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Utrecht University “Cancer Genomics and Developmental Biology” master’s programme Thesis: Cancer Stem Cells Giannis Ampatziadis-Michailidis, 3311864 Supervisor: Dr. R. Vries Clevers Laboratory, Hubrecht Institute Acknowledgments I am truly grateful for the help and insightful comments of Dr. R. Vries and his inspiring supervision. Moreover I have to thank in depth Prof. H. Clevers for approving my application and for being the second reviewer of this thesis. Lastly I have to pay my respects to Sofia and Nitsa for always being supportive and encouraging and to Sofoklis because without his financial support I would not be able to follow my dreams. Utrecht, January 2010 2 Index: Chapter 1. Introduction 2. Stem cells 3. The niche 4. The identity of Cancer Stem Cell 5. Self-renewal and differentiation 6. Identification assays 6.1 Side population 6.2 Markers 6.3.1 In vitro demonstration of tumorigenicity – sphere formation 6.3.2 In vivo demonstration of tumorigenicity - xenotransplantation 7. Frequency of CSCs 8. Stochastic model versus CSCs model 9. CSCs and therapy resistance 10. Specific tumor types 10.1 CSCs and leukemia 10.2 CSCs and solid tumors 10.2.1 CSCs and breast cancer 10.2.2 CSCs and brain cancer 10.2.3 CSCs and colorectal cancer 10.2.4 CSCs in other solid tumors 11. CSCs and signal transduction 12. Cancer treatment – is there hope? 13. Future directions 14. References Pages 4-5 5-6 6-7 7-8 9-10 10 10-11 11-13 13 13-15 15-16 16-17 18 19 19-21 22 22-24 24-26 26-29 29 29 30 30-31 32-36 3 1. Introduction Cancer is a disease that is caused by uncontrolled cell growth. It starts with the generation of one or more tumor cells (oncogenesis) which have the potential to divide infinitively followed by the formation of a mass of abnormally proliferating cells. Next some of the cells from the tumor acquire extra mutations which allow some of the tumor cells to spread in other parts of the body of the patient and establish secondary tumors (metastasis)1,2. Exactly this feature of cancer, being able to spread to distant places, is characteristic of the disease’s aggressiveness and is lethal for the patients. The current view on the oncogenesis field suggests that for the first tumor cell to arise 6-10 events have to take place. These can be grouped into two categories, mutations on tumor suppressor genes, or mutation on protooncogenes. The role of tumor suppressors is to regulate cell cycle and mutations causing loss of function of these genes can lead to uncontrolled cell growth. On the other hand gain of function mutations on proto-oncogenes that under normal conditions promote cell growth can lead to their excessive over activation and then as oncogenes will drive uncontrolled cell proliferation. In the case of tumor suppressors both alleles need to be mutated and hence deactivated in order for the gene’s function to be lost but for a proto-oncogene to become oncogene one mutated - overactivate allele is sufficient. Importantly the first of the series of mutations needs to confer survival benefit on a normal cell and in that why allow for more mutations to accumulate3,4. Several ways to treat cancer have been discovered during the last century but in most cases they do not lead to completely cure of the patients. Inspite of the increase of cancer incidents during the last century, mainly because of higher life expectancy rates and life style parameters, such as smoking, improvement of screening methods and public awareness together with discovery of new drugs and targeted therapy have actually increased the cure rate since the last 50 years5. Different methods to treat cancer exist as chemotherapy, radiotherapy, immunotherapy and surgery2. Surgery has the best success rates although is most of times combined with one or more of the other options. Chemotherapy has also high success rates but has major side-effects for the patiens6,7. The main reason that cancer is incurable is that even when surgically removed and targeted by some kind of therapy the tumor reappear most of the times with an even more aggressive phenotype. In general tumors tend to be resistant to all possible combination of treatments. Some tumors are not being 4 affected by the application of treatment and some others even if they respond initially, they finally develop acquired resistance. It is obvious that scientists are missing an important piece of the puzzle and efforts are being made to discover the Achille’s heel of the cancer. Towards this direction the last decade a theory has been brought to light, suggesting that in each tumor only certain cells have tumorigenic potential and they are the ones that sustain the tumor mass and cause metastasis. This theory, called the Cancer Stem Cells (CSCs) theory is not news for the scientific community8-14. If the CSCs theory holds true then it will revolutionize the oncology field and change radically the way of cancer treatment. Hopefully time will bring more insightful publications and scientists will critically review new insights remaining critical in order to prevent rushing into wrong conclusions. 2. Stem Cells Before deciphering the cancer stem cell field it is important to be familiar with the stem cell (SCs) area. According to the Collins english dictionary a stem cell is “a type of cell that can produce other cells which are able to develop into any kind of cell in the body”. A stem cell can be described as a cell that has self renewal properties and gives rise to more differentiated populations, called the progenitor cells12,15-22. The progenitor or transit amplifying cells will give rise to the differentiated cells and they still exhibit self renewal properties but in a less extent, meaning that they will eventually stop proliferating after a certain number of divisions. It is believed that SCs divide extremely rarely in order to preserve the integrity of DNA by minimizing accumulation of mutations by cell division. The burden of further extending the pool of differentiated cell is curried by the progenitor cells. Furthermore the stem cells can be divided into two broad categories, embryonic and adult SCs. The embryonic SCs are present in early embryos and originate from the zygote. The second category adult SCs, which can be alternatively named tissue or somatic SCs characterize SCs from fully developed tissues and organs. These cells are committed to producing a more restricted repertoire of cells16-24. Embryonic SCs are pluripotent and adult SCs are multipotent. These terms are used to determine the differentiation potential of the cell. Topipotent cells are at the top of the hierarchy giving rise to the 3 germ layers, the placenta and the primordial germ cells. Next in the hierarchy are the pluripotent cells that can give rise to all 3 germ layers and further down are multipotent cells which have the ability to produce various cell types but only within the 5 context of one germ layer. In mammals, topipotency show cells from the zygote until the 8 cells morula stage and pluripotency cells from the inner mass of the blastocyst, whereas multipotent are the SCs that will give rise to a specific germ layer like the endoderm16-21. Multipotent cells are needed in the adult organism in order to maintain the number of differentiated cells in constant levels, by replacing the cells that die under normal or stress conditions15,23,25. An example of multipotent cells are the hematopoietic SCs which constantly renew the population of differentiated blood cells. The main feature of SCs, self renewal is sustained by the asymmetric divisions of these cells. In an asymmetric division one SC produces a copy of itself (thus complying with the self renewal) and another more committed cell following the differentiation pathway. SCs can also undergo symmetric division and produce either two SCs or two differentiated cells when there is a need to expand the SCs pool or the progenitors pool respectively16-20,23,26-28. Figure 1: Stem cells self-renewal and differentiation properties, adapted from29 3. The niche Niche is a term used to characterize an ill-defined population of cells which are not SCs but provide the actual SCs with an essential homing microenvironment. It is proposed that the niche works as a nest for the SCs, physically interacting with them and providing growth factors required for their survival8,10,11,15,25,28,30. Interactions include paracrine signaling pathways, suppression of the differentiation programme and pathways that inhibit mitogenic stimuli15. Since CSCs exhibit properties of normal SCs scientists believe that the requirement of niche applies to them as well. Moreover the niche has been implicated in the seed and soil 6 theory which claims that the tumor cells that metastasize are able to nest and establish a distant metastasis only when they meet the appropriate microenvironment – niche11,13,30. Lately the niche has been under investigation because it has been proposed that targeting the niche cells, could have an effect on the viability of CSCs. So the hypothesis is that the destruction of the niche could indirectly lead to cancer regression by making the CSCs incompetent13. Finally if the niche hypothesis is true then some of the available in vitro data might have to be re-examined to include the interactions between the tumor cells and their microenvironment10. Figure 2: Possible carcinogenesis pathways involving the niche (a, b, c) or the progenitor cells (d). Adapted from 25 4. The identity of Cancer Stem Cell According to the Cancer Stem Cell theory, a cancer stem cell (CSC) is the cell that gives rises to a tumor. Based on the findings that CSCs share common characteristics with normal SCS the original hypothesis was that CSCs arise from normal SCs that acquire genetic mutations. Nonetheless according to the literature a CSC does not necessarily come from a stem cell, but in fact it can originate from different cell populations as diverse as normal stem cells, progenitor cells or even differentiated cells8,9,12-15,21,31-33. The initial hypothesis that CSCs originate from normal SCs was based on the fact that SCs have a longer life expectancy and 7 hence they have more chances to acquire mutations that will transform them into CSCs. Additionally normal SCs by themselves exhibit the self renewal and differentiation properties that characterize CSCs. For a progenitor cell the process is more complicated because it needs to turn the differentiation clock backwards and acquire mutations that will enable it to show the previously mentioned features. On the other hand SCs numbers are comparably low and statistically it is more probably one of the non-SCs to gain mutations that will transform them in to CSCs13. Figure 3: Cancer stem cells can originate from normal stem cells, progenitor cells or even from differentiated cells. It is commonly appreciated that for a sell to be marked as CSC it needs to meet two criteria: be able to self-renew and differentiate to the whole spectrum of cancer cells found in the tumor from where they were isolated11-13,23,24,28,34. In other words only one CSC removed from tumor biopsy should in theory have the potential to regrow an identical tumor in a suitable environment. An alternative name for the cells called CSCs, broadly used in the literature is Tumor Initiating Cells (TICs). It has been suggested that this might be a more accurate term for the cells in question because it does not lead to the assumption that these cells originate exclusively from normal stem cells12,13,22,27,33. On the other hand using the term TIC can be also misleading because one can believe that the reference is for the initial “unique” cell that acquired the first mutation and lead to the formation of the tumor8. In this review the term CSCs will be used to describe cells isolated from the tumor, primary or metastatic, that have the ability to self-renew, differentiate and are experimentally identified by exclusively recapitulating the tumor of origin with all its heterogeneity, mainly in NOD/SCID mice8,9,13,31. 8 5. Self-renewal and differentiation CSCs are special because they exhibit two special properties, self-renewal and differentiation potential, properties that they share with normal stem cells23. Self-renewal can be described as the ability to unlimited proliferation, in contrast with the conventional proliferation, where a committed cell has a limited numbers of proliferation cycles after whom it stops dividing and undergoes apoptosis. There are various mechanisms that dictate this behavior in the cell, with the most dominant being the shrinkage of telomeres with each division. CSCs and stem cells have developed mechanisms to circumvent this, by maintaining active telomerase for example, allowing them to undergo unlimited rounds of divisions11,12,23,27. If a stem cell (cancer or not) every time it divides produces two identical clones (symmetrical division) then this will result in the expansion of the stem cell pool. For this reason stem cells mostly undergo asymmetrical divisions which result in one daughter cell being identical clone of the parental cell and a second daughter cell that has lost some of the properties of the parental cell and deviates forming a new more differentiated cell subpopulation termed progenitor cells. By following the asymmetrical division model constant numbers of stem cells are maintained through time8,11,22,28. Differentiation is very important in developmental biology because it makes feasible to get a complex multicellular organism by only one cell, the zygote. The differentiation programme for each cell is very complex and delicate with a plethora of molecules working on creating a diversity of cells, each one with one particular task and astoundingly different from each other although one common parental can be trashed back for all of them. The general principle is that after zygote formation a pool of stem cells is established that will give rise to a subpopulation of progenitor cells which next will produce a rich variety of fully differentiated cells that eventually will form the diversity of tissues and organs.35 As discussed previously CSCs can originate from stem cells, progenitor cells or differentiated cells 8,9,13,31 . A stem cell can acquire mutations and become CSC since it already demonstrates the two essential properties, self-renewal and differentiation potential and needs only to be deregulated. In addition stem cells live longer than normal cells and thus they have statistically more chances to accumulate mutations. By being present for longer periods they are exposed to various oncogenic factors and for longer periods. Progenitor cells are evolutionary close to stem cells and they need some additional events which will allow them 9 to change their phenotype back to stem cells. Differentiated cells need to go through multiple events in order to become CSCs, they need to dedifferentiate, regain self-renewal and differentiation potential and accumulate mutations which will make them tumorigenic. This is why initially it was thought that CSCs derive exclusively from stem cells but today it is established that progenitors and differentiated cells can be a source of origin as well. 8,9,13,31. 6. Identification assays Right in the core of the CSCs theory lays the issue of the identification of such cells. To be able to raise the case of CSCs scientists need to prove that such a subpopulation actually exists. Furthermore to prove that the isolated population corresponds to the CSCs it is necessary to demonstrate that these cells can self-renew and differentiate, resulting in the formation of a tumor resembling the one that these cells were taken from. For the identification of CSCs three methods are available to date36,37. A. isolation of a side population (SP) based on the efflux of the Hoechst dye. B. use of cell surface proteins – antigens, markers. These methods are being currently used for the identification of CSCs resulting in different degrees of enrichment and each one of them has certain advantages and disadvantages. However the use of expression markers is the most popular. After the identification of cells by either of the previously mentioned techniques these cells will be further analyzed by in vitro sphere culture or in vivo xenotransplantation in mice model in order to demonstrate their true CSCs properties. 6.1 Side population Side population (SP) is a term used to describe the specific subpopulation of cells that upon administration of the Hoechst 33342 dye, shows no accumulation of the dye, in contrast with the rest of the cell population. The Hoechst 33342 dye binds to A-T regions in the DNA and when excited by ultraviolet light emits a blue light. This is very useful because using flow cytometry (FACS, Fluorescence Activated Cell Scanning38,39) analysis is easy to isolate this SP. The interesting characteristic of this specific subpopulation is that SP from a variety of 10 tumors exhibit CSCs properties8,21,23. The conclusion is that when the SP assay is applied in tumors it can enrich for CSCs although the enrichment rate may vary36,37,40. The explanation as to why the SP cells are not accumulating the Hoechst dye needs further validation but at present two possible scenarios try to interpret the findings based on molecular functions. Firstly the SP population has been suggested to be in a quiescence state, a property of SCs in general and because of that low or inefficient dye is entering the cells in comparison with the rapid proliferating counterparts. An alternative hypothesis is that SP although it originally takes up the dye, has the potential to pump it out due to the presence of overactive pumps like the ABC drug transporters (ATP binding cassette family). None of the hypotheses has been firmly proven although there is evidence to support both of them and perhaps they are both valid. As for the disadvantages selecting the SP assay as a way to enrich for CSCs, despite the enrichment the identified cells are not homogeneous and thus only a minority within the SP is true CSCs. Moreover in some tissues the SP assay fails completely to identify CSCs. Of course as for all methods, the precise protocol used is very important and differences in the dye concentration, staining time and temperature may cause misleading results and explain inconsistencies between different experiments. However the major argument against the use of the SP assay is that the Hoechst dye itself has been reported to be toxic and thus might interfere with the outcome of the experiments, perhaps by selecting for resistant cells36,37. 6.2 Markers Taking advantage of the similarities of SCs and CSCs unique phenotypes the following method identifies cells based on the expression of special proteins on their cell surface. Some markers are linked to the tumorigenic activity of the CSCs, in a way that they drive tumor growth themselves, but others do not show to be functionally involved in the oncogenic procedure. Most if not all CSCs markers have been identified as SCs markers in the past but since these two categories share similar phenotypes the SCs markers have been extended in the CSCs field as well36,37. Moreover for each tumor type there are specific markers which are known to identify CSCs and a list of them is available in table 1, Nonetheless some markers like CD133 and CD44 can be used for several tissues.9-11,13. The method to separate the content of a biopsy into different subpopulations is to dissolve the whole mass and then sort the cells by Fluorescence activated cell scanning (FACS38,39) analysis. 11 CD133 (Prominin 1) is broadly used in a variety of tumor types for identification of CSCs although the function of the protein remains elusive. CD44, a cell surface glycoprotein is also used for CSCs identification in several tumor types. The molecular function of this protein is known and correlates with an oncogenic phenotype. CD44 activates many receptor tyrosine kinases (RTKs) like EGFR and HER2 upon binding of hyaluroan (HA), leading to activation of proliferation (MAPK) and survival (PI3K/AKT) pathways. The two markers CD133 and CD44 will be reviewed later in the context of specific tumor types. More markers do exist and ongoing efforts are aiming at making the selection criteria even stricter so that the markers used could lead to enrichment up to 100% if possible. The main disadvantage of the use of markers for identification of CSCs is that the number of cells isolated each time is significantly low41 . This makes the identification of any CSC impossible when it comes to tumor biopsies relatively small in size. Moreover for solid tumors to be able to be screened for marker expression the tumor mass needs to be dissociated so that all cells will become accessible. The treatment itself is chemically severe -proteinasesand can interfere with the expression phenotype41. Finally the main argument against the use of this method is that the available proteins are not specific enough and more markers with stringer expression are needed10. Cancer type Blood malignancies Cell surface proteins CD34+ CD38- Breast CD44+ CD24-/low Lin- EpCam+ CD90+ Brain CD133+ Colon CD133+ CD44+ EpCam+ CD166+ CD117+ Pancreatic CD44+ EpCam+ CD24+ CD133+ CD14+ Prostate CD44+ α2β1+ CD133+ CD24- Multiple myeloma CD138- CD34+ CD20+ 12 Melanoma CD20+ CD44+ CD133+ ABCB5+ Lung CD133+ CD90+ Head and Neck CD44+ Lin- Liver CD90+ CD133+ CD44+ Table 1: Based on data gathered from the following reviews: 14,26,36,40,42-49. 6.3.1 In vitro demonstration of tumorigenicity - sphere formation When cultured in vitro in serum free media rich in EGF and FGF, the SCs tend to form a characteristic structure called the sphere (from the greek word σφαίρα which means globe). When viewed under the microscope these spheres are easily detectable and can be a helpful tool to identify SCs or CSCs in a further extent8,14,23,34,36,37,50. This assay was first demonstrated for cells from the central nervous system (CNS) and the structure was called neurosphere, but since then it has been shown that SCs from other tissues follow this pattern by forming mammospheres (breast tissue) and colonspheres (colon). The main concern for the use of sphere culture for enrichment of CSCs is that the sphere itself shows heterogeneity, meaning that there are issues of contamination by non CSCs. Again protocol matters like culture medium, passages that he cells have undergone, sphere size may differ between experiments and interfere with the outcome. Additionally the growth factors present in the media have been charged with de-differentiation propertieas and may cause artifacts generating CSCs by differentiated cells rather than selecting the CSCs present in the biopsy taken13. 6.3.2 In vivo demonstration of tumorigenicity - xenotransplantation No matter which technique is for enrichment of CSCs the next step is to prove that the isolated subpopulation demonstrates in fact CSCs properties: self renewal, tumorigenic and differentiation potential. The best way up to date to show that a cell is in fact a CSC is to transplant it to immunocompromised mice models8,9,13,14,31,36,37,50. If truly a CSC should be able to proliferate and give rise to a tumor that recapitulates the histological phenotype of the 13 parental one40. The main mice models currently used for this purpose are the SCID (severe combined immunodeficiency) mouse and the NOD/SCID (nonobese diabetic SCID) mouse. Both lacking T and B lymphocytes but the latter is additionally deficient in producing natural killer cells (NKs) and antigen presenting cells (APCs). This means that SCID mice show deficiency in the adaptive immune system and NOD/SCID in the adaptive and innate immune system8,23,27. The most useful feature of both mice models is that they are unable to reject xenografts and thus candidate CSCs can be transplanted in orthotopic sites - the same area where the parental tumor was located-. Usually in experiments with mice models after an initial successful tumor formation, cells from the mice’s tumor this time are being isolated based on the same criteria that were used for the initial selection. Those cells are transplanted in other mice - syngenic transplanation- and then the new cells in other mice (with the same genotype although) and undergo what is called serial transplantation, rounds of several transplantations, usually 2-313,23. This is to ensure that the cells isolated from the parental tumor can self renew, differentiate and reconstitute the original tumor’s heterogeneity not only upon xenotransplantation but also when transplanted from one mouse to another40. Self renewal properties can alternatively be tested in vitro with non adherent sphere culture or culture in soft agar. However culture in 3D matrix gel has the potential to cause epigenetic changes and to promote tumorigenesis50,51. Taking everything into account mice models although more laborious, timing consuming and expensive still give insightful information by resembling the condition of a tumor growing inside patients. . Figure 4: Cells are isolated from solid tumors and the sorted cells can be next tested in vivo for demonstration of CSCs properties. Here only the cells depicted in green are true CSCs and 14 can reproduce the heterogeneity of the original tumor upon xenotransplantation. Adapted from44 Up to date scientists have managed to enrich for CSCs so that as few as 100 selected cells can induce tumor formation in mice models when more than 105 non-selected cells are needed to have the same outcome. These results can be interpreted in two ways. Either the isolation methods are not sensitive enough, reaching only up to 0.1% or there is a niche dependency issue. The latter hypothesis is based on the assumption that CSCs as SCs are dependent on their microenvironment, so upon xenotransplantation even if the selected subpopulation are indeed CSCs only 1% can adopt to the new mouse microenvironment and actually establish a tumor(xenotransplantation issue)13,31. Moreover the debate against the use of mice models in the CSCs area lies in the fact that although these mice resemble the conditions in a human organism still are not optimal and other factors may interfere in the survival of a specific pool of cells For example there is incompatibility in respect of the growth factors and chemokines and their receptors between the human and mouse10,13,28,50. All considered, the NOD/SCID mouse model is very useful in the CSCs field making possible to test for self renewal, tumorigenic and differentiation potential resulting in formation of a tumor histologically similar to the parental. Despite all the arguments against their use, mice models are the model which is closest to human patients. Overall until reliable methods for isolating cells from a tumor, without interfering with the cells, and a accurate, suitable assay for self-renewal and differentiation are found all data gathered in the field of CSCs need to looked at with great care. Needless to say working with biopsies means that scientists are allowed to “see” the tumor only during only one particular stage, most of the times when it has already grown and has made its presence known. This also adds more uncertainty to the experiments done because it introduces more variability. 7. Frequency of CSCs Based on the theory of CSCs, those cells are rarely found in a tumor. This is of course not definite and numbers greatly depend on the tumor type and vary from patient to patient. But in principle CSCs represent a minor subpopulation of the tumor. This assumption derives from the CSCs theory itself. Contradicting the stochastic model where each cell has the potential to lead to carcinogenesis, the CSCs suggests that the cells capable to cause tumor formation can 15 only be found in the CSCs pool. Therefore the CSCs need to be a subpopulation of the whole cell number of a tumor. Furthermore based on the majority of available data, the CSCs detected until now suggest that the CSCs represent a minor subpopulation. Hence CSCs are characterized as rare based on the or their low frequency. As the tumor mass expands and tumor cells continue proliferating it makes sense that the stage and aggressiveness of a tumor will correlate with the percentage of CSCs in the tumor. The number of CSCs identified in the first attempts was indeed low but some groups lately have discovered that the percentage of CSCs may vary significantly and may rich levels that do not comply with the “rare” description9,28. The key behind this inconsistency is mainly the felicitous selection of the markers for the selection of the CSCs. It has to be mentioned that the finding that in some cases the percentage of CSCs is very high is one of the main arguments against the existence of CSCs10. 8. Stochastic model vs CSCs model Before the CSCs theory was introduced the main theory for tumor formation was the stochastic model. According to the stochastic model each cell in the organism has the potential to acquire mutations, gain of function for oncogenes and loss of function for tumorsuppressors, that will lead to tumor formation1,2,11,13,15,23,40. Moreover within the tumor accumulative mutations happening in a random fashion lead to clonal expansion of a subpopulation of cells that is the most capable of surviving under the given conditions in each case. Based on this assumption one can not predict which cell is going to start the oncogenic process, because the model is characterized by randomness. According to the CSCs theory there is a specific subpopulation of cells that is inducing tumor formation and sustains the tumor’s growth. The main principle of this theory is that there is a hierarchical organization within the tumor which could also explain the functional heterogeneity observed. Based on the CSCs theory each tumor consists of a subpopulation of cells that are the mother cells of all tumor cells. All other cells form the bulk of the tumor and do not have the potential to sustain the tumor mass. In conclusion not all cells are important for studying cancer induction but only the few with CSCs properties. 16 Figure 5: The two models explaining tumor heterogeneity and tumorigenic potential. Adapted from 52 Finally both theories the clonal expansion and the CSCs may be applicable and may even work together promoting carcinogenesis by not being necessarily mutually exclusive13. In other words oncogenesis could be started only by the CSCs subpopulation but then stochastic events will select for the CSC that will establish the tumor or even the CSC that will be implicated in metastasis and tumor resistance, figure 5 (d) Figure 6: Cellular hierarchy (a) and oncogenesis explained by the stochastic (b) and CSCs model (c). CSCs model incorporating the clonal evolution theory (d). Adapted from50 17 9. CSCs and therapy resistance The CSCs theory has drawn the attention of scientists because it indicates which subpopulation of the tumor is the one that will be resistant to therapy and will further expand and differentiate to repopulate the tumor mass, resembling the phenotype of the original tumor. If the CSCs model holds true, then resistance to therapy could be circumvent by specifically targeting the CSCs pool11,13-15,21-23,40. It is assumed that chemotherapy and radiotherapy fail to kill CSCs48 and only cause shrinkage of the tumor because they target the bulk cells11,12,27,34. CSCs are resistant to chemotherapy because they are proliferating less frequently than the normal cells and so chemotherapeutics whose mechanism of function depends on interferences with the cells cycle, efficiently kill dividing cells but fail to kill CSCs11,12,21,23. Moreover CSCs are resistant to radiotherapy because their DNA damage response is more efficient compared to differentiated cells. It is like conventional therapy can only destroy the differentiated cancer cells when the CSCs being resistant by nature can give birth to the tumor again and again. This raises serious questions concerning the routine treatment that patients get and also the reliability of the assays used in laboratories and clinical trials to measure tumor regression11-13,27. There is a possibility that the majority of the drugs used in the clinic and radiotherapy are unsuitable for cancer treatment and in no way they aim at full recovery of the patient10,12. They only save time but reducing the tumor size (which is the standard criteria of a successful treatment12) but the “source of all evil” the CSCs are still present after the treatment and they strike back as soon as the treatment has stopped or even during the administration. In some cases the new tumor is even more aggressive. Figure 7: Cancer stem cells involved in carcinogenesis, tumorigenesis and resistance to current therapies. Adapted from 43 18 10. Specific tumor types As mentioned the CSCs field has still a lot of unclear aspects. Each tumor type exhibits specific characteristics and this contributes to the variability of the data. Historically the malignancy that first provided evidence for the CSCs theory was leukemia 8,13,31,53 .The last couple of years more information has been brought to light from the examination of solid tumors like breast54,brain55 and colorectal56,57 cancer. Many of the issues of the CSCs model, like the origin of the CSCs, the most appropriate markers and assays used to identify them should more appropriately be addressed in specific tumor types. This is the reason certain malignancies will be discussed here based on their location. 10.1 CSCs and leukemia Why do blood malignancies play such a pivotal role in CSCs field? Cells with tumor initiating properties were historically first presented in blood tumors11-13,40,58. The hematopoietic system is a perfect candidate to investigate the CSCs hypothesis because the heterogeneity and hierarchy among the normal blood cells has long been established23,27,59. The various cell subpopulations across the differentiation lineage are represented in figure 8 where the hierarchical differentiation is depicted35. Figure 8: Hematopoietic stem cells differentiate and lead to the formation of the variety of blood cells. Adapted from25 19 Hematopoietic stem cells (HSCs) are on the top of the differentiation pyramid, producing myeloid and lymphoid progenitor cells. Those are more restricted in their developmental potential and will go through cycles of differentiation generating at the end the whole spectrum of blood cells. In more details the myeloid precursors will establish the lines of gralulocytes, erythrocyes, monocytes and megacaryocytes whereas the lymphoid precursors will differentiate leading to the formation of all lymphocyte populations. As differentiation continues the cells become more morphologically distinct, acquiring special properties distinctive of the their cell identity35. The question that remains to be answered is if the same hierarchy can be observed in leukemias. Efforts to identify one single leukemic cell that can propagate and transmit the systemic disease upon transplantation in mice are tracing back to the past. Taking advantage of the development of tools for identifying HSCs59, several groups demonstrated that only a minority of the malignant blood cells can induce tumor formation in vivo60, in NOD/SCID mice models or form colonies in vitro61. These experiments might be subjected to the survival of particular cells due to the nature of the assay used. This is why the fact that hierarchy exists in blood malignancies was first addressed when diverse blood diseases like acute myeloid leukemia (AML), chronic myeloid leukemia (CML), essential thrombocythemia and polycythemia vera, were found to be characterized as clonal diseases58,62,63. These publications contributed to the popularity of the CSCs theory, but the pioneering work in the CSCs field performed by J. Dick and his colleagues53,64. The main issue in the CSCs field was to identify the specific subpopulation that bears the tumorigenic properties. The approach of J. Dick’s group was to use FACS analysis to sort the various subtypes of cell populations from AML patient’s peripheral blood,. Several antibodies were used with known targets, anti-CD15 (granulocytes), anti-CD14 (monocytes), anti-CD34 (primitive progenitors and stem cells), anti-CD33 and anti-CD38 (myeloid cells). So after the subpopulations of blood cells were sorted out they transplanted them into NOD/SCID mice. The fascinating finding was that only one subpopulation that had the capacity to engraft in the mouse model was characterized as CD34+CD38- . These cells were able to recapitulate the phenotypic heterogeneity of the original tumor, and showed self-renewal and differentiation properties. This subpopulation was named leukemic stem cells (LSCs) and such cells were found in low frequency in the original pool of cells, only 1 in 250.00053. Altogether this work was the first experimental proof of the CSCs model because in a human malignancy the cancer population was found to follow a hierarchic organization (as is the case with normal 20 hematopoiesis and HSC). Furthermore a study on the CD34+CD38− population showed that the number of these cells could be of important prognostic significance for diagnosis in AML patients. The hypothesis of the authors is that as larger the CSCs number the higher the percentage of cells resistant to chemotherapy which predicts poor survival65. A very important issue is the origin of the CSCs in hematological malignancies. Originally it was believed that the LSCs derive exclusively from normal HSCs because they shared phenotypic characteristics, both being CD34+CD38-. Later it was observed that LSCs phenotype actually resembles more the progenitor cells rather than HSCs66. On the other hand it has been suggested that is reasonable for HSCs and LSCs to have distinct phenotypic differences due to the oncogenic events that lead to the formation of the latter and subsequently change the repertoire of the cell’s markers67. Studying the CSCs in hematological cases, various articles have been published, experimentally approaching the subject mainly by transducing mouse models with leukemia related genes23. However the main conclusion is that progenitor cells are able to become LSCs but, as expected they first have to go through some additional rounds of mutagenesis in order to gain the self-renewal properties. Studies on AML tend to show a HSC origin of the LSC23 but different types of blood malignancies should be examined separately as they may have constitutional differences and the most probable scenario is that both normal SCs and progenitor cells are involved in the origin of the LSCs Following disease progression, this time with experiments focused on human CML (mainly cells from patients with chronic phase versus blast crisis) revealed that the LSC might be changing during the time course of the disease68. It was observed that in chronic phase CML the cells with oncogenic potential were similar to normal blood SCs whereas in blast crisis these cells they resembled the blood progenitor cells. These findings led to the assumption that the two phases of CML are driven by different cell subpopulations. The chronic phase seems to be coordinated by cells originating from the normal SCs compartment and the latter phase of blast crisis by cells originating from more differentiated cells like the progenitor cells69 21 10.2 Solid tumors Blood malignancies were the first to be examined for the CSCs theory. The next step was to further validate the theory in solid tumors, something that has been more complicated8,42,50,70. Experimentally the main obstacle is to get access to all cells in a tumor mass, since the mass itself is rather heterogeneous23,27. Within the mass the cells are attached to one another and some are well hidden in the core. The inevitable dissociation of the tumor mass in order to get single cells may interfere with the experiments, as it can induce anoikis71. Moreover the physicochemical procedures used to break the cell – cell interaction within a tumor may alter the phenotype of the tumor cells41. Furthermore in solid tumors it has been suggested that the CSCs are dependent on the presence of a special microenvironment called the niche, which mainly consists of normal cells. So by removing them from their niche scientists select for niche independent cells13,41,71. Finally it is common practice to use normal SCs marker’s to identify CSCs50. Nonetheless the field of normal SCs in solid tumors is not as much investigated as in the hematopoietic system and there is a lack of proper markers for SCs which makes things even more complicated for the identification of CSCs8,13,41. All in all the CSCs theory is more laborious to be verified in solid tumors in comparison with blood malignancies. However efforts have been made and the available data so far are more than promising. Since each tumor type demonstrates special features, the main tumor types in which progress has been made in the CSCs field will be individually reviewed. 10.2.1 CSCs and breast cancer Breast cancer was the first solid tumor that exhibited the presence of a CSC population8,14,23,31,40,42,44,50,70,72-76. Cells taken from human tumors, pleural effusion samples, were first FACS sorted according to their phenotype and then xenografted in NOD/SCID mice54. From this experiment it was observed that only one subpopulation had the potential to regenerate tumors in mice which were phenocopies of the original cancer, even after being serially passaged several times. These cells were reported to be CD44+CD24- actually in the original article CD44+CD24-/lowLineage-) and as few as 100 in number could induce tumor formation when as much as 104 from the rest of the cancer cells failed to54. This was the first in vivo proof that in solid tumors, a subpopulation of cells from the tumor exclusively posses 22 self renewal and tumorigenic potential while at the same time they are able to produce the whole spectrum of cells present in the parent tumor. Research is still ongoing in this field and scientists try to gain more insights into both the normal breast SCs and CSCs and in some cases exciting data come into light. Work performed on mouse models revealed that a subpopulation of cells exists from which even only one cell is able to generate a functional mammary gland in vivo77.In this publication these stem cells were called mammary stem cells (MaSCs) and were CD29hiCD24+Lin- and single cells with this phenotype (marked with a LacZ transgene) could restructure a complete mammary gland when transplanted in mice with surgically cleared mouse breast fat pads77. Moreover these particular cells were both multipotent and self renewing since a single cells could recapitulate the whole spectrum of heterogeneity of the breast tissue and it could also divide indefinitely proved by serial transplantation clonal outgrowths77. In another article the same cells were characterized as mammary repopulating units (MRUs) and were CD24medCD49fhi 78. In addition the latter group demonstrated that these cells are SCs that can differentiate into progenitor cells which can form adherent colonies in vitro. Just recently more information about the issue of SCs and CSCs in breast has come under the spotlight. When breast cancer patients where examined after treatment, the tumor cells that remained exhibited a distinctive signature in their gene expression profile79. The subpopulation of the resistant tumor cells showed the same signature independently from the kind of treatment, chemotherapy or hormone (endocrine) treatment. This indicates that the mechanism of resistance is a general feature of these cells and is not treatment specific. Interestingly this signature is claimed to resemble the signatures of the CD44+CD24/low Lineage- cells, identified as the CSCs54 and of self renewing cells, as proven by in-vitro mammosphere assays80. Furthermore this specific signature is proposed to work as a prognostic tool for metastasis77 since it has been correlated with a specific uncommon breast cancer type, claudin low, where the cells undergo epithelial mesenchymal transition (EMT)81. These findings are in agreement with previous efforts comparing normal and cancerous breast tissue82. A second publication in the same month brought more data about the phenotypic resemblance of normal breast SCs, breast CSCs and embryonal carcinoma cells. All these subtypes showed a down regulation of some micro-RNAs with the most important being miR-200c83 which normally inhibits the BMI1 protein which itself is repressing apoptotic and differentiation 23 signals84. Elevating dose of this micro-RNA suppressed self-renewal and induced differentiation in not only normal CSs but also in CSCs opening new ways for treatment. Furthermore there were identified more than 30 micro-RNAs that were differentially expressed between normal SCs and CSCs which also can lead to the delineation of molecules that only interfere with CSCs and not normal SCs. These findings can lead to therapies targeting exclusively the CSCs and leaving the SCs unaffected. 10.2.2 CSCs and brain cancer Soon after the publication of evidence for CSCs in breast cancer54 more information was brought for CSCs in solid tumors by work focused in brain tumors8,11,14,23,27,40,46-48,50,55,85-89. Initially the CSCs were defined in vitro, with the help of the neurosphere assay, in fact from various types of human brain tumors (medulloblastoma, ganglioglioma, pilocytic astrocytoma, ependydoma). CSCs were exclusively found in the subpopulation expressing the CD133 marker, a glycoprotein also known as prominin-155. These subpopulation demonstrated proliferation, self renewal and differentiation properties reconstituting the phenotype of the patient’s tumor in vitro when the CD133 - failed to do so. With further experiments the same group tested the CD133+ cells in vivo using a xenograft assay85. The CD133+ was the only subpopulation that could initiate tumor formation in NOD/SCID mice models that was a phenocopy of the parental tumor, even after serial transplantation. In fact, as few as 100 CD133 positive cells were enough, when as many as 105 did not lead to carcinogenesis. For these studies only one marker was used, CD13386,87. In the future the need for more markers to validate the identity of the CSCs and to achieve even better enrichment rates is imperative. CD133 was not randomly selected for the prospective isolation of CSCs. It had been established before as a marker for SCs or even progenitors cells in the hematopoietic90 and central nervous system (CNS)87,91,92. Since it is characteristic for both stem and progenitor cells the cell or origin of the CSCs in brain cancer remains elusive89. Moreover when the induced tumors in the NOD/SCID mice were investigated CD133- were present, meaning that CD133+ not only capable to self renew, proliferate and induce tumor formation but in addition they can differentiate and give rise to more committed cells23. Furthermore the number of CD133+ varies among the available publications, 5-30 %27, 19-22 %23, 0.2-10.4 %45. This 24 difference in number might be explained based on the various brain tumor types and aggressiveness of each individual tumor85. Finally it has to be highlighted that the CD133 positive population most probably contains another subpopulation that are the true CSCs87. Since the number of CD133+ cells needed to induce tumor formation in NOD/SCID mice is unreasonably high, 100 cells, it has been proposed that the CSCs can be found in the CD133 + population in a frequency of 0.1-1%. In chronological order CD133 marker has been found to be associated with CSCs properties in cells isolated from ependymoma in combination with other markers, in the context of CD133+/Nestin+/RC2+/BLB2+ cells93. Arguing that the CSCs are found exclusively in the CD133+ pool, CD133- cells isolated from glioblastomas were shown to be as much tumorigenic as CD133+ using xenograft methods94. Further on CD133 has been correlated with the patient’s outcome in glioma patients. In fact not only has the proportion of CD133 positive cells been shown to be an important prognostic factors for adverse progression free survival and overall survival, independently from tumor grade, extent of resection, or the patient’s age, but at the same time to work as an independent risk factor for tumor regrowth and period to tumor progression for grade 2-3 tumors95. Additional evidence for correlation of CD133 expression and poor prognosis for patients with gliomas has been published short after the last publication96. In the same year work on glioblastomas revealed that the CD133- can be tumorigenic as well, using mice models and specifically this subpopulation can produce upon passaging CD133+ cells, an event that accompanied angiogenesis and marked aggressiveness. In conclusion the authors claim that their results suggest that a positive CD133 cells is not prerequisite for tumor induction but that its detection rather confirms tumor progression97. Further evidence linking CSCs and angiogenesis have been presented with CD133+ forming tumors which exhibit elevated levels of angiogenesis via an VEGF mediated mechanism98 and most recently, for CSCs selected based on the expression of an alternative marker (serine)99. Finally a recent review apart from questioning the special identity of CD133+ being the solely CSCs cells in brain, based on publications that prove that cells lacking the marker show CSCs characteristics, introduces a new controversy by gathering data that CD133 is also expressed in normal cells, in contrast with the belief that marks only stem cells and progenitors45. Additional work done on brain CSCs, isolated based on CD133 expression has revealed the important clinical significance of these cells in terms of radioresistance100. In gliomas these 25 cells are enriched after radiation treatment and contribute to tumor radioresistance via a mechanism that activate two responses. The DNA damage checkpoint and that of DNA repair. After the initial experiments in brain cancer that showed that CD133+ in contrast to the CD133- are the cells that harbor the ability to induce tumor formation in vitro55 and in vivo85 some publications presented evidence that suggest that the later category has also tumorigenic potential, at least in some brain tumor types. At the same point data have been gathered that prove the CD133+ protein as a CSCs marker. The most probably explanation is that in brain tumors the phenotype of the CSCs is specific to each subtype and also that the methods used for the identification of the CSCs need to be further optimized50,87. The important message from all the above articles is that no publication claimed that the CD133+ are not CSCs but the main argument against them is being the sole CSCs subpopulation and that CD133- can also exhibit CSCs properties. 10.2.3 CSCs and colorectal cancer Colorectal cancer was the third solid cancer, after breast and brain to be investigated for the presence of CSCs14,48,76,101. Colorectal cancer has been examined in details and a model describing the process of tumorigenesis is depicted in figure 9. Some publication have shed light on the CSCs model but still the identification assays are under criticism because of the use of the debatable marker CD133. However recent publications using more CSCs markers seem to validate the CSCs model in the gut tumorigenesis. Figure 9: The adenoma-carcinoma model describing colorectal carcinogenesis according to4. Adapted from 76 26 The first evidence of the presence of human CSCs in colon was based on their isolation using the CD133 marker56, which as mentioned before is used also in brain cancer. According to the results of this group all CSCs in this publication referred to as colon cancer initiating cells (CC-IC) were CD133+. As in previous experiments the criteria that CSCs had to meet were self-renewal, differentiation potential and recapitulation of the original tumor’s heterogeneity. In contrast the CD133- cells, although were redundant within the tumor mass did not have the potential to induce tumor formation when xenotransplanted in NOD/SCID mice under the renal capsule. Moreover the researchers calculated the frequency of the CC-IC cells which was enriched more than 200-fold within the CD133+ subpopulation. This means that not every cell in the CD133+ pool is a true CSC. On the next article in the same issue of the Nature journal the theory of tumor organization in colon in a hierarchic way was addressed57. The main finding of the second publication was that CD133+ show CSCs properties whereas their counterparts CD133- did not, when xenotransplanted in SCID mice (and not NOD/SCID this time) subcutaneously. Additional in vitro work proved that the CD133+ cells could proliferate continuously for one year as undifferentiated tumor spheres in serum-free medium, still being able to demonstrate tumor formation upon transplantation. Furthermore CD133+cells were found in areas of the tumor which demonstrated high cellular density and not frequently found in normal colon tissues. Finally CD133 positive cells would lose their oncogenic ability once started to differentiate but in contrast would exhibit a more aggressive phenotype after serial in vivo passages. Moving forward, the third publication presented evidence about the CSCs, not using the CD133 marker but this time the cells that showed CSCs properties were isolated based on the EpCAMhighCD44+ phenotype102. In fact according to the authors using these two markers instead of the CD133 is a more reliable method for the identification of CSCs based on the facts that CD133- cells did show CD44 expression and moreover the CD44 marker could be used for further enrichment within the CD133+ subpopulation. Furthermore by analyzing the EpCAMhighCD44+ cells, which were also positive for CD49f and ALDH (regarded also as CSCs marker14,103) activity, a new marker was identified in colon, the CD166 protein. In summary the result of this work is that CD133+ cells can be enriched for CSCs by using EpCAM and then the new subpopulation can be enriched a step further with the use of CD166. The EpCAM and CD44 proteins have been also utilized in isolation of breast CSCs 27 and CD166, a mesenchymal SC marker has been associated with poor clinical outcome in colon cancer104. As in brain CSCs the use of CD133 as a marker for CSCs has been a field of controversy. After the previous publications more followed that supported the case that CD133+ are enriched for CSCs105 and that the marker is of high prognostic significance106, but evidence started to appear suggesting that CD133+ is the not the most appropriate marker for the identification of CSCs107 with some publications suggesting that the use of the CD133 antigen for this purpose should be avoided108. The last article revealed new data concerning the CD133 marker that strongly argued against its use for identification of colorectal CSCs. In more details the researchers found that the expression of CD133 in colon is not restricted to stem cells but is largely spread in all differentiated epithelial cells and furthermore they showed that CD133+ but also CD133- from samples isolated from metastases can induce tumor formation. So, according to their data, selecting for CD133+ is not necessary leading to enrichment for CSCs in colon cancer. Perhaps in the metastatic population some of the CD133+ cells have differentiated into CD133- but they still are CSCs. That would mean that different markers should be applied in biopsies taken by metastatic sites. Finally the model that they propose is that CD133 antigen can be used for identification of mature ciliated ductal and luminal epithelia cells and to distinguish cells from the CD133- stromal tumor components. Moreover they believe that CD133 expression is reduced in the metastatic compartments and both CD133 positive and negative cells isolated from metastatic sites can induce tumor formation, both being candidates for CSCs in colon cancer. This last publication raises some important issues about the selection of the CSCs markers and how strict the criteria need to be. However the methods used differ from the ones used in the previous publications. In particular the expression of CD133 in cells was measured using a mice model which was designed to have a knock-in lacZ reporter whose expression was driven by endogenous CD133 promoters (CD133lacZ/+). This is entirely different from using antibodies against CD133. It is possible that mRNA and/or antibody affinity issues have caused misleading results by missing out CD133+ cells in the previous experiments. Alternative markers need to be discovered in colon cancer in order to be positive that the cells identified in each experiment are true CSCs. Towards this direction work on colon stem cells is necessary. In fact the knowledge in this field is constantly expanding especially with the discovery of Lgr5, the leucine rich repeat containing G-protein coupled receptor 5. Lgr5 has 28 been found to be solely expressed in cycling columnar cells in the base of the colon crypt109. These cells were demonstrated to be stem cells with lineage trace experiments using an inducible Cre knock-in allele and as a reporter the Rosa26-lacZ. The Lgr5 positive stem cells could generate the whole spectrum of epithelial cells (enterocytes, goblet and paneth cells) within 2 months. Taking advantage of the identification of this new SCs marker, the same group proved that the Lgr5 protein can be of great value for the identification of CSCs in colon cancer110. 10.2.4 CSCs in other solid tumors Apart from the before mentioned tumor types, leukemias, breast, brain and colorectal there has been research on others trying to identify CSCs to sustain a universal model of their existence. Such efforts include work on prostate14,111 and pancreatic cancer14,112,113, head neck squamous cell carcinoma114, lung cancer115, melanoma14,116, liver14,117 and ovarian cancer118. Evidence from all studies should be viewed as specific cases, especially because of the different methods or markers used each time. However certain general conclusions can be drawn indicating that the CSCs theory is valid. 11. CSCs and signal transduction Several developmental pathways are implicated in the CSCs field which makes perfect sense because of the self renewal properties of these cells. Cancer specimens show over activation of many molecular pathways like the Notch, Wnt/β-catenin, mTOR, Bmi-1, Pten and Hedgehog pathways whose role has long been established in developmental processes9,14,23. In more details, the Notch pathway is found to be over activated (mainly by mutations) in blood malignancies. The Wnt signaling is up regulated in many human cancers and accumulation of β-catenin is depicted in melanoma, sarcoma, breast and brain cancer. Especially mutation in the β-catenin and APC genes are commonly found in colon cancer. Pten a tumor suppressor gene shows loss of function mutations in various malignancies whereas over activation of Hedgehog is associated with skin and brain cancer whereas Bmi-1 is over expressed in LSCs23. Targeted inhibition of these pathways could lead to tumor regression although their regulation should be monitored closely since they are important for normal cells as well. 29 12. Cancer treatment - is there hope? There have been theories about the resistance of the CSCs and SCs in general, to apoptotic stimuli and how chemotherapy fails to kill them and chemo-resistance emerges as a result119. This is why some scientists believe that is impossible to kill the CSCs without killing all other cells too, since it would be necessary to take very drastic measurements in terms of the toxicity of chemotherapeutics. A new publication just last year seems to give an alternative way to selectively kill CSCs, in particular breast CSCs and brings more optimism for the future120. The interesting point about this work is that the researchers developed a method to create artificially CSCs in the laboratory. This is most convenient because CSCs are both rare in numbers but also difficult to extract from patients. Practically the method they used allowed them to grow immortal human breast cells and then induce mutations until the cells would become CSCs. Actually it is an artificial condition but allows for experimentation when samples of CSCs are not available. Furthermore, when the CSCs population was established this way, a screening was performed for a variety of chemical compounds. In total 16000 chemicals, commercial and natural, were tested for epithelial CSCs specific toxicity on the breast CSCs. In fact only 32 were selectively toxic and 4 out of them showed consistency. One of them in particular, salinomycin proved to be 100 times more effective than paclitaxel, a drug widely used in the clinic. Salinomycin was next tested in mice models and proved to cause inhibition of the tumorigenesis, induction of the differentiation and correlated with loss of expression of genes related to poor prognosis. The future goal is to test salinomycin in clinical trials to determine if it has clinical significance and then to optimize the conditions for treatment of human patients. 13. Future directions The CSCs theory has drawn a lot of attention during the last decade and many research laboratories have invested significant amounts of time and money in order to delineate the model. The necessity of finding new treatments for cancer may have caused an early excitement and high expectations from the public after the first evidence of the existence of CSCs. Many people idealize CSCs as the root of all evil, the reason of tumor formation and recurrence and believe that by specifically targeting them, the true cure of cancer can be found. Humanity has gained a great tool to fight the disease of the century and may even win 30 the war against cancer, declared several years back. However many issues in CSCs research need to be refined and optimized, especially the issue of the identification assays. Known markers should be used wisely and only after their validation by several experiments. Needless to say the search for more optimal markers for the CSCs is one of the main goals for the future. However in the case that CSCs actually exist then new treatments should be designed taking into consideration the CSC role in tumor resistance. Such treatments would be mainly be targeted therapy against the CSC subpopulation. Apart from the direct targeting treatment, alternative ways are differentiation of all CSCs which then could be targeted by conventional therapies as differentiated cells and therapies that target the niche leaving the CSCs unable to survive and proliferate. Figure 10: strategies to eliminate the CSCs population by directly targeting them, forcing them to differentiate or targeting their niche adapted from 71 The CSCs theory seems very fascinating and promising but scientists need to stay rational and test the hypothesis before rushing into any conclusions. There is also the possibility that the CSCs is not applicable for all tumor types and thus has to be investigated if it is a universal model or not. Time will show how solid and universal the CSCs theory is and if in fact will help in the elimination of treatment resistance. 31 14. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Alberts. Molecular Biology Of The Cell, 4th edition, (2002). Pelengaris. The molecular biology of cancer. (2006). Kinzler, K.W. & Vogelstein, B. Lessons from hereditary colorectal cancer. Cell 87, 159-70 (1996). Fearon, E.R. & Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 61, 759-67 (1990). Parkin, D.M., Bray, F., Ferlay, J. & Pisani, P. Global cancer statistics, 2002. CA Cancer J Clin 55, 74-108 (2005). Cancer-Monthly. http://www.cancermonthly.com. Cancer-ResearchUK. http://info.cancerresearchuk.org/cancerstats/survival/latestrates/. Clarke, M.F. et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66, 9339-44 (2006). Marotta, L.L. & Polyak, K. Cancer stem cells: a model in the making. Curr Opin Genet Dev 19, 44-50 (2009). Tomasson, M.H. Cancer stem cells: a guide for skeptics. J Cell Biochem 106, 745-9 (2009). Wicha, M.S., Liu, S. & Dontu, G. Cancer stem cells: an old idea--a paradigm shift. Cancer Res 66, 1883-90; discussion 1895-6 (2006). Sagar, J., Chaib, B., Sales, K., Winslet, M. & Seifalian, A. Role of stem cells in cancer therapy and cancer stem cells: a review. Cancer Cell Int 7, 9 (2007). Vermeulen, L., Sprick, M.R., Kemper, K., Stassi, G. & Medema, J.P. Cancer stem cells--old concepts, new insights. Cell Death Differ 15, 947-58 (2008). Sarkar, B., Dosch, J. & Simeone, D.M. Cancer stem cells: a new theory regarding a timeless disease. Chem Rev 109, 3200-8 (2009). Clarke, M.F. & Fuller, M. Stem cells and cancer: two faces of eve. Cell 124, 1111-5 (2006). Bongso-Lee. Stem Cells, From bench to bedside. (2005 World Scientific Publishing). Sell. Stem Cells Handbook, (2004 Humana Press). Stem-Cell-Information. stemcells.nih.gov. Wobus-Boheler. Stem Cells, (2006 Springer). Mathews, L.A., Crea, F. & Farrar, W.L. Epigenetic gene regulation in stem cells and correlation to cancer. Differentiation 78, 1-17 (2009). Trosko, J.E. Review paper: cancer stem cells and cancer nonstem cells: from adult stem cells or from reprogramming of differentiated somatic cells. Vet Pathol 46, 17693 (2009). Mittal, S., Mifflin, R. & Powell, D.W. Cancer stem cells: the other face of Janus. Am J Med Sci 338, 107-12 (2009). Lobo, N.A., Shimono, Y., Qian, D. & Clarke, M.F. The biology of cancer stem cells. Annu Rev Cell Dev Biol 23, 675-99 (2007). Clarke, M.F. Self-renewal and solid-tumor stem cells. Biol Blood Marrow Transplant 11, 14-6 (2005). Clarke, M.F. & Becker, M.W. Stem cells: the real culprits in cancer? Sci Am 295, 52-9 (2006). Boman, B.M. & Wicha, M.S. Cancer stem cells: a step toward the cure. J Clin Oncol 26, 2795-9 (2008). Dalerba, P., Cho, R.W. & Clarke, M.F. Cancer stem cells: models and concepts. Annu Rev Med 58, 267-84 (2007). 32 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. McDonald, S.A., Graham, T.A., Schier, S., Wright, N.A. & Alison, M.R. Stem cells and solid cancers. Virchows Arch 455, 1-13 (2009). Marciniak-Czochra, A., Stiehl, T. & Wagner, W. Modeling of replicative senescence in hematopoietic development. Aging (Albany NY) 1, 723-32 (2009). Li, L. & Neaves, W.B. Normal stem cells and cancer stem cells: the niche matters. Cancer Res 66, 4553-7 (2006). Gupta, P.B., Chaffer, C.L. & Weinberg, R.A. Cancer stem cells: mirage or reality? Nat Med 15, 1010-2 (2009). Reya, T., Morrison, S.J., Clarke, M.F. & Weissman, I.L. Stem cells, cancer, and cancer stem cells. Nature 414, 105-11 (2001). van Staveren, W.C. et al. Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells? Biochim Biophys Acta 1795, 92-103 (2009). Al-Hajj, M. & Clarke, M.F. Self-renewal and solid tumor stem cells. Oncogene 23, 7274-82 (2004). Gilbert. Developmental biology, 7th edition, (2003). Farrar, W.L. Cancer Stem Cells, (2010, Cambridge University Press). Rajasekhar, V.K. Analytical methods for cancer stem cells. Methods Mol Biol 407, 8395 (2007). Urbanits, S. et al. FACS analysis-a new and accurate tool in the diagnosis of lymphoma in the cerebrospinal fluid. Clin Chim Acta 317, 101-7 (2002). Masters-Palsson-Thomson. Embryonic Stem Cells, (2007, Springer). Tang, C., Ang, B.T. & Pervaiz, S. Cancer stem cell: target for anti-cancer therapy. Faseb J 21, 3777-85 (2007). Hill, R.P. Identifying cancer stem cells in solid tumors: case not proven. Cancer Res 66, 1891-5; discussion 1890 (2006). Ailles, L.E. & Weissman, I.L. Cancer stem cells in solid tumors. Curr Opin Biotechnol 18, 460-6 (2007). Frank, N.Y., Schatton, T. & Frank, M.H. The therapeutic promise of the cancer stem cell concept. J Clin Invest 120, 41-50 (2010). Cho, R.W. & Clarke, M.F. Recent advances in cancer stem cells. Curr Opin Genet Dev 18, 48-53 (2008). Wu, Y. & Wu, P.Y. CD133 as a marker for cancer stem cells: progresses and concerns. Stem Cells Dev 18, 1127-34 (2009). Hide, T., Takezaki, T., Nakamura, H., Kuratsu, J. & Kondo, T. Brain tumor stem cells as research and treatment targets. Brain Tumor Pathol 25, 67-72 (2008). Nakano, I. & Kornblum, H.I. Brain tumor stem cells. Pediatr Res 59, 54R-8R (2006). O'Brien, C.A., Kreso, A. & Dick, J.E. Cancer stem cells in solid tumors: an overview. Semin Radiat Oncol 19, 71-7 (2009). Fabian, A., Barok, M., Vereb, G. & Szollosi, J. Die hard: are cancer stem cells the Bruce Willises of tumor biology? Cytometry A 75, 67-74 (2009). Visvader, J.E. & Lindeman, G.J. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8, 755-68 (2008). Battista, S. et al. The effect of matrix composition of 3D constructs on embryonic stem cell differentiation. Biomaterials 26, 6194-207 (2005). Dick, J.E. Stem cell concepts renew cancer research. Blood 112, 4793-807 (2008). Lapidot, T. et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645-8 (1994). Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J. & Clarke, M.F. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983-8 (2003). 33 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. Singh, S.K. et al. Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821-8 (2003). O'Brien, C.A., Pollett, A., Gallinger, S. & Dick, J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445, 106-10 (2007). Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer-initiating cells. Nature 445, 111-5 (2007). Fialkow, P.J. et al. Acute nonlymphocytic leukemia: heterogeneity of stem cell origin. Blood 57, 1068-73 (1981). Till, J.E. & Mc, C.E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14, 213-22 (1961). Bruce, W.R. & Van Der Gaag, H. A Quantitative Assay for the Number of Murine Lymphoma Cells Capable of Proliferation in Vivo. Nature 199, 79-80 (1963). Hamburger, A.W. & Salmon, S.E. Primary bioassay of human tumor stem cells. Science 197, 461-3 (1977). Fialkow, P.J. Stem cell origin of human myeloid blood cell neoplasms. Verh Dtsch Ges Pathol 74, 43-7 (1990). Fialkow, P.J., Jacobson, R.J. & Papayannopoulou, T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Am J Med 63, 125-30 (1977). Bonnet, D. & Dick, J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3, 730-7 (1997). van Rhenen, A. et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 11, 6520-7 (2005). Tavil, B., Cetin, M. & Tuncer, M. CD34/CD117 positivity in assessment of prognosis in children with myelodysplastic syndrome. Leuk Res 30, 222-4 (2006). Blair, A., Hogge, D.E. & Sutherland, H.J. Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR. Blood 92, 4325-35 (1998). Jamieson, C.H., Weissman, I.L. & Passegue, E. Chronic versus acute myelogenous leukemia: a question of self-renewal. Cancer Cell 6, 531-3 (2004). Jamieson, C.H. et al. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 351, 657-67 (2004). Scopelliti, A. et al. Therapeutic implications of Cancer Initiating Cells. Expert Opin Biol Ther 9, 1005-16 (2009). Zhou, B.B. et al. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8, 806-23 (2009). Dick, J.E. Breast cancer stem cells revealed. Proc Natl Acad Sci U S A 100, 3547-9 (2003). Morrison, B.J., Schmidt, C.W., Lakhani, S.R., Reynolds, B.A. & Lopez, J.A. Breast cancer stem cells: implications for therapy of breast cancer. Breast Cancer Res 10, 210 (2008). Er, O. Cancer stem cells in solid tumors. Onkologie 32, 605-9 (2009). Shipitsin, M. & Polyak, K. The cancer stem cell hypothesis: in search of definitions, markers, and relevance. Lab Invest 88, 459-63 (2008). Ricci-Vitiani, L., Pagliuca, A., Palio, E., Zeuner, A. & De Maria, R. Colon cancer stem cells. Gut 57, 538-48 (2008). Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84-8 (2006). 34 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993-7 (2006). Creighton, C.J. et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci U S A 106, 13820-5 (2009). Grimshaw, M.J. et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 10, R52 (2008). Herschkowitz, J.I. et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol 8, R76 (2007). Shipitsin, M. et al. Molecular definition of breast tumor heterogeneity. Cancer Cell 11, 259-73 (2007). Shimono, Y. et al. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell 138, 592-603 (2009). Park, I.K. et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423, 302-5 (2003). Singh, S.K. et al. Identification of human brain tumour initiating cells. Nature 432, 396-401 (2004). Dirks, P.B. Brain tumor stem cells. Biol Blood Marrow Transplant 11, 12-3 (2005). Dirks, P.B. Brain tumor stem cells: bringing order to the chaos of brain cancer. J Clin Oncol 26, 2916-24 (2008). Tunici, P. et al. Brain tumor stem cells: new targets for clinical treatments? Neurosurg Focus 20, E27 (2006). Singh, S.K., Clarke, I.D., Hide, T. & Dirks, P.B. Cancer stem cells in nervous system tumors. Oncogene 23, 7267-73 (2004). Yin, A.H. et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90, 5002-12 (1997). Uchida, N. et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 97, 14720-5 (2000). Tamaki, S. et al. Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. J Neurosci Res 69, 976-86 (2002). Taylor, M.D. et al. Radial glia cells are candidate stem cells of ependymoma. Cancer Cell 8, 323-35 (2005). Beier, D. et al. CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67, 4010-5 (2007). Zeppernick, F. et al. Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 14, 123-9 (2008). Rebetz, J. et al. Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma. PLoS One 3, e1936 (2008). Wang, J. et al. CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. Int J Cancer 122, 761-8 (2008). Bao, S. et al. Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res 66, 7843-8 (2006). Folkins, C. et al. Glioma tumor stem-like cells promote tumor angiogenesis and vasculogenesis via vascular endothelial growth factor and stromal-derived factor 1. Cancer Res 69, 7243-51 (2009). Bao, S. et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-60 (2006). 35 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. Klonisch, T. et al. Cancer stem cell markers in common cancers - therapeutic implications. Trends Mol Med 14, 450-60 (2008). Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 104, 10158-63 (2007). Douville, J., Beaulieu, R. & Balicki, D. ALDH1 as a Functional Marker of Cancer Stem and Progenitor Cells. Stem Cells Dev (2008). Weichert, W., Knosel, T., Bellach, J., Dietel, M. & Kristiansen, G. ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Pathol 57, 1160-4 (2004). Vermeulen, L. et al. Single-cell cloning of colon cancer stem cells reveals a multilineage differentiation capacity. Proc Natl Acad Sci U S A 105, 13427-32 (2008). Horst, D. et al. The cancer stem cell marker CD133 has high prognostic impact but unknown functional relevance for the metastasis of human colon cancer. J Pathol 219, 427-34 (2009). Ieta, K. et al. Biological and genetic characteristics of tumor-initiating cells in colon cancer. Ann Surg Oncol 15, 638-48 (2008). Shmelkov, S.V. et al. CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. J Clin Invest 118, 2111-20 (2008). Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-7 (2007). Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608-11 (2009). Patrawala, L. et al. Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25, 1696-708 (2006). Li, C. et al. Identification of pancreatic cancer stem cells. Cancer Res 67, 1030-7 (2007). Li, C., Lee, C.J. & Simeone, D.M. Identification of human pancreatic cancer stem cells. Methods Mol Biol 568, 161-73 (2009). Prince, M.E. et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104, 973-8 (2007). Eramo, A. et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15, 504-14 (2008). Schatton, T. et al. Identification of cells initiating human melanomas. Nature 451, 345-9 (2008). Yang, Z.F. et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology 47, 919-28 (2008). Zhang, S. et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 68, 4311-20 (2008). Testa, U. & Riccioni, R. Deregulation of apoptosis in acute myeloid leukemia. Haematologica 92, 81-94 (2007). Gupta, P.B. et al. Identification of selective inhibitors of cancer stem cells by highthroughput screening. Cell 138, 645-59 (2009). 36