently many investigators have turned to NOD/SCID/Il-2r [4] and Rag2/ c/ mice [5] as these mice lack B, T, and NK lymphocytes. Commonly, this in vivo assay is complemented by an ex vivo clonogenicity assay called a sphere-forming assay that measures the frequency with which these prospectively isolated cells form colonies (eg mammospheres, neurospheres, colonospheres) when plated at clonal density in non-adherent culture.
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Stem cells, both normal and malignant, are classically dened as cells with the capacity for limitless self-renewal and the ability to produce differentiated progeny [6]. To demonstrate stem cell-derived clones, the gold standard in mice involves lineage tracing, commonly employing hormone-dependent Cre recombinase expression. Using this genetic marking technology in the mouse small intestine, it has been shown that crypt base columnar cells (CBCCs), cells that express the Wnt target gene lgr5 (encoding an orphan G-protein-coupled receptor [7]), are most denitely multipotential stem cells [8]. Moreover, targeted deletion of the tumour suppressor gene Apc (adenomatous polyposis coli) in these Lgr5-expressing CBCCs resulted in rapidly growing adenomas, whereas targeted deletion of Apc in the higher-positioned transitamplifying cells (TACs) failed to induce signicant adenoma growth [9], illustrating that a mutation in a stem cell population is most effective for tumour initiation in the short term. It is commonly assumed that normal stem cells are the founder cells of tumours and even of TICs because they already have a self-renewal mechanism in place. So the Barker et al study [9] is consistent with this assumption and since their adenomas also contained a small population of Lgr5-positive cells that might be TICs, it begs the question as to whether normal cells and cancer stem cells can have the same identity. Methods of stem cell identication other than lineage tracing are notably less robust; stem cells are supposed to have inherent properties such as being slow cycling, enabling DNA label retention after a pulse of the likes of BrdU, but label retention may simply be because of imminent cell cycle exit; moreover, a slow cycling nature has not been found in all stem cells. The cell surface phenotype is often used to identify putative adult stem cells, but without subsequent lineage tracing the unique attributes of stem cells cannot be demonstrated. Moreover, many of these so-called markers of adult stem cells lack specicity; for example, in colonic crypts, Musashi1, CD44, and CD133 have all been proposed as suitable stem cell markers, but expression can also be seen outwith the stem cell zone extending to the transit-amplifying cells higher up the crypt [10,11]. Other proposed markers of stem cells include particularly high levels of cell adhesion molecules, especially in the basal layers of squamous epithelia, and also the ability to evade cytotoxic insults. With regard to the latter, there appear to be two major cytoprotective strategies. One involves high expression of ATP-binding cassette proteins (ABC transporters) which efux xenobiotics across cell membranes against a concentration gradient, identied as cells with the ability to efux a uorescent dye, usually Hoechst 33 342, described as the side population, rst reported by Goodell et al [12] for the isolation of murine haematopoietic stem cells (HSCs) with longterm multi-lineage repopulating potential. This review concerns the other cytoprotective mechanism, namely
Copyright 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

the existence of an efcient enzyme-based detoxication system based on high aldehyde dehydrogenase (ALDH) activity. Since ALDH is an effective detoxifying enzyme, high expression of ALDH can also provide a route for tumours to resist chemotherapy [1315]; for example, cyclophosphamide treatment of human colonic xenografts enriches for CD44+ ALDH+ cells, and these double-positive cells are more tumourigenic than cells selected solely on the basis of CD44 positivity [16].

Aldehyde dehydrogenases (ALDHs)

ALDHs are a family of NAD(P)+ -dependent enzymes involved in detoxifying a wide variety of aldehydes to their corresponding weak carboxylic acids [17]. They serve to detoxify both xenobiotic aldehydes (eg cyclophosphamide) and many other intracellular aldehydes, eg ethanol and vitamin A [18,19]. There are 20 Aldh genes in the mouse and 19 ALDH genes in humans, the latter being organized into 11 groups (19, 16, and 18), the largest being group 1 with six members comprising three subfamilies (A, B, and L); thus, ALDH1A1 is the rst ALDH gene in group 1, subfamily A (see http://www.aldh.org and http://www.genenames.org/genefamily/aldh.php). Most isoforms are widely distributed in the body, though the highest expression is seen in the kidney and liver [20]. In the liver, ALDH1 mainly functions as a retinoic acid biosynthetic enzyme, catalysing the conversion of vitamin A (retinol) to retinoic acid (RA). In human HSCs, where the utility of ALDH as a stem cell marker was rst recognized [21], RA signalling is believed to be crucial for cell fate determination, and inhibiting ALDH activity leads to an expansion of HSC numbers [2225]. Notably, ALDH1A1 and ALDH2 participate in alcohol metabolism [19] and the ALDHs with retinaldehyde dehydrogenase activity (ALDH1A1, ALDH1A2, ALDH1A3, and ALDH8A1) oxidize retinaldehydes to their corresponding retinoic acids, some of which are essential for embryogenesis. Cells expressing ALDH1 or other ALDH family members may be identied by immunohistochemistry using specic antibodies, but the functional activity of all ALDHs may be accurately assessed in living cells using the commercial reagent ALDEFLUOR (STEMCELL Technologies Inc, Vancouver, BC, Canada) (Figure 1). The ALDEFLUOR substrate, BODIPY aminoacetaldehyde (BAAA) is taken up by live cells through passive diffusion, whereupon it is converted in the cytoplasm into a uorescent molecule (the nega tively charged BODIPY aminoacetate; BAA) under the action of ALDH [21]. This uorescent product accumulates in cells, partly due to the presence of ABC-transporter inhibitors in the assay buffer, which prevent active efux, allowing cells with high ALDH activity to be identied by their bright green uorescence and subsequently isolated by ow cytometry.
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Figure 1. The basis of the ALDEFLUOR reaction (see ref 19 for further details). Cells are incubated with BODIPY aminoacetaldehyde (BAAA) in the presence of verapamil to inhibit multidrug resistance. (A) Cells with high ALDH activity convert BAAA in the cytoplasm into a uorescent molecule (the negatively charged BODIPY aminoacetate; BAA); these cells appear as a distinct cohort of cells exhibiting green uorescence and low side scatter (SSC)lower panel. (B) In the presence of diethylaminobenzaldehyde (DEAB), ALDH activity is abolished and no highly green uorescent subpopulation can be detected. Both plots are theoretical FACS histograms.

ALDH activity can be inhibited by the addition of diethylaminobenzaldehyde (DEAB), allowing the differentiation of cells with high ALDH activity from those with low or no activity. During ow cytometry, BAA is excited at 488 nm and uorescence may be detected using a standard 530/30 bandpass lter, similarly to uorescein isothiocyanate (FITC). In the haematopoietic system, the ALDHbri stem cell fraction exhibits a distinctive low side-scatter (SSC) distribution [26,27] as depicted in Figure 1 and appears as a discrete subpopulation in the lower right of the SSCarea versus 530/30 blue FACS scatter plot, whereas in primary tumours and tumour cell lines, ALDHbri cells typically display a range of side scatter properties (Figure 2). Targeted deletion of Aldh1a1 (a member of the retinaldehyde dehydrogenase family) has been shown not to affect the stem cell status or the degree of ALDEFLUOR uorescence of murine haematopoietic or neural stem cells [28], indicating not only functional overlap between ALDH family members, but also that evaluation of pan-ALDH activity via ALDEFLUOR uorescence may correlate more accurately with ALDH-mediated regulation of stem cell phenotype. The ALDEFLUOR substrate molecule BAAA and its reaction product BAA are both reportedly nontoxic, do not require potentially damaging UV-light
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for excitation or detection, and are efuxed effectively from treated cells following removal of the verapamil-containing assay buffer [21]. The necessity for ABC-transporter inhibitors in the ALDEFLUOR assay buffer precludes the simultaneous identication and co-isolation of SP cells, which may represent the stem cell fraction for some tissues; however, removal of the assay buffer and subsequent side-population analysis of isolated ALDHbri cells are feasible.

ALDH activity in normal adult tissues

Since the markers of normal tissue stem cells may be similar to those in their malignant counterparts, for example CD34+ CD38 identies both normal and malignant haematopoietic cells [29], it is appropriate to review the signicance of high ALDH activity for stem cell behaviour in normal tissues.

ALDH activity in haematopoietic cells

ALDH activity has by far the greatest utility in sorting for stem/progenitor cells within the various haematopoietic systems. In conjunction with other markers, high levels of ALDH activity have been shown to characterize highly clonogenic, undifferentiated multipotential stem/progenitor cells in both human
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Figure 2. Analysis of two pancreatic cancer cell lines for ALDH activity using the ALDEFLUOR kit. ALDH (area to the far left of each plot), ALDH+ (middle pink gates), and ALDH2+ cells (far-right pink gates) were detected in Capan-2 and Panc-1 cell lines using ALDEFLUOR. The percentage of live single cell population contained in each gate is shown. Data were captured with BD LSRII with subsequent FlowJo analysis; 100k events are shown per plot. Typically malignant cells such as these have a wide range of side scatter, whereas normal cells would be more tightly distributed as depicted in Figure 1.

bone marrow [30] and blood [3133]. High ALDH activity has also been advocated for selection of progenitors amongst the mouse Lin bone marrow population [34], but other studies [35] have suggested that ALDH activity via the ALDEFLUOR kit is not suitable for the detection of murine Lin Sca-1+ HSCs and that the SP is more representative of murine HSCs. Within the human Lin bone marrow mononuclear cell population, Lin CD34+ CD38low/ cells can be found throughout the ALDH activity spectrum, but they are especially enriched within the combined SP/ALDHbri subpopulation [36]. In human umbilical cord blood (h.UCB), elevated ALDH activity markedly enriches for HSCs with repopulating ability in immunodecient mice and in fact there is a declining gradient of ALDH activity as haematopoietic cells differentiate [32]. Other studies have suggested that the most primitive HSCs in h.UCB reside in the ALDHbri CD34+ cell fraction [37], while the ALDHbri CD34 cell fraction does not reliably repopulate NOD/SCID mice; in the bone marrow, this latter subset appears to be committed to erythroid differentiation [30]. Lin ALDHbri cells in h.UCB also comprise the CD34+ CD38 and the CD34+ CD133+
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subpopulations [38]; thus, the ALDHbri /CD133+ subpopulation will also produce robust haematopoietic reconstitution of immunodecient mice [33]. The numbers of ALDHbri cells also correlate with CD133+ /CD34+ cell numbers in fresh human peripheral blood, even after cryopreservation; so the marker clearly has a therapeutic utility with respect to endothelial progenitor cells (EPCs) [39], although only 65% of CD133+ /CD34+ cells actually expressed high levels of ALDH [40]. Somewhat surprisingly, in a ap ischaemia model in immunocompromised mice, it was in fact the ALDHlo rather than the ALDHbri subpopulation from sorted h.UCB EPCs that was best able to minimize the necrosis within the most hypoxic area of the ap [41]. This ability appeared to be related to the hypoxic up-regulation of the likes of VEGF, CXCR4, and GLUT-1 by HIFs in the ALDHlo rather than the ALDHbri EPCs. A growing body of evidence suggests that various haematopoietic cells selected on the basis of high ALDH activity can be used in a variety of cell therapy applications. For example, human bone marrow mononuclear cells, sorted on the basis of
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SSClo ALDHbri , make up 1.2% of all nucleated cells and these cells are massively enriched in haematopoietic colony-forming ability as well as in endothelial progenitors and CFU-F [27]. Results from preclinical trials conducted by Aldagen Inc. (Durham, NC, USA) also suggest that the ALDHbri cell population in human cord blood might provide chemical signals that promote angiogenesis (see http://www.aldagen.com). Likewise, the ALDHbri subpopulation from human bone marrow was found to be most effective at promoting revascularization in an ischaemic limb model in immunodecient mice, although there was no integration of these cells into the neovessels [42]. Likewise, improved vascularization has been seen in an immunodecient mouse model of myocardial infarction when ALDHbri cells from h.UCB were injected intravenously 1 day after infarction, again with little or no integration of these cells into the neovessels [43].

High ALDH activity was also found to be a feature of neural stem cells [47]. An ALDHbr side-scatter low (SSClo ) population can be isolated from murine cultured neurospheres capable of further highly efcient neurosphere formation and tri-lineage potential; these cells were generally negative for differentiation antigens, but the majority expressed nestin, Sox2, and Musashi [47]. In the prostate, Burger et al [48] have isolated an ALDHbr population from mouse prostate that was predominantly basally located, comprising 8% of the total population, and it was enriched for cells that efciently generated complete prostatic tissue in an in vivo reconstitution assay, being transplanted along with urogenital sinus mesenchyme under the kidney capsule. Most cells expressed Sca-1 to a varying degree, but one-third expressed very high levels of this murine stem cell marker.

ALDH activity in non-haematopoietic organs

High ALDH activity has also been used to detect putative stem/progenitor cells in a variety of solid organs. In normal human mammary epithelia, the ALDHbri population averages 8% and exclusively contains the clonogenic cells that can generate both CD10-positive (myoepithelial) and EpCAM-positive (luminal) cells, as well as double-negative cells in mammosphere culture [44]; additionally, only the ALDHbri , and not the ALDHlo , cells were capable of in vivo outgrowth in the cleared and humanized fat pads of NOD/SCID mice. These ALDH1-positive cells appeared to be located at the bifurcation points of terminal duct lobular units (TDLUs) and did not express CK18 (a luminal marker) or smooth muscle actin (SMA; a myoepithelial marker). In the murine pancreas, centroacinar/terminal duct cells have been proposed as a pancreatic precursor population that can be identied by high activity of ALDH: these cells did not show lineage-specic markers, but were enriched for progenitor markers such as Sox9, Sca-1, c-Met, and nestin [45]. Furthermore, these cells were shown to be capable of multi-lineage differentiation in vitro, forming pancreatospheres at clonal density containing both exocrine (amylase-positive) and endocrine (C-peptide-positive) lineages, and their numbers expanded dramatically in the regenerating tubular complexes observed during caerulein-induced pancreatitis. In the human colon, immunohistochemistry has been used to locate ALDH1+ cells; one study found that up to 15% of colonic epithelial cells were immunoreactive and although some were at the crypt base, worryingly (for a supposed stem cell marker) some were more than ten cell positions higher [46]. On the other hand, Huang et al [11] observed a much tighter distribution of ALDH1+ cells at the crypt base, comprising 6% of all epithelial cells, and these appeared to be a sub-set of the more widely distributed CD44+ and CD133+ cell populations.
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ALDH activity as a marker of human cancer stem cells

As with a variety of so-called markers of cancer stem cells, the level of ALDH positivity can vary not only between tumour types, but also between cell lines of the same tumour type [49]; thus, ALDH is probably not a universal marker for cancer stem cells in any malignancy. ALDH activity as a cancer stem cell marker has come under greatest scrutiny in the breast. Since breast cancer is not a single disease but a heterogeneous group of diseases with different aetiologies, pathologies, and clinical behaviours, it is not surprising that the identity of their cancer stem cells varies from tumour to tumour, and indeed ALDH1-positive cells are more frequent in basallike and HER2-positive tumours than in the luminal subtypes [50], but most studies have not made these distinctions. The rst major study of breast cancer examined invasive ductal carcinomas, nding that ALDHbri cells made up 310% of the epithelial cells and that these cells were greatly enriched for tumourigenic capacity [44]; there was only a small overlap with the CD44+ CD24 fraction, but in one case as few as 20 cells having both phenotypes were tumourigenic. About a quarter of a large series of breast cancers actually expressed ALDH, with an average of 5% positive cells in the expressing cases; these cases were positively associated with a high histological grade, oestrogen receptor (ER) negativity, human epidermal growth factor receptor type 2 (HER2) positivity, and, importantly, poorer patient survival. Other studies have also found ALDH-positive cells in a minority of breast cancers (10%), but again these tended to be associated with features that characterize the aggressive phenotype, such as being ER and HER2+ [51]. So what of the ALDH-negative cases? As noted by Hwang-Verslues et al [52], in some breast cancer cell lines, selection based on ALDH activity does not always select for the most clonogenic cells.
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Most malignant breast cancer cell lines, particularly those of basal type, contain an ALDHbri subpopulation enriched for clonogenicity, tumourigenicity, and metastatic capacity [53]. Expression of ALDH1 is also more common in basal-like tumours within African populations [54]. In two inammatory breast cancer cell lines, the ALDHbri subpopulation is enriched for tumourigenicity and invasive and metastatic capacity; furthermore, ALDH expression in primary tumours was an independent prognostic predictor of metastasis and poor overall survival [55]. ALDH may also be a useful marker of circulating tumour cells (CTCs), with the majority of metastatic cancer patients with CTCs having ALDH expression [56]. A review of existing literature has also concluded that ALDH expression is an indicator of poorer survival and furthermore is positively associated with high histological grade, ER and PR negativity, but HER2 positivity [57]. Although some or all of the cancer stem cells in many breast cancers may have the CD44+ CD24 phenotype, it seems that it is the number of ALDH-positive cells that is a more useful predictor of outcome. In response to neoadjuvant therapy with paclitaxel and epirubicin, it was only the frequency of ALDH1immunopositive cells that was signicantly associated with a lower complete response rate [58]. Moreover, in the group of patients not achieving a pathological complete response, the proportions of ALDHpositive cells had increased signicantly, whereas there was no change in the frequencies of CD44+ CD24 cells. The activity of the PI3-K/Akt pathway seems crucial to the maintenance of the ALDH-positive population in breast cancer, as illustrated by the up- and down-regulation of the negative regulator of the pathway PTEN (phosphatase and tensin homologue deleted on chromosome 10) in cultured cells [59]. Moreover, the Akt inhibitor perifosine not only retards xenograft growth, but also drastically reduces the frequency of ALDH-positive cells in the tumours. In fact, not all ALDH-positive putative cancer stem cells are resistant to chemotherapy, as shown by only a 64% reduction in ALDH-positive cells following cyclophosphamide treatment in a xenograft model [60]. Nevertheless, this was correlated with a signicant reduction in tumour-initiating ability of the treated cells upon further xenografting. The dietary polyphenols circumin and piperine are able to effectively abolish the mammosphere-forming ability of both normal and malignant breast cells [61], also severely reducing the frequency of ALDHbr cells amongst unsorted normal breast epithelial cells. Potentiation of retinoic acid signalling through exposure to all-trans-retinoic acid (ATRA) can reduce the sphere-forming efciency of a number of breast cancer cell lines, while the ALDH inhibitor DEAB actually increases efciency [62]. From gene expression proling, these observations were interpreted as retinoic acid signalling inducing differentiation and thus reducing the cancer stem cell population, while
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inhibition of ALDH activity by DEAB increases the population, in line with the observations that ALDH inhibition increases the pool of human haematopoietic stem cells [22]. Similarly, lung cancer cells exposed to ATRA have reduced ALDH1A1 and ALDH3A1 enzyme activity, making the cells more sensitive to an active derivative of cyclophosphamide [63]. The sphere-forming ability of breast cancer cell lines can also be severely reduced by exposure to sulforaphane, a molecule found in cruciferous vegetables such as broccoli [64], related to a large reduction in ALDHbri cells; moreover, sulforaphane also reduced their numbers in xenografted tumours that were signicantly retarded in growth, all observations linked to a suppression of the Wnt/-catenin self-renewal pathway. Butein, a tetrahydroxychalcone, a plant polyphenol, can also reduce the number of ALDHbri cells in a number of breast cancer cell lines, reducing their sphere-forming capacities after treatment [65], and presumed to be mediated through an inhibition of NK-B signalling, required amongst other things for EMT. ALDH activity also appears to be a useful marker in haematological malignancies. In acute myelogenous leukaemia (AML), ALDHbri cells seem to represent the putative CD34+ CD38 leukaemic stem cell population in about one-third of cases [31], but many cases have no detectable ALDH activity and this could be because these negative cases have their origins in more committed progenitor cells. In another study of AML, two-thirds of the ALDHbri cells expressed CD34 [66], and ALDH expression was linked to poorer survival in this and other studies [67]. ALDH activity also features in lymphomas. A small (0.48%), relatively quiescent, highly clonogenic, and drug-resistant ALDHbri population has been observed in mantle cell lymphoma [68], and in two Hodgkin lymphoma (HL) cell lines a very small (0.20.3%) CD27+ /ALDH+ subpopulation has been found that could self-renew and give rise to the classic Hodgkin and ReedSternberg cells of this disease [69]; similar (clonal) populations could also be found in the blood of most newly diagnosed HL patients. Given the plethora of markers recently proposed for cancer stem cells in malignant melanoma (ABCB5, CD271, and JARID1B) or even up to 1 in 4 cells with apparently no specic phenotype [4], it is perhaps not too surprising that cells with high ALDH activity, although comprising up to 70% of the cells in some melanoma biopsies, had neither superior clonogenic nor tumourigenic abilities compared with the ALDHnegative cells [70]. Moreover, both subpopulations had a similar response to anti-melanoma drugs. On the other hand, much smaller (<7%) subpopulations of ALDHbri cells have been isolated from head and neck squamous cell carcinomas (HNSCCs) that were greatly enriched for tumourigenic activity [71]most likely a subset of a larger CD44-positive population. Similar results were reported in another study of HNSCC [72] that found that the CD44+ CD24 ALDHbri subpopulation was enriched for clonogenic, tumourigenic, and
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radioresistant cells, and that the cancer stem cell properties of these cells could be blunted by transfection with Snail siRNA (inhibiting EMT). In a malignant prostate cell line, ALDHbri cells are greatly enriched for clonogenicity and tumourigenicity [73], and ALDH expression in patient samples is positively correlated with Gleason score and pathological stage, and negatively correlated with patient survival. ALDHbri cells in other prostatic cancer cell lines are also enriched for clonogenicity and tumourigenicity and, more importantly, in metastatic ability in animal models [74]; in primary patient samples, ALDHbri cells were found at an average frequency of 8%. CD133 has often been used to enrich for TICs in hepatocellular carcinoma (HCC), but studies of numerous cell lines have now suggested that the ALDHbri subset of the CD133-positive fraction represents a more clonogenic and tumourigenic population [75]. In primary HCC, these doubly positive cells occur with a frequency of 5% and their location, often in associated connective tissue and within vascular lumina associated with HCC invasion, suggested a role in tumour dissemination, but as a word of caution these hepatocellular phenotypes could also be seen in cases of viral hepatitis [76]. Amongst ovarian epithelial tumours, the selection of ALDHbri cells has greatly enriched for clonogenic, tumourigenic, and metastatic cells in adenoid cystic carcinoma [77]. On the other hand, ALDH1 immunohistochemical staining of a large series of ovarian epithelial tumours suggested that high expression was a favourable prognostic indicator [78], the converse of what is found for many tumour types, particularly the breast. A high frequency (up to 50%) of ALDHbri cells has been noted in some cervical carcinoma cell lines [79], increasing up to 80% in spheres, and these sphereforming cells were more tumourigenic than the parental cell line. In patients undergoing resection for early-stage pancreatic cancer, ALDH expression is an indicator of poorer survival [80]; moreover, some patients had ALDH-negative primary tumours but ALDH-positive metastases, suggesting a signicant role of ALDHpositive cells in disease progression. In two pancreatic cancer cell lines, there was only partial overlap between a slow cycling, highly clonogenic, and tumourigenic population and the ALDHbri population [81]. In a xenograft model, pancreatic tumour debulking with gemcitabine leads to an increase in the frequency of ALDH-positive cells [82]. In some pancreatic cancer cell lines, TICs, identied by combined CD44+ CD24 and ALDH expression, can be sensitized to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by sulforaphane, a molecule that interrupts anti-apoptotic NF-B signalling [83]. A similar mechanism appears to underlie the dramatic reductions in clonogenicity, tumourigenicity, and ALDH expression seen when pancreatic cancer cells are exposed to the multi-kinase
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inhibitor sorafenib in combination with sulforaphane [84]. In a large series of non-small cell lung cancer (NSCLC), ALDH expression was positively correlated with grade and stage and related to poorer prognosis in early-stage disease [85]; in cell lines, the ALDHbri subpopulation (of which two-thirds was also CD133positive) was also the most clonogenic, tumourigenic, and invasive, and more resistant to a range of frontline chemotherapeutic drugs. In small cell lung cancer (SCLC), both CD133 and ALDH expression are regulated by the bHLH transcription factor achaetescute complex homologue 1 (ASCL1), and knock-down of ASCL1 greatly reduces the clonogenicity and tumourigenicity of these cells [86]. Similarly, in NSCLC cell lines, knock-down of ALDH1A1 and ALDH3A1 by siRNA results in large decreases in clonogenicity and motility [87], and increased sensitivity to 4-hydroperoxycyclophosphamide [88]. In the lung cancer cell line H522, the ALDHbri subpopulation is a cytogenetically distinct cell clone, and while both ALDHbri and ALDHlo were tumourigenic, the ALDHlo population actually grew faster on rst passage, but on subsequent passaging into secondary and tertiary NOD/SCID mice it was the cells from the ALDHbri derived xenografts that grew more quickly, observations consistent with ALDHbri having the stem cell phenotype of slow cycling but long-term repopulating capacity [89]. In colon cancer cell lines, the majority of cancer stem cells recognized by CD44 and EpCAM expression also express high ALDH activity, and these cells increase in frequency after exposure to cyclophosphamide [16]; inhibition of ALDH1 by DEAB or shRNA markedly improved the drug sensitivity of these cancer stem cells. CD44 also selects for TICs from primary colonic tumour and in some patients, a ten-fold increase in tumourigenicity can be achieved by selection of cells positive for both CD44 and ALDH [90]. On the other hand, other studies [11] have found that as few as 25 ALDHbri cells from primary colon cancers can be tumourigenic and no real enrichment of TICs was achieved by combining with CD44. Very interestingly, some colitic patients (3/22) with no evidence of dysplasia harbour TICs based on EpCAM and high ALDH expression, with as few as 50 cells being tumourigenic [46], perhaps indicating that ALDH could be a useful marker for colonic cancer surveillance in colitic patients. ALDHbri cells have been found with a frequency of 10% in osteosarcoma and brosaroma cell lines, exhibiting enhanced resistance to the front-line antisarcoma drugs doxorubicin and cisplatin [91]. Other osteosarcoma lines have very variable ALDHbri subpopulations [92]; nevertheless, these cells were highly enriched for so-called stemness genes and clonogenic and tumourigenic efciency. Tumours in which high ALDH activity has been signicantly correlated with patient prognosis are listed in Table 1.
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Table 1. Examples of human tumours where ALDH expression has been positively correlated with overall patient survival
Tumour type AML AML Breast (invasive ductal) Breast (inammatory) HNSCC Lung (NSCLC) Ovary (endometrioid) Pancreas Prostate Poor prognostic indicator Yes Yes Yes Yes Yes Yes Good prognosis Yes Yes Reference 66 67 44 55 72 85 78 80 73

Summary
This review has examined the cancer stem cell credentials of malignant cells from a variety of tumours expressing high ALDH activity. In normal tissues, there is some evidence that high ALDH activity does select for cells with stem/progenitor activity, particularly within haematopoietic cell populations; this bias may be a technical issue since it is difcult to obtain a monodispersed cell population from solid organs which is, of course, required for FACS analysis using the ALDEFLUOR reagent. Resistance to oxazaphosphorines (eg cyclophosphamide) appears to be one of the key features of cells expressing high ALDH activity and strategies to block ALDH activity such as antisense, siRNA, diethylaminobenzaldehyde (DEAB), and disulram may be useful for reducing the cancer stem cell burden, with the important caveat that simply blocking ALDH activity with DEAB may simply increase stem cell numbers in some tissues by virtue of inhibiting retinoic acid-mediated cell differentiation. Clearly, ALDH activity is not a one cap ts all for cancer stem cells, even in a given tumour type, but in those malignancies where it seems to be a valid marker, the frequency of ALDH-positive cells appears to be an independent prognostic indicator of poor survival.

Author contribution statement

All authors contributed to the synthesis and writing of this review.

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