Toxicologic Pathology, 38: 62-71, 2010 Copyright # 2010 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623309354109

Mouse Models for Cancer Stem Cell Research LE CHENG, ANIRUDH V. RAMESH, ANDREA FLESKEN-NIKITIN, JINHYANG CHOI,

AND

ALEXANDER YU. NIKITIN

Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA ABSTRACT The cancer stem cell concept assumes that cancers are mainly sustained by a small pool of neoplastic cells, known as cancer stem cells or tumor initiating cells, which are able to reproduce themselves and produce phenotypically heterogeneous cells with lesser tumorigenic potential. Cancer stem cells represent an appealing target for development of more selective and efficient therapies. However, direct testing of the cancer stem cell concept and assessment of its therapeutic implications in human cancers have been complicated by the use of immunocompromised mice. Genetically defined immunocompetent autochthonous mouse models of human cancer provide a valuable tool to address this problem. Furthermore, they allow for a better understanding of the relevance of mechanisms controlling normal stem cell compartment to carcinogenesis. Advantages and disadvantages of some of the existing mouse models are reviewed, and future challenges in cancer stem cell research are outlined. Keywords:

breast cancer; cancer stem cell; colon cancer; genetically modified mouse models; ovarian cancer; prostate cancer; soft tissue sarcomas.

The concept of cancer stem cells (CSC) or tumor-initiating cells can be traced back to the ‘‘embryonal rest’’ theory proposed by Conheim and Durante in the nineteenth century (reviewed in Sell 2004). The modern version stipulates that CSC represent a subpopulation of neoplastic cells that may be responsible for cancer initiation and/or progression (reviewed in Cho and Clarke 2008; Clarke and Fuller 2006; Matoso and Nikitin 2008; Nafus and Nikitin, in press; Visvader and Lindeman 2008; Wicha, Liu, and Dontu 2006). CSC have stem cell–like properties that include the active expression of telomerase and common stem cell genes, the activation of antiapoptotic pathways, and an increased ability to migrate and metastasize. Furthermore, CSC may remain relatively quiescent and have mechanisms enhancing their survival and multidrug resistance, enabling them to evade traditional cancer therapies that target rapidly dividing cells (reviewed in Matoso and Nikitin 2008; Visvader and Lindeman 2008). It is important to emphasize that the term CSC does not refer to the cell of origin. Rather, CSC have been named as such in reference to the properties that CSC share with normal stem cells (Wicha et al. 2006). Cancer stem cells may originate from mutated stem, transit-amplifying, or differentiated cells (Figure 1). It is also possible that an initial mutation occurs within an adult stem cell, but subsequent mutations occur in its downstream progeny that gain stem cell properties during neoplastic transformation and function as CSC. As discussed below, an accumulating body of evidence indicates that CSC origin may vary in the context of target lineages and initiating genetic mutations. As accurately pointed out by Visvader and Lindeman (2008), the CSC concept is easily reconcilable with a model of clonal evolution of neoplastic populations (Fialkow 1976; Nowell 1976). Traditionally, CSC have been identified through sphere formation in cell culture with matrigel or extra-low attachment conditions and serial transplantation of cellular subpopulation isolated with fluorescence-activated cell sorting (FACS) into

INTRODUCTION Cancers, whether solid or hematopoietic, from different patients often exhibit heterogeneity in morphology, genetic lesions, molecular profile, cell surface markers, cell proliferation kinetics, and response to therapy (Buerger et al. 1999; Heppner and Miller 1983). This heterogeneity may be a result of cells with stem cell properties that can differentiate to yield different cell types within a tumor. It is now generally accepted that the vast majority of tissues, if not all, have rare tissue-specific multipotent adult stem cells. The adult stem cell is capable of renewing itself and differentiating into one or more specialized cells (reviewed in Fuchs 2009; Orkin and Zon 2008; Rossi, Jamieson, and Weissman 2008). Proliferating progeny of stem cells fated for differentiation are usually called transit-amplifying cells (Smith 2006 and Figure 1). At the same time, the term ‘‘progenitor cell’’ is commonly used to identify any dividing cell with the capacity to differentiate. This term includes putative stem cells and transit-amplified cells in which presence of long-term selfrenewal potential has not yet been determined or excluded, respectively.

Address correspondence to Alexander Yu. Nikitin, Department of Biomedical Sciences, Cornell University, Veterinary Research Tower T2 014A VRT, Ithaca, NY 14853, USA; e-mail: [email protected]. Abbreviations: AR, androgen receptor; Bcrp1, breast cancer - resistance protein 1; CBC, crypt-based columnar; CK, cytokeratin; CSC, cancer stem cells; GFAP, glial fibrillary acidic protein; FACS, fluorescence-activated cell sorting; LRC, label-retaining cells; Lgr5, leucine-rich-repeat–containing G-protein coupled receptor 5; MFH, malignant fibrous histiocytoma; MSC, mesenchymal stem cells; Nf1, neurofibromatosis 1; NOD/SCID, nonobese diabetic/severe combined immunodeficient; NSP, non-side population; OSE, ovarian surface epithelium; PIN, prostatic intraepithelial neoplasia; Shh, Sonic hedgehog; SP, side population; SVZ, subventricular zone; UGSM, urogenital sinus mesenchyme. 62

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FIGURE 1.—Cancer stem cell hypothesis. Cancer stem cells (CSC, dark purple) have been defined as a subset of tumor cells with stem cell–like properties that are thought to be responsible for the growth, progression, metastasis, and recurrence of a tumor. Initiating mutation (yellow bolts) can occur in stem cells (red), transit-amplifying cells (blue), and possibly even their fully differentiated progeny (green).

animal models (reviewed in Nafus and Nikitin, in press). Both assays are designed to determine the self-renewal and differentiation potential of a cell population and are adapted from adult stem cell research, where they have been used to characterize adult stem cells in neural tissues (Doetsch et al. 1999), the prostate (Tsujimura et al. 2002; Xin et al. 2003), and the mammary gland (Shackleton et al. 2006; Stingl et al. 2006). After transplantation of the CSC population, the resulting tumor is expected to mirror the phenotypic heterogeneity of the original tumor and contain CSC with preserved ability to self-renew in subsequent serial transplantations. It is commonly anticipated that CSC have higher tumorigenicity as compared to other neoplastic cells (reviewed in Nafus and Nikitin, in press). The tumorigenic potential is usually tested by transplantations of serially diluted cell populations. To date, in humans, CSC have been identified in a number of malignancies, including leukemia (Bonnet and Dick 1997; Lapidot et al. 1994), breast cancer (Al-Hajj et al. 2003), prostate cancer (Collins et al. 2005), brain tumors (Bao et al. 2006), ovarian cancer (Alvero et al. 2009; Szotek et al. 2006; S. Zhang et al. 2008), and colon cancer (O’Brien et al. 2007; Ricci-Vitiani et al. 2007) (Table 1). These studies typically rely upon xenotransplantation of the CSC subpopulation of a human tumor into mice with a compromised immune system (typically nonobese diabetic/severe combined immunodeficient (NOD/ SCID) mice). Albeit useful, these systems do not accurately reflect factors critical for carcinogenesis, such as the microenvironment and immune response (Frese and Tuveson 2007). Consistent with these limitations of xenotransplantations, a recent study by Quintana and colleagues (2008) demonstrated

that by using severely immunocompromised (IL2rg null NOD/SCID) mice, about 25% of unselected melanoma cells formed tumors as compared to 0.1% to 0.0001% of cells that are able to form tumors in less immunocompromised NOD/ SCID mice. Improved models of xenotransplantation that employ humanized mice may alleviate some pitfalls. For example, in some xenotransplantation models, human cytokines are exogenously administered, or human stromal cells are co-injected to provide the transplanted CSC with a more native growth environment (reviewed in Pearson, Greiner, and Shultz 2008). However, complete understanding of cancer pathogenesis, particularly its earliest stages, is impossible without accurate immunocompetent autochthonous mouse models of human cancer. Recent advances in genetic engineering and genomics have provided the necessary tools to generate genetically defined mouse models that replicate such essential features of human cancers as molecular and histological characteristics, the process of cancer initiation and progression, and response to therapeutics (Table 2) (reviewed in Frese and Tuveson 2007; Jonkers and Berns 2002; Van Dyke and Jacks 2002). Importantly, the mouse has been among the leading model systems to study normal adult stem cell biology, which makes them particularly useful for furthering our understanding of the role of the stem cell compartment in carcinogenesis. Mouse models to study hematopoietic CSC are well described elsewhere (Jordan 2004; Neering et al. 2007; Stubbs and Armstrong 2007; J. Zhang et al. 2006) and will not be discussed in this review. Instead, we focus on recent advances in CSC research in genetically modified mouse models of solid

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TABLE 1.—Identification and characterization of human and mouse cancer stem cells.

Organ

Human/mouse model

Markers

Brain

Human

CD133þ CD15þ GFAPþ

Intestine

Ptcþ/- mouse p53þ/, Nf1fl x/fl x, hGFAP-Cre mouse Human

CD133þ

Lgr5-CreERT2, Apcfl x/fl x mouse Lgr5þ Bmi1Cre-ER/þ Ctnnb1lox(ex3) Bmi1þ mouse Prom1þ/C-L Ctnnb1lox(ex3) mouse CD133þ Mammary gland

Ovary

Prostate

% of CSC

References

3.5-46.3 Sphere formation; xenograft (brain); serial transplantation *15-20 Transplantation N/A* Lineage tracing; anatomic location

Singh et al. 2003; Singh et al. 2004 Read et al. 2009 Y. Zhu et al. 2005

2.5+1.4 Sphere formation; xenograft (subcutaneous; renal); serial transplantation N/A Lineage tracing; anatomic location N/A Lineage tracing; anatomic location

O’Brien et al. 2007; RicciVitiani et al. 2007 Barker et al. 2009 Sangiorgi and Capecchi 2008

N/A

L. Zhu et al. 2009

2-4

p53-/- mouse

CD44þ CD24-/l w CD326þ; CD133þ CD24hi CD29hi

MMTV-Wnt1 mouse MMTV-Wnt1 mouse Human

Thyþ CD24þ CD29low CD24þ CD61þ CD44þ

1-4 *15

MISRII-TAg and LSL-K-rasG12D/ þ Ptenfl x/fl x mice Human Probasin-Cre, Ptenfl x/fl x mouse

6.28 Hoechst 33342low Bcrp1þ CD44þ a2b1hi CD133þ 0.1 Sca1þ, BCL2þ N/A

Probasin-Cre, p53fl x/fl xRbfl x/ fl x mouse

Sca1þ, CK8þ/Synþ

Human

Assays

5-10

6.3-19

N/A

Lineage tracing; anatomic location, clone formation Sphere formation; xenograft (mammary fat pad); serial transplantation Sphere formation; serial transplantation; serial dilution Serial transplantation; serial dilution Serial transplantation; serial dilution

Al-Hajj et al. 2003 S. Zhang et al. 2008 Cho et al. 2008 Vaillant et al. 2008

Sphere formation; xenograft (subcutaneous); serial transplantation Transplantation

Alvero et al. 2009 Szotek et al. 2006

Colony-formation; long-term serial culture Anatomic location

Collins et al. 2005 Wang et al. 2006

Anatomic location

Zhou, Flesken-Nikitin, and Nikitin 2007

Abbreviation: N/A, not available.

cancers, which account for approximately 80% of all human cancers (Jemal et al. 2009). MOUSE MODELS TO STUDY CANCER STEM CELLS Breast Cancer There are many advantages of using mice as a model to study the mammary gland. Mammary stem cells can be relatively easily isolated and have been well characterized (Shackleton et al. 2006; Stingl et al. 2006). Furthermore, it is easy to amass a large quantity of mammary epithelial cells for protein, RNA, or DNA isolation, given that mice have ten mammary glands. In addition, orthotopic transplantation into the cleared fat pad has been a standard technique in mammary gland biology. For this assay, the mammary epithelium is cleared from a three- to four-week-old mouse, leaving only the fat pad. Mammary stem cells transplanted into this fat pad can regenerate the mammary gland. Thus, efficient serial transplantation and limiting dilution assays can be easily performed. In the context of mammary tumors, the CSC subpopulation has been described in several models of mammary carcinogenesis using different cell surface markers (Cho et al. 2008; Vaillant et al. 2008; Zhang, Behbod et al. 2008). For example, in the p53-null mammary tumor model described by Zhang and colleagues (M. Zhang et al. 2008), most tumors coexpressed luminal cell markers (cytokeratin 8 [CK8] and ERa) and the

myoepithelial cell marker CK14. A subpopulation expressing high levels of CD24 and CD29 (Lin-CD24HCD29H), which accounts for 5% to 10% of the total mammary epithelial cell population, has been identified as CSC by limiting dilution in cell culture mammosphere assays and subsequent transplantation in vivo (M. Zhang et al. 2008). Upon transplantation, this CSC subpopulation generated heterogeneous tumors that showed properties similar to the primary tumor, suggesting that this tumor-initiating population is able to regenerate the heterogeneous characteristics of its parent tumor. As few as 100 CD45-, Ter119-, CD31-, and CD140a- (Lin-) CD24HCD29H cells were needed for tumor initiation in eight of fourteen transplantations, whereas no tumors were observed from an equal number of cells from the other Lin- cell populations (CD24LCD29L, CD24HCD29L, or CD24LCD29H). Additionally, mammospheres formed by culturing Lin-CD24HCD29H were larger in size and number compared to other cell surface marker combinations. Microarray gene expression showed that the Lin-CD24HCD29H CSC differentially expressed genes involved in DNA damage response and repair and stem cell self-renewal when compared to the other populations. However, it should be noted that all of the above experiments were performed on tumors derived by transplantation of p53-null mammary epithelium cells into cleared mammary fat pads. Thus, it remains to be demonstrated that observed results can be reproduced in an autochthonous mouse model of mammary carcinogenesis.

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FIGURE 2.—Aldh1 expression in mouse mammary carcinoma initiated by mammary epithelium–specific inactivation of tumor suppressor genes p53 and Rb. A group of putative cancer stem cells contains a large amount of Aldh1 (brown color, also shown in the insert). Paraformaldehydefixed, paraffin-embedded material. ABC Elite method, hematoxylin counterstaining. Calibration bar: 100 mm, insert 40 mm.

In the MMTV-Wnt1 mouse model of mammary cancer, a Thy1þCD24þ CSC population was found to be highly enriched for CSC relative to the non-Thy1þCD24þ population (Cho et al. 2008). Upon mammary fat pad transplantation, one in every 200 cells from this basal CSC population generated tumors that were phenotypically similar to the original tumor and could be serially transplanted. Vaillant and colleagues reported a different set of markers (CD29, CD24, and CD61) that can be used to isolate a CSC population and results in a twenty-fold enrichment tumor–initiating capacity in MMTV–Wnt1 mammary tumors (Vaillant et al. 2008). These studies suggested that the Wnt–bcatenin signaling pathway has an important role in governing the self-renewal of CSC in these tumors. Notably, this group has not found CSC in mammary neoplasms of the MMTV-neu/erbB2 mouse model, suggesting existence of an alternative model of carcinogenesis (Vaillant et al. 2008). Mouse models of mammary tumors do possess some limitations. For example, the exact locations of the mammary stem cell and its niche have yet to be identified. The recent discovery that normal and cancer stem cells have increased activity and expression of aldehyde dehydrogenase (Aldh1) may allow identification of putative stem cell populations in tissue sections (Ginestier et al. 2007) and (Figure 2). Additionally, further work must be done to establish the hierarchy of mammary stem and progenitor cells (reviewed in Stingl and Caldas 2007). Identification of specific markers that can distinguish cells in this hierarchy is crucial for future studies of mammary carcinogenesis.

TABLE 2.—Guidelines for validation of mouse models for human cancer and their specific properties beneficial for cancer stem cell research. General features

Stem-cell–related features

Common molecular mechanisms. Pathology mimics human cancer. Neoplastic progression, including metastasis, recapitulates human cancer. Therapeutic and host immune responses resemble human. Involvement of a specific organ or cell lineage resembles human.

Normal adult stem cells are well characterized. Controlled cancer initiation is possible. Location of stem cell compartment is anatomically defined. Putative cancer stem cells are identified. Normal stem cells are abundant.

Prostate Cancer Anatomically, the mouse prostate consists of a series of branching ducts, and each duct consists of a proximal region attached to the urethra, an intermediate region, and a distal region or acinus (reviewed in Nikitin et al., 2009). Recent experiments have shown that transit-amplifying progenitor cells and differentiated cells are located in the distal region, whereas prostate epithelial stem cells are densely concentrated in the proximal region of the prostatic ducts (Leong et al. 2008; Tsujimura et al. 2002; Xin et al. 2005). A very useful system for the analysis of prostate stem cells is the urogenital tissue

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recombination procedure (Cunha and Lung 1978; Xin et al. 2003). In this assay, prostate epithelial cells are combined with mouse or rat urogenital sinus mesenchyme (UGSM), which is located near the caudal part of the embryo and provides a strong inductive influence on the prostate epithelium. This system presents the opportunity to study proliferation and regeneration of prostatic branching tubules after transplantation under the renal capsule. In the probasin-Cre–induced Pten knockout model, deletion of tumor suppressor Pten in a mouse model of prostate cancer replicates the disease progression seen in humans, from hyperplasia to prostatic intraepithelial neoplasia (PIN), invasive adenocarcinoma, and sometimes to metastasis (Wang et al. 2006). Pten-inactivated p63þ basal cells were positive for CK5 (basal cell marker) and had increased proliferation. Expansion of p63þ basal cells was concomitant with the expansion of cells positive for stem cell marker Sca-1, which has been found to be enriched in proximal regions of the prostate gland (Xin et al. 2005). This finding demonstrated that basal cell proliferation can be an initiating event for precancerous lesions. In another mouse model of prostate cancer, prostate epithelium-specific p53 and Rb deficiency cooperate in prostate carcinogenesis and lead to highly aggressive, poorly differentiated, and metastatic carcinomas (Zhou et al. 2006). Use of stage-by-stage analysis of carcinogenesis demonstrated that malignant neoplasms arise from the stem/progenitor cell– enriched proximal region of prostatic ducts (Zhou, FleskenNikitin, and Nikitin 2007). The earliest lesions express Sca-1 and coexpress luminal markers CK8 and AR, and neuroendocrine marker synaptophysin, but not basal marker CK5, which indicates that a common progenitor for luminal and neuroendocrine differentiation is a potential cell of origin in these tumors. In the same study, inactivation of p53 and Rb has also been found in transit-amplifying and/or differentiated cells of the distal part of prostatic ducts. However, only PINs were found in those areas, and these PINs never progressed by the time of mouse death. These observations demonstrate that p53 and Rb play an important role in controlling the prostate stem cell compartment, the transformation of which may lead to particularly aggressive cancers. Comparing the Pten and p53/Rb models presented, it is important to note that both models use the same transgenic mouse strain, PB-Cre4, for prostatespecific Cre-loxP-mediated gene inactivation. However, whereas neoplasms in the Pten model contain basal, luminal, and neuroendocrine cells (Liao et al. 2007), only luminal and neuroendocrine differentiation is observed in the p53/Rb model. This finding indicates that different initiating genetic events may have unequal transforming effects on stem cells and their progeny. Studies of multiple genetically modified mouse models are likely to lead to better appreciation of genetically based variations in the morphology observed in human prostate cancer. Although mouse prostate is an excellent organ for serial sectioning followed by three-dimensional reconstitution, which is essential for studies of early stages of carcinogenesis and its relation to the stem cell compartment, its small size represents

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a challenge for projects requiring large number of stem cells. Additionally, its location complicates intravital imaging approaches. Intestinal Cancer Epithelial cells in the intestine and colon experience rapid turnover. To maintain tissue homeostasis, it has been proposed that multipotent stem cells reside in the base of the colon crypts and are capable of regenerating all intestinal cell types (Booth and Potten 2000; Brittan and Wright 2002; Radtke and Clevers 2005). Differentiation is thought to occur along the crypt axis; stem cells reside at the base of the crypts in their niche and differentiate upward to produce actively proliferating transitamplifying cells. These cells can differentiate into committed progenitors that yield all the colonic cell lineages (reviewed in Humphries and Wright 2008). Several experiments have been conducted that confirm a colonic crypt has a monoclonal origin (reviewed in Booth and Potten 2000; Humphries and Wright 2008; Marshman, Booth, and Potten 2002; Potten, Booth, and Pritchard 1997). Colon cancer is thought to develop commonly because of mutations in the APC/Wnt pathway. Mutations in the epithelial cells lining the colon result in the development of adenomatous polyps, which can develop into invasive carcinomas (Heyer et al. 1999). Common events leading to polyp formation include mutations in the tumor suppressor APC and in the proto-oncogene K-RAS. Inflammation in the colon can also lead to cancer through dysregulation of the TGFb pathway (Humphries and Wright 2008). Of these two main classes of colon cancer, many existing mouse models employ mutations in APC/Wnt or IL-10 to mirror polyp formation and inflammation, respectively. Recently, the leucine-rich-repeat–containing G-protein coupled receptor 5 (Lgr5) was identified as a stem cell marker in the small intestine and colon (Barker et al. 2007). This marker was expressed solely at the base of intestinal crypts and correlated strongly with long-term label-retaining crypt-based columnar (CBC) cells. Using mice with an inducible Cre knock-in allele (Lgr5CreERT2) and the Rosa26-lacZ reporter, lineage-tracing experiments demonstrated that the Lgr5þ CBC cells generated all epithelial lineages over a sixty-day period. Identification of Lgr5þ CBC cells as the stem cells of the small intestine and colon allowed testing of whether these cells may represent a cell of origin of intestinal cancer. To achieve inactivation of Apc specifically in Lgr5þ cells, Lgr5CreERT2 mice were crossed with Apcfl x/fl x mice, resulting in a targeted knockout of Apc in Lgr5þ colon stem cells upon tamoxifen induction (Barker et al. 2009). It was determined that stem cells with deletion of Apc remained at crypt bottoms and led to rapid growth of microadenomas followed by formation of macroscopic adenomas within three to five weeks after Apc inactivation. Interestingly, deletion of Apc in short-lived transitamplifying cells rarely resulted in large adenomas, even after thirty weeks. These results suggest that inactivation of Apc in stem but not transit-amplifying cells leads to neoplastic

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progression. Furthermore, the distribution of Lgr5þ cells within stem-cell–derived adenomas indicates preservation of a stem cell/progenitor cell hierarchy in early neoplastic lesions. Consistent with a notion that transformed intestinal stem cells may lead to neoplasia, it has been demonstrated that Bmi1 may also serve as a small intestine stem cell marker and that activation of b-catenin by expression of tamoxifen-inducible Bmi1CreER leads to rapidly growing adenomas (Sangiorgi and Capecchi 2008). Similarly, it has been reported that another stem cell marker, Prominin-1 (also known as CD133) marks intestinal stem cells that are susceptible to neoplastic transformation (L. Zhu et al. 2009). It should be noted that although human colon CSC were isolated from colon cancers based on their expression of the marker CD133/Prominin-1, this marker, similar to Bmi1, labels stem cells in the small intestine of the adult mouse. Furthermore, the majority of mouse intestinal neoplasms are adenomas and adenocarcinomas of the small intestine (Heyer et al. 1999). Considering that the majority of human intestinal neoplasms are colon cancers, further identification of colon stem cells as well as development of an accurate mouse model of colon cancer are of critical importance.

Brain Tumors In the 1990s, the field of neuroscience went through a paradigm shift owing to the discovery of neural stem cells. Several groups identified persistent adult neurogenesis within discrete brain regions (reviewed in Lie et al. 2004). The subventricular zone (SVZ) of the lateral ventricles, the subgranular layer of the hippocampus, and the olfactory bulbs have been identified as locations of adult mouse neural stem cells (Sanai et al. 2004). In particular, the largest population of adult neural stem cells was shown to reside in the SVZ (Doetsch et al. 1999). These putative SVZ neural stem cells are a subset of the astrocyte population, called type B astrocytes, and express glial fibrillary acidic protein (GFAP). These cells are thought to differentiate into progenitors and migrate to the olfactory bulbs, where they integrate as mature neurons (reviewed in Zaidi et al. 2009). An analogous process of neurogenesis is thought to occur in humans (reviewed in Ma, Ming, and Song 2005). The implication of continued neurogenesis is the presence of undifferentiated, quiescent stem and progenitor cells that are capable of renewing brain tissue. These neural stem and progenitor cells may be targets of transformation, giving rise to brain tumors. The two most common brain tumors are gliomas and medulloblastomas. The most aggressive primary brain tumor, glioblastoma multiforme, has a median survival of approximately fourteen months (Vescovi, Galli, and Reynolds 2006). Numerous mouse models of brain tumors have been created to study the various kinds of brain tumors. Many of these models are able to phenotypically and histologically mimic corresponding human cancers (reviewed in Holland 2001; Huse and Holland 2009). For example, transgenic expression of src kinase driven by the GFAP promoter is able to recapitulate glioblastoma multiforme (Holland 2004). Common mouse

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models of gliomas and medulloblastomas also include genetically engineered mutations in Pten, Ras, Wnt, and other genes. A mouse glioma model was generated by Zhu and colleagues (Y. Zhu et al. 2005) by crossing p53 mutant heterozygous (p53þ/) mice with a floxed neurofibromatosis 1 (Nf1) allele mouse and a GFAP promoter–driven Cre-expressing mouse. The Nf1 tumor suppressor is a Ras GTPase activating protein that serves to moderate Ras signaling. Targeted loss of NF1 in astrocytes and glial precursors caused malignant, high-grade gliomas and astrocytomas with complete penetrance. Furthermore, longitudinal MRI imaging of asymptomatic mutant mice identified lesions in the SVZ that developed into astrocytomas expressing the neural stem cell markers GFAP and nestin (Y. Zhu et al. 2005). This model provides evidence that the cell of origin of some gliomas may be a neural stem or progenitor cell in the SVZ. Kessler and colleagues (Kessler et al. 2009) used a patched (ptc) mutant mouse model of medulloblastoma. Ptc is an antagonist of the Sonic hedgehog (Shh) signaling pathway, and its loss results in increased Shh signaling. About 15% of Ptc-heterozygous mice develop medulloblastomas that resemble the corresponding human disease. Using this Ptc mouse model, Read and colleagues (2009) found that expression of CD15 but not CD133, a marker typically used for isolation of human brain CSC, can be used for enrichment of tumorigenic neoplastic cells. These CD15þ cells form tumors that resemble the original tumor after transplantation into the mouse cerebellum. Interestingly, these CD15þ cells did not form neurospheres in culture, indicating a deficiency in self-renewal. Therefore, CD15þ cells are likely to be transformed progenitor but not stem cells. These authors also observed CD15 expression in a subset of human medulloblastomas (Read et al. 2009). It should be noted that some of the existing mouse models of brain tumors represent phenocopies of developed human neoplasms but do not necessarily accurately recapitulate tumor-predisposing conditions and initiating genetic alterations (Huse and Holland 2009). Development of more accurate models, as well as identification of markers allowing for the discrimination of stem cells from transit-amplifying cells should significantly accelerate advances in understanding the cell of origin and early pathogenesis of brain tumors. Ovarian Cancer The ovarian surface epithelium (OSE) covers the ovary as a single layer of squamous or cuboidal cells. During folliculogenesis, the OSE is ruptured and a series of molecular events initiates and executes repair of the epithelia (Clow, Hurst, and Fleming 2002; Tan and Fleming 2004). Ninety percent of ovarian cancers, the most deadly gynecological disease for the last three decades (Jemal et al. 2009), are of epithelial origin, and the majority of them are believed to arise from the OSE lining the ovary or epithelial inclusion cysts (reviewed in Auersperg et al. 2001; Nikitin and Hamilton 2005). Unlike the majority of epithelial tissues, presence of epithelial ovarian stem cells remains insufficiently established,

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and no unique markers have been identified. Recently, using pulsechase experiments with BrdU/IdU (5-bromo-20 -deoxyuridine/ 5-iodo-20 deoxyuridine) and tetracycline-regulated (doxycycline responsive) tetO-H2B-GFP transgenic mice, Szotek and colleagues (2008) have identified existence of ovarian epithelial label–retaining cells (LRCs). This putative somatic stem/progenitor cell population exhibits properties of quiescence, functional response to estrous cycling by proliferation in the mouse, enhanced colony-forming ability in tissue culture, and cytoprotection (Szotek et al. 2008). The location of these putative stem cells, as well as their ability for self-renewal, remains to be determined. In an attempt to isolate ovarian CSC, Szotek and colleagues conducted experiments to identify ‘‘side population’’ (SP) cells in mouse and human ovarian cancer cell lines (Szotek et al. 2006). SP cells are detected by differential efflux of the DNA-binding dye Hoechst 33342. A subset of verapamilsensitive SP cells that express breast cancer–resistance protein 1 (Bcrp1) was identified in two distinct, genetically engineered mouse epithelial ovarian cancer cell lines (MOVCAR7 and 4306), three human ovarian cancer cell lines (IGROV-1, SK-OV3, and OVCAR-3), and four out of six EOC primary ascites. These studies are in agreement with other recent studies reporting identification of CSC in human EOC (Alvero et al. 2009; S. Zhang et al. 2008). Notably, CD44 and CD117 were found to be markers for human EOC CSC (Alvero et al. 2009; S. Zhang et al. 2008). It remains to be determined whether any of those markers are suitable for isolation of CSC from already existing genetically modified mouse models of human EOC, including its serous (Flesken-Nikitin et al. 2003) and endometrioid (Dinulescu et al. 2005; Wu et al. 2007) variants. Since many of the same functional properties that characterize somatic stem cells also define CSC, it will be of interest to see whether any of already available ovarian CSC markers may be used for detection of normal OSE stem cells. Further studies aiming to confirm existence of OSE stem cells and to provide their complete characterization will allow evaluation of the link between normal and CSC of the OSE. Soft Tissue Sarcomas Soft tissue sarcomas are categorized as mesenchymal neoplasms because of their similarities to mesenchymal cells. Indeed, recent studies strongly support the hypothesis that mesenchymal stem cells (MSC) are the cell of origin of sarcomas (Mackall, Meltzer, and Helman 2002). Somatic MSC are present in a variety of tissues and are able to give rise to many cell lineages, such as bone, cartilage, muscle, fat, marrow, stroma, and a variety of other connective tissues (Bianco, Robey, and Simmons 2008; Caplan and Bruder 2001). These fibroblast-like cells were first isolated with the ability to adhere from bone marrow and to proliferate without differentiating for up to forty passages in culture. MSC can be isolated with the unique immunophenotypical profile, being positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106,

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CD120a, and CD124 in humans, and positive for Sca-1, CD29, CD44, c-kit, and CD105 in mice (Charytonowicz et al. 2009; Sun et al. 2003). Several recent experiments indicate that transformed MSC progress to sarcomas. Mesenchymal stem cells expanded in culture and transplanted into the bone marrow of mice resulted in the development of sarcomas (Tolar et al. 2007). Similar results have been demonstrated by implantation of MSC/ bioscaffold constructs into syngeneic and immunodeficient recipients (Tasso et al. 2009). Consistent with these observations, a study by Matushansky and colleagues showed that a stem cell–specific gene expression pattern was significantly associated with that of the malignant fibrous histiocytoma (MFH), which is a common type of sarcoma (Matushansky et al. 2007). Transplantation of human MSC with inhibited Wnt signaling into mice resulted in high-grade, MFH-like tumors (Matushansky et al. 2007). The development of myxoid liposarcomas and Ewing sarcomas, characterized by the unique chromosomal translocation involving fusion proteins FUS-CHOP and EWS-FLI1, respectively, can be induced by expression of those fusion proteins in MSC (Riggi et al. 2005; Riggi et al. 2006). Likewise, alveolar rhabdomyosarcomas develop in mice after xenografts of MSC carrying the PAX3-FKHR fusion proteins. However, p53 inactivation often occurs as a cooperative genetic event in this model (Charytonowicz et al. 2009; Ren et al. 2008). Taken together, these results suggest that MSC may be the cell of origin of some sarcomas and that aberrant differentiation of MSC by molecular alterations may lead to the development of histologically diversified sarcomas. The histological diversity of sarcomas and lack of reliable markers have hindered identification of their CSC. However, CSC have recently been identified in Ewing’s sarcoma (Suva et al. 2009) and embryonal rhabdomyosarcoma (Langenau et al. 2007). Connection between MSC and CSC in various sarcomas remains to be determined. Autochthonous mouse models of soft tissue sarcomas that permit the study of CSC are relatively underdeveloped. However, initiation of sarcomas by conditional gene inactivation, such as a model based on Cre-loxP–mediated deletion of p53 and activation of K-ras (Kirsch et al. 2007), is a welcome development. The generation of mouse models allowing selective targeting of either MSC or more differentiated transitamplifying cells will eventually allow the determination of the role of MSC compartment in sarcoma pathogenesis. CONCLUSION During recent years, there have been extensive efforts to identify, isolate, and target CSC in human cancers. However, such fundamental questions as specific contributions of stem cell– related molecular mechanisms to pathogenesis of various cancers, the role of the normal stem cell compartment in cancer initiation and effects of the immune system and microenvironment on stem cells during carcinogenesis have only just began to be addressed. Rapid advances in genetic engineering and mouse modeling have provided a favorable ground for

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generation of accurate immunocompetent autochthonous models of human cancer. Application of these models has already significantly facilitated stem cell research in general and its cancerrelated aspects in particular. However, since each organ site and tissue has certain limitations, further refinement of existing mouse models and generation of new ones are required. These models will provide the rigorous testing of the clinical relevance of CSC in a broad spectrum of human tumors and provide a solid basis for development of new individualized diagnostic, predictive, and therapeutic approaches to their full extent. ACKNOWLEDGMENTS We thank David C. Corney for critical reading of this manuscript. This work was supported by NIH grants CA96823 and CA112354, and by the Empire State Stem Cell Fund through New York State Department of Health Contract #C023050. REFERENCES Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., and Clarke, M. F. (2003). Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983–88. Alvero, A. B., Chen, R., Fu, H. H., Montagna, M., Schwartz, P. E., Rutherford, T., Silasi, D. A., Steffensen, K. D., Waldstrom, M., Visintin, I., and Mor, G. (2009). Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance. Cell Cycle 8, 158–66. Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., and Leung, P. C. (2001). Ovarian surface epithelium: Biology, endocrinology, and pathology. Endocr Rev 22, 255–88. Bao, S., Wu, Q., McLendon, R. E., Hao, Y., Shi, Q., Hjelmeland, A. B., Dewhirst, M. W., Bigner, D. D., and Rich, J. N. (2006). Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–60. Barker, N., Ridgway, R. A., van Es, J. H., van de Wetering, M., Begthel, H., van den Born, M., Danenberg, E., Clarke, A. R., Sansom, O. J., and Clevers, H. (2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608–11. Barker, N., van Es, J. H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P. J., and Clevers, H. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–7. Bianco, P., Robey, P. G., and Simmons, P. J. (2008). Mesenchymal stem cells: Revisiting history, concepts, and assays. Cell Stem Cell 2, 313–19. Bonnet, D., and Dick, J. E. (1997). Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3, 730–37. Booth, C., and Potten, C. S. (2000). Gut instincts: Thoughts on intestinal epithelial stem cells. J Clin Invest 105, 1493–99. Brittan, M., and Wright, N. A. (2002). Gastrointestinal stem cells. J Pathol 197, 492–509. Buerger, H., Otterbach, F., Simon, R., Poremba, C., Diallo, R., Decker, T., Riethdorf, L., Brinkschmidt, C., Dockhorn-Dworniczak, B., and Boecker, W. (1999). Comparative genomic hybridization of ductal carcinoma in situ of the breast-evidence of multiple genetic pathways. J Pathol 187, 396–402. Caplan, A. I., and Bruder, S. P. (2001). Mesenchymal stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med 7, 259–64. Charytonowicz, E., Cordon-Cardo, C., Matushansky, I., and Ziman, M. (2009). Alveolar rhabdomyosarcoma: is the cell of origin a mesenchymal stem cell? Cancer Lett 279, 126–36.

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