Lung cancer stem cells - Nature

Oncogene (2010) 29, 4625–4635

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REVIEW

Lung cancer stem cells: tools and targets to fight lung cancer A Eramo1, TL Haas1,2 and R De Maria1,2 1

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome, Italy and 2Mediterranean Institute of Oncology, Via Penninazzo 7, Viagrande, Catania, Italy

Cancer stem cell (CSC) theory states that tumors are organized in a similar hierarchical manner as normal tissues, with a sub-population of tumorigenic stem-like cells that generate the more differentiated nontumorigenic tumor cells. CSCs are chemoresistant and seem to be responsible for tumor recurrence and formation of metastases. Therefore, the study of these cells may lead to crucial advances in the understanding of tumor biology as well as to innovative and more effective therapies. Lung cancer represents the leading cause of cancer-related mortality worldwide. Despite improvements in medical and surgical management, patient survival rates remain stable at B15%, calling for innovative strategies that may contribute to improve patient outcome. The discovery of lung CSCs and the possibility to characterize their biological properties may provide powerful translational tools to improve the clinical outcome of patients with lung cancer. In this report, we review what is known about lung CSCs and discuss the diagnostic, prognostic and therapeutic prospective of these findings. Oncogene (2010) 29, 4625–4635; doi:10.1038/onc.2010.207; published online 7 June 2010 Keywords: lung cancer; cancer stem cells; therapy; stem cells

Cancer stem cells Tumor cells display a broad spectrum of functional and morphological heterogeneity. Even cells of a single tumor lesion vary in their differentiation level, proliferation capacity and tumorigenicity. This phenomenon could be explained by the presence of ‘cancer stem cells’ (CSCs) (Reya et al., 2001; Wang and Dick, 2005; Clarke et al., 2006) as the driving force of tumorigenesis. The CSC model implies a hierarchical organization within the tumor in which a limited number of CSCs represents the apex of the hierarchy. Similar to normal stem cells, CSCs have the ability to self-renew and undergo asymmetric divisions, thereby giving rise to a differentiated progeny that represents most of the tumor Correspondence: Professor R De Maria, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome 00161, Italy. E-mail: [email protected] Received 3 February 2010; revised 22 April 2010; accepted 25 April 2010; published online 7 June 2010

population. These key features enable CSCs to initiate tumors and promote cancer progression. The first direct experimental evidence supporting the idea of CSC-driven tumorigenesis came from acute myeloid leukemia (Lapidot et al., 1994), the initiating cells of which have a phenotype (that is, CD34 þ ; CD38) similar to normal hematopoietic stem cells (Bonnet and Dick, 1997). Despite some technical limitations, a growing number of reports support the existence of CSCs in solid tumors. Specific biomarkers for detection and isolation of CSCs have been suggested for all major tumor types, including lung cancer (Eramo et al., 2008; Visvader and Lindeman, 2008). Besides the expression of surface proteins, CSCs may also share functional features with tissue stem cells, such as their ability to actively exclude the dye Hoechst 33342, which defines them as side-population (SP) cells in flow cytometric assays (Storms et al., 1999) and high aldehyde dehydrogenase (ALDH) activity (Ginestier et al., 2007). In parallel to the growing number of potential surface markers, it is getting evident that the known markers are not always ideal to sort for the CSC population. A good example is the expression of CD133, which was initially reported to be a reliable marker for glioblastoma and medulloblastoma CSCs (Singh et al., 2003, 2004). However, independent studies have shown that CD133negative glioblastoma cells can also establish a tumor in recipient mice with similar efficiencies compared with CD133 þ cells (Beier et al., 2007) and that CD133 is highly expressed on nontransformed neural progenitor cells (Lee et al., 2005). In line with tumor heterogeneity, the phenotype of CSCs is not uniform, even when they originate from the same tumor subtype. This is underlined by studies using colorectal carcinoma as the model system. We along with others found that colorectal CSCs are enriched in the CD133 þ sub-population (O’Brien et al., 2007; Ricci-Vitiani et al., 2007; Todaro et al., 2007), whereas Dalerba et al. (2007) reported an enrichment of CSCs in the EpCAMhi, CD44hi subpopulation of colon cancer cells. The picture is further complicated by a recent study showing that colorectal CSCs are highly enriched in the ALDH-positive cell population. In this report, the tumorigenicity of ALDHpositive cells is only modestly increased using CD133 and CD44 expression as a second marker for a more stringent selection (Huang et al., 2009). On the other hand, a combination of markers may be required to isolate a highly enriched breast CSC population. Breast CSCs are reported to be enriched in both CD44 þ CD24/low

Tools and targets to fight lung cancer A Eramo et al

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(Al-Hajj et al., 2003) and ALDH-positive subfractions. Although the overlap of the two populations was surprisingly low (0, 1–1.2%), cells displaying the combined phenotype turned out to be highly tumorigenic when transplanted into a recipient mouse (Ginestier et al., 2007). The marked heterogeneity within CSC sub-populations underlines the necessity to find more specific single markers, or to define new marker combinations for the prospective isolation of CSCs in solid tumors. Recently developed transgenic mouse models provided more hints about the potential identity of the CSC population in colon and lung cancer. Targeted oncogenic transformation of adult stem or progenitor cells led to the formation of colon adenomas (Barker et al., 2009; Zhu et al., 2009) or lung adenocarcinomas (ACs) (Kim et al., 2005). Although these results underline the pivotal role of stem/progenitor cells in carcinogenesis, the term ‘cancer stem cell’ does not imply that tumorigenic cells necessarily have to derive from tissue stem cells. It is also likely that CSCs develop from more restricted progenitor cells that receive reprogramming ‘hits’, and thereby regain the stem cell trait of unlimited self-renewal capacity (Clarke et al., 2006). Although there is substantial evidence for CSC relevance in human carcinomas, many issues need to be considered, as deeply discussed in a recent review (Visvader and Lindeman, 2008). First of all, the best functional in vivo assay for CSCs is their ability to recapitulate the patient tumor in animal models. However, animal models do not entirely mimic the human tumor microenvironment and poorly tumorigenic CSCs may be inadvertently selected during xenotransplantation. Besides the issue of species specificity, tissue specificity also has to be considered for in vivo transplantation of CSCs. Therefore, orthotopic tumors may better reproduce the CSC niche, especially when the human microenvironment is mimicked by the use of humanized supports or patient-derived accessory cells, such as fibroblasts or mesenchymal stem cells (Kuperwasser et al., 2004). The impact of the microenvironment on tumor growth is underlined by the fact that subcutaneous implantation of CSCs results in less efficient engraftment than brain or renal capsule injection for gliomas or colorectal CSCs, respectively. Finally, the presence of residual immune effector cells in recipient mice may also influence the efficiency of human cell engraftment in NOD/SCID mice, whereas NOD/SCIDIL2Rgnull mice, recently introduced for CSC transplantation, represent a more permissive environment (Shultz et al., 2005). On the other hand, it is also true that immune cells have a role in the progression of human tumors. These limitations of mouse models should be kept in mind when CSC behavior, frequency in tumors, growth requirements or other properties linked to niche interaction are investigated. Moreover, until now, no complete agreement regarding CSC frequency or phenotype has been reached. However, although some controversies about CSCs in solid tumors have been raised, their biological relevance and the immense therapeutic potential of their targeting are not seriously questioned (Hill, 2006; Visvader and Lindeman, 2008; Zhou et al., 2009). Oncogene

Lung cancer Lung cancer is the most common cause of cancer-related mortality worldwide, with >160 000 deaths in the United States in 2008 and a poor 5-year survival rate, which remains stable at 15% (Jemal et al., 2008). It has been estimated that 9 out of 10 lung cancer cases are directly caused by smoking. Other risk factors for lung cancer are exposure to asbestos and, to a lesser extent, to radon, arsenic, chromium, nickel, vinyl chloride and ionizing radiation or some preexisting nonmalignant lung diseases (Dubey and Powell, 2009). Different known or unknown etiologies might account for the occurrence of different forms of lung cancer, the heterogeneity of which has critical diagnostic, prognostic and therapeutic implications (Borczuk et al., 2009). To facilitate treatment and prognostic decisions, lung cancer has been categorized into small cell lung carcinoma (SCLC) and non-SCLC (NSCLC). NSCLC can be further distinguished into three major histological classes: AC, squamous cell carcinoma and large cell carcinoma (Collins et al., 2007). SCLC accounts for B20% of the pulmonary tumors. Despite a generally good initial response to chemotherapy, it has a particularly poor prognosis, because of early extra thoracic dissemination and frequent disease relapse. SCLC predominately localizes to midlevel bronchioles and displays neuroendocrine differentiation, suggesting that transformed pulmonary neuroendocrine cells might give rise to this form of lung cancer (Giangreco et al., 2007). With B40% prevalence, adenocarcinoma (AC) is the most common form of NSCLC in both smokers and nonsmokers. It develops mostly from the junction between the terminal bronchiole and the alveolus, termed ‘bronchoalveolar duct junction’, and displays either airway or alveolar differentiation, or both (Giangreco et al., 2007). Squamous cell carcinoma has a 25% prevalence rate and is highly associated with tobacco smoking. It arises in the proximal airways down to the second or third bifurcation and is rarely observed distally. Compared with other forms of NSCLCs, large cell carcinoma (10% prevalence) comprises a class of rather poorly differentiated and less aggressive tumors, the most frequent subtype of which is large cell neuroendocrine carcinoma (Giangreco et al., 2007). Analysis of the genetic alterations occurring in lung cancer has shown that histopathological differences are in line with genetic heterogeneity of the disease (Minna et al., 2002; Sekido et al., 2003). Additional genetic differences have been found among the three main forms of NSCLCs (Minna et al., 2002; Sekido et al., 2003). Consistent with the genetic heterogeneity, expression studies have not only unveiled profound differences between SCLC and NSCLC but also within different NSCLC subtypes (Bianchi et al., 2008). However, until recently, all NSCLC lung cancer forms have been treated with similar approaches regardless of their biological differences (Minna et al., 2002; Collins et al., 2007; Dubey and Powell, 2008; Tiseo et al., 2009). Thus, it is likely that the poor lung cancer response rates may be due, at least in part, to a too homogeneous


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treatment approach used in the past for a highly heterogeneous disease (Borczuk et al., 2009). Only recently, lung cancer heterogeneity has started to gain therapeutic relevance, as documented by the attempt to propose preferential chemotherapeutic options for each tumor histotype (Tiseo et al., 2009) (Scagliotti et al., 2009). Besides histology-based approaches, ongoing efforts are underway to identify clinically relevant biological properties of lung tumors that will facilitate individualized therapy. In this direction, different expression profiles have also been found within the same tumor subtype, and could be correlated with patient survival (Bhattacharjee et al., 2001). To define more personalized therapies, both genetic and proteomic signatures of different lung tumor subtypes have to be determined and correlated with tumor histotype, clinical outcome and treatment response. For instance, it has been found that, within ACs, KRAS mutations are more common in tumors from patients with tobacco exposure and are associated with worse prognosis and primary resistance to treatment. In contrast, tumors from nonsmokers are more frequently associated with molecular alterations involving the epidermal growth factor receptor and with longer overall survival (Pao et al., 2004; Janne et al., 2005; Riely et al., 2009). From the therapeutic point of view, patients with epidermal growth factor receptor mutations display increased sensitivity to epidermal growth factor receptor inhibitors, whereas KRASbearing tumors are resistant (Pao et al., 2005). Therefore, epidermal growth factor receptor and KRAS mutations define two distinct populations of NSCLC patients with different natural histories and responses to targeted therapy (Bianchi et al., 2008; Lantuejoul et al., 2009; Riely et al., 2009). These evidences strongly suggest that a deep knowledge of each patient tumor can predict treatment response and improve patient outcome, calling for a strong effort toward personalized therapy. In the attempt to improve lung cancer patient outcome, CSC research might have a central role not only in the search for new anticancer drugs but also in the diagnosis and monitoring of the therapeutic success as discussed later. However, the relative low frequency of CSCs in tumors may complicate their investigation. For instance, the current methodology for molecular profiling of tumors does not analyze cell sub-populations and may provide misleading results because the signal related to CSC gene expression is extremely diluted. Adult progenitor cells of the lung CSCs share a number of features with normal tissue stem or progenitor cells, regardless of whether they originate directly from stem cells gaining tumorigenic hits or by reprogramming of more differentiated cells. In adult organisms, tissue homeostasis is maintained by stem and progenitor cells that have the ability to divide throughout life and replace dying or damaged cells. Stem cell populations have been well characterized in several organs with a high rate of homeostatic proliferation, such as the hematopoietic system, skin,

gut and hair follicle (Blau et al., 2001; Barker et al., 2008; Fuchs, 2009). A classical model of a ‘stem cell hierarchy’ is well established for the maintenance of the hematopoietic system (Orkin and Zon, 2002; Iwasaki and Akashi, 2007) and the small intestine (Barker et al., 2008; Fuchs, 2009). In organs with slow turnover rates, such as the lung, liver and pancreas, the terminology developed to define the ‘hierarchical stem cell’ model of rapid renewing tissues has to be refined. Cellular turnover in the adult lung tissue is very slow when compared with the small intestine (Blenkinsopp, 1967; Barker et al., 2008; Rawlins, 2008), and only extreme conditions such as pollutant- or pathogen-induced injuries lead to a massive lung cell proliferation. During organ regeneration, lung cells which usually fulfil differentiated functions in the normal tissue actively start to proliferate and act as progenitor cells that can give rise to several cell types (Stripp and Reynolds, 2008). In a popular lung injury model, mice are treated with naphthalene, which leads to the ablation of Clara cells (Mahvi et al., 1977). Cell proliferation starts 48–72 h after injury and the epithelium is completely regenerated after 2–4 weeks (Stripp et al., 1995; Van Winkle et al., 1995). The mechanism of how the Clara cell population is regenerated depends on the region of the lung (see Figure 1). In the trachea and bronchi, cytokeratin 14-positive cells are most probably basal cells that can act as self-renewing progenitors and give rise to Clara-like and ciliated cells (Hong et al., 2004a, b). In the more distal regions of the lung, naphthalene-resistant Clara cells start to proliferate and regenerate the damaged tissue. These rare ‘variant’ Clara cells (ClaraV), accumulate in specialized stem cell niches close to the neuroendocrine bodies and at the bronchoalveolar duct junctions (Reynolds et al., 2000; Giangreco et al., 2002). Upon injury, a subset of ClaraV cells found in the bronchoalveolar duct junctions show a unique expression pattern comprising both Clara and alveolar cell proteins (CCSP and SP-C) in combination with CD34 and Sca-1, two common stem cell markers. These cells were named bronchoalveolar stem cells (BASCs), as they have a substantial clonogenic survival capacity, are able to self-renew and can give rise to Clara and alveolar cells in vitro (Kim et al., 2005). In this report, we will refer to CCSP/SP-C positive cells as BASCs, although the ability of these cells to self-renew and give rise to different lineages in vivo has not been formally proven, and recent cell-tracing experiments have suggested that BASCs contribute to the maintenance and repair of the proximal airways, but not of the alveolar epithelium (Giangreco et al., 2009; Rawlins et al., 2009). However, even though their exact function in maintenance and repair of the lung is not properly understood yet, there is growing evidence that adenomas and ACs arise from transformed BASCs, at least in mouse models. Potential lung cancer-initiating cells in the mouse Although the identity of tissue stem cells in the lung is controversial, BASCs drive the tumorigenic process in Oncogene


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Figure 1 Airways, stem cells and lung cancers. Schematic representation of the airways and the different cell types present in each region. The red arrows indicate cell types with stem cell potential, possibly linked to tumor initiation. Moreover, the preferential location of different lung cancer forms in different regions of the airways is schematically indicated.

several mouse models of lung ACs. As described before, KRAS is mutated in a substantial number of NSCLCs. Therefore, transgenic mice expressing oncogenic variants of KRAS (such as KRAS(G12D)) represent important animal models for lung cancer, especially ACs. Several lines of evidence point toward the importance of BASCs for the induction of KRAS(G12D)-induced ACs: (1) It was shown that the number of BASCs was expanded in AC precursors of mice expressing an inducible form of KRAS(G12D) (Jackson et al., 2001; Kim et al., 2005). (2) Isolated transgenic BASCs displayed enhanced in vitro proliferation capacity when compared with more differentiated cells of the same tissue. (3) When the number of BASCs were elevated by naphthalene injury before the induction of the transgene, tumor growth was strongly enhanced compared with control animals (Jackson et al., 2001; Kim et al., 2005). Such results suggest that these progenitor cells have a key role in the onset of Oncogene

KRAS-induced lung cancer. In line with these experiments, it was shown that inducible p38 knockout mice were more susceptible to KRAS-induced formation of ACs. p38 supports the differentiation and suppresses the proliferation of BASCs, which accumulated in the lungs of p38-deficient mice even without the ectopic expression of mutated KRAS (Ventura et al., 2007). This accumulation could explain the higher susceptibility of KRAS-transgenic/p38-deficient mice for lung ACs when compared with p38-proficient mice. BASCs also have an important role in other tumor model systems. For example, mice knocked in for an oncogenic form of p27KIP (p27CK) displayed a high incidence of spontaneous hyperplastic lesions in several tissues, including the retina, pituitary gland, ovary, adrenal gland, spleen and lung. When compared with wild-type mice, these mice had an abnormal high number of BASCs in terminal bronchioles and eventually developed AC (Besson et al., 2007). Moreover, in a mouse


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model in which PTEN (phosphatase and tensin homolog) can be deleted in SP-C-expressing cells, the number of BASCs and SP cells was significantly increased. Almost all mice in whom PTEN was deleted spontaneously developed lung cancer and, strikingly, like in the human system, KRAS was mutated in 30% of ACs (Yanagi et al., 2007). In summary, these reports suggest an important role for BASCs in the early steps of lung tumorigenesis. There is a similar behavior between normal lung stem cells in injured tissues and transformed BASCs in tumors. After lung injury, normal lung stem cells survive and proliferate while giving rise to specialized cells that repair the damaged tissues. This process is induced by external stimuli but strictly regulated within the stem cell niche, which guarantees that stem cell expansion occurs only if required. In contrast, the expansion of transformed CSCs during lung tumorigenesis appears rather unrestrained, and poorly regulated by signals coming from the niche. However, it still has to be proven whether transformed BASC-like cells also fulfill all criteria of being CSCs, as there is no evidence of a sub-population with the BASC phenotype able to establish a histophenocopy of the initial tumor in secondary and tertiary hosts. Human lung CSCs Following the growing enthusiasm for the CSC model in solid tumors and the new insights into the biology of normal cells and CSCs in the mouse airway, research interest has recently started to rise for stem cells in

Table 1

human lung cancer. Tumorigenic human lung cancer cells have been isolated using different approaches from both cell lines and primary tumors (see Table 1). The first approach was based on the SP phenotype (low Hoechst 33342 staining pattern) of stem cells. SP lung cancer cells isolated from H460, H23, HTB-58, A549, H441 and H2170 cell lines, displayed increased invasiveness, higher resistance to chemotherapeutic drugs and were more tumorigenic in vivo when compared with non-SP cells (Ho et al., 2007). On the basis of the widely accepted hypothesis that CSCs of different origins are endowed with increased drug resistance, the second approach used to enrich human lung CSCs from cell lines was based on their inherent resistance to cisplatin, doxorubicin or etoposide treatment. Drug-surviving cells exhibited several CSC features, such as high clonogenic capacity, enrichment in SP cells, expression of embryonic stem cell markers, capacity for self-renewal and generation of differentiated progeny and high tumorigenicity (Levina et al., 2008). The third approach leading to the isolation of lung CSCs was based on increased ALDH activity previously shown in stem cell populations of several human cancers. ALDH-positive cells isolated from lung cancer cell lines displayed features of CSCs both in vitro (invasive properties, expression of stem cell markers) and in vivo (ability to generate tumors) (Jiang et al., 2009). The first isolation and expansion of lung CSCs from primary patient tumors was reported by our group (Eramo et al., 2008). In our study, human lung CSCs were isolated on the basis of their ability to survive under serum-free conditions and proliferate as cellular

Reports on lung cancer stem cell isolation

Study

Stem cell property used for isolation

Lung cancer cell lines Ho et al., Side population 2007 Levina et al., Chemoresistance 2008 Jiang et al., 2009

Aldehyde dehydrogenase 1 (ALDH1) activity

Primary lung cancer samples Eramo et al., Ability to grow as spheres 2008 in serum-free medium Chen et al., 2008 Bertolini et al., 2009

CD133 expression CD133/ESA expression

Cancer stem cell properties

Marker expression

Increased invasiveness, chemoresistance and tumorigenic potential High clonogenic capacity, enrichment with SP cells, capacity for self-renewal and differentiation and high tumorigenic potential Invasive properties, expression of stem cell markers, resistance to chemotherapy and in vivo ability to recapitulate original tumors

ABCG2 low Hoechst staining Expression of embryonic stem cell markers

High self-renewal potential, extensive proliferation ability, differentiation capacity, increased chemoresistance and capacity to recapitulate tumor heterogeneity and mimic the histology of the specific tumor subtype in mice xenografts Self-renewal capacity invasive chemoresistance and radioresistance Increased expression of stemness, adhesion, motility and drug efflux genes Resistance to cisplatin treatment in vitro and in vivo Frequency of CD133 þ cells in patient tumor predictive of patient chemoresponse

CD133 Oct4,Nanog EpCAM/NCAM CEA

ALDH1 CD133

Oct4 enriched ABCG2 CD133/ESA ABCG2 CXCR4

Abbreviations: CSC, cancer stem cell; SP, side population. List of the current reports about isolation and characterization of lung CSCs. For each study, the source of lung CSCs (that is, cell lines or primary patient tumors), the stem cell property exploited for their isolation, physiological characteristics of the isolated cells and CSC-related antigen expression are indicated. Oncogene


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clusters known as ‘tumor spheres’. This experimental strategy represents the best approach so far to obtain the unlimited expansion of the tumorigenic lung cancer cell population from primary patients, providing a powerful tool to allow extensive studies on these cells. Owing to the low frequency of lung CSCs within primary tumor tissues, extensive investigation on these cells would not be possible in the absence of their in vitro expansion. On the basis of our approach, high numbers of lung CSCs could be generated and extensively characterized. Spheres from the major subtypes of lung cancer (such as SCLC, AC, squamous cell carcinoma and large cell carcinoma) were found to possess CSC properties, both in vitro (expression of the CSC marker CD133, unlimited proliferative potential, extended selfrenewal and differentiation ability) and in vivo (high tumorigenic potential, capacity to recapitulate tumor heterogeneity and mimic the histology of the specific tumor subtype from which CSCs were derived). In addition, lung cancer ‘spheres’ were extremely resistant to most conventional drugs currently used to treat lung cancer patients, calling for an intense effort in testing both conventional and innovative drugs on the tumorigenic lung cancer cell population. This tumor cell population was also characterized by the expression of embryonic genes, such as Oct-4 and nanog, thus confirming the undifferentiated phenotype of isolated cancer cells. Oct-4 expression was also recently reported to characterize CD133-positive tumor cells in freshly isolated tumors, and to be essential in maintaining the stem-like properties of isolated cells, such as invasion and self-renewal (Chen et al., 2008). Recently, the relevance of CD133-positive cells in tumorigenicity and chemoresistance of lung cancer has been further confirmed (Bertolini et al., 2009). CD133 þ cells isolated from primary tumors displayed increased tumorigenicity and expression of stemness, adhesion, motility and drug efflux genes in comparison with the corresponding CD133 tumor cells. In addition, CD133 þ cells survived cisplatin treatment after in vitro drug exposure of the A549 cell line or in primary tumor-derived mouse xenograft after cisplatin administration. Importantly, the expression of CD133 in tumors was linked to shorter progression-free survival of NSCLC patients treated with platinum-based regimens, thus providing the first evidence of the relevance of CD133 þ tumor-initiating cells for prediction of chemoresponse and prognosis of lung cancer patients. Although these and other encouraging results are starting to clarify different aspects of lung CSCs, further investigation is still required to obtain a deep elucidation on the biology of these important cells.

CSCs and their possible implications for the development of innovative cancer treatments Advances in CSC research might help to establish successful therapies against leukemia and solid tumors. Conventional antineoplastic agents used to fight cancer Oncogene

mainly hit the malignant cells by two different mechanisms. One class of conventional chemotherapeutic drugs induces DNA damage, whereas the second drug impairs mitosis or DNA replication. Unfortunately, this therapeutic combination can only prolong the patient’s survival for a few months. One reason for this poor medical outcome might be the presence of drug- and radiation-resistant CSCs. There are several indications for the higher resistance of CSCs to conventional chemotherapy or radiotherapy in vivo. In tumor biopsies from breast cancer patients treated with conventional chemotherapy, a higher frequency of potential CSCs (CD24low/CD44 þ ) was detected when compared with pretreatment biopsies (Yu et al., 2007; Li et al., 2008; Creighton et al., 2009). Similar results were obtained in mouse xenograft models of glioblastoma and colorectal cancer (Bao et al., 2006; Dylla et al., 2008). We previously reported that lung CSCs isolated from primary tumors also display high resistance to standard chemotherapy in vitro (Eramo et al., 2008), suggesting that these cells exhibit highly efficient intrinsic resistance mechanisms. Moreover, lung cancer mouse xenografts treated with cisplatin regimens in vivo showed enrichment of specific sub-populations with stem cell phenotype (CD133 þ /ABCG2 þ /CXCR4 þ ) (Bertolini et al., 2009). The combinations of slow cell-cycle progression and efficient DNA repair machinery are potential resistance mechanisms of CSCs. In contrast to the majority of tumor progenitors and precursors, CSCs showed increased quiescence in vivo and in vitro, suggesting poor responses to conventional treatments, which primarily kill proliferating cells (Holyoake et al., 1999; Guan et al., 2003; Ishikawa et al., 2007; Cicalese et al., 2009). In addition, it has been reported that glioblastoma stem cells exhibit active DNA repair mechanisms, which allow them to recognize and repair radiation-induced DNA damage efficiently (Bao et al., 2006). The accumulating evidence that the CSC sub-population of tumor cells is resistant to conventional anticancer therapy can explain the frequent relapse after a good initial response to chemotherapy was observed in patients (see Figure 2). For this reason, innovative therapeutic approaches to kill or disarm CSCs might be the key to cure solid cancers. Targeted treatment of CSCs is an ambitious approach, as they most probably represent a heterogeneous population of cells with different sensitivities to chemotherapeutic drugs that have to be combined to achieve full antitumor effects. In contrast to the induction of apoptosis in the majority of tumor cells observed with conventional chemotherapeutic agents, targeted elimination of CSCs might lead to slow but sustainable tumor eradication. This implicates that most preclinical xenograft models used to test the efficacy of chemical compounds or biomolecules have to be modified. These adjustments include not only longer observation periods but also a faithful analysis of treated tumors for cells expressing potential CSC markers at intermediate end points. Monitoring druginduced alterations in the CSC compartment might be a useful indicator for general anticancer efficacy of the


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Figure 2 The role of cancer stem cells in tumor response to current or innovative anticancer therapies. (a) Conventional chemotherapy kills differentiated cells, while sparing CSCs. Tumor is debulked; however, surviving CSCs may drive tumor regrowth and the patient eventually relapses. (b) CSC-targeting therapeutic approaches destroy CSCs. The surviving tumor cells, endowed with limited proliferation potential and lacking self-renewal capacity, are unable and maintain the tumor, and the tumor is eradicated.

compound. As CSCs share several resistance mechanisms with normal tissue stem cells, there is a high risk to hit normal stem cells by a targeted anti-CSC therapy with disastrous consequences for the patient. Therefore, it is important to design new therapeutic approaches to selectively hit CSC-specific pathways, while sparing normal stem cells. So far, a number of preclinical studies have been performed using different strategies to target CSCs. These include the specific elimination of CSCs with selective targeting (Schatton et al., 2009) or sensitization of CSCs to conventional chemotherapy and differentiation therapies. In some of these innovative therapeutic approaches, antibodies were used to neutralize autocrine signaling mediators important for CSC growth and chemoresistance, such as CD123 (interleukin3 receptor) in acute myeloid leukemia (Jin et al., 2009) and interleukin-4 in colorectal cancer (Todaro et al., 2007, 2008). Other antibodies interfere with the communication of CSCs with noncancerous tissue, and thereby prevent CSC localization in their niche (Jin et al., 2006; Yang et al., 2008). Similarly, inhibitors blocking CXCR4 impair the interaction of acute myeloid leukemia stem cells with the microenvironment in the bone marrow, with a consequent sensitization to targeted therapy (Zeng et al., 2009). In a glioblastoma model, it was shown that endothelial cells promote the propagation of glioblastoma

stem cells. Subsequently, antiangiogenic therapy using vascular endothelial growth factor inhibitors depleted not only tumor vascularization but also ablated CSCs in the xenograft (Calabrese et al., 2007). Another approach for innovative therapy is the targeted inhibition of important signal transduction pathways and transcription factors highly active in CSCs, including embryonic pathways. Pharmacological inhibition of the hedgehog pathway reduced the growth of lung tumor cells in xenograft models, suggesting an important role of this pathway in lung CSCs also (Watkins et al., 2003). A recent study showed that knock down of the transcription factor Oct-4 led to apoptosis of a CSC-like population of lung cancer cells (Hu et al., 2008). This finding is of particular interest as Oct-4 is expressed in potential human lung progenitor cells that display some characteristics of murine BASCs (Ling et al., 2006; Chen et al., 2007). CSCs can divide asymmetrically and give rise to more differentiated progeny that lose the ability to divide indefinitely. Differentiation therapy aims at converting tumorigenic CSCs in their nontumorigenic progeny. It was shown that treatment with bone morphogenic protein 4 reduced the tumor-initiating cell pool in a glioma model and markedly slowed down tumor growth in vivo without toxic side effects (Piccirillo et al., 2006). Oncogene


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Finally, CSCs represent a good model system to test new drugs for antitumor treatments. However, until recently, several technical difficulties limited the use of CSCs for high throughput screenings of chemical compounds. The biggest problem is probably the rarity of CSCs. Therefore, it is virtually impossible to use sorted CSCs from primary human tumors for large-scale experiments. To overcome these technical limitations, one potential solution is the generation of ‘induced’ CSCs. Gupta et al. (2009) demonstrated that immortalized human mammary epithelial cells underwent epithelial– mesenchymal transition, acquired tumorigenicity, gained the ability to grow as mammospheres and acquired the expression of CSC marker proteins when the expression of E-cadherin was knocked down with short hairpin RNA. These cells were used for high-content screenings and identified compounds selectively abolishing the CD44 þ / CD24low cell population in vitro, leading to a marked reduction in tumorigenicity of pretreated cell lines in vivo. Other experimental strategies can overcome the need to genetically modify cells to achieve sufficient numbers of CSC-like cells for in vitro assays. For several tumors, it is possible to propagate primary cancer cells in spheroid cultures (Reynolds and Weiss, 1992), which may allow extensive CSC characterization in vitro. These cultures contain defined growth factors supporting the proliferation of undifferentiated stem-like cells endowed with high clonogenic capacity and the ability to generate tumor xenografts recapitulating the initial tumor (Singh et al., 2004; Ricci-Vitiani et al., 2007, Todaro et al., 2007). Recently, Pollard et al. described a cultivation method for primary glioblastoma cells using laminincoated flasks and a similar medium used for spheroid cultures. Under these conditions, the cells adhere but maintain a CSC-like character (Pollard et al., 2009). However, these approaches also harbor a number of technical difficulties. At present, CSC spheres from epithelial tumors are difficult to establish, particularly in the case of lung cancer. Thus, the use of CSC spheroids from primary tumors may be limited to a few specialized laboratories. Another potential limitation is that an average of 15–20% of cells in tumor spheres are CSCs (Eramo et al., 2008). As elegantly shown in mouse breast cancer, the rate of symmetric divisions depends on the genomic alterations present in tumors. Thus, although a normal sphere may contain as low as one stem cell, the percentage of CSCs in tumor spheres may be very variables (Cicalese et al., 2009; Pece et al., 2010). Despite all of these limitations, CSC cultures represent an interesting in vitro model system for systematic highcontent drug-screening approaches aiming to find new compounds targeting CSCs and their direct progeny. Lung CSCs: implications for diagnosis, prognosis and monitoring of therapeutic response As discussed in the previous paragraph, lung CSC research may provide a relevant contribution to the establishment of innovative tumor eradicating therapies. Besides therapy, knowledge derived from the CSC field Oncogene

of investigation is going to lead to crucial advances in lung cancer diagnosis, prognosis and monitoring of patient response to treatment. On the basis of the assumption that CSCs are more aggressive than the bulk tumor cell population, tumor malignancy may be related to the grade of cell differentiation and to the abundance of the CSC fraction within the bulk tumor cells. The possibility to isolate, expand and characterize CSCs from different tissues has enabled the identification of CSC-associated antigens and consequently the immunodetection of tumorigenic cells within patient tumors. The estimation of CSC frequency in the tumor mass may be used as a diagnostic tool to define tumor grade and staging more precisely. Recent studies have highlighted the growing interest in the exploitation of lung CSC knowledge for the improvement of lung cancer diagnosis. It has been reported that the expression of CSC-associated markers ALDH1 or Sox2 positively correlated with higher stage and grade in different lung tumors (Jiang et al., 2009; Sholl et al., 2009). Additional CSC-associated markers, embryonic genes or combinations of multiple markers might be relevant to generate more informative lung cancer diagnoses. Highly descriptive, CSC-considering diagnoses might be the starting point for improved and more tailored lung cancer treatments as the therapeutic choice has to be strictly linked to the type and stage of the tumor. It is noteworthy that a correct and detailed diagnosis is relevant to define tumor stage also to predict the chance of metastatic dissemination of tumors. In this context, the so-called circulating tumor cells can be of high importance. Circulating tumor cells are cancer cells that are detectable in patient blood and indicative of tumor dissemination potential (Pantel et al., 2009). An early spread of tumor cells is usually undetected by current imaging technologies in patients with cancer. Therefore, different detection approaches are currently being set up as early metastasis-sensitive methods. These methods rely on the immunological, cytometrical or molecular detection of circulating tumor cells (Pantel et al., 2009). Each type of tumor cell may be ‘circulating’. However, it is likely that only a restricted fraction of them may migrate through the blood stream, colonize other tissues and initiate a new tumor mass in another location, thereby giving rise to metastatic lesions. As shown for breast cancer, these cells are now largely believed to be compatible with CSCs (Ross and Slodkowska, 2009; Theodoropoulos et al., 2009). These findings confer key relevance to CSCs also in the topic of circulating tumor cells and in the differential diagnosis of localized or premetastatic disease. Therefore, circulating CSCs might be used as prognostic tools, representing predictors of tumor progression. (Aktas et al., 2009). The investigation on CSC-related factors linked to patient prognosis and also predictive of patient response to treatments will be crucial for the choice of alternative therapeutic options. Several publications suggest that CSC-related antigen expressions in the tumor mass represent relevant prognostic indicators in various cancers (Maeda et al., 2008; Pallini et al., 2008; Song et al., 2008; Zeppernick et al., 2008).


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In pulmonary tumors, higher ALDH1 expression was linked to poor prognosis in patients with early-stage lung cancer (Jiang et al., 2009). When treatment has started, several biomarkers are used to determine the effect of the therapeutic regimen on both, tumor and patient at early time points, so that ineffective or highly toxic approaches may be rapidly substituted with a second-line therapy. At the end of treatment, specific biomarkers are currently used to monitor over time disease regression, progression or relapse. Current biomarkers to monitor treatment response are the abundance of tumor-derived cells in patient blood or plasma levels of tumor-derived proteins, such as prostate-specific antigen for prostate cancer, CA19.9 for gastric cancer, CA125 for ovarian cancer or CEA and cytokeratins for other tumors, including lung cancer (Pantel et al., 2009). As tumor relapse depends on the presence of tumorigenic cells, a careful monitoring of tumor response to treatments should include the detection of CSC-related biomarkers. In fact, treatment of a tumor with conventional anticancer drugs might result in partial reduction of the tumor mass, representing an indicator of good response using the current standards of tumor monitoring. However, as the drug might preferentially kill the differentiated cells, it is likely that the CSC frequency would not be reduced within the tumor, but rather increased (Bao et al., 2006; Ishikawa et al., 2007). Thus, the evaluation of CSCbased biomarkers seems crucial to dissect among transient tumor-debulking activity and long-lasting tumor-eradicating treatments.

Conclusions Despite the development of targeted therapies, clinical oncologists have experienced a limited improvement in the prognosis of lung cancer patients. It is likely that an improvement in lung cancer patient outcome may require the introduction of more effective innovative agents with increased activity against drug-resistant tumorigenic lung CSCs. We envision that future investigations on CSCs routinely isolated from cancer patients would pave the way to innovative knowledge, leading in turn to novel approaches to fight lung cancer. This may result in considerable changes into the clinical practice, which will require a more intense role of molecular pathologists to provide information for more effective and less destructive personalized therapies.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We thank Giuseppe Loreto for technical assistance with figures and tables. We thank the Italian Association for Cancer Research (AIRC) and the Marie Curie network project ‘ApopTrain’ (MRTN-CT-2006-035624) for supporting the lung cancer stem cell research.

References Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. (2009). Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res 11: R46. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. (2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100: 3983–3988. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB et al. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444: 756–760. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M et al. (2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611. Barker N, van de Wetering M, Clevers H. (2008). The intestinal stem cell. Genes Dev 22: 1856–1864. Beier D, Hau P, Proescholdt M, Lohmeier A, Wischhusen J, Oefner PJ et al. (2007). CD133(+) and CD133() glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67: 4010–4015. Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L et al. (2009). Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA 106: 16281–16286. Besson A, Hwang HC, Cicero S, Donovan SL, Gurian-West M, Johnson D et al. (2007). Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype. Genes Dev 21: 1731–1746. Bhattacharjee A, Richards WG, Staunton J, Li C, Monti S, Vasa P et al. (2001). Classification of human lung carcinomas by mRNA

expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 98: 13790–13795. Bianchi F, Nicassio F, Di Fiore PP. (2008). Unbiased vs biased approaches to the identification of cancer signatures: the case of lung cancer. Cell Cycle 7: 729–734. Blau HM, Brazelton TR, Weimann JM. (2001). The evolving concept of a stem cell: entity or function? Cell 105: 829–841. Blenkinsopp WK. (1967). Proliferation of respiratory tract epithelium in the rat. Exp Cell Res 46: 144–154. Bonnet D, Dick JE. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3: 730–737. Borczuk AC, Toonkel RL, Powell CA. (2009). Genomics of lung cancer. Proc Am Thorac Soc 6: 152–158. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B et al. (2007). A perivascular niche for brain tumor stem cells. Cancer Cell 11: 69–82. Chen Y, Chan VS, Zheng B, Chan KY, Xu X, To LY et al. (2007). A novel subset of putative stem/progenitor CD34+Oct-4+ cells is the major target for SARS coronavirus in human lung. J Exp Med 204: 2529–2536. Chen YC, Hsu HS, Chen YW, Tsai TH, How CK, Wang CY et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One 3: e2637. Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B et al. (2009). The tumor suppressor p53 regulates polarity of selfrenewing divisions in mammary stem cells. Cell 138: 1083–1095. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL et al. (2006). Cancer stem cells—perspectives on current status and Oncogene


4634 future directions: AACR Workshop on cancer stem cells. Cancer Res 66: 9339–9344. Collins LG, Haines C, Perkel R, Enck RE. (2007). Lung cancer: diagnosis and management. Am Fam Physician 75: 56–63. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A et al. (2009). Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA 106: 13820–13825. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW et al. (2007). Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 104: 10158–10163. Dubey S, Powell CA. (2008). Update in lung cancer 2007. Am J Respir Crit Care Med 177: 941–946. Dubey S, Powell CA. (2009). Update in lung cancer 2008. Am J Respir Crit Care Med 179: 860–868. Dylla SJ, Beviglia L, Park IK, Chartier C, Raval J, Ngan L et al. (2008). Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. PLoS One 3: e2428. Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A et al. (2008). Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15: 504–514. Fuchs E. (2009). The tortoise and the hair: slow-cycling cells in the stem cell race. Cell 137: 811–819. Giangreco A, Arwert EN, Rosewell IR, Snyder J, Watt FM, Stripp BR. (2009). Stem cells are dispensable for lung homeostasis but restore airways after injury. Proc Natl Acad Sci USA 106: 9286–9291. Giangreco A, Groot KR, Janes SM. (2007). Lung cancer and lung stem cells: strange bedfellows? Am J Respir Crit Care Med 175: 547–553. Giangreco A, Reynolds SD, Stripp BR. (2002). Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. Am J Pathol 161: 173–182. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M et al. (2007). ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1: 555–567. Guan Y, Gerhard B, Hogge DE. (2003). Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML). Blood 101: 3142–3149. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA et al. (2009). Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138: 645–659. Hill RP. (2006). Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 66: 1891–1895. Ho MM, Ng AV, Lam S, Hung JY. (2007). Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67: 4827–4833. Holyoake T, Jiang X, Eaves C, Eaves A. (1999). Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 94: 2056–2064. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. (2004a). Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium. Am J Pathol 164: 577–588. Hong KU, Reynolds SD, Watkins S, Fuchs E, Stripp BR. (2004b). in vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations. Am J Physiol Lung Cell Mol Physiol 286: L643–L649. Hu T, Liu S, Breiter DR, Wang F, Tang Y, Sun S. (2008). Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis. Cancer Res 68: 6533–6540. Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H et al. (2009). Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 69: 3382–3389. Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S et al. (2007). Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat Biotechnol 25: 1315–1321. Oncogene

Iwasaki H, Akashi K. (2007). Myeloid lineage commitment from the hematopoietic stem cell. Immunity 26: 726–740. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R et al. (2001). Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15: 3243–3248. Janne PA, Engelman JA, Johnson BE. (2005). Epidermal growth factor receptor mutations in non-small-cell lung cancer: implications for treatment and tumor biology. J Clin Oncol 23: 3227–3234. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM et al. (2008). Annual report to the nation on the status of cancer, 1975–2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 100: 1672–1694. Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L et al. (2009). Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res 7: 330–338. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. (2006). Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med 12: 1167–1174. Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L et al. (2009). Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 5: 31–42. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S et al. (2005). Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121: 823–835. Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L et al. (2004). Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci USA 101: 4966–4971. Lantuejoul S, Salameire D, Salon C, Brambilla E. (2009). Pulmonary preneoplasia—sequential molecular carcinogenetic events. Histopathology 54: 43–54. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, CaceresCortes J et al. (1994). A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367: 645–648. Lee A, Kessler JD, Read TA, Kaiser C, Corbeil D, Huttner WB et al. (2005). Isolation of neural stem cells from the postnatal cerebellum. Nat Neurosci 8: 723–729. Levina V, Marrangoni AM, DeMarco R, Gorelik E, Lokshin AE. (2008). Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One 3: e3077. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF et al. (2008). Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100: 672–679. Ling TY, Kuo MD, Li CL, Yu AL, Huang YH, Wu TJ et al. (2006). Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARSCoV) infection in vitro. Proc Natl Acad Sci USA 103: 9530–9535. Maeda S, Shinchi H, Kurahara H, Mataki Y, Maemura K, Sato M et al. (2008). CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer. Br J Cancer 98: 1389–1397. Mahvi D, Bank H, Harley R. (1977). Morphology of a naphthaleneinduced bronchiolar lesion. Am J Pathol 86: 558–572. Minna JD, Roth JA, Gazdar AF. (2002). Focus on lung cancer. Cancer Cell 1: 49–52. O’Brien CA, Pollett A, Gallinger S, Dick JE. (2007). A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445: 106–110. Orkin SH, Zon LI. (2002). Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nat Immunol 3: 323–328. Pallini R, Ricci-Vitiani L, Banna GL, Signore M, Lombardi D, Todaro M et al. (2008). Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme. Clin Cancer Res 14: 8205–8212. Pantel K, Alix-Panabieres C, Riethdorf S. (2009). Cancer micrometastases. Nat Rev Clin Oncol 6: 339–351.


4635 Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I et al. (2004). EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 101: 13306–13311. Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M et al. (2005). KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2: e17. Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S et al. (2010). Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 140: 62–73. Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G et al. (2006). Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444: 761–765. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R et al. (2009). Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4: 568–580. Rawlins EL. (2008). Lung epithelial progenitor cells: lessons from development. Proc Am Thorac Soc 5: 675–681. Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, Hasegawa H et al. (2009). The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4: 525–534. Reya T, Morrison SJ, Clarke MF, Weissman IL. (2001). Stem cells, cancer, and cancer stem cells. Nature 414: 105–111. Reynolds BA, Weiss S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255: 1707–1710. Reynolds SD, Giangreco A, Power JH, Stripp BR. (2000). Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol 156: 269–278. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C et al. (2007). Identification and expansion of human colon-cancer-initiating cells. Nature 445: 111–115. Riely GJ, Marks J, Pao W. (2009). KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc 6: 201–205. Ross JS, Slodkowska EA. (2009). Circulating and disseminated tumor cells in the management of breast cancer. Am J Clin Pathol 132: 237–245. Scagliotti G, Hanna N, Fossella F, Sugarman K, Blatter J, Peterson P et al. (2009). The differential efficacy of pemetrexed according to NSCLC histology: a review of two phase III studies. Oncologist 14: 253–263. Schatton T, Frank NY, Frank MH. (2009). Identification and targeting of cancer stem cells. Bioessays 31: 1038–1049. Sekido Y, Fong KM, Minna JD. (2003). Molecular genetics of lung cancer. Annu Rev Med 54: 73–87. Sholl LM, Long KB, Hornick JL. (2009). Sox2 expression in pulmonary non-small cell and neuroendocrine carcinomas. Appl Immunohistochem Mol Morphol 18: 55–61. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S et al. (2005). Human lymphoid and myeloid cell development in NOD/ LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174: 6477–6489. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J et al. (2003). Identification of a cancer stem cell in human brain tumors. Cancer Res 63: 5821–5828. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al. (2004). Identification of human brain tumour initiating cells. Nature 432: 396–401. Song W, Li H, Tao K, Li R, Song Z, Zhao Q et al. (2008). Expression and clinical significance of the stem cell marker CD133 in hepatocellular carcinoma. Int J Clin Pract 62: 1212–1218.

Storms RW, Trujillo AP, Springer JB, Shah L, Colvin OM, Ludeman SM et al. (1999). Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci USA 96: 9118–9123. Stripp BR, Maxson K, Mera R, Singh G. (1995). Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 269: L791–L799. Stripp BR, Reynolds SD. (2008). Maintenance and repair of the bronchiolar epithelium. Proc Am Thorac Soc 5: 328–333. Theodoropoulos PA, Polioudaki H, Agelaki S, Kallergi G, Saridaki Z, Mavroudis D et al. (2009). Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett. 288: 99–106. Tiseo M, Bartolotti M, Gelsomino F, Ardizzoni A. (2009). First-line treatment in advanced non-small-cell lung cancer: the emerging role of the histologic subtype. Expert Rev Anticancer Ther 9: 425–435. Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L, Iovino F et al. (2007). Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1: 389–402. Todaro M, Perez Alea M, Scopelliti A, Medema JP, Stassi G. (2008). IL-4-mediated drug resistance in colon cancer stem cells. Cell Cycle 7: 309–313. Van Winkle LS, Buckpitt AR, Nishio SJ, Isaac JM, Plopper CG. (1995). Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice. Am J Physiol 269: L800–L818. Ventura JJ, Tenbaum S, Perdiguero E, Huth M, Guerra C, Barbacid M et al. (2007). p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation. Nat Genet 39: 750–758. Visvader JE, Lindeman GJ. (2008). Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8: 755–768. Wang JC, Dick JE. (2005). Cancer stem cells: lessons from leukemia. Trends Cell Biol 15: 494–501. Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB. (2003). Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422: 313–317. Yanagi S, Kishimoto H, Kawahara K, Sasaki T, Sasaki M, Nishio M et al. (2007). Pten controls lung morphogenesis, bronchioalveolar stem cells, and onset of lung adenocarcinomas in mice. J Clin Invest 117: 2929–2940. Yang ZJ, Ellis T, Markant SL, Read TA, Kessler JD, Bourboulas M et al. (2008). Medulloblastoma can be initiated by deletion of patched in lineage-restricted progenitors or stem cells. Cancer Cell 14: 135–145. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C et al. (2007). Let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131: 1109–1123. Zeng Z, Shi YX, Samudio IJ, Wang RY, Ling X, Frolova O et al. (2009). Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML. Blood 113: 6215–6224. Zeppernick F, Ahmadi R, Campos B, Dictus C, Helmke BM, Becker N et al. (2008). Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 14: 123–129. Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. (2009). Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8: 806–823. Zhu L, Gibson P, Currle DS, Tong Y, Richardson RJ, Bayazitov IT et al. (2009). Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 457: 603–607.

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