[Frontiers in Bioscience 16, 1927-1938, January 1, 2011]
Metastatic potential of tumor-initiating cells in solid tumors
Amit S. Adhikari, Neeraj Agarwal, Tomoo Iwakuma
Department of Genetics, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
TABLE OF CONTENTS
1. ABSTRACT
The lethality of cancer is mainly caused by its properties of metastasis, drug resistance, and subsequent recurrence. Understanding the mechanisms governing these properties and developing novel strategies to overcome them will greatly improve the survival of cancer patients. Recent findings suggest that tumors are comprised of heterogeneous cell populations, and only a small fraction of these are tumorigenic with the ability to self-renew and produce phenotypically diverse tumor cell populations. Cells in this fraction are called tumor-initiating cells (TICs) or cancer stem cells (CSCs). TICs have been identified from many types of cancer. They share several similarities with normal adult stem cells including sphere-forming ability, self-renewability, and expression of stem cell surface markers and transcription factors. TICs have also been proposed to be responsible for cancer metastasis, however, scarce evidence for their metastatic potential has been provided. In this review article, we have attempted to summarize the studies which have examined the metastatic potential of TICs in solid tumors.
2. INTRODUCTION
Earlier findings from the Dick's laboratory have led to the proposal of a new concept for the origin of cancer; using human acute myeloid leukemia (AML) cells they demonstrated that tumors consists of heterogeneous cellular populations, of which, only a small fraction, called tumor-initiating cells (TICs) or cancer stem cells (CSCs), have the ability to initiate tumors (1, 2). Comparisons between stem cells from normal haematopoietic and leukemic tissues successfully demonstrate their phenotypic similarities including stem cell surface marker profiles and the capabilities of proliferation, differentiation, and self-renewal (2). These results suggest that normal haematopoietic primitive cells are the targets for leukemic transformation and that TICs originate from the transformation of normal stem cells. However, there might be alternative mechanisms that give rise to CSCs; cancer cells could acquire stem cell properties via de-differentiation or by fusion with progenitor cells, although there is no evidence for these mechanisms (3, 4). Thus, the origin of TICs remains unresolved.
Compared to the haematopoietic system, cell surface markers or functional assays for identifying and evaluating normal adult stem cells from other tissues are underdeveloped. Further, cells within solid tumors are less accessible than those from haematologic malignancies. Nonetheless, for the last several years, TICs have been identified from solid tumors of diverse origins, such as breast (5), brain (6), colon (7), pancreas (8), prostate (9), lungs (10, 11), ovaries (12), liver (13), and bone and soft tissues (14-18). In this review, we have considered cells as TICs, only when isolated cells were shown to initiate tumors in vivo as compared with other cellular populations or in studies that used already established methods for TIC isolation. Findings from these studies confirm that TICs share many properties with normal adult stem cells including self-renewability, multi-lineage differentiation potential, sphere formation, and the expression of genes related to stem cell maintenance and proliferation (19, 20). Another important property observed in both normal adult stem cells and TICs is the ability to efflux Hoechst 33342 and rhodamine dyes, a property that is used to identify a subpopulation of cells, called the side population (SP) (21, 22). This property is mainly mediated by ATP-binding cassette (ABC) transporters and confers a drug-resistance phenotype. The SP cells in many types of cancer have been shown to be enriched in tumorigenic stem-like cancer cells (23).
TICs have also been proposed to be responsible for cancer metastasis. This is mainly due to the following reasons: 1) TICs are believed to possess an increased ability to survive and grow in a foreign environment (24, 25); 2) if TICs are the only population that can initiate tumors, tumor formation at secondary sites should be initiated by the TICs (26); and, 3) cancer cells use the same molecular machinery for invasion and metastasis as normal stem cells do for homing or mobilization (27-31). Direct evidence for metastatic potential of TICs, however, is just beginning to emerge. In this review, we focus on the recent studies which provide evidence of the metastatic property of TICs with the hope of understanding the mechanisms behind cancer metastasis as well as accelerating the development of novel therapies that target metastatic cancer.
3. METHODS TO ENRICH TUMOR INITIATING CELLS
Based on the hypothesis that TICs might originate from normal adult stem cells and share many properties with them (2), methods to isolate normal adult stem cells have been used to enrich TICs from solid tumors. These include the presence of stem cell surface markers, ABC transporters, aldehyde dehydrogenase (ALDH) activity, and stem cell transcription factors. Additionally, biological properties of stem cells, such as sphere formation and resistance to chemotherapeutic drugs have also been used (Figure 1). Properties of TICs identified using one method may be different from those identified by other methods, because of the possible heterogeneous nature of TICs. In this section, we summarize the methods for enriching TICs from various types of cancer.
3.1. Stem cell surface markers
TICs in acute myeloid leukemia were enriched with CD34+CD38- cells similar to the haematopoietic stem cells. When transplanted into immunocompromised mice, CD34+CD38- cells initiated tumors, whereas CD34-CD38+ cells did not (1, 2). Since then, cell surface markers present in normal adult stem cells such as CD44, CD133, and CD117, in combination with tumor-type specific or lineage markers have been used to enrich TICs from solid tumors. CD44, a receptor for hyaluronic acid (HA), is a transmembrane glycoprotein involved in cell growth, survival, differentiation, cell-cell interaction, and motility (32, 33). CD44 is expressed in both embryonic and adult stem cells (34). TICs from solid tumors were first identified from breast cancer using CD44+CD24-Lineage- (5). In breast cancer cells, CD24 is expressed in more differentiated cells, whereas CD44 is expressed in more progenitor-like cells (35). In pancreatic adenocarcinoma, CD44+CD24+ epithelial specific antigen (ESA)+ was used to identify the TICs (8). To enrich prostate TICs, CD44+alpha2beta1+ and CD44+CD24- were used (9, 36). CD133 is a glycoprotein also known as Prominin 1 (PROM1), is expressed in haematopoietic stem cells, endothelial progenitor cells, and neuronal/glial stem cells (29, 37). Therefore, CD133 was used to identify TICs from many different types of cancer including brain tumor (6), prostate cancer (38), colon cancer (7), lung cancer (10, 11), and melanoma (39). CD117/c-kit is a 145 kDa transmembrane glycoprotein and is expressed in both haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Recently, our group has shown that CD117 in combination with another MSC-specific marker Stro-1 can identify osteosarcoma TICs (40). Thus, TICs can be identified from various types of cancer using stem cell surface markers. However, it remains unclear whether the stem cell surface markers play an active role in the biological properties of TICs in addition to serving as their identification markers. Further studies are required to clarify this issue.
3.2. Side population (SP)/ABC transporters
The ABC transporters, which are expressed in a wide variety of stem cells and are associated with cellular drug resistance, are molecular determinants of SP cells (23). SP cells can be identified as a cellular fraction that effluxes the fluorescent dyes Hoechst 33342 and rhodamine 123 (41). Specifically, ABCG2 effluxes Hoechst 33342, while ABCB1 and ABCB5 efflux rhodamine 123 (42, 43). Since the SP is used to identify stem-like cells in various normal tissues (44), this population along with ABC transporters have also been used to identify TICs from various types of tumors including gastric cancer (45-47), hepatocellular carcinoma (48, 49), nasopharyngeal carcinoma (50), lung cancer (51), esophageal carcinoma (52), glioma/glioblastoma (53, 54), pancreatic cancer (55, 56), and breast cancer (57, 58). SP cells have also been used to isolate TICs from bone and soft tissue sarcomas, such as malignant fibrous histiocytoma, rhabdomyosarcoma, and osteosarcoma (16). Additionally, ABCB5 was used to identify melanoma TICs (59). However, cells that are non-SP or negative for ABC transporters can occasionally initiate tumors in some types of cancer (46, 60, 61), suggesting that these markers are not universal for all tumor types and that TICs cannot exclusively be enriched by these methods.
3.3. Aldehyde dehydrogenase (ALDH)
The enzyme aldehyde dehydrogenase (ALDH) is responsible for the oxidation of intracellular aldehydes. Since ALDH is highly expressed in haematopoietic stem/progenitor cells (62, 63) and also in primitive cells from other lineages including neuronal and mammary epithelial cells (64), high levels of ALDH activity measured by the ALDEFLUOR system have been used as a novel approach for the identification of stem/progenitor cells. Cells with high ALDH activity become brightly fluorescent and can be identified and enumerated using a standard flow cytometer. Since cells exclusively having an intact cellular membrane can retain the ALDEFLUOR reaction product, only viable ALDH+/hi cells are identified and the isolated cells are readily available for both in vitro and in vivo studies. ALDH+/hi cells have been isolated from several types of cancer and show high tumorigenic stem cell-like properties including breast cancer (64-68), hepatocellular carcinoma (69), colorectal cancer (70), osteosarcoma (71), and pancreatic cancer (72). It should be noted that several studies show little correlation between ALDH expression and the CD44+CD24- phenotype in breast cancer cells (64, 65, 68). Thus, it remains unclear if TICs identified by ALDH activity possess similar stem cell-like properties to those identified by other methods and whether ALDH activity can be used as a universal method for all types of cancer.
3.4. Sphere formation
Sphere formation is the ability of progenitor cells to propagate in the absence of serum and in an anchorage-independent manner (73). This assay was first introduced by Reynolds and Weiss to identify normal neural stem cells in a defined sphere-specific medium whereby striatal embryonic progenitors could be isolated and maintained (74, 75). By culturing tumor cells in a sphere-specific condition, formed spheres have been shown to be enriched in TICs. These studies include glioblastoma (76), ovarian cancer (77), melanoma (78), cervical cancer (79), and osteosarcoma (40, 80). However, sphere formation requires prolonged culture times, amounting to about two weeks, which could give rise to non-TICs after cell division. Another caveat of the sphere formation assay is that the observed spheres could be just aggregated cells, unless performed using a single cell clonogenic assay. For these reasons, this method is mainly used to test the in vitro tumorigenic potential and self-renewability of the TICs isolated by other methods.
3.5. Other methods
A novel approach to isolate TICs is based on the previous observation of enhanced Oct-4 expression in osteosarcoma-derived spheres (81). Levings et al. (18) generated a transgenic human osteosarcoma cell line stably expressing an Oct-4 promoter-driven green fluorescent protein (GFP). Cells expressing Oct-4/GFP showed high tumor-initiating potential. Rhabdomyosarcoma TICs were identified by the expression of fibroblast growth factor receptor 3 (FGFR3) (17). The FGFR3-positive cells showed an elevated expression of progenitor-related genes, such as CD34, Pax3, Oct-4, Nanog, and Sox2, and initiated tumors more efficiently than the FGFR3-negative cells. Based on the idea that TICs are responsible for tumor regeneration after chemotherapy, they can be enriched and maintained following the treatment of particular chemotherapeutic drugs. Levina et al. (82) treated a lung cancer cell line with several chemotherapeutic drugs and demonstrated that the drug surviving cells (DSCs) were enriched in TICs having high metastatic potential.
4. METASTATIC PROPERTY OF TICS IN SOLID TUMORS
Metastasis is the ability of cells to detach from a primary tumor, migrate into lymphatic or blood vessels, disseminate and survive in the lymphatic or blood systems, and initiate new tumors at secondary sites. Most solid cancers develop metastases, which are directly responsible for the majority of cancer-related deaths. Although developing therapies to target cancer metastasis is essential, a lack of complete understanding of the underlying mechanisms remains a major hurdle. Recent studies suggest that the molecular machinery for cancer invasion and metastasis is similar to that involved in the activation, mobilization, and homing of normal stem cells (27-31). Since non-TICs do not have the ability to initiate tumors at secondary sites and because TICs share several molecular and biological properties with normal stem cells, TICs have been proposed to be responsible for metastasis (26, 83). Unfortunately, till date, only a small number of studies have investigated the invasive and metastatic properties of TICs. In this section, we summarize the recent studies that demonstrate the metastatic potential of TICs in solid tumors (Table 1).
4.1. Breast cancer
Since the first report of breast cancer TICs using CD44+CD24-Lineage- (5), several other studies have also identified breast cancer TICs using different methods (57, 58, 64). Many of them, however, lack investigation on the metastatic property of TICs. The first report suggesting the high metastatic nature of TICs was made by Balic et al. (84). They demonstrated significant enrichment of breast cancer cells, having stem cell-like phenotypes within the bone marrow micrometastases. Later studies providing evidence for the metastatic property of breast cancer TICs include those from Wicha's group (66). They demonstrated that HER2 overexpression increased the ALDH+ population in several human breast cancer cell lines and that ALDH+ cells showed higher abilities of tumor formation and invasion compared with ALDH- cells (Table 1). Furthermore, the same group used in vitro assays and intracardiac injections to show that ALDH+ TICs from human breast cancer cell lines or a xenograft possessed higher metastatic potential (67, 68). Interestingly, CXCR1/IL-8RA signaling increased the ALDH+ population and self-renewability (67). ALDH1 expression was also associated with the development of early metastasis, as well as poor clinical outcome in inflammatory breast cancer patients (68).
Croker et al. (65) identified a subpopulation of human breast cancer TICs having high metastatic potential by combining two methods for TIC identification, namely ALDH activity and the presence of stem cell surface markers CD44 or CD133 (Table 1). ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ TICs showed greater abilities of colony formation on soft agar as well as tumor formation following orthotopic injections. Furthermore, ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells displayed enhanced metastasis compared with ALDHlowCD44low/-CD24+ and ALDHlowCD44low/-CD133- cells. Most notably, they examined the metastatic behavior of these cells by performing in vitro cell adhesion, migration, and invasion assays, as well as in vivo tail vein and orthotopic (mammary fat pad) injections. This is one of the few studies that determined the metastatic potential of TICs using orthotopic injections.
4.2. Pancreatic cancer
TICs in pancreatic cancer were first reported by Simeone's group in 2007 using CD44+CD24+ESA+ (8), however, this study did not investigate the metastatic ability of these cells. Hermann et al. (85) used CD133 as a marker for the isolation of human pancreatic adenocarcinoma TICs. They showed that CD133+ cells were tumorigenic and highly resistant to the chemotherapeutic drug gemcitabine when compared with the CD133- cells. Immunohistochemistry using human pancreatic tumors revealed a distinct subpopulation of CD133+CXCR4+ cells at the invasive front of tumors. They further performed orthotopic injections using a CD133+CXCR4+ group (equal number of CD133+CXCR4+ and CD133+CXCR4- cells) and a CD133+CXCR4- group. Although both groups efficiently formed tumors, only the CD133+CXCR4+ group showed liver metastasis, while no metastasis was observed in CD133+CXCR4- cells. This in vivo result was supported by in vitro invasion assays where the CXCR4 neutralizing antibody inhibited the high invasive potential of CD133+ pancreatic TICs (Table 1). Thus, these findings provide direct in vivo evidence for the heterogeneity of CD133+ TICs and high metastatic property of CD133+CXCR4+ TICs.
SP cells isolated from human pancreatic cancer cell lines also showed higher liver metastases following intrasplenic injections, compared to the main population (MP) cells (55). Interestingly, SP cells showed superior potential of TGF-β-induced epithelial to mesenchymal transition (EMT) and invasion in relation to MP cells (Table 1).
ALDH activity was also used to identify pancreatic TICs (72). ALDH+, CD44+CD24+, and ALDH+CD44+CD24+ cells initiated subcutaneous tumors more efficiently than unsorted cells. These enriched cells showed higher migratory potential with altered expression of EMT-associated genes, including reduced CDH1 (E-cadherin) expression and increased SNAI2 (SLUG) expression (Table 1). Interestingly, the high migratory potential and reduced CDH1 (E-cadherin) expression were more obvious in ALDH+ and ALDH+CD44+CD24+ cells than those in CD44+CD24+ cells, suggesting that ALDH+ and CD44+CD24+ cells are biologically distinct. It should also be noted that patients with ALDH+ primary tumors had worse survival rates than patients having ALDH- primary tumors. Immunohistochemical staining further revealed that ALDH+ cells were observed more frequently in metastatic lesions compared to those in matched primary tumors. These results suggest the crucial role of ALDH activity in the progression of pancreatic cancer.
4.3. Lung cancer
Lung TICs were identified using CD133 as a marker (10, 11). Although these studies showed the tumor-initiating property of lung CD133+ TICs, their metastatic property was not investigated. Levina et al. (82) enriched lung TICs by treating a human lung cancer cell line H460 with doxorubicin, cisplatin, or etoposide. DSCs expressed high levels of several embryonic stem cell markers and showed an ability to self-renew and initiate tumors when compared with the parental cells, suggesting that the DSCs functioned as TICs. Morphological analyses revealed that the DSCs displayed a greater migratory phenotype, such as lamellipodia extensions and actin spikes at the leading edge, compared with the parental cells. Consistent with these observations, DSCs also showed high migratory and invasive potential by in vitro assays using IL-8 as an attractant. In tail vein injections, DSCs displayed more metastatic nodules than the parental cells (Table 1). Interestingly, DSCs expressed higher levels of human VEGFR2, FGFR2, CXCR1, 2, and 4 receptors than the parental cells. Further, the DSC-derived tumors stimulated murine stroma to produce elevated levels of angiogenic and growth factors. These upregulations in the cytokine network could serve as the basis for the enhanced tumorigenic and metastatic potential of the TICs (82).
4.4. Gastric cancer
Takahashi et al. (86) first demonstrated that CD44+ cells in gastric cancer possessed TIC properties such as high sphere-forming and tumor-initiating abilities. Additionally, these cells were resistant to chemotherapy and -irradiation. However, the metastatic potential of the isolated CD44+ cells was not studied. TICs from gastric carcinoma were also isolated using the Hoechst 33342 dye exclusion method (45). The SP cells were shown to initiate tumors more efficiently and possessed higher ability of peritoneal metastasis following intraperitoneal injections, as compared with unsorted cells (Table 1). Consistently, the SP cells showed higher expression of adhesion molecules 2-, 5-, 3-, and 5-integrins than unsorted cells (45).
4.5. Prostate cancer
Prostate cancer TICs were identified using CD44+α2β1+ and CD44+CD24- markers by Patrawala et al. (9) and Hurt et al. (36) respectively, yet these studies lacked exploration of their metastatic potential. Klarmann et al. (87) recently demonstrated a higher invasive potential of CD44+ prostate cancer TICs compared to that of CD44- cells. CD44+CD24- cells showed similar RNA expression profile to that of the Matrigel-invasive cells, including expression of EMT-related genes and those associated with stem cell maintenance, proliferation, and differentiation (Table 1).
The crucial role of CD44 in prostate cancer metastasis is also supported by the study of Eaton et al. (88), where CD44 expression, but not the expression of CD133 or 21 integrin, was observed more frequently in metastases than in primary tumors.
4.6. Osteosarcoma
Although several groups suggested the presence of TICs in osteosarcomas (81, 89-91), the tumor-initiating potential of osteosarcoma TICs was not demonstrated until recently. Wu et al. (15) used a dye-excluding SP for enriching TICs from cultured cells of one human osteosarcoma, two malignant fibrous histiocytoma, and one synovial sarcoma. Interestingly, SP cells excluding the Hoechst 33342 dye initiated serially transplantable tumors following subcutaneous injections, whereas SP cells excluding the rhodamine 123 dye did not form tumors, suggesting the involvement of a specific fraction of the SP cells in the TIC properties.
Based on the previous observation that an embryonal transcriptional regulator Oct-4 was expressed in spheres from osteosarcoma cells (81), Levings et al. (18) generated a transgenic cell line where cells from an osteosarcoma biopsy (OS521) were stably transfected with a plasmid containing the human Oct-4 promoter-driven GFP reporter gene. The Oct-4/GFP(+) cells were over 100-fold more tumorigenic by subcutaneous injections than the GFP-depleted cells. Interestingly, serial transplantation of the Oct-4/GFP(+) cells into immunocompromised mice resulted in acquired metastatic ability, suggesting a selective adaptation analogous to the process of tumor progression (18).
Wang et al. (71) used ALDH activity to enrich TICs from a human osteosarcoma cell line OS99-1. Cells having a high activity of ALDH (ALDHbr) showed more efficient tumor initiation by subcutaneous injections and higher levels of expression of the stem cell-associated genes Oct-3/4a, Nanog, and Sox-2 than cells possessing low ALDH activity (ALDHlo). Although various methods were used to demonstrate the presence of TICs in osteosarcoma, none of them demonstrated orthotopic tumor formation or provided direct evidence for their high metastatic potential.
Recently, our group (40) showed in vitro as well as in vivo evidence of the high metastatic potential of osteosarcoma TICs (Table 1). Using murine primary and established cell lines, we demonstrated that mesenchymal stem cell markers CD117 and Stro-1, but not markers such as CD44, CD105, and CD49b, were preferentially expressed in spheres and doxorubicin-resistant cells. As low as 200 CD117+Stro-1+ (double positive, DP) cells from several mouse and human osteosarcoma cell lines efficiently formed serially transplantable tumors following subcutaneous injections, whereas CD117-Stro-1- (double negative, DN) cells rarely initiated tumors. Consistently, orthotopic injections of 200 and 2,000 DP cells from a primary mouse cell line revealed that DP cells possessed significantly higher ability of tumor initiation than DN cells. Importantly, we found multiple metastatic nodules in the lungs and liver of the majority of mice orthotopically injected with DP cells. When DN cells were injected with ten times or higher numbers (20,000 and 200,000) than DP cells, DN cells did form tumors. However, it took a much longer time period than the DP cells. When average numbers of metastatic nodules were compared, DN-derived tumors developed a significantly less number of metastatic nodules than DP-derived tumors (6 vs. 29). Results of in vitro invasion assays revealed that DP cells possessed a two-fold higher invasive potential than DN cells, supporting our in vivo observations. We further compared the expression patterns of stem cell markers CD117, Stro-1, ABCG2, and CXCR4 between DP-derived primary tumors and their corresponding lung metastases by immunohistochemistry. Although we detected cells positive for all four markers in both primary and metastatic tumors, the intensely stained cells were more frequent at the metastatic sites when compared to those at their primary sites. These results support previous findings that a high expression of TIC markers positively correlates with cancer metastasis (68, 84, 92). Moreover, we demonstrated that DP cells were enriched with cells expressing CXCR4 and ABCG2 in both murine and human model systems. CXCR4 enrichment in DP cells was over 80% and 20% in mice and human cell lines respectively, whereas that in unsorted and DN cells was less than 5%. Regarding ABCG2, over 60% and 80% of DP cells contained ABCG2+ cells in mice and human cell lines, respectively, whereas only a few unsorted and DN cells were positive for ABCG2. Consistent with enrichment of ABCG2+ population in DP cells, these cells also showed a higher drug-resistant property than DN cells. Thus, CD117 and Stro-1 identify osteosarcoma TICs associated with metastasis and drug resistance.
5. PROSPECTIVES
TICs have been identified using diverse techniques in various types of cancer. Different methods have also been used for the characterization of TICs. Since TICs are defined experimentally by their abilities of in vivo tumor initiation and self-renewability, investigators need to have a consensus regarding the methods to isolate and evaluate TICs according to specific tumor types (4).
Considering the possible heterogeneous nature of TICs (65, 72, 93-95), the use of different TIC-isolation methods might result in enriching TICs with different properties, regarding drug resistance and metastasis. The biological significance of stem cell markers for enriching TICs remains unclear. The methods to evaluate TICs should also be carefully chosen. Different types of immunocompromised mice (NOD/SCID vs. NOD/SCID IL2r-/-) or use of Matrigel during the TIC injections resulted in dramatically different outcomes regarding the efficacy of tumor initiation (93). NOD/SCID IL2r-/- mice lack T, B, and NK cells and are most appropriate for xenografting human tumors. However, the development of mouse models of cancer enables us to test the tumor-initiating ability of TICs in syngeneic or congenic systems having intact immune environment and without any concerns of tumor rejection (96, 97). Additionally, since the tumor microenvironment varies depending on the tissue of origin, the injection of tumor cells into their orthotopic sites is ideal to test the tumor-initiating ability and metastatic potential of TICs. Although in vivo assays such as tumor-initiating ability and serial transplantability are the gold standard for evaluating the TICs, these assays with a small number of cells are time-consuming. Developing simpler and more reliable assays would be helpful for the progress of this field.
Several recent studies suggest the functional interaction between TICs and their specialized microenvironment called a niche (98). Tumor cells are actively involved in the creation of their own future premetastatic niche for distant metastasis by secreting cytokines, metalloproteinases, and growth factors or by recruiting bone marrow derived cells (BMDC). The premetastatic niche provides a suitable environment to recruit the tumor cells20, 29, 99, 100). Calabrese et al. (101) showed that tumor endothelial cells increased the number of TICs in brain tumors, whereas their depletion reduced the TIC numbers, suggesting that niche plays a crucial role in the formation and maintenance of TICs. Further studies are required to gain a better understanding of the interaction between TICs and their niche.
Epithelial to mesenchymal transition (EMT) is an important mechanism for metastasis of epithelial tumor cells. Recent studies suggest that EMT endows normal and transformed mammary epithelial cells with stem cell-like properties, including the ability to self-renew and to initiate tumors (102). A recent report by Kabashima et al. (55) demonstrated that SP cells from a pancreatic cancer cell line were more responsive to TGF-β-mediated EMT than main population (MP) cells. These observations showing a functional link between TICs and EMT may suggest that the EMT process is involved in the generation of TICs, acquisition of metastatic potential, and formation of metastatic nodules at secondary sites (103).
In addition to the stem cell marker profiles and biological properties, TICs also share expression pattern of genes related to stem cell maintenance, proliferation, and differentiation with those of normal stem cells (19, 20). Comparative analyses of DNA and RNA profiling of TICs with that of normal adult stem cells or between different tumor types will help dissect their properties at molecular levels, thereby accelerating TIC-targeted therapy.
To develop a novel therapy targeting TICs, altering their properties would be crucial. This can be executed by inhibiting their high tumorigenic, drug-resistant, and metastatic properties, as well as manipulating their cell cycle phase, differentiation status, and niche environment. Further, finding strategies that discriminate TICs or their niche from normal cells will accelerate the development of novel drug delivery systems to specifically target TICs.
6. ACKNOWLEDGMENTS
We would like to thank Dr. Saloni Pasta, Jonna L. Ellis, Swathi V. Iyer, Kristy-Le T. Nguyen, Byron M. Wood, and Kaushiki Sen for editing the manuscript.
7. REFERENCES
1. Lapidot, T., C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M. A. Caligiuri & J. E. Dick: A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 367, 645-8 (1994) 34. Corbel, C., A. Lehmann & F. Davison: Expression of CD44 during early development of the chick embryo. Mech Dev, 96, 111-4 (2000) 65. Croker, A. K., D. Goodale, J. Chu, C. Postenka, B. D. Hedley, D. A. Hess & A. L. Allan: High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med, 13, 2236-52 (2009) Key Words: Tumor-initiating cells, Cancer stem cells, Metastasis, Solid tumors, Osteosarcoma, CXCR4, ABCG2, Side population, CD117, Stro-1, Review
Send correspondence to: Tomoo Iwakuma, Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB, Room 439, LA 70112, U.S.A., Tel: 504-568-3235, Fax: 504-568-8500, E-mail:tiwaku@lsuhsc.edu
doi:10.1038/367645a0
2. Bonnet, D. & J. E. Dick: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 3, 730-7 (1997)
doi:10.1038/nm0797-730
3. Bjerkvig, R., B. B. Tysnes, K. S. Aboody, J. Najbauer & A. J. Terzis: Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer, 5, 899-904 (2005)
doi:10.1038/nrc1740
4. Clarke, M. F., J. E. Dick, P. B. Dirks, C. J. Eaves, C. H. Jamieson, D. L. Jones, J. Visvader, I. L. Weissman & G. M. Wahl: Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res, 66, 9339-44 (2006)
doi:10.1158/0008-5472.CAN-06-3126
5. Al-Hajj, M., M. S. Wicha, A. Benito-Hernandez, S. J. Morrison & M. F. Clarke: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A, 100, 3983-8 (2003)
doi:10.1073/pnas.0530291100
6. Singh, S. K., C. Hawkins, I. D. Clarke, J. A. Squire, J. Bayani, T. Hide, R. M. Henkelman, M. D. Cusimano & P. B. Dirks: Identification of human brain tumour initiating cells. Nature, 432, 396-401 (2004)
doi:10.1038/nature03128
PMid:15549107
7. O'Brien, C. A., A. Pollett, S. Gallinger & J. E. Dick: A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 445, 106-10 (2007)
doi:10.1038/nature05372
8. Li, C., D. G. Heidt, P. Dalerba, C. F. Burant, L. Zhang, V. Adsay, M. Wicha, M. F. Clarke & D. M. Simeone: Identification of pancreatic cancer stem cells. Cancer Res, 67, 1030-7 (2007)
doi:10.1158/0008-5472.CAN-06-2030
9. Patrawala, L., T. Calhoun-Davis, R. Schneider-Broussard & D. G. Tang: Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer Res, 67, 6796-805 (2007)
doi:10.1158/0008-5472.CAN-07-0490
10. Eramo, A., F. Lotti, G. Sette, E. Pilozzi, M. Biffoni, A. Di Virgilio, C. Conticello, L. Ruco, C. Peschle & R. De Maria: Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ, 15, 504-14 (2008)
doi:10.1038/sj.cdd.4402283
11. Bertolini, G., L. Roz, P. Perego, M. Tortoreto, E. Fontanella, L. Gatti, G. Pratesi, A. Fabbri, F. Andriani, S. Tinelli, E. Roz, R. Caserini, S. Lo Vullo, T. Camerini, L. Mariani, D. Delia, E. Calabro, U. Pastorino & G. Sozzi: Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci U S A, 106, 16281-6 (2009)
doi:10.1073/pnas.0905653106
12. Curley, M. D., V. A. Therrien, C. L. Cummings, P. A. Sergent, C. R. Koulouris, A. M. Friel, D. J. Roberts, M. V. Seiden, D. T. Scadden, B. R. Rueda & R. Foster: CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem Cells, 27, 2875-83 (2009)
13. Ma, S., K. W. Chan, L. Hu, T. K. Lee, J. Y. Wo, I. O. Ng, B. J. Zheng & X. Y. Guan: Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology, 132, 2542-56 (2007)
doi:10.1053/j.gastro.2007.04.025
14. Suva, M. L., N. Riggi, J. C. Stehle, K. Baumer, S. Tercier, J. M. Joseph, D. Suva, V. Clement, P. Provero, L. Cironi, M. C. Osterheld, L. Guillou & I. Stamenkovic: Identification of cancer stem cells in Ewing's sarcoma. Cancer Res, 69, 1776-81 (2009)
doi:10.1158/0008-5472.CAN-08-2242
15. Wu, C., Q. Wei, V. Utomo, P. Nadesan, H. Whetstone, R. Kandel, J. S. Wunder & B. A. Alman: Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. Cancer Res, 67, 8216-22 (2007)
doi:10.1158/0008-5472.CAN-07-0999
16. Murase, M., M. Kano, T. Tsukahara, A. Takahashi, T. Torigoe, S. Kawaguchi, S. Kimura, T. Wada, Y. Uchihashi, T. Kondo, T. Yamashita & N. Sato: Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas. Br J Cancer, 101, 1425-32 (2009)
doi:10.1038/sj.bjc.6605330
17. Hirotsu, M., T. Setoguchi, Y. Matsunoshita, H. Sasaki, H. Nagao, H. Gao, K. Sugimura & S. Komiya: Tumour formation by single fibroblast growth factor receptor 3-positive rhabdomyosarcoma-initiating cells. Br J Cancer, 101, 2030-7 (2009)
doi:10.1038/sj.bjc.6605407
18. Levings, P. P., S. V. McGarry, T. P. Currie, D. M. Nickerson, S. McClellan, S. C. Ghivizzani, D. A. Steindler & C. P. Gibbs: Expression of an exogenous human Oct-4 promoter identifies tumor-initiating cells in osteosarcoma. Cancer Res, 69, 5648-55 (2009)
doi:10.1158/0008-5472.CAN-08-3580
19. Lobo, N. A., Y. Shimono, D. Qian & M. F. Clarke: The biology of cancer stem cells. Annu Rev Cell Dev Biol, 23, 675-99 (2007)
doi:10.1146/annurev.cellbio.22.010305.104154
20. Malanchi, I. & J. Huelsken: Cancer stem cells: never Wnt away from the niche. Curr Opin Oncol, 21, 41-6 (2009)
doi:10.1097/CCO.0b013e32831d1faf
21. Hirschmann-Jax, C., A. E. Foster, G. G. Wulf, J. G. Nuchtern, T. W. Jax, U. Gobel, M. A. Goodell & M. K. Brenner: A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A, 101, 14228-33 (2004)
doi:10.1073/pnas.0400067101
22. Borst, P., J. Jonkers & S. Rottenberg: What makes tumors multidrug resistant? Cell Cycle, 6, 2782-7 (2007)
doi:10.4161/cc.6.22.4936
23. Hadnagy, A., L. Gaboury, R. Beaulieu & D. Balicki: SP analysis may be used to identify cancer stem cell populations. Exp Cell Res, 312, 3701-10 (2006)
doi:10.1016/j.yexcr.2006.08.030
24. Li, F., B. Tiede, J. Massague & Y. Kang: Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res, 17, 3-14 (2007)
doi:10.1038/sj.cr.7310118
25. Pandit, T. S., W. Kennette, L. Mackenzie, G. Zhang, W. Al-Katib, J. Andrews, S. A. Vantyghem, D. G. Ormond, A. L. Allan, D. I. Rodenhiser, A. F. Chambers & A. B. Tuck: Lymphatic metastasis of breast cancer cells is associated with differential gene expression profiles that predict cancer stem cell-like properties and the ability to survive, establish and grow in a foreign environment. Int J Oncol, 35, 297-308 (2009)
26. Marotta, L. L. & K. Polyak: Cancer stem cells: a model in the making. Curr Opin Genet Dev, 19, 44-50 (2009)
doi:10.1016/j.gde.2008.12.003
27. Lapidot, T. & O. Kollet: The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice. Leukemia, 16, 1992-2003 (2002)
doi:10.1038/sj.leu.2402684
28. Kang, H., G. Watkins, A. Douglas-Jones, R. E. Mansel & W. G. Jiang: The elevated level of CXCR4 is correlated with nodal metastasis of human breast cancer. Breast, 14, 360-7 (2005)
doi:10.1016/j.breast.2004.12.007
29. Kaplan, R. N., R. D. Riba, S. Zacharoulis, A. H. Bramley, L. Vincent, C. Costa, D. D. MacDonald, D. K. Jin, K. Shido, S. A. Kerns, Z. Zhu, D. Hicklin, Y. Wu, J. L. Port, N. Altorki, E. R. Port, D. Ruggero, S. V. Shmelkov, K. K. Jensen, S. Rafii & D. Lyden: VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature, 438, 820-7 (2005)
doi:10.1038/nature04186
30. Dewan, M. Z., S. Ahmed, Y. Iwasaki, K. Ohba, M. Toi & N. Yamamoto: Stromal cell-derived factor-1 and CXCR4 receptor interaction in tumor growth and metastasis of breast cancer. Biomed Pharmacother, 60, 273-6 (2006)
doi:10.1016/j.biopha.2006.06.004
31. Ratajczak, M. Z., E. Zuba-Surma, M. Kucia, R. Reca, W. Wojakowski & J. Ratajczak: The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis. Leukemia, 20, 1915-24 (2006)
doi:10.1038/sj.leu.2404357
32. Nagano, O. & H. Saya: Mechanism and biological significance of CD44 cleavage. Cancer Sci, 95, 930-5 (2004)
doi:10.1111/j.1349-7006.2004.tb03179.x
33. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill & B. Seed: CD44 is the principal cell surface receptor for hyaluronate. Cell, 61, 1303-13 (1990)
doi:10.1016/S0925-4773(00)00347-6
35. Shipitsin, M., L. L. Campbell, P. Argani, S. Weremowicz, N. Bloushtain-Qimron, J. Yao, T. Nikolskaya, T. Serebryiskaya, R. Beroukhim, M. Hu, M. K. Halushka, S. Sukumar, L. M. Parker, K. S. Anderson, L. N. Harris, J. E. Garber, A. L. Richardson, S. J. Schnitt, Y. Nikolsky, R. S. Gelman & K. Polyak: Molecular definition of breast tumor heterogeneity. Cancer Cell, 11, 259-73 (2007)
doi:10.1016/j.ccr.2007.01.013
36. Hurt, E. M., B. T. Kawasaki, G. J. Klarmann, S. B. Thomas & W. L. Farrar: CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br J Cancer, 98, 756-65 (2008)
doi:10.1038/sj.bjc.6604242
37. Corbeil, D., K. Roper, A. Hellwig, M. Tavian, S. Miraglia, S. M. Watt, P. J. Simmons, B. Peault, D. W. Buck & W. B. Huttner: The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem, 275, 5512-20 (2000)
doi:10.1074/jbc.275.8.5512
38. Miki, J., B. Furusato, H. Li, Y. Gu, H. Takahashi, S. Egawa, I. A. Sesterhenn, D. G. McLeod, S. Srivastava & J. S. Rhim: Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res, 67, 3153-61 (2007)
doi:10.1158/0008-5472.CAN-06-4429
39. Monzani, E., F. Facchetti, E. Galmozzi, E. Corsini, A. Benetti, C. Cavazzin, A. Gritti, A. Piccinini, D. Porro, M. Santinami, G. Invernici, E. Parati, G. Alessandri & C. A. La Porta: Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential. Eur J Cancer, 43, 935-46 (2007)
doi:10.1016/j.ejca.2007.01.017
40. Adhikari, A. S., N. Agarwal, B. M. Wood, C. Porretta, B. Ruiz, R. R. Pochampally & T. Iwakuma: CD117 and Stro-1 Identify Osteosarcoma Tumor-Initiating Cells Associated with Metastasis and Drug Resistance. Cancer Res, 70, 4602-12 (2010)
doi:10.1158/0008-5472.CAN-09-3463
41. Wolf, N. S., A. Kone, G. V. Priestley & S. H. Bartelmez: In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol, 21, 614-22 (1993)
42. Zhou, S., J. D. Schuetz, K. D. Bunting, A. M. Colapietro, J. Sampath, J. J. Morris, I. Lagutina, G. C. Grosveld, M. Osawa, H. Nakauchi & B. P. Sorrentino: The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med, 7, 1028-34 (2001)
doi:10.1038/nm0901-1028
43. Wagner-Souza, K., H. R. Diamond, M. H. Ornellas, B. E. Gomes, A. Almeida-Oliveira, E. Abdelhay, L. F. Bouzas & V. M. Rumjanek: Rhodamine 123 efflux in human subpopulations of hematopoietic stem cells: comparison between bone marrow, umbilical cord blood and mobilized peripheral blood CD34+ cells. Int J Mol Med, 22, 237-42 (2008)
44. Moserle, L., M. Ghisi, A. Amadori & S. Indraccolo: Side population and cancer stem cells: therapeutic implications. Cancer Lett, 288, 1-9 (2010)
doi:10.1016/j.canlet.2009.05.020
45. Nishii, T., M. Yashiro, O. Shinto, T. Sawada, M. Ohira & K. Hirakawa: Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma. Cancer Sci, 100, 1397-402 (2009)
doi:10.1111/j.1349-7006.2009.01211.x
46. Patrawala, L., T. Calhoun, R. Schneider-Broussard, J. Zhou, K. Claypool & D. G. Tang: Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res, 65, 6207-19 (2005)
doi:10.1158/0008-5472.CAN-05-0592
47. Fukuda, K., Y. Saikawa, M. Ohashi, K. Kumagai, M. Kitajima, H. Okano, Y. Matsuzaki & Y. Kitagawa: Tumor initiating potential of side population cells in human gastric cancer. Int J Oncol, 34, 1201-7 (2009)
48. Chiba, T., K. Kita, Y. W. Zheng, O. Yokosuka, H. Saisho, A. Iwama, H. Nakauchi & H. Taniguchi: Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology, 44, 240-51 (2006)
doi:10.1002/hep.21227
49. Shi, G. M., Y. Xu, J. Fan, J. Zhou, X. R. Yang, S. J. Qiu, Y. Liao, W. Z. Wu, Y. Ji, A. W. Ke, Z. B. Ding, Y. Z. He, B. Wu, G. H. Yang, W. Z. Qin, W. Zhang, J. Zhu, Z. H. Min & Z. Q. Wu: Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials. J Cancer Res Clin Oncol, 134, 1155-63 (2008)
doi:10.1007/s00432-008-0407-1
50. Wang, J., L. P. Guo, L. Z. Chen, Y. X. Zeng & S. H. Lu: Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res, 67, 3716-24 (2007)
doi:10.1158/0008-5472.CAN-06-4343
51. Ho, M. M., A. V. Ng, S. Lam & J. Y. Hung: Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res, 67, 4827-33 (2007)
doi:10.1158/0008-5472.CAN-06-3557
52. Huang, D., Q. Gao, L. Guo, C. Zhang, W. Jiang, H. Li, J. Wang, X. Han, Y. Shi & S. H. Lu: Isolation and identification of cancer stem-like cells in esophageal carcinoma cell lines. Stem Cells Dev, 18, 465-73 (2009)
doi:10.1089/scd.2008.0033
53. Harris, M. A., H. Yang, B. E. Low, J. Mukherjee, A. Guha, R. T. Bronson, L. D. Shultz, M. A. Israel & K. Yun: Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res, 68, 10051-9 (2008)
doi:10.1158/0008-5472.CAN-08-0786
54. Fukaya, R., S. Ohta, M. Yamaguchi, H. Fujii, Y. Kawakami, T. Kawase & M. Toda: Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1. Cancer Lett, 291, 150-7 (2010)
doi:10.1016/j.canlet.2009.10.010
55. Kabashima, A., H. Higuchi, H. Takaishi, Y. Matsuzaki, S. Suzuki, M. Izumiya, H. Iizuka, G. Sakai, S. Hozawa, T. Azuma & T. Hibi: Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer, 124, 2771-9 (2009)
doi:10.1002/ijc.24349
56. Wang, Y. H., F. Li, B. Luo, X. H. Wang, H. C. Sun, S. Liu, Y. Q. Cui & X. X. Xu: A side population of cells from a human pancreatic carcinoma cell line harbors cancer stem cell characteristics. Neoplasma, 56, 371-8 (2009)
doi:10.4149/neo_2009_05_371
57. Han, J. S. & D. L. Crowe: Tumor initiating cancer stem cells from human breast cancer cell lines. Int J Oncol, 34, 1449-53 (2009)
58. Nakanishi, T., S. Chumsri, N. Khakpour, A. H. Brodie, B. Leyland-Jones, A. W. Hamburger, D. D. Ross & A. M. Burger: Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling. Br J Cancer, 102, 815-26 (2010)
doi:10.1038/sj.bjc.6605553
59. Schatton, T., G. F. Murphy, N. Y. Frank, K. Yamaura, A. M. Waaga-Gasser, M. Gasser, Q. Zhan, S. Jordan, L. M. Duncan, C. Weishaupt, R. C. Fuhlbrigge, T. S. Kupper, M. H. Sayegh & M. H. Frank: Identification of cells initiating human melanomas. Nature, 451, 345-9 (2008)
doi:10.1038/nature06489
60. Burkert, J., W. R. Otto & N. A. Wright: Side populations of gastrointestinal cancers are not enriched in stem cells. J Pathol, 214, 564-73 (2008)
doi:10.1002/path.2307
61. Mitsutake, N., A. Iwao, K. Nagai, H. Namba, A. Ohtsuru, V. Saenko & S. Yamashita: Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology, 148, 1797-803 (2007)
doi:10.1210/en.2006-1553
62. Armstrong, L., M. Stojkovic, I. Dimmick, S. Ahmad, P. Stojkovic, N. Hole & M. Lako: Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells, 22, 1142-51 (2004)
doi:10.1634/stemcells.2004-0170
63. Hess, D. A., T. E. Meyerrose, L. Wirthlin, T. P. Craft, P. E. Herrbrich, M. H. Creer & J. A. Nolta: Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity. Blood, 104, 1648-55 (2004)
doi:10.1182/blood-2004-02-0448
64. Ginestier, C., M. H. Hur, E. Charafe-Jauffret, F. Monville, J. Dutcher, M. Brown, J. Jacquemier, P. Viens, C. G. Kleer, S. Liu, A. Schott, D. Hayes, D. Birnbaum, M. S. Wicha & G. Dontu: ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell, 1, 555-67 (2007)
doi:10.1016/j.stem.2007.08.014
doi:10.1111/j.1582-4934.2008.00455.x
66. Korkaya, H., A. Paulson, F. Iovino & M. S. Wicha: HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene, 27, 6120-30 (2008)
doi:10.1038/onc.2008.207
67. Charafe-Jauffret, E., C. Ginestier, F. Iovino, J. Wicinski, N. Cervera, P. Finetti, M. H. Hur, M. E. Diebel, F. Monville, J. Dutcher, M. Brown, P. Viens, L. Xerri, F. Bertucci, G. Stassi, G. Dontu, D. Birnbaum & M. S. Wicha: Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res, 69, 1302-13 (2009)
doi:10.1158/0008-5472.CAN-08-2741
68. Charafe-Jauffret, E., C. Ginestier, F. Iovino, C. Tarpin, M. Diebel, B. Esterni, G. Houvenaeghel, J. M. Extra, F. Bertucci, J. Jacquemier, L. Xerri, G. Dontu, G. Stassi, Y. Xiao, S. H. Barsky, D. Birnbaum, P. Viens & M. S. Wicha: Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res, 16, 45-55 (2010)
doi:10.1158/1078-0432.CCR-09-1630
69. Ma, S., K. W. Chan, T. K. Lee, K. H. Tang, J. Y. Wo, B. J. Zheng & X. Y. Guan: Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res, 6, 1146-53 (2008)
doi:10.1158/1541-7786.MCR-08-0035
70. Huang, E. H., M. J. Hynes, T. Zhang, C. Ginestier, G. Dontu, H. Appelman, J. Z. Fields, M. S. Wicha & B. M. Boman: 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, 3382-9 (2009)
doi:10.1158/0008-5472.CAN-08-4418
71. Wang, L., P. Park, H. Zhang, F. La Marca & C. Y. Lin: Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99-1 cells based on high aldehyde dehydrogenase activity. Int J Cancer, In press (2010)
doi:10.1002/ijc.25331
72. Rasheed, Z. A., J. Yang, Q. Wang, J. Kowalski, I. Freed, C. Murter, S. M. Hong, J. B. Koorstra, N. V. Rajeshkumar, X. He, M. Goggins, C. Iacobuzio-Donahue, D. M. Berman, D. Laheru, A. Jimeno, M. Hidalgo, A. Maitra & W. Matsui: Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst, 102, 340-51 (2010)
doi:10.1093/jnci/djp535
73. Hirschhaeuser, F., H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser & L. A. Kunz-Schughart: Multicellular tumor spheroids: An underestimated tool is catching up again. J Biotechnol, 148, 3-15 (2010)
doi:10.1016/j.jbiotec.2010.01.012
74. Reynolds, B. A., W. Tetzlaff & S. Weiss: A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci, 12, 4565-74 (1992)
75. Reynolds, B. A. & S. Weiss: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 255, 1707-10 (1992)
doi:10.1126/science.1553558
76. Ghods, A. J., D. Irvin, G. Liu, X. Yuan, I. R. Abdulkadir, P. Tunici, B. Konda, S. Wachsmann-Hogiu, K. L. Black & J. S. Yu: Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells. Stem Cells, 25, 1645-53 (2007)
doi:10.1634/stemcells.2006-0624
77. Lai, D. M., T. Liu, Y. Huang, L. H. Wang, J. Zhang & W. W. Cheng: (Identification and characterization of ovarian cancer stem-like cells from primary tumor.). Zhonghua Fu Chan Ke Za Zhi, 44, 936-40 (2009)
78. Na, Y. R., S. H. Seok, D. J. Kim, J. H. Han, T. H. Kim, H. Jung, B. H. Lee & J. H. Park: Isolation and characterization of spheroid cells from human malignant melanoma cell line WM-266-4. Tumour Biol, 30, 300-9 (2009)
doi:10.1159/000261073
79. Feng, D., C. Peng, C. Li, Y. Zhou, M. Li, B. Ling, H. Wei & Z. Tian: Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri. Oncol Rep, 22, 1129-34 (2009)
80. Zhou, S., F. Li, J. Xiao, W. Xiong, Z. Fang, W. Chen & P. Niu: Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs. J Huazhong Univ Sci Technolog Med Sci, 30, 81-4 (2010)
doi:10.1007/s11596-010-0114-4
81. Gibbs, C. P., V. G. Kukekov, J. D. Reith, O. Tchigrinova, O. N. Suslov, E. W. Scott, S. C. Ghivizzani, T. N. Ignatova & D. A. Steindler: Stem-like cells in bone sarcomas: implications for tumorigenesis. Neoplasia, 7, 967-76 (2005)
doi:10.1593/neo.05394
82. Levina, V., A. M. Marrangoni, R. DeMarco, E. Gorelik & A. E. Lokshin: Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One, 3, e3077 (2008)
doi:10.1371/journal.pone.0003077
83. Li, L. & W. B. Neaves: Normal stem cells and cancer stem cells: the niche matters. Cancer Res, 66, 4553-7 (2006)
doi:10.1158/0008-5472.CAN-05-3986
84. Balic, M., H. Lin, L. Young, D. Hawes, A. Giuliano, G. McNamara, R. H. Datar & R. J. Cote: Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res, 12, 5615-21 (2006)
doi:10.1158/1078-0432.CCR-06-0169
85. Hermann, P. C., S. L. Huber, T. Herrler, A. Aicher, J. W. Ellwart, M. Guba, C. J. Bruns & C. Heeschen: Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell, 1, 313-23 (2007)
doi:10.1016/j.stem.2007.06.002
86. Takaishi, S., T. Okumura, S. Tu, S. S. Wang, W. Shibata, R. Vigneshwaran, S. A. Gordon, Y. Shimada & T. C. Wang: Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells, 27, 1006-20 (2009)
doi:10.1002/stem.30
87. Klarmann, G. J., E. M. Hurt, L. A. Mathews, X. Zhang, M. A. Duhagon, T. Mistree, S. B. Thomas & W. L. Farrar: Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature. Clin Exp Metastasis, 26, 433-46 (2009)
doi:10.1007/s10585-009-9242-2
88. Eaton, C. L., M. Colombel, G. van der Pluijm, M. Cecchini, A. Wetterwald, J. Lippitt, I. Rehman, F. Hamdy & G. Thalman: Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer. Prostate, 70, 875-82 (2010)
89. Tirino, V., V. Desiderio, R. d'Aquino, F. De Francesco, G. Pirozzi, U. Galderisi, C. Cavaliere, A. De Rosa & G. Papaccio: Detection and characterization of CD133+ cancer stem cells in human solid tumours. PLoS ONE, 3, e3469 (2008)
doi:10.1371/journal.pone.0003469
90. Di Fiore, R., A. Santulli, R. D. Ferrante, M. Giuliano, A. De Blasio, C. Messina, G. Pirozzi, V. Tirino, G. Tesoriere & R. Vento: Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment. J Cell Physiol, 219, 301-13 (2009)
doi:10.1002/jcp.21667
91. Fujii, H., K. Honoki, T. Tsujiuchi, A. Kido, K. Yoshitani & Y. Takakura: Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines. Int J Oncol, 34, 1381-6 (2009)
92. Maeda, S., H. Shinchi, H. Kurahara, Y. Mataki, K. Maemura, M. Sato, S. Natsugoe, T. Aikou & S. Takao: CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer. Br J Cancer, 98, 1389-97 (2008)
doi:10.1038/sj.bjc.6604307
93. Quintana, E., M. Shackleton, M. S. Sabel, D. R. Fullen, T. M. Johnson & S. J. Morrison: Efficient tumour formation by single human melanoma cells. Nature, 456, 593-8 (2008)
doi:10.1038/nature07567
94. Hays, L. E.: Heterogeneity in the AML stem cell pool. Blood, 114, 3976-7 (2009)
doi:10.1182/blood-2009-09-239285
95. Heuser, M., L. M. Sly, B. Argiropoulos, F. Kuchenbauer, C. Lai, A. Weng, M. Leung, G. Lin, C. Brookes, S. Fung, P. J. Valk, R. Delwel, B. Lowenberg, G. Krystal & R. K. Humphries: Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal. Blood, 114, 3983-93 (2009)
doi:10.1182/blood-2009-06-227603
96. Zhang, M., F. Behbod, R. L. Atkinson, M. D. Landis, F. Kittrell, D. Edwards, D. Medina, A. Tsimelzon, S. Hilsenbeck, J. E. Green, A. M. Michalowska & J. M. Rosen: Identification of tumor-initiating cells in a p53-null mouse model of breast cancer. Cancer Res, 68, 4674-82 (2008)
doi:10.1158/0008-5472.CAN-07-6353
97. Kelly, P. N., A. Dakic, J. M. Adams, S. L. Nutt & A. Strasser: Tumor growth need not be driven by rare cancer stem cells. Science, 317, 337 (2007)
doi:10.1126/science.1142596
98. Iwasaki, H. & T. Suda: Cancer stem cells and their niche. Cancer Sci, 100, 1166-72 (2009)
doi:10.1111/j.1349-7006.2009.01177.x
99. Hiratsuka, S., K. Nakamura, S. Iwai, M. Murakami, T. Itoh, H. Kijima, J. M. Shipley, R. M. Senior & M. Shibuya: MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell, 2, 289-300 (2002)
doi:10.1016/S1535-6108(02)00153-8
100. Veeravagu, A., S. R. Bababeygy, M. Y. Kalani, L. C. Hou & V. Tse: The cancer stem cell-vascular niche complex in brain tumor formation. Stem Cells Dev, 17, 859-67 (2008)
doi:10.1089/scd.2008.0047
101. Calabrese, C., H. Poppleton, M. Kocak, T. L. Hogg, C. Fuller, B. Hamner, E. Y. Oh, M. W. Gaber, D. Finklestein, M. Allen, A. Frank, I. T. Bayazitov, S. S. Zakharenko, A. Gajjar, A. Davidoff & R. J. Gilbertson: A perivascular niche for brain tumor stem cells. Cancer Cell, 11, 69-82 (2007)
doi:10.1016/j.ccr.2006.11.020
102. Mani, S. A., W. Guo, M. J. Liao, E. N. Eaton, A. Ayyanan, A. Y. Zhou, M. Brooks, F. Reinhard, C. C. Zhang, M. Shipitsin, L. L. Campbell, K. Polyak, C. Brisken, J. Yang & R. A. Weinberg: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell, 133, 704-15 (2008)
103. Hollier, B. G., K. Evans & S. A. Mani: The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies. J Mammary Gland Biol Neoplasia, 14, 29-43 (2009)
doi:10.1007/s10911-009-9110-3
Abbreviations: TIC: tumor-initiating cell, CSC: cancer stem cell, ABC: ATP-binding cassette, HSC: haematopoietic stem cell, MSC: mesenchymal stem cell, AML: human acute myeloid leukemia, SP: side population, MP: main population, GFP: green fluorescent protein, MMP: metalloproteinase, BMDC: bone marrow derived cells, ALDH: aldehyde dehydrogenase, EMT: epithelial to mesenchymal transition, DSC: drug surviving cell, DP: double positive, DN: double negative, NOD/SCID: non-obese diabetic/severe combined immunodeficiency, REF: references