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Keywords:

  • cancer stem cells;
  • cancer stem cell markers;
  • cancer stem cell niche;
  • therapy resistance

Abstract

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

Cancer stem cell (CSC) biology is a rapidly developing field within cancer research. CSCs are postulated to be a unique cell population exclusively capable of infinite self renewal, multilineage differentiation and with ability to evade conventional cytotoxic cancer therapy. These traits distinguish CSCs from their more differentiated counterparts, which possess only limited or no potential for self renewal and tumor initiation. Therefore, CSCs would be the driving motor of malignant growth and therapy resistance. Accordingly, successful cancer treatment would need to eliminate this highly potent group of cells, since even small residual numbers would suffice to recapitulate the disease after therapy. Putative CSCs has been identified in a broad range of human malignancies and several cell surface markers have been associated with their stem cell phenotype. Despite all efforts, a pure CSC population has not been isolated and often in vitro clonogenic and in vivo tumorigenic potential is found in several cell populations with occasionally contradictory surface marker signatures. Here, we give a brief overview of recent advances in CSC theory, including the signaling pathways in CSCs that also appear crucial for stem cells homeostasis in normal tissues. We discuss evidence for the interaction of CSCs with the stromal tumor environment. Finally, we review the emerging potentially effective CSC-targeted treatment strategies and their future role in therapy. © 2012 International Society for Advancement of Cytometry

Cancer stem cell (CSC) theory hypothesizes that heterogeneity within tumors is not a mere consequence of random mutation and clonal evolution, but results from an intrinsic hierarchy of cells, with the putative CSC at the apex of the hierarchy (1, 2). The CSC is thought to share several key features with normal stem cells: unlimited capacity for self renewal, including maintenance of the CSC population through asymmetric division, the ability to differentiate into several cell lineages and intrinsic resistance against cytotoxic therapies through drug-efflux mechanisms and slow cell cycling (2). CSC theory postulates that not all tumor cells are equal with regard to self-renewal, tumor initiation, and maintenance potential, these traits being reserved for the CSC population. Additionally, cellular heterogeneity and hierarchy within the tumor originates from CSCs, which give rise to daughter cells that proliferate and differentiate into the cell mass that compromises a significant portion of the bulk tumor (1). Further, CSCs are thought to be responsible for therapy resistance, minimal residual disease and relapse after initial successful therapy, their stem-like features making them able to evade conventional treatment modalities (3). It has also been implied that these stem cell traits allow CSCs to play a leading role in metastasis (4–6).

Since the first experiments verifying that a specific phenotype within AML cells enriches for cells that recapitulate the original disease upon transplantation into NOD/SCID mice (7, 8), putative CSCs have been identified in several human malignancies such as CML (9, 10), brain (11–15), breast (14, 16–21), colon (22–26), endometrial (27), head and neck (28, 29), gastric (30, 31), liver (32–36), lung (19, 37–39), melanoma (40–42), ovarian (43–47), pancreatic (48–51), prostate (14, 52), renal cell (53, 54) and thyroid carcinomas (55). Despite the large body of work in CSC research, there is still a considerable amount of confusion and controversy surrounding the CSC hypothesis. Part of this stems from the fact, that to date, the CSC phenotype is a functional one, based on the cell's behavior in an array of in vitro and in vivo experiments designed to test for renewal and differentiation capacity, as well as tumorigenic potential. Recent addition of testing for stemness-related gene expression patterns (31, 36, 38, 39, 53, 54, 56, 57) has helped to further verify stem-like properties at the molecular level, but there is still not a set of surface markers in any malignancy that not only enriches, but is exclusive for cells displaying CSC behavior. Often, cells lacking the surface marker phenotype of putative CSCs still retain various degrees of tumor initiation capacity (14, 20, 27, 31, 41, 49, 58). In extremis, mutually exclusive surface marker phenotypes have been shown to harbor cells with CSC traits (12, 59–61). To further obscure the picture, a significant amount of evidence is mounting, that CSC behavior is substantially influenced by the tumor microenvironment (62) and CSCs can alter their expression profiles, resulting in a “moving target” for CSC research and raising questions about the adequacy of the assays used in vitro and in vivo. Whether CSCs have exclusive tumorigenic potential within the tumor population and are responsible for all the heterogeneity and the establishment of all cell lineages, remains to be seen.

Nevertheless, there is clearly a functionally distinct population within most malignancies with an intrinsic resistance against conventional cytotoxic therapy (63, 64). This in vitro and in vivo resistance is in line with recent findings that CSC-associated expression patterns in bulk tumors have clinical significance and are independent prognostic markers for patient survival (65–70). Further, treatment modalities developed along the lines of CSC theory and targeting putative CSCs show promising results and are effective in preliminary studies (71, 72). Taken together, the CSC population isolated with the admittedly limited functional assays and surface markers has biological relevance and broadens our appreciation for the complexity of tumor biology.

EVIDENCE SUPPORTING CANCER STEM CELL THEORY

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

The CSC theory, although widely accepted, has had to face justified criticism. Several caveats are known, starting with the identification of putative CSCs (discussed in detail later). CSCs were originally thought to arise from normal stem cells of tissues through malignant transformation, but little irrevocable evidence exists linking CSCs to their normal counterparts. One major reason for CSCs having remained elusive is that CSC isolation techniques only select an enriched, nonhomogeneous population. Therefore, a direct investigation of CSCs and their possible evolution from nonmalignant tissue has largely not been possible. At the same time, the dysregulation of several key pathways that are important in the maintenance and differentiation of normal stem cells were shown to play crucial roles in carcinogenesis. Amongst others, pathways regulated by Wnt (73), Hedgehog (74, 75), Notch (76), Nestin (77), Nanog (78, 79), and transforming growth factor beta (TGF-β) (80, 81) were shown to be functionally active and relevant in cancer. More and more active stemness-associated pathways are also identified in putative CSCs (82–84). Not only are these pathways active in CSCs, but they also seem to be essential as evidenced by experiments in which selective inhibition led to loss of CSC function. Invasion and migration of prostate CSCs was not possible after knockdown of sex determining region of Y box 2 (SOX-2) and octamer-binding transcription factor 3/4 (OCT3/4) (85), while small interfering RNA (siRNA) knockdown of SOX-2 eliminates tumorigenicity of side population (SP) cells in lung adenocarcinoma (86). Knockdown of β-catenin led to tumor regression and loss of CSCs in chemically induced murine skin tumors and in human squamous cell carcinoma, and reduced the growth of xenografted human tumor cell lines (87). Combined inhibition of sonic hedgehog and mammalian target of rapamycin (mTOR) together with gemcitabine depleted CSCs in human pancreatic cancer cell lines (88), while antagonism of hedgehog signaling induced apoptosis in CSCs (89). Downregulation of SOX-2 or twist-related protein 1 (TWIST-1) lead to differentiation of tumorsphere forming CD133+ glioblastoma cells (90). Blocking hedgehog signaling at the level of Smoothened by cyclopamine inhibited chronic myeloid leukemia (91) and prostate CSCs (92). Although active stemness-associated pathways do not necessarily prove stem cell origin of CSCs, they do show a common functional heritage.

Biological tumor behavior does not always conform to the linear-hierarchical organization dictated by the strictly taken CSC theory. For instance, Quintana et al. showed that the cells in various types of melanoma tumors are not hierarchically organized and have a reversible phenotype with tumorigenic potential equal among different phenotypes (93). Similarly, in BCR-ABL1 lymphoblastic leukemia, functionally defined CSC populations proved to be genetically diverse, often with multiple genetically distinct CSC subclones in the same patient, indicating a branching multi-clonal evolution (94). While these cases demonstrate that not all tumors adhere to the CSC model, there is mounting evidence that clonal expansion and multi-lineage differentiation is sufficient to drive tumor growth and heterogeneity (95, 96). Lathia et al. separated CSCs and non-CSCs from glioblastomas and then lentivirally labeled the cells with either GFP or YFP (97). After inoculating mice with a cell suspension containing CSCs and non-CSCs in a 1:10 ratio, they monitored tumor growth with intravital microscopy. The CSCs initially localized in the perivascular region and then outgrew the non-CSC population, with cells of dominantly CSC origin constituting the bulk tumor. Since tumors evolving in this manner eventually resembled the parent tumors at the histological level, the case can be considered exemplary of the potential of CSCs for intratumor evolution and diversification. The same group also found CSCs capable of symmetric or asymmetric cell divisions, depending on the environmental conditions (98). The coexistence of these division types is a long standing cornerstone of CSC theory and was thought to be a prerequisite for CSC derived tumor heterogeneity and CSC renewal. Interestingly, both differentiated progeny and CSCs could be generated by either division type, questioning the dogma, that one type is responsible for self-maintenance and the other for cellular expansion. In a truly groundbreaking work, Zhu et al. first identified Prominin-1 (Prom1 or CD133) cells as normal tissue stem cells in the mouse small intestine (99). Then endogenous Wnt signaling was activated in the mice using a mutant beta-catenin allele, leading to gross dysplasia and intraepithelial neoplasia. Through lineage tracking with a YFP encoding reporter allele, they showed a disproportionate expansion of the Prom1+ cells and that cells within the neoplastic lesion were progeny of Prom1+ cells. In line with CSC theory, only a fraction of cells retained the Prom1+ phenotype and only a fraction of these cells were proliferating. This, to our knowledge, was the first direct proof that a solid tumor can arise from malignant transformation of a tissue stem cell and that all tumor cells are descendents of CSCs. Previously, leukemia CSCs were found to be heterogeneous in self renewal potential and to follow a hierarchy reminiscent of the normal hematopoietic stem cell compartment (100). While this can be interpreted as indirect evidence of stem cell origin, a recent study suggests that CSCs need not originate from the pinnacle of cellular hierarchy. Goardon et al. showed that in human acute myeloid leukemia, two molecularly distinct CSC harboring populations are present (101). These populations are hierarchically ordered, in so far as one can give rise to the other, but not vice versa. Global gene expression profiling showed that the CSC populations resembled normal progenitors and not hemopoetic stem cells. Therefore, stem cell origin is not necessary for CSC functionality. Based on these data it is highly probable, that depending on the type and origin of malignancy, a spectrum of cells can be responsible for giving birth to the common CSC phenotype. Newly developed in vivo imaging modalities could aid in tracking and identifying putative CSCs (102–104) and thereby bring us closer to the origins of CSCs. Recent advances in detecting circulating tumor cells (105–108) and monitoring minimal residual disease (109, 110) should also help establish the role of CSCs in metastasis formation and disease propagation.

IDENTIFICATION AND MARKERS OF PUTATIVE CSCS

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

The in vitro and in vivo assays for identifying cell populations harboring CSCs have been extensively discussed elsewhere (2, 111–113). Briefly, colony forming capacity and tumorsphere formation in specific nonattached culturing conditions, maintained through serial passaging, has been associated with a stem-like phenotype. Activity of pathways associated with stemness is increasingly being used to verify stem-like behavior. Additionally, loss of stemness-associated molecular and surface markers parallel to induced differentiation is also used. In this respect, growth of daughter cell lineages with differentiated marker signatures distinct from that of parent cells and/or other progeny are hallmarks of multi-lineage differentiation capacity.

Xenotransplantation has become the gold standard of in vivo testing. Putative CSCs should be tumorigenic in vivo, reproducibly giving rise to tumors when transplanted into immune-compromised mice. The cell numbers required for tumor initiation are preferentially lower than when inoculating unsorted or non-CSC phenotype cells. The tumors should ideally recapitulate the morphology and histological characteristics of parent tumors, verifying multi-lineage differentiation and hierarchical heterogeneity. The tumors should also be capable of serial passaging, that is, CSCs derived from primary xenograft tumors should initiate tumor growth and give rise to secondary tumors. While exemplifying the special capabilities of CSCs, xenotransplantation has also come under the most scrutiny as an adequate method for assessing tumorigenic potential (114–116). The question that frequently arises is whether we are indentifying all cells capable of tumor initiation or only a subset that has the unique capacity to propagate in mice. Experiments have shown that even non-CSCs can engraft and remain viable in the mouse environment, but fail to proliferate and form tumors (11, 23, 97). As more and more information surfaces about the active role of the tumor microenvironment in tumor growth, concerns amount that otherwise tumorigenic cells may lack stromal and interstitial factors in the xeno-environment and therefore fail to thrive (115). While one can argue that CSCs identified under these circumstances still have survival advantage and growth autonomy over the non-CSC population, whether this automatically translates into the same biological behavior and tumorigenic potential in the human body is an open question. The efficacy of the xenotransplant system was also stressed in an elegant work by Quintana et al. (117), who showed that the addition of Matrigel lowers the number of melanoma cells needed for tumor initiation in NOD/SCID mice, indirectly lending support to the significance of the inoculation environment. More importantly, by using NOD/SCID IL2Rγnull mice, which lack the natural killer activity of wild type NOD/SCID mice, the number of melanoma cells required for tumor initiation was drastically reduced to almost the single cell level and tumor initiation capability did not show an association with any particular cell surface phenotype. While this might be a unique property of melanoma, which is a highly aggressive malignancy known to metastasize early and at small tumor size; it clearly signifies that even in the immune-compromised NOD/SCID mice tumorigenic cells are lost due to immune-surveillance. Again, the biological significance and specific human relevance of cells with tumorigenic potential that cannot escape the immune-surveillance of NOD/SCID mice can be debated. One way to circumvent the potential negative influence of the xeno-environment is by using syngeneic murine model systems of human disease (118, 119). Of course, in these cases care needs to be taken to verify that the relevant cellular markers and signaling pathways are also present and implicated in the human counterpart.

Using the methods mentioned above, CSC populations with a characteristic cell surface expression profile were discovered in a plethora of human malignancies (for a summary and references, please see Table 1). Several of the most commonly used CSC markers are CD133, CD44, CD24, and ALDH1 in solid tumors and CD34 in hematological malignancies. Recently, several new markers have been identified, including CD105 (53), granulin-epithelin precursor (GEP) (36), c-Met (50), and integrin α6 (120). It is important to realize that all the markers used only enrich for the putative CSC population and the signature is not unique to CSCs. Often, the population negative for one or more CSC markers also has the potential for tumor initiation, although usually a very limited capability. One of the most controversial markers is CD133 (Prominin-1). Initial studies proved CD133 as a marker for brain and colon CSCs and later on also liver, lung, ovarium, pancreatic and prostate cancer. Several authors also found CSC activity in CD133 populations of glioblastoma and colon tumors (12, 61, 121), in some instances CSC functionality being exclusive to the CD133 population (122, 123). Günther et al. showed that under stem cell culturing conditions, cell lines derived from patient glioblastomas can form tumorspheres which, depending on the cell line, are either formed by CD133+ or CD133 cells (60). Although the cell lines with CD133+ tumorspheres proved to be more tumorigenic, the CD133 tumorspheres were also capable of initiating tumor growth. This controversy of CD133 expression in glioblastoma CSCs can be reconciled through the work of Chen et al., whom from PTEN-deficient glioblastomas isolated three subtypes of neurosphere forming cells, all capable of multi-lineage differentiation (59). Type I cells were CD133, produced CD133+ and CD133 daughter cells, showed the most aggressive tumor growth in xenotransplants and gave rise to histologically less differentiated tumors; Type II cells were CD133+, produced CD133+ and CD133 daughter cells, showed intermediate tumor growth and differentiation; Type III cells were also CD133, but produced only CD133 daughter cells, showed the slowest tumor growth and produced the most differentiated tumors. The authors concluded that in PTEN-deficient glioblastomas, several CSC lineages may be present, which are hierarchically organized and have different cell surface markers. An example of CSC marker plasticity was supplied by the work of Bae et al. (85). They showed that E-cadherin, which is expressed by prostate CSCs, was transiently downregulated during the migration of CSCs in a Matrigel assay and only returned to premigration levels several hours afterwards. It should also be mentioned that CSC markers are not necessarily universal in a given tumor type. For instance, the CD44+CD24 phenotype thought specific for CSCs in breast tumors has been shown to be only present in basal-type breast cancers and ALDH1+ is only applicable as a CSC marker in basal-type and HER2+ breast tumors (124).

Table 1. Cancer stem cell markers and associated tumor types
Tumor typeCSC marker
CD133CD44CD24ALDHCD90CD34OtherRef.
  1. GEP, granulin-epithelin precursor; SP, side population.

Brain+/–   + SP, integrin-α6(11–15, 120)
Breast++++ SP(14, 16–21)
Colon/colorectal++ +   (22–26)
Endometrium+      (27)
Gastric ++    (30, 31)
Head and Neck + +   (28, 29)
Leukemia (AML, CML)     +CD38(7–10)
Liver++ ++ GEP(32–36)
Lung+  +++SP, CD117(19, 37–39, 86)
Melanoma   +  CD20, ABCB5(40–42, 117)
Ovarian++    SP, CD117(43–47)
Pancreatic++++  c-Met, EpCam(48–51)
Prostate++    SP(14, 52)
Renal cell      CD105(53–54)
Thyroid      SP(55)

It also remains to be seen whether all CSC markers have an essential part in maintaining the functional CSC phenotype or their expression is just a byproduct of stemness. The role of CD44 is crucial for cell viability of hepatocellular carcinoma CSCs (33), intestinal carcinogenesis of mice (125), and bone marrow homing of chronic myeloid leukemia (CML) cells (126). Du et al. found that in colorectal cancer, CD44+ cells were enriched for CSCs with clonogenic and in vivo tumorigenic potential and both properties were impaired after siRNA knockdown of CD44 (58). Similarly, Pham et al. showed in primary cultures of malignant breast tumors that small hairpin RNA (shRNA) knockdown of CD44 in CD44+/CD24 breast CSCs reduced expression levels of Wnt, Hedgehog and Akt pathway associated markers to that seen in non-CSCs (57). The in vivo tumorigenicity of CSCs was also impaired with CD44 knockdown. In gastric CSCs, lentiviral knockdown of CD44 was shown to impair tumorsphere formation and tumor growth (30). Recently, Li et al. reported that they identified c-Met, a receptor tyrosine kinase, as a marker for CSCs in human pancreatic adenocarcinomas (50). In their study, lentiviral knockdown of c-Met inhibited in vivo tumor formation and pharmacological blockade of c-Met inhibited tumorsphere formation, induced CSC apoptosis and decreased tumor growth while concurrently reducing the CSC population of the tumor. In glioblastoma multiforme, integrin α6 was shown to enrich for CSCs (120). The shRNA knockdown of integrin α6 inhibited in vitro CSC growth and tumorsphere formation as well as in vivo tumor formation and tumor growth. The same effects were achieved using a blocking antibody against integrin α6. It has also been suggested that the ABCG2 multidrug-transporter not only causes the SP phenotype of putative CSCs through dye-efflux mechanisms, but also regulates Hedgehog signaling in these cells (127).

In general, whenever feasible, knockdown or blockade of a new CSC marker should be attempted to determine whether it influences CSC behavior and function. Ideally, gene expression should be monitored simultaneously, preferentially focusing on known stemness-associated pathways.

CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

While initial research focused on the autonomous tumor initiation and growth properties of CSCs, recent studies have highlighted the important influence of the tumor environment not only on tumor behavior, but on CSCs themselves. Early on it was hypothesized that similar to their normal stem cell counterparts, a niche may exist that provides CSCs with environmental cues that help maintain stemness and may partially regulate CSC fate (128). Support for this theory was gained from histological and immunohistochemical slides showing cells with a CSC-associated marker phenotype preferentially enriched in perivascular regions of tumors (15, 120, 129, 130). As previously mentioned, initial perivascular localization of xenografted CSCs was recently visualized with intravital microscopy (97). There is mounting evidence that this is not a mere spatial coincidence, but also harbors a functional relationship. Beck et al. found that vascular endothelial growth factor receptor 2 (VEGFR-2) blockade decreased tumor microvascular density and also proportionally reduced CSC pool size in papillomas (130). Most remarkably, this was not solely owed to the reduced availability of nutrients, but also to a direct inhibition of CSC proliferation. They successfully verified a neurolipin-dependent autocrine VEGF loop, in which VEGF regulates CSC stemness-associated gene expression and proliferation. Similarly, underscoring the crucial interaction of tumor cells with surrounding stroma, paracrine hedgehog signaling with stromal elements was found to be essential for carcinogenesis and tumor angiogenesis (131–134). Even the normal stem cell compartment is implicated, as mesenchymal stem cells were shown to affect the phenotype of tumor cells, as reviewed by Grisendi et al. (135). In a mouse breast cancer model, Malanchi et al. verified that putative CSCs are the only cell population capable of forming distant metastasis (136). This function of CSCs, however, was dependent on inducing periostin (POSTN) expression in the host stroma via transforming growth factor β3 (TGF-β3). POSTN was shown to augment Wnt signaling in CSCs, and loss of POSTN or blockade of TGF-β3 action inhibited metastasis formation. Further evidence that CSCs are susceptible to environmental factors was provided by a study showing that stemness of glioma CSCs can be inhibited by human umbilical cord blood stem cells (HUCBSC), resulting in reduced in vitro migration and in vivo reduction of tumor volumes (90). The inhibitory effect of HUCBSCs was found to arise from reducing SOX-2 and TWIST1 expression in CSCs, which induced differentiation of the cells. The tumor environment can also significantly contribute to therapy resistance of tumors. Lonardo et al. found that despite successful targeting of Nodal signaling in CSCs in a pancreatic cancer cell line leading to diminished in vivo tumor growth in combination with gemcitabine, initial results with primary tumor xenografts were discouraging (137). They discovered that efficient accumulation of the therapeutic agent was inhibited; however, blocking the hedgehog pathway in stromal cells increased drug delivery by a factor of 10 and restored therapeutic efficacy, in line with a previous study showing stromal depletion as an effective measure for enhancing target site delivery of chemotherapeutics (138).

Hypoxia within tumors has been suggested to be a determinant of CSC behavior (139). It was shown that in glioblastomas, CSCs differentially express hypoxia inducible factors (86), with the HIF2α subtype preferentially expressed in CSCs (140). While knockdown of HIF1α inhibited both CSCs and non-CSCs, knockdown of HIF2α selectively decreased in vitro CSC cell survival and impaired in vivo tumor formation. Another group identified a set of signature genes differentially expressed in SP cells from glioblastoma cell lines and primary glioblastomas (141). The expression of signature genes as well as of CD133 was upregulated by culturing under hypoxic conditions. Overexpression of HIF2α had a similar effect, while knockdown of HIF2α inhibited hypoxic upregulation of genes. In human pancreatic cancer cell lines, hypoxia also elevated the expression of CD133 and increased invasiveness of cancer cells (142). In this study, overexpression of HIF1α increased metastatic and tumorigenic ability. Finally, in another study with prostate cancer cell lines, culturing under hypoxic conditions induced expression of HIF1α and HIF2α with an upregulation of Oct 3/4 and Nanog (143). Hypoxia also induced expression of CD44 and ABCG2 and led to higher clonogenicity and increased sphere formation. The hypoxia induced changes were already evident at a moderate oxygen level of 7%. This oxygen level, thought to be representative of physiological concentrations in human tissues, was also shown to increase HIF2α expression in glioblastoma CSCs (144). Upregulation of Oct 4and Sox2, and an increase in proliferation rate and self-renewal potential of CD133+ cells were also witnessed. An interesting signaling pathway was identified by Feldman et al., who found leptin receptor to be expressed via an OCT4/SOX2 mechanism on hepatocellular carcinoma cell line CSCs (145). In CSCs, leptin induced phosphorylation of STAT3 and induction of Oct4 and Sox2. Additionally, intraperitoneal injection of leptin into xenografted mice led to faster tumor growth and CSCs formed tumors faster in obese mice than in lean littermates. Immunostaining of hepatocellular carcinoma specimens demonstrated leptin receptor expression in putative CD133+ CSCs. Leptin driven Notch-1 signaling has also been identified in breast cancer cell lines (146). The clinical relevance of leptin augmented tumor development could be especially high in developed countries with obesity-prone populations. The diversity of identified environmental factors shows that CSC-niche interactions are at least as complex as the CSCs themselves. It has also been hypothesized, that several different CSC niches may coexist, with different pathways active in perivascular, hypoxic, and metastatic environments (62).

In summary, the stromal compartment may play as much a pivotal role in determining tumor growth and metastasis, as the intrinsic properties of CSCs. However, the CSC niche need not necessarily evolve from nontumorous cell populations. In two studies focusing on angiogenesis in glioblastomas, vasculature of tumors was shown to be of partially neoplastic origin. Ricci-Vitiani et al. showed a portion of endothelial cells in glioblastomas carry the same genetic alterations as tumor cells (147). They also found that in xenograft tumors grown from glioblastoma CSCs, the endothelial lining of vessels is primarily composed of cells of human origin. Wang et al. also showed somatic mutations to be shared by tumor cells and a fraction of endothelial cells within the tumor (148). Through lineage analyses they were able to trace the endothelial cells to a special subset of CD133+ glioblastoma CSCs, which can differentiate into endothelial progenitors which in turn give rise to the endothelial population. They found the transition from CSC to endothelial progenitor to be VEGF dependent, whereas further maturation to endothelial cells relied on effective Notch-1 signaling. A new layer of complexity is added if we consider that hedgehog signaling was shown to induce VEGF production in fibroblasts (131), which in this context may not only promote angiogenesis per se, but also the cross-differentiation of CSCs. Results similar to those with glioblastoma were seen in xenografts of human renal cell carcinoma, where tumor vasculature was also of human origin (53). These studies shed light on a new facet of CSC function and hint that the role of CSCs in tumor biology may go beyond maintaining the strictly taken tumor cell population. Interestingly, the capacity of CSCs to differentiate into endothelial cells seems to be a trait shared with normal pluripotent- and stem cells, which were shown to be capable of cross-lineage differentiation (149–152). Therefore, cross-differentiation might be a further normal stem cell property conserved in CSCs.

PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

The clinical significance of CSCs has been a subject of much debate. It was speculated that stemness features of CSCs would allow them to evade conventional anticancer therapy and maintain minimal residual disease, eventually leading to tumor relapse (153, 154). While the therapy resistance in vitro, and in xenotransplanted tumors has been well documented (64, 155, 156), there was little proof that poor therapy outcomes in patients can be directly linked to CSCs. Recently, it has been shown that immunohistochemically verifiable CSC marker expression in histological slides from patient tumors has prognostic value and is associated with a poorer clinical outcome (65–70, 157, 158). Similarly to circulating cancer cells (159–162), the presence of circulating CSCs was proven to be a prognostic factor for faster disease progression (163–165). Further, a relationship was established between myeloma CSC numbers in patients and progression free survival after treatment with rituximab (153). In breast cancer, the number of breast CSCs in patient's core needle biopsies increased after chemotherapy, demonstrating a survival advantage of CSCs over the bulk tumor population (166). While CSCs were thought to resist therapy through drug efflux mechanisms, antiapoptotic ability, efficient DNA repair and slow cycling (167), two recent studies have suggested other coping mechanisms that may be active in the CSC population. Chaterjee et al. found that breast CSCs can enter dormancy upon treatment with farnesyl transferase inhibitors, a class of drugs designed to inhibit proper maturation of Ras (168). This induced dormancy was maintained by CSCs through autophagy and the cells were able to reenter the cell cycle upon withdrawal of the drugs. Viale et al. showed p21 dependent cell cycle restriction to be functional in CSCs in a mouse leukemia model (169). The cell cycle restriction not only prevented DNA damage accumulation, but also functional exhaustion of CSCs and therefore contributed to maintenance of stemness. The role of CSCs in minimal residual disease and relapse was supported by the work of Hamilton et al., who showed that a subpopulation of Bcr-Abl positive CML CSCs are able to survive the withdrawal of growth factors in addition to combined Bcr-Abl knockdown and pharmacological inhibition by dasatinib, a tyrosine kinase inhibitor (170). These cells were quiescent under the harsh conditions and although they did not proliferate, remained viable. Upon withdrawal of dasatinib and addition of growth factors, the cells reentered the cell cycle and began to proliferate.

It has become clear that traditional treatment regiments are inefficient at eliminating the CSC population and new therapeutic approaches are needed that take into account the special properties of CSCs. Basically, a reduction in the CSC population can be achieved by inducing differentiation/apoptosis in CSCs by targeting receptors or signaling pathways crucial for CSC self-maintenance and viability. This approach has started to show promising results. The paracrine signaling of colon CSCs through interleukin 4 (IL-4) was successfully blocked by anti-IL-4 antibody treatment, reducing in vitro and in vivo chemotherapy resistance (24). In human renal cell carcinomas, IL-15 treatment of CSCs abolished tumorigenic potential and blocked CSC self-renewal (54). A heterodimer of bone morphogenetic protein efficiently inhibited TGF-β dependent breast CSC function, decimating CSCs in vitro and inhibiting metastases formation in vivo (171). Reduction in Nanog-1 expression in breast CSCs by 3-O-methylfunicone inhibited sphere formation and colony formation (172). An off-target shRNA against neostemin, lentivirally transfected into glioblastoma cells, led to preferential apoptosis of CD133+ putative CSCs and significantly slowed xenotransplanted tumor growth (173). Another study found shRNA knockdown of ubiquitine ligases to induce apoptosis and differentiation in cell line derived glioblastoma CSCs (174). As previously discussed, blockade of TGF-β3 action with a secreted decoy receptor inhibited metastasis of mouse breast CSCs (136) and antibody targeting of integrin α6 in glioblastoma inhibited CSC driven in vivo tumor initiation and growth (120). Recent development of subtype-specific Notch antibodies demonstrated efficacy by inhibiting Notch-dependent xenograft tumor growth without the side effects of pan-Notch blockade (175). A selective antibody targeting Notch-1 induced apoptosis in a breast cancer cell line, reduced the CSC population and irreversibly impaired CSC mammosphere formation (176). Oxymatrine was also shown to diminish the side population in a breast cancer cell line by decreasing β-catenin levels and activation (177).

The successful targeting of CSCs may, however, not be sufficient to achieve adequate disease control. A computational modeling of CSC and non-CSC population dynamics concluded that differentiation therapy of slow cycling, self-renewing populations or cytotoxic therapy of a larger, faster proliferating population alone cannot eliminate tumorous growth (178). These calculations did not utilize any specific assumptions about tumor growth regulation, they only hypothesized that relative population densities regulate proliferation and asymmetric cycling of CSCs. A recent study demonstrated the viability of this assumption, with CSC densities reaching the same level in in vitro mixed cultures of CSCs and non-CSCs, irrespective of the initial ratio of the populations after plating (179). Therefore, efficient tumor therapy is suggested to be dependent on parallel administration of cytotoxic agents in combination with targeted CSC elimination. Several studies have found a systematic synergistic effect between traditional chemotherapeutics and newer, CSC-targeted agents. Checkpoint kinase 1 inhibition in combination with chemotherapy greatly enhanced in vitro cytotoxity and in vivo efficacy of chemotherapy against non-small cell lung CSCs (180). Acting through ataxia telangiectasia mutated, an oncolytic virus and the alkylating agent temozolomide synergistically killed glioblastoma CSCs while sparing neurons (181). In pancreatic cancer, inhibition of c-Met reduced the CSC fraction in xenograft tumors; however, full growth inhibition was only achieved after adding gemcitabine to the treatment regimen (50). Similarly, stable disease and growth inhibition of primary pancreatic cancer xenografts was only accomplished with triple therapy consisting of gemcitabine, knockdown of Nodal in CSCs and blockade of smoothened in stromal elements (137). Also in pancreatic cancer, combined gemcitabine, sonic hedgehog- and mTOR inhibition abolished metastatic activity and reduced xenograft tumor growth (88). Finally, retinoid compounds blocked the increase in ALDH1+ cells after carboplatin therapy of ovarian cancer cell lines and in combination with carboplatin significantly reduced in vivo tumor growth (182). While the novel combination therapies show great potential, tolerability in the clinical setting and especially the long term effects on normal stem cells still need to be determined. In a special setting, the evolving approach of tumor therapy with the patient's reprogrammed and expanded own T cells (183) can be ideally exploited to target CSC specific surface antigens (72). This approach has first been confirmed for targeting CD20 on CSCs in a xenograft model of human melanoma cells (184) and then successfully applied in a clinical case (185).

CONCLUSION

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED

Our understanding of CSC biology is rapidly expanding. Attention is gradually shifting from merely identifying markers that enrich for CSCs to untangling the complex web of signaling pathways that determine the stem-like behavior. These pathways are often dysregulated counterparts of networks functionally important in the maintenance of normal tissue stem cells. We are also beginning to recognize that CSCs are not lonely fighters, they often interact with and modulate signaling of stromal elements to elucidate a paracrine environment favoring tumor growth and invasion. To what extent stromal factors influence CSC behavior and how essential these factors are for tumor propagation still remains to be seen. An intriguing new aspect of CSC biology is their potential to not only give rise to the various malignant cell lines within a tumor, but also to produce progeny that can differentiate into various supporting tissues and structures. The clinical significance of CSCs is growing, with the CSC phenotype representing an independent prognostic factor for poorer patient outcome. Indirect and direct evidence for therapeutic consequences of CSC behavior, such as therapy resistance, minimal residual disease and relapse are mounting. Luckily, every identified marker and pathway represents a new potential target for effective therapy. Initial experience with targeted CSC therapy is promising and hopefully the preliminary in vitro and in vivo results, whether alone or in combination with traditional cytotoxic agents, can be carried over into clinical trials and eventually lead to successful cancer therapy.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. EVIDENCE SUPPORTING CANCER STEM CELL THEORY
  4. IDENTIFICATION AND MARKERS OF PUTATIVE CSCS
  5. CSC INTERACTIONS WITH THE TUMOR MICROENVIRONMENT
  6. PROGNOSTIC VALUE OF CSCs AND THEIR IMPLICATIONS FOR THERAPY
  7. CONCLUSION
  8. LITERATURE CITED
  • 1
    Reya T,Morrison SJ,Clarke MF,Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105111.
  • 2
    Clarke MF,Dick JE,Dirks PB,Eaves CJ,Jamieson CH,Jones DL,Visvader J,Weissman IL,Wahl GM. Cancer stem cells—Perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006; 66: 93399344.
  • 3
    Al-Hajj M,Becker MW,Wicha M,Weissman I,Clarke MF. Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004; 14: 4347.
  • 4
    Wicha MS. Cancer stem cells and metastasis: Lethal seeds. Clin Cancer Res 2006; 12: 56065607.
  • 5
    Li F,Tiede B,Massague J,Kang Y. Beyond tumorigenesis: Cancer stem cells in metastasis. Cell Res 2007; 17: 314.
  • 6
    Dalerba P,Clarke MF. Cancer stem cells and tumor metastasis: First steps into uncharted territory. Cell Stem Cell 2007; 1: 241242.
  • 7
    Lapidot T,Sirard C,Vormoor J,Murdoch B,Hoang T,Caceres-Cortes J,Minden M,Paterson B,Caligiuri MA,Dick JE. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645648.
  • 8
    Bonnet D,Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730737.
  • 9
    Sirard C,Lapidot T,Vormoor J,Cashman JD,Doedens M,Murdoch B,Jamal N,Messner H,Addey L,Minden M, et al. Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis. Blood 1996; 87: 15391548.
  • 10
    Wang JC,Lapidot T,Cashman JD,Doedens M,Addy L,Sutherland DR,Nayar R,Laraya P,Minden M,Keating A, et al. High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase. Blood 1998; 91: 24062414.
  • 11
    Singh SK,Hawkins C,Clarke ID,Squire JA,Bayani J,Hide T,Henkelman RM,Cusimano MD,Dirks PB. Identification of human brain tumor initiating cells. Nature 2004; 432: 396401.
  • 12
    Beier D,Hau P,Proescholdt M,Lohmeier A,Wischhusen J,Oefner PJ,Aigner L,Brawanski A,Bogdahn U,Beier CP. CD133(+) and CD133(–) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 2007; 67: 40104015.
  • 13
    Shu Q,Wong KK,Su JM,Adesina AM,Yu LT,Tsang YT,Antalffy BC,Baxter P,Perlaky L,Yang J, et al. Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma. Stem Cells 2008; 26: 14141424.
  • 14
    Patrawala L,Calhoun T,Schneider-Broussard R,Zhou J,Claypool K,Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 2005; 65: 62076219.
  • 15
    He J,Liu Y,Zhu T,Zhu J,Dimeco F,Vescovi AL,Heth JA,Muraszko KM,Fan X,Lubman DM. CD90 is identified as a marker for cancer stem cells in primary high-grade gliomas using tissue microarrays. Mol Cell Proteomics 2012;11:M111.010744.
  • 16
    Al-Hajj M,Wicha MS,Benito-Hernandez A,Morrison SJ,Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 39833988.
  • 17
    Ponti D,Costa A,Zaffaroni N,Pratesi G,Petrangolini G,Coradini D,Pilotti S,Pierotti MA,Daidone MG. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005; 65: 55065511.
  • 18
    Sheridan C,Kishimoto H,Fuchs RK,Mehrotra S,Bhat-Nakshatri P,Turner CH,Goulet RJr,Badve S,Nakshatri H. CD44+/CD24– breast cancer cells exhibit enhanced invasive properties: An early step necessary for metastasis. Breast Cancer Res 2006; 8: R59.
  • 19
    Donnenberg VS,Landreneau RJ,Donnenberg AD. Tumorigenic stem and progenitor cells: Implications for the therapeutic index of anti-cancer agents. J Controlled Release 2007; 122: 385391.
  • 20
    Ginestier C,Hur MH,Charafe-Jauffret E,Monville F,Dutcher J,Brown M,Jacquemier J,Viens P,Kleer CG,Liu S, et al. ALDH1 Is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007; 1: 555567.
  • 21
    Wright MH,Calcagno AM,Salcido CD,Carlson MD,Ambudkar SV,Varticovski L. Brca1 breast tumors contain distinct CD44+/CD24– and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res 2008; 10: R10.
  • 22
    Ricci-Vitiani L,Lombardi DG,Pilozzi E,Biffoni M,Todaro M,Peschle C,De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007; 445: 111115.
  • 23
    O'Brien CA,Pollett A,Gallinger S,Dick JE. A human colon cancer cell capable of initiating tumor growth in immunodeficient mice. Nature 2007; 445: 106110.
  • 24
    Todaro M,Alea MP,Di Stefano AB,Cammareri P,Vermeulen L,Iovino F,Tripodo C,Russo A,Gulotta G,Medema JP, et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 2007; 1: 389402.
  • 25
    Dalerba P,Dylla SJ,Park IK,Liu R,Wang X,Cho RW,Hoey T,Gurney A,Huang EH,Simeone DM, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 2007; 104: 1015810163.
  • 26
    Huang EH,Hynes MJ,Zhang T,Ginestier C,Dontu G,Appelman H,Fields JZ,Wicha MS,Boman BM. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 2009; 69: 33823389.
  • 27
    Rutella S,Bonanno G,Procoli A,Mariotti A,Corallo M,Prisco MG,Eramo A,Napoletano C,Gallo D,Perillo A, et al. Cells with characteristics of cancer stem/progenitor cells express the CD133 antigen in human endometrial tumors. Clin Cancer Res 2009; 15: 42994311.
  • 28
    Prince ME,Sivanandan R,Kaczorowski A,Wolf GT,Kaplan MJ,Dalerba P,Weissman IL,Clarke MF,Ailles LE. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 2007; 104: 973978.
  • 29
    Chen YC,Chen YW,Hsu HS,Tseng LM,Huang PI,Lu KH,Chen DT,Tai LK,Yung MC,Chang SC, et al. Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochem Biophys Res Commun 2009; 385: 307313.
  • 30
    Takaishi S,Okumura T,Tu S,Wang SS,Shibata W,Vigneshwaran R,Gordon SA,Shimada Y,Wang TC. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells 2009; 27: 10061020.
  • 31
    Zhang C,Li C,He F,Cai Y,Yang H. Identification of CD44+CD24+ gastric cancer stem cells. J Cancer Res Clin Oncol 2011; 137: 16791686.
  • 32
    Ma S,Chan KW,Hu L,Lee TK,Wo JY,Ng IO,Zheng BJ,Guan XY. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007; 132: 25422556.
  • 33
    Yang ZF,Ngai P,Ho DW,Yu WC,Ng MN,Lau CK,Li ML,Tam KH,Lam CT,Poon RT, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology 2008; 47: 919928.
  • 34
    Yin S,Li J,Hu C,Chen X,Yao M,Yan M,Jiang G,Ge C,Xie H,Wan D, et al. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007; 120: 14441450.
  • 35
    Ma S,Chan KW,Lee TK,Tang KH,Wo JY,Zheng BJ,Guan XY. Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res 2008; 6: 11461153.
  • 36
    Cheung PF,Cheng CK,Wong NC,Ho JC,Yip CW,Lui VC,Cheung AN,Fan ST,Cheung ST. Granulin-epithelin precursor is an oncofetal protein defining hepatic cancer stem cells. PLoS One 2011; 6: e28246.
  • 37
    Kim CF,Jackson EL,Woolfenden AE,Lawrence S,Babar I,Vogel S,Crowley D,Bronson RT,Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005; 121: 823835.
  • 38
    Bertolini G,Roz L,Perego P,Tortoreto M,Fontanella E,Gatti L,Pratesi G,Fabbri A,Andriani F,Tinelli S, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA 2009; 106: 1628116286.
  • 39
    Eramo A,Lotti F,Sette G,Pilozzi E,Biffoni M,Di Virgilio A,Conticello C,Ruco L,Peschle C,De Maria R. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 2008; 15: 504514.
  • 40
    Fang D,Nguyen TK,Leishear K,Finko R,Kulp AN,Hotz S,Van Belle PA,Xu X,Elder DE,Herlyn M. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 2005; 65: 93289337.
  • 41
    Boonyaratanakornkit JB,Yue L,Strachan LR,Scalapino KJ,LeBoit PE,Lu Y,Leong SP,Smith JE,Ghadially R. Selection of tumorigenic melanoma cells using ALDH. J Invest Dermatol 2010; 130: 27992808.
  • 42
    Schatton T,Murphy GF,Frank NY,Yamaura K,Waaga-Gasser AM,Gasser M,Zhan Q,Jordan S,Duncan LM,Weishaupt C, et al. Identification of cells initiating human melanomas. Nature 2008; 451: 345349.
  • 43
    Ferrandina G,Bonanno G,Pierelli L,Perillo A,Procoli A,Mariotti A,Corallo M,Martinelli E,Rutella S,Paglia A, et al. Expression of CD133-1 and CD133-2 in ovarian cancer. Int J Gynecol Cancer 2008; 18: 506514.
  • 44
    Zhang S,Balch C,Chan MW,Lai HC,Matei D,Schilder JM,Yan PS,Huang TH,Nephew KP. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 2008; 68: 43114320.
  • 45
    Baba T,Convery PA,Matsumura N,Whitaker RS,Kondoh E,Perry T,Huang Z,Bentley RC,Mori S,Fujii S, et al. Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells. Oncogene 2009; 28: 209218.
  • 46
    Szotek PP,Pieretti-Vanmarcke R,Masiakos PT,Dinulescu DM,Connolly D,Foster R,Dombkowski D,Preffer F,Maclaughlin DT,Donahoe PK. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci USA 2006; 103: 1115411159.
  • 47
    Wang L,Mezencev R,Bowen NJ,Matyunina LV,McDonald JF. Isolation and characterization of stem-like cells from a human ovarian cancer cell line. Mol Cell Biochem 2012; 363: 257268.
  • 48
    Hermann PC,Huber SL,Herrler T,Aicher A,Ellwart JW,Guba M,Bruns CJ,Heeschen C. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007; 1: 313323.
  • 49
    Li C,Heidt DG,Dalerba P,Burant CF,Zhang L,Adsay V,Wicha M,Clarke MF,Simeone DM. Identification of pancreatic cancer stem cells. Cancer Res 2007; 67: 10301037.
  • 50
    Li C,Wu JJ,Hynes M,Dosch J,Sarkar B,Welling TH,Pasca di Magliano M,Simeone DM. c-Met is a marker of pancreatic cancer stem cells and therapeutic target. Gastroenterology 2011; 141: 22182227e5.
  • 51
    Rasheed ZA,Yang J,Wang Q,Kowalski J,Freed I,Murter C,Hong SM,Koorstra JB,Rajeshkumar NV,He X, et al. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma. J Natl Cancer Inst 2010; 102: 340351.
  • 52
    Collins AT,Berry PA,Hyde C,Stower MJ,Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 2005; 65: 1094610951.
  • 53
    Bussolati B,Bruno S,Grange C,Ferrando U,Camussi G. Identification of a tumor-initiating stem cell population in human renal carcinomas. FASEB J 2008; 22: 36963705.
  • 54
    Azzi S,Bruno S,Giron-Michel J,Clay D,Devocelle A,Croce M,Ferrini S,Chouaib S,Vazquez A,Charpentier B, et al. Differentiation therapy: Targeting human renal cancer stem cells with interleukin 15. J Natl Cancer Inst 2011; 103: 18841898.
  • 55
    Mitsutake N,Iwao A,Nagai K,Namba H,Ohtsuru A,Saenko V,Yamashita S. Characterization of side population in thyroid cancer cell lines: Cancer stem-like cells are enriched partly but not exclusively. Endocrinology 2007; 148: 17971803.
  • 56
    Choijamts B,Jimi S,Kondo T,Naganuma Y,Matsumoto T,Kuroki M,Iwasaki H,Emoto M. CD133+ cancer stem cell-like cells derived from uterine carcinosarcoma (malignant mixed Mullerian tumor). Stem Cells 2011; 29: 14851495.
  • 57
    Pham PV,Phan NL,Nguyen NT,Truong NH,Duong TT,Le DV,Truong KD,Phan NK. Differentiation of breast cancer stem cells by knockdown of CD44: Promising differentiation therapy. J Transl Med 2011; 9: 209.
  • 58
    Du L,Wang H,He L,Zhang J,Ni B,Wang X,Jin H,Cahuzac N,Mehrpour M,Lu Y. et al. CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res 2008; 14: 67516760.
  • 59
    Chen R,Nishimura MC,Bumbaca SM,Kharbanda S,Forrest WF,Kasman IM,Greve JM,Soriano RH,Gilmour LL,Rivers CS, et al. A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell 2010; 17: 362375.
  • 60
    Gunther HS,Schmidt NO,Phillips HS,Kemming D,Kharbanda S,Soriano R,Modrusan Z,Meissner H,Westphal M,Lamszus K. Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 2008; 27: 28972909.
  • 61
    Shmelkov SV,Butler JM,Hooper AT,Hormigo A,Kushner J,Milde T,St Clair R,Baljevic M,White I,Jin DK, et al. CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors. J Clin Invest 2008; 118: 21112120.
  • 62
    Lathia JD,Heddleston JM,Venere M,Rich JN. Deadly teamwork: Neural cancer stem cells and the tumor microenvironment. Cell Stem Cell 2011; 8: 482485.
  • 63
    O'Brien CA,Kreso A,Dick JE. Cancer stem cells in solid tumors: An overview. Semin Radiat Oncol 2009; 19: 7177.
  • 64
    Beier D,Schulz JB,Beier CP. Chemoresistance of glioblastoma cancer stem cells—Much more complex than expected. Mol Cancer 2011; 10: 128.
  • 65
    Kawamoto M,Ishiwata T,Cho K,Uchida E,Korc M,Naito Z,Tajiri T. Nestin expression correlates with nerve and retroperitoneal tissue invasion in pancreatic cancer. Hum Pathol 2009; 40: 189198.
  • 66
    Maeda S,Shinchi H,Kurahara H,Mataki Y,Maemura K,Sato M,Natsugoe S,Aikou T,Takao S. CD133 expression is correlated with lymph node metastasis and vascular endothelial growth factor-C expression in pancreatic cancer. Br J Cancer 2008; 98: 13891397.
  • 67
    Sakakibara M,Fujimori T,Miyoshi T,Nagashima T,Fujimoto H,Suzuki HT,Ohki Y,Fushimi K,Yokomizo J,Nakatani Y, et al. Aldehyde dehydrogenase 1-positive cells in axillary lymph node metastases after chemotherapy as a prognostic factor in patients with lymph node-positive breast cancer. Cancer 2012; 118: 38993910.
  • 68
    Toll AD,Boman BM,Palazzo JP. Dysplastic lesions in inflammatory bowel disease show increased positivity for the stem cell marker aldehyde dehydrogenase. Hum Pathol 2012; 43: 238242.
  • 69
    Vogler T,Kriegl L,Horst D,Engel J,Sagebiel S,Schaffauer AJ,Kirchner T,Jung A. The expression pattern of aldehyde dehydrogenase 1 (ALDH1) is an independent prognostic marker for low survival in colorectal tumors. Exp Mol Pathol 2011; 92: 111117.
  • 70
    Wang T,Ong CW,Shi J,Srivastava S,Yan B,Cheng CL,Yong WP,Chan SL,Yeoh KG,Iacopetta B, et al. Sequential expression of putative stem cell markers in gastric carcinogenesis. Br J Cancer 2011; 105: 658665.
  • 71
    Zhou BB,Zhang H,Damelin M,Geles KG,Grindley JC,Dirks PB. Tumor-initiating cells: Challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 2009; 8: 806823.
  • 72
    Schmidt P,Abken H. The beating heart of melanomas: A minor subset of cancer cells sustains tumor growth. Oncotarget 2011; 2: 313320.
  • 73
    Reya T,Clevers H. Wnt signalling in stem cells and cancer. Nature 2005; 434: 843850.
  • 74
    Ng JM,Curran T. The Hedgehog's tale: Developing strategies for targeting cancer. Nat Rev Cancer 2011; 11: 493501.
  • 75
    Singh S,Wang Z,Liang Fei D,Black KE,Goetz JA,Tokhunts R,Giambelli C,Rodriguez-Blanco J,Long J,Lee E, et al. Hedgehog-producing cancer cells respond to and require autocrine Hedgehog activity. Cancer Res 2011; 71: 44544463.
  • 76
    Roy M,Pear WS,Aster JC. The multifaceted role of Notch in cancer. Curr Opin Genet Dev 2007; 17: 5259.
  • 77
    Ishiwata T,Matsuda Y,Naito Z. Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis. World J Gastroenterol 2011; 17: 409418.
  • 78
    Jeter CR,Badeaux M,Choy G,Chandra D,Patrawala L,Liu C,Calhoun-Davis T,Zaehres H,Daley GQ,Tang DG. Functional evidence that the self-renewal gene NANOG regulates human tumor development. Stem Cells 2009; 27: 9931005.
  • 79
    Jeter CR,Liu B,Liu X,Chen X,Liu C,Calhoun-Davis T,Repass J,Zaehres H,Shen JJ,Tang DG. NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene 2011; 30: 38333845.
  • 80
    Bierie B,Moses HL. Tumor microenvironment: TGFbeta: The molecular Jekyll and Hyde of cancer. Nat Rev Cancer 2006; 6: 506520.
  • 81
    Massague J. TGFbeta in Cancer. Cell 2008; 134: 215230.
  • 82
    Clement V,Sanchez P,de Tribolet N,Radovanovic I,Ruiz i Altaba A. HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 2007; 17: 165172.
  • 83
    Izrailit J,Reedijk M. Developmental pathways in breast cancer and breast tumor-initiating cells: Therapeutic implications. Cancer Lett 2012; 317: 115126.
  • 84
    Liu S,Dontu G,Mantle ID,Patel S,Ahn NS,Jackson KW,Suri P,Wicha MS. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 2006; 66: 60636071.
  • 85
    Bae KM,Parker NN,Dai Y,Vieweg J,Siemann DW. E-cadherin plasticity in prostate cancer stem cell invasion. Am J Cancer Res 2011; 1: 7184.
  • 86
    Nakatsugawa M,Takahashi A,Hirohashi Y,Torigoe T,Inoda S,Murase M,Asanuma H,Tamura Y,Morita R,Michifuri Y, et al. SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity. Lab Invest 2011; 91: 17961804.
  • 87
    Malanchi I,Peinado H,Kassen D,Hussenet T,Metzger D,Chambon P,Huber M,Hohl D,Cano A,Birchmeier W, et al. Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling. Nature 2008; 452: 650653.
  • 88
    Mueller MT,Hermann PC,Witthauer J,Rubio-Viqueira B,Leicht SF,Huber S,Ellwart JW,Mustafa M,Bartenstein P,D'Haese JG, et al. Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer. Gastroenterology 2009; 137: 11021113.
  • 89
    Singh BN,Fu J,Srivastava RK,Shankar S. Hedgehog signaling antagonist GDC-0449 (Vismodegib) inhibits pancreatic cancer stem cell characteristics: Molecular mechanisms. PLoS One 2011; 6: e27306.
  • 90
    Velpula KK,Dasari VR,Tsung AJ,Dinh DH,Rao JS. Cord blood stem cells revert glioma stem cell EMT by down regulating transcriptional activation of Sox2 and Twist1. Oncotarget 2011; 2: 10281042.
  • 91
    Zhao C,Chen A,Jamieson CH,Fereshteh M,Abrahamsson A,Blum J,Kwon HY,Kim J,Chute JP,Rizzieri D, et al. Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 2009; 458: 776779.
  • 92
    Zhou Y,Yang J,Kopecek J. Selective inhibitory effect of HPMA copolymer-cyclopamine conjugate on prostate cancer stem cells. Biomaterials 2012; 33: 18631872.
  • 93
    Quintana E,Shackleton M,Foster HR,Fullen DR,Sabel MS,Johnson TM,Morrison SJ. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell 2010; 18: 510523.
  • 94
    Notta F,Mullighan CG,Wang JC,Poeppl A,Doulatov S,Phillips LA,Ma J,Minden MD,Downing JR,Dick JE. Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating cells. Nature 2011; 469: 362367.
  • 95
    Dalerba P,Kalisky T,Sahoo D,Rajendran PS,Rothenberg ME,Leyrat AA,Sim S,Okamoto J,Johnston DM,Qian D, et al. Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nat Biotechnol 2011; 29: 11201127.
  • 96
    Navin N,Kendall J,Troge J,Andrews P,Rodgers L,McIndoo J,Cook K,Stepansky A,Levy D,Esposito D, et al. Tumor evolution inferred by single-cell sequencing. Nature 2011; 472: 9094.
  • 97
    Lathia JD,Gallagher J,Myers JT,Li M,Vasanji A,McLendon RE,Hjelmeland AB,Huang AY,Rich JN. Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells. PLoS One 2011; 6: e24807.
  • 98
    Lathia JD,Hitomi M,Gallagher J,Gadani SP,Adkins J,Vasanji A,Liu L,Eyler CE,Heddleston JM,Wu Q, et al. Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions. Cell Death Dis 2011; 2: e200.
  • 99
    Zhu L,Gibson P,Currle DS,Tong Y,Richardson RJ,Bayazitov IT,Poppleton H,Zakharenko S,Ellison DW,Gilbertson RJ. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 2009; 457: 603607.
  • 100
    Hope KJ,Jin L,Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 2004; 5: 738743.
  • 101
    Goardon N,Marchi E,Atzberger A,Quek L,Schuh A,Soneji S,Woll P,Mead A,Alford KA,Rout R, et al. Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia. Cancer Cell 2011; 19: 138152.
  • 102
    Hermann PC,Bhaskar S,Cioffi M,Heeschen C. Cancer stem cells in solid tumors. Semin Cancer Biol 2010; 20: 7784.
  • 103
    Wessels JT,Yamauchi K,Hoffman RM,Wouters FS. Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo. Cytometry A 2010; 77: 667676.
  • 104
    Morgan SP. Can new optical techniques for in vivo imaging and flow cytometry of the microcirculation benefit sickle cell disease research? Cytometry A 2011; 79: 766774.
  • 105
    Li Y,Guo J,Wang C,Fan Z,Liu G,Gu Z,Damm D,Mosig A,Wei X. Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry. Cytometry A 2011; 79: 848854.
  • 106
    Takao M,Takeda K. Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination-free flow cytometry. Cytometry A 2011; 79: 107117.
  • 107
    Fiser K,Sieger T,Schumich A,Wood B,Irving J,Mejstrikova E,Dworzak MN. Detection and monitoring of normal and leukemic cell populations with hierarchical clustering of flow cytometry data. Cytometry A 2012; 81: 2534.
  • 108
    Scholtens TM,Schreuder F,Ligthart ST,Swennenhuis JF,Greve J,Terstappen LW. Automated identification of circulating tumor cells by image cytometry. Cytometry A 2012; 81: 138148.
  • 109
    Liu X,Hsieh HB,Campana D,Bruce RH. A new method for high speed, sensitive detection of minimal residual disease. Cytometry A 2012; 81: 169175.
  • 110
    Solly F,Angelot F,Garand R,Ferrand C,Seilles E,Schillinger F,Decobecq A,Billot M,Larosa F,Plouvier E, et al. CD304 is preferentially expressed on a subset of B-lineage acute lymphoblastic leukemia and represents a novel marker for minimal residual disease detection by flow cytometry. Cytometry A 2012; 81: 1724.
  • 111
    Fabian A,Barok M,Vereb G,Szollosi J. Die hard: Are cancer stem cells the Bruce Willises of tumor biology? Cytometry A 2009; 75: 6774.
  • 112
    Mannelli G,Gallo O. Cancer stem cells hypothesis and stem cells in head and neck cancers. Cancer Treat Rev 2012; 38: 515539.
  • 113
    Greve B,Kelsch R,Spaniol K,Eich HT,Gotte M. Flow cytometry in cancer stem cell analysis and separation. Cytometry A 2012; 81: 284293.
  • 114
    Kelly PN,Dakic A,Adams JM,Nutt SL,Strasser A. Tumor growth need not be driven by rare cancer stem cells. Science 2007; 317: 337.
  • 115
    Hill RP. Identifying cancer stem cells in solid tumors: Case not proven. Cancer Res 2006; 66: 1891–1895; discussion 1890.
  • 116
    Shackleton M,Quintana E,Fearon ER,Morrison SJ. Heterogeneity in cancer: Cancer stem cells versus clonal evolution. Cell 2009; 138: 822829.
  • 117
    Quintana E,Shackleton M,Sabel MS,Fullen DR,Johnson TM,Morrison SJ. Efficient tumor formation by single human melanoma cells. Nature 2008; 456: 593598.
  • 118
    Cheng L,Ramesh AV,Flesken-Nikitin A,Choi J,Nikitin AY. Mouse models for cancer stem cell research. Toxicol Pathol 2010; 38: 6271.
  • 119
    Yilmaz OH,Valdez R,Theisen BK,Guo W,Ferguson DO,Wu H,Morrison SJ. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006; 441: 475482.
  • 120
    Lathia JD,Gallagher J,Heddleston JM,Wang J,Eyler CE,Macswords J,Wu Q,Vasanji A,McLendon RE,Hjelmeland AB, et al. Integrin alpha 6 regulates glioblastoma stem cells. Cell Stem Cell 2010; 6: 421432.
  • 121
    Prestegarden L,Svendsen A,Wang J,Sleire L,Skaftnesmo KO,Bjerkvig R,Yan T,Askland L,Persson A,Sakariassen PO, et al. Glioma cell populations grouped by different cell type markers drive brain tumor growth. Cancer Res 2010; 70: 42744279.
  • 122
    Ogden AT,Waziri AE,Lochhead RA,Fusco D,Lopez K,Ellis JA,Kang J,Assanah M,McKhann GM,Sisti MB, et al. Identification of A2B5+CD133– tumor-initiating cells in adult human gliomas. Neurosurgery 2008; 62: 505514; discussion 514–515.
  • 123
    Wang J,Sakariassen PO,Tsinkalovsky O,Immervoll H,Boe SO,Svendsen A,Prestegarden L,Rosland G,Thorsen F,Stuhr L, et al. CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. Int J Cancer 2008; 122: 761768.
  • 124
    Nakshatri H,Srour EF,Badve S. Breast cancer stem cells and intrinsic subtypes: Controversies rage on. Curr Stem Cell Res Ther 2009; 4: 5060.
  • 125
    Zeilstra J,Joosten SP,Dokter M,Verwiel E,Spaargaren M,Pals ST. Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis. Cancer Res 2008; 68: 36553661.
  • 126
    Krause DS,Lazarides K,von Andrian UH,Van Etten RA. Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. Nat Med 2006; 12: 11751180.
  • 127
    Balbuena J,Pachon G,Lopez-Torrents G,Aran JM,Castresana JS,Petriz J. ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties. Cytometry A 2011; 79: 672683.
  • 128
    Li L,Neaves WB. Normal stem cells and cancer stem cells: The niche matters. Cancer Res 2006; 66: 45534557.
  • 129
    Calabrese C,Poppleton H,Kocak M,Hogg TL,Fuller C,Hamner B,Oh EY,Gaber MW,Finklestein D,Allen M, et al. A perivascular niche for brain tumor stem cells. Cancer Cell 2007; 11: 6982.
  • 130
    Beck B,Driessens G,Goossens S,Youssef KK,Kuchnio A,Caauwe A,Sotiropoulou PA,Loges S,Lapouge G,Candi A, et al. A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumors. Nature 2011; 478: 399403.
  • 131
    Chen W,Tang T,Eastham-Anderson J,Dunlap D,Alicke B,Nannini M,Gould S,Yauch R,Modrusan Z,DuPree KJ, et al. Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells. Proc Natl Acad Sci USA 2011; 108: 95899594.
  • 132
    Tian H,Callahan CA,DuPree KJ,Darbonne WC,Ahn CP,Scales SJ,de Sauvage FJ. Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis. Proc Natl Acad Sci USA 2009; 106: 42544259.
  • 133
    Yauch RL,Gould SE,Scales SJ,Tang T,Tian H,Ahn CP,Marshall D,Fu L,Januario T,Kallop D, et al. A paracrine requirement for hedgehog signalling in cancer. Nature 2008; 455: 406410.
  • 134
    Dosch JS,Pasca di Magliano M,Simeone DM. Pancreatic cancer and hedgehog pathway signaling: New insights. Pancreatology 2010; 10: 151157.
  • 135
    Grisendi G,Bussolari R,Veronesi E,Piccinno S,Burns JS,De Santis G,Loschi P,Pignatti M,Di Benedetto F,Ballarin R, et al. Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: The Mesenkillers. Am J Cancer Res 2011; 1: 787805.
  • 136
    Malanchi I,Santamaria-Martinez A,Susanto E,Peng H,Lehr HA,Delaloye JF,Huelsken J. Interactions between cancer stem cells and their niche govern metastatic colonization. Nature 2012; 481: 8589.
  • 137
    Lonardo E,Hermann PC,Mueller MT,Huber S,Balic A,Miranda-Lorenzo I,Zagorac S,Alcala S,Rodriguez-Arabaolaza I,Ramirez JC, et al. Nodal/activin signaling drives self-renewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy. Cell Stem Cell 2011; 9: 433446.
  • 138
    Olive KP,Jacobetz MA,Davidson CJ,Gopinathan A,McIntyre D,Honess D,Madhu B,Goldgraben MA,Caldwell ME,Allard D, et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009; 324: 14571461.
  • 139
    Axelson H,Fredlund E,Ovenberger M,Landberg G,Pahlman S. Hypoxia-induced dedifferentiation of tumor cells—A mechanism behind heterogeneity and aggressiveness of solid tumors. Semin Cell Dev Biol 2005; 16: 554563.
  • 140
    Li Z,Bao S,Wu Q,Wang H,Eyler C,Sathornsumetee S,Shi Q,Cao Y,Lathia J,McLendon RE, et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 2009; 15: 501513.
  • 141
    Seidel S,Garvalov BK,Wirta V,von Stechow L,Schanzer A,Meletis K,Wolter M,Sommerlad D,Henze AT,Nister M, et al. A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain 2010; 133: 983995.
  • 142
    Hashimoto O,Shimizu K,Semba S,Chiba S,Ku Y,Yokozaki H,Hori Y. Hypoxia induces tumor aggressiveness and the expansion of CD133-positive cells in a hypoxia-inducible factor-1alpha-dependent manner in pancreatic cancer cells. Pathobiology 2011; 78: 181192.
  • 143
    Ma Y,Liang D,Liu J,Axcrona K,Kvalheim G,Stokke T,Nesland JM,Suo Z. Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties. PLoS One 2011; 6: e29170.
  • 144
    McCord AM,Jamal M,Shankavaram UT,Lang FF,Camphausen K,Tofilon PJ. Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro. Mol Cancer Res 2009; 7: 489497.
  • 145
    Feldman DE,Chen C,Punj V,Tsukamoto H,Machida K. Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells. Proc Natl Acad Sci USA 2011; 109: 829834.
  • 146
    Guo S,Liu M,Gonzalez-Perez RR. Role of Notch and its oncogenic signaling crosstalk in breast cancer. Biochim Biophys Acta 2011; 1815: 197213.
  • 147
    Ricci-Vitiani L,Pallini R,Biffoni M,Todaro M,Invernici G,Cenci T,Maira G,Parati EA,Stassi G,Larocca LM, et al. Tumor vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 2010; 468: 824828.
  • 148
    Wang R,Chadalavada K,Wilshire J,Kowalik U,Hovinga KE,Geber A,Fligelman B,Leversha M,Brennan C,Tabar V. Glioblastoma stem-like cells give rise to tumor endothelium. Nature 2010; 468: 829833.
  • 149
    Wurmser AE,Nakashima K,Summers RG,Toni N,D'Amour KA,Lie DC,Gage FH. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature 2004; 430: 350356.
  • 150
    Joo KM,Jin J,Kang BG,Lee SJ,Kim KH,Yang H,Lee Y-A,Cho YJ,Im Y-S,Lee D-S, et al. Trans-differentiation of neural stem cells: A therapeutic mechanism against the radiation induced brain damage. PLoS One 2012; 7: e25936.
  • 151
    Montiel-Eulefi E,Nery AA,Rodrigues LC,Sanchez R,Romero F,Ulrich H. Neural differentiation of rat aorta pericyte cells. Cytometry A 2012; 81: 6571.
  • 152
    Zimmerlin L,Donnenberg VS,Donnenberg AD. Pericytes: A universal adult tissue stem cell? Cytometry A 2012; 81: 1214.
  • 153
    Ghiaur G,Gerber J,Jones RJ. Concise review: Cancer stem cells and minimal residual disease. Stem Cells 2012; 30: 8993.
  • 154
    Raimondi C,Gianni W,Cortesi E,Gazzaniga P. Cancer stem cells and epithelial-mesenchymal transition: Revisiting minimal residual disease. Curr Cancer Drug Targets 2010; 10: 496508.
  • 155
    Koch U,Krause M,Baumann M. Cancer stem cells at the crossroads of current cancer therapy failures—Radiation oncology perspective. Semin Cancer Biol 2010; 20: 116124.
  • 156
    Dylla SJ,Beviglia L,Park IK,Chartier C,Raval J,Ngan L,Pickell K,Aguilar J,Lazetic S,Smith-Berdan S, et al. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. PLoS One 2008; 3: e2428.
  • 157
    Strojnik T,Rosland GV,Sakariassen PO,Kavalar R,Lah T. Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: Correlation of nestin with prognosis of patient survival. Surg Neurol 2007; 68: 133143; discussion 143–144.
  • 158
    Zeppernick F,Ahmadi R,Campos B,Dictus C,Helmke BM,Becker N,Lichter P,Unterberg A,Radlwimmer B,Herold-Mende CC. Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 2008; 14: 123129.
  • 159
    Cohen SJ,Punt CJ,Iannotti N,Saidman BH,Sabbath KD,Gabrail NY,Picus J,Morse M,Mitchell E,Miller MC, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26: 32133221.
  • 160
    Dawood S,Broglio K,Valero V,Reuben J,Handy B,Islam R,Jackson S,Hortobagyi GN,Fritsche H,Cristofanilli M. Circulating tumor cells in metastatic breast cancer: From prognostic stratification to modification of the staging system? Cancer 2008; 113: 24222430.
  • 161
    Hu Y,Fan L,Zheng J,Cui R,Liu W,He Y,Li X,Huang S. Detection of circulating tumor cells in breast cancer patients utilizing multiparameter flow cytometry and assessment of the prognosis of patients in different CTCs levels. Cytometry A 2010; 77: 213219.
  • 162
    Wang FB,Yang XQ,Yang S,Wang BC,Feng MH,Tu JC. A higher number of circulating tumor cells (CTC) in peripheral blood indicates poor prognosis in prostate cancer patients—A meta-analysis. Asian Pac J Cancer Prev 2011; 12: 26292635.
  • 163
    Fan ST,Yang ZF,Ho DW,Ng MN,Yu WC,Wong J. Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells: A prospective study. Ann Surg 2011; 254: 569576.
  • 164
    Iinuma H,Watanabe T,Mimori K,Adachi M,Hayashi N,Tamura J,Matsuda K,Fukushima R,Okinaga K,Sasako M, et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes' stage B and C colorectal cancer. J Clin Oncol 2011; 29: 15471555.
  • 165
    Pilati P,Mocellin S,Bertazza L,Galdi F,Briarava M,Mammano E,Tessari E,Zavagno G,Nitti D. Prognostic value of putative circulating cancer stem cells in patients undergoing hepatic resection for colorectal liver metastasis. Ann Surg Oncol 2012; 19: 402408.
  • 166
    Li X,Lewis MT,Huang J,Gutierrez C,Osborne CK,Wu MF,Hilsenbeck SG,Pavlick A,Zhang X,Chamness GC, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 2008; 100: 672679.
  • 167
    Signore M,Ricci-Vitiani L,De Maria R. Targeting apoptosis pathways in cancer stem cells. Cancer Lett 2011. DOI: 10.1016/j.canlet.2011.01.013.
  • 168
    Chaterjee M,van Golen KL. Breast cancer stem cells survive periods of farnesyl-transferase inhibitor-induced dormancy by undergoing autophagy. Bone Marrow Res 2011; 2011: 362938.
  • 169
    Viale A,De Franco F,Orleth A,Cambiaghi V,Giuliani V,Bossi D,Ronchini C,Ronzoni S,Muradore I,Monestiroli S, et al. Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature 2009; 457: 5156.
  • 170
    Hamilton A,Helgason GV,Schemionek M,Zhang B,Myssina S,Allan EK,Nicolini FE,Muller-Tidow C,Bhatia R,Brunton VG, et al. Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival. Blood 2012; 119: 15011510.
  • 171
    Buijs JT,van der Horst G,van den Hoogen C,Cheung H,de Rooij B,Kroon J,Petersen M,van Overveld PG,Pelger RC,van der Pluijm G. The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation. Oncogene 2012; 31: 21642174.
  • 172
    Buommino E,Tirino V,De Filippis A,Silvestri F,Nicoletti R,Ciavatta ML,Pirozzi G,Tufano MA. 3-O-methylfunicone, from Penicillium pinophilum, is a selective inhibitor of breast cancer stem cells. Cell Prolif 2011; 44: 401409.
  • 173
    Gil-Ranedo J,Mendiburu-Elicabe M,Garcia-Villanueva M,Medina D,del Alamo M,Izquierdo M. An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells. PLoS One 2011; 6: e28753.
  • 174
    Low J,Blosser W,Dowless M,Ricci-Vitiani L,Pallini R,de Maria R,Stancato L. Knockdown of ubiquitin ligases in glioblastoma cancer stem cells leads to cell death and differentiation. J Biomol Screen 2012; 17: 152162.
  • 175
    Wu Y,Cain-Hom C,Choy L,Hagenbeek TJ,de Leon GP,Chen Y,Finkle D,Venook R,Wu X,Ridgway J, et al. Therapeutic antibody targeting of individual Notch receptors. Nature 2010; 464: 10521057.
  • 176
    Sharma A,Paranjape AN,Rangarajan A,Dighe RR. A monoclonal antibody against human Notch1 ligand-binding domain depletes subpopulation of putative breast cancer stem-like cells. Mol Cancer Ther 2012; 11: 7786.
  • 177
    Zhang Y,Piao B,Hua B,Hou W,Xu W,Qi X,Zhu X,Pei Y,Lin H. Oxymatrine diminishes the side population and inhibits the expression of beta-catenin in MCF-7 breast cancer cells. Med Oncol 2011; 28( Suppl 1): 99107.
  • 178
    Vainstein V,Kirnasovsky OU,Kogan Y,Agur Z. Strategies for cancer stem cell elimination: Insights from mathematical modeling. J Theor Biol 2011; 298: 3241.
  • 179
    Agur Z,Kogan Y,Levi L,Harrison H,Lamb R,Kirnasovsky OU,Clarke RB. Disruption of a quorum sensing mechanism triggers tumorigenesis: A simple discrete model corroborated by experiments in mammary cancer stem cells. Biol Direct 2010; 5: 20.
  • 180
    Bartucci M,Svensson S,Romania P,Dattilo R,Patrizii M,Signore M,Navarra S,Lotti F,Biffoni M,Pilozzi E, et al. Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy. Cell Death Differ 2012; 19: 768778.
  • 181
    Kanai R,Rabkin SD,Yip S,Sgubin D,Zaupa CM,Hirose Y,Louis DN,Wakimoto H,Martuza RL. Oncolytic virus-mediated manipulation of DNA damage responses: Synergy with chemotherapy in killing glioblastoma stem cells. J Natl Cancer Inst 2012; 104: 4255.
  • 182
    Whitworth JM,Londono-Joshi AI,Sellers JC,Oliver PJ,Muccio DD,Atigadda VR,Straughn JM Jr,Buchsbaum DJ. The impact of novel retinoids in combination with platinum chemotherapy on ovarian cancer stem cells. Gynecol Oncol 2012; 125: 226230.
  • 183
    Hawkins RE,Gilham DE,Debets R,Eshhar Z,Taylor N,Abken H,Schumacher TN,Consortium A. Development of adoptive cell therapy for cancer: A clinical perspective. Hum Gene Ther 2010; 21: 665672.
  • 184
    Schmidt P,Kopecky C,Hombach A,Zigrino P,Mauch C,Abken H. Eradication of melanomas by targeted elimination of a minor subset of tumor cells. Proc Natl Acad Sci USA 2011; 108: 24742479.
  • 185
    Schlaak M,Schmidt P,Bangard C,Kurschat P,Mauch C,Abken H. Regression of metastatic melanoma in a patient by antibody targeting of cancer stem cells. Oncotarget 2012; 3: 2230.