evidence shows that the association represents an actual phenomenon, although specific cancers have been linked to a specific cause. de-differentiation de-differentiation of mutated cells may create stem cell-like characteristics, suggesting that any cell might become a cancer stem cell. in other words, fully differentiated cell undergoes mutations or extracellular signals that drive it back to a stem-like state. this concept has been demonstrated most recently in prostate cancer models, whereby cells undergoing androgen deprivation therapy appear to transiently alter their transcriptome to that of a neural crest stem-like cell, with the invasive and multipotent properties of this class of stem-like cells. hierarchy the concept of tumor hierarchy claims that a tumor is a heterogeneous population of mutant cells, all of which share some mutations, but vary in specific phenotype. a tumor hosts several types of stem cells, one optimal to the specific environment and other less successful lines. these secondary lines may be more successful in other environments, allowing the tumor to adapt, including adaptation to therapeutic intervention. if correct, this concept impacts cancer stem cell-specific treatment regimes. such a hierarchy would complicate attempts to pinpoint the origin. identification cscs, now reported in most human tumors, are commonly identified and enriched using strategies for identifying normal stem cells that are similar across studies. these procedures include fluorescence-activated cell sorting (facs), with antibodies directed at cell-surface markers and functional approaches including side population assay or aldefluor assay. the csc-enriched result is then implanted, at various doses, in immune-deficient mice to assess its tumor development capacity. this in vivo assay is called a limiting dilution assay. the tumor cell subsets that can initiate tumor development at low cell numbers are further tested for self-renewal capacity in serial tumor studies. cscs can also be identified by efflux of incorporated hoechst dyes via multidrug resistance (mdr) and atp-binding cassette (abc) transporters. another approach is sphere-forming assays. many normal stem cells such as hematopoietic or stem cells from tissues, under special culture conditions, form three-dimensional spheres that can differentiate. as with normal stem cells, the cscs isolated from brain or prostate tumors also have the ability to form anchor-independent spheres. recent years have seen an advent of genetic approaches to identify cancer stem cells in experimental rodents. in such studies, following the induction of cancer (usually through the application of mutagens), a genetic cassette is activated resulting in the expression of an easily identifiable marker, for instance green fluorescent protein (gfp). this overcomes the limitations of traditional approaches (e.g. the classic bromodeoxyuridine (brdu) labeling technique has been used to identify slow-cycling cells in animals) as genetic approaches are cell cycle independent and can be used for in vivo pulse-chase labeling to identify quiescent/slow-cycling cells. this strategy, for instance, was instrumental for identifying the so-called lgr5+ compartment as a cancer stem cell compartment in liver cancer and showing its potential as a viable therapeutic target. heterogeneity (markers) cscs heterogeneity is a pool of differentiated and undifferentiated tumour cells that are replenished by cells possessing both tumour and stem cell like properties and having phenotypic and metabolic heterogeneity inside the single tumour mass. there are two theories to explain the phenotypic and metabolic heterogeneity of cscs; clonal variation and cancer stem cell theory. while former theory dictates the role of genetic, epigenetic and micro environment where tumour cell resides to acquire undifferentiated tumorigenic traits. the latter theory focus more on the malignancy traits acquired by stem cells where these undifferentiated and highly tumorigenic stem cells repopulate the differentiated tumour mass. cscs have been identified in various solid tumors. commonly, markers specific for normal stem cells are used for isolating cscs from solid and hematological tumors. markers most frequently used for csc isolation include: cd133 (also known as prom1), cd44, aldh1a1, cd34, cd24 and epcam (epithelial cell adhesion molecule, also known as epithelial specific antigen, esa). cd133 (prominin 1) is a five-transmembrane domain glycoprotein expressed on cd34+ stem and progenitor cells, in endothelial precursors and fetal neural stem cells. it has been detected using its glycosylated epitope known as ac133. epcam (epithelial cell adhesion molecule, esa, trop1) is hemophilic ca2+-independent cell adhesion molecule expressed on the basolateral surface of most epithelial cells. cd90 (thy1) is a glycosylphosphatidylinositol glycoprotein anchored in the plasma membrane and involved in signal transduction. it may also mediate adhesion between thymocytes and thymic stroma. cd44 (pgp1) is an adhesion molecule that has pleiotropic roles in cell signaling, migration and homing. it has multiple isoforms, including cd44h, which exhibits high affinity for hyaluronate and cd44v which has metastatic properties. cd24 (hsa) is a glycosylated glycosylphosphatidylinositol-anchored adhesion molecule, which has co-stimulatory role in b and t cells. cd200 (ox-2) is a type 1 membrane glycoprotein, which delivers an inhibitory signal to immune cells including t cells, natural killer cells and macrophages. leptin receptor (lepr) is a type i cytokine receptor for the fat associate hormone leptin, which plays an important role in promoting malignant progression. aldh is a ubiquitous aldehyde dehydrogenase family of enzymes, which catalyzes the oxidation of aromatic aldehydes to carboxyl acids. for instance, it has a role in conversion of retinol to retinoic acid, which is essential for survival. the first solid malignancy from which cscs were isolated and identified was breast cancer and they are the most intensely studied. breast cscs have been enriched in cd44+cd24−/low, sp and aldh+ subpopulations. breast cscs are apparently phenotypically diverse. csc marker expression in breast cancer cells is apparently heterogeneous and breast csc populations vary across tumors. both cd44+cd24− and cd44+cd24+ cell populations are tumor initiating cells; however, csc are most highly enriched using the marker profile cd44+cd49fhicd133/2hi. cscs have been reported in many brain tumors. stem-like tumor cells have been identified using cell surface markers including cd133, ssea-1 (stage-specific embryonic antigen-1), egfr and cd44. the use of cd133 for identification of brain tumor stem-like cells may be problematic because tumorigenic cells are found in both cd133+ and cd133− cells in some gliomas and some cd133+ brain tumor cells may not possess tumor-initiating capacity. cscs were reported in human colon cancer. for their identification, cell surface markers such as cd133, cd44 and abcb5, functional analysis including clonal analysis and aldefluor assay were used. using cd133 as a positive marker for colon cscs generated conflicting results. the ac133 epitope, but not the cd133 protein, is specifically expressed in colon cscs and its expression is lost upon differentiation. in addition, cd44+ colon cancer cells and additional sub-fractionation of cd44+epcam+ cell population with cd166 enhance the success of tumor engraftments. multiple cscs have been reported in prostate, lung and many other organs, including liver, pancreas, kidney or ovary. in prostate cancer, the tumor-initiating cells have been identified in cd44+ cell subset as cd44+α2β1+, tra-1-60+cd151+cd166+ or aldh+ cell populations. putative markers for lung cscs have been reported, including cd133+, aldh+, cd44+ and oncofetal protein 5t4+. metastasis metastasis is the major cause of tumor lethality. however, not every tumor cell can metastasize. this potential depends on factors that determine growth, angiogenesis, invasion and other basic processes. epithelial-mesenchymal transition in epithelial tumors, the epithelial-mesenchymal transition (emt) is considered to be a crucial event. emt and the reverse transition from mesenchymal to an epithelial phenotype (met) are involved in embryonic development, which involves disruption of epithelial cell homeostasis and the acquisition of a migratory mesenchymal phenotype. emt appears to be controlled by canonical pathways such as wnt and transforming growth factor β. emt's important feature is the loss of membrane e-cadherin in adherens junctions, where β-catenin may play a significant role. translocation of β-catenin from adherens junctions to the nucleus may lead to a loss of e-cadherin and subsequently to emt. nuclear β-catenin apparently can directly, transcriptionally activate emt-associated target genes, such as the e-cadherin gene repressor slug (also known as snai2). mechanical properties of the tumor microenvironment, such as hypoxia, can contribute to csc survival and metastatic potential through stabilization of hypoxia inducible factors through interactions with ros (reactive oxygen species). tumor cells undergoing an emt may be precursors for metastatic cancer cells, or even metastatic cscs. in the invasive edge of pancreatic carcinoma, a subset of cd133+cxcr4+ (receptor for cxcl12 chemokine also known as a sdf1 ligand) cells was defined. these cells exhibited significantly stronger migratory activity than their counterpart cd133+cxcr4− cells, but both showed similar tumor development capacity. moreover, inhibition of the cxcr4 receptor reduced metastatic potential without altering tumorigenic capacity. epithelial-mesenchymal transition requires iron, which is taken up in cells via cd44. these resulting mesenchymal cells have properties of cancer stem cells. two-phase expression pattern in breast cancer cd44+cd24−/low cells are detectable in metastatic pleural effusions. by contrast, an increased number of cd24+ cells have been identified in distant metastases in breast cancer patients. it is possible that cd44+cd24−/low cells initially metastasize and in the new site change their phenotype and undergo limited differentiation. the two-phase expression pattern hypothesis proposes two forms of cancer stem cells - stationary (scs) and mobile (mcs). scs are embedded in tissue and persist in differentiated areas throughout tumor progression. mcs are located at the tumor-host interface. these cells are apparently derived from scs through the acquisition of transient emt (figure 7).figure 7: the concept of migrating cancer stem cells (msc). stationary cancer stem cells are embedded in early carcinomas and these cells are detectable in the differentiated central area of a tumor. the important step toward malignancy is the induction of epithelial mesenchymal transition (emt) in the stationary cancer stem cells (scs), which become mobile or migrating cancer stem cells. stem cells divide asymmetrically. one daughter cell will begin proliferation and differentiation. the remaining mcs migrates a short distance before undergoing a new asymmetric division, or disseminates through blood vessels or lymphatic vessels and produces a metastasis. implications cscs have implications for cancer therapy, including for disease identification, selective drug targets, prevention of metastasis and intervention strategies. treatment cscs are inherently more resistant to chemotherapeutic agents. there are 5 main factors that contribute to this: 1. their niche protects them from coming into contact with large concentrations of anti-cancer drugs. 2. they express various transmembrane proteins, such as mdr1 and bcrp, that pump drugs out of the cytoplasm. 3. they divide slowly, like adult stem cells tend to do, and are thus not killed by chemotherapeutic agents that target rapidly replicating cells via damaging dna or inhibiting mitosis. 4. they upregulate dna damage repair proteins. 5. they are characterized by an overactivation of anti-apoptotic signaling pathways. after chemotherapy treatment, surviving cscs are able to repopulate the tumor and cause a relapse. additional treatment targeted at removing cscs in addition to cancerous somatic cells must be used to prevent this. targeting selectively targeting cscs may allow treatment of aggressive, non-resectable tumors, as well as prevent metastasis and relapse. the hypothesis suggests that upon csc elimination, cancer could regress due to differentiation and/or cell death. the fraction of tumor cells that are cscs and therefore need to be eliminated is unclear. studies looked for specific markers and for proteomic and genomic tumor signatures that distinguish cscs from others. in 2009, scientists identified the compound salinomycin, which selectively reduces the proportion of breast cscs in mice by more than 100-fold relative to paclitaxel, a commonly used chemotherapeutic agent. it was later shown in 2017 that salinomycin kills cancer stem cells by sequestering iron in lysosomes and killing the cells by exploiting their vulnerability to ferroptosis. some types of cancer cells can survive treatment with salinomycin through autophagy, whereby cells use acidic organelles such as lysosomes to degrade and recycle certain types of proteins. the use of autophagy inhibitors can kill cancer stem cells that survive by autophagy. the cell surface receptor interleukin-3 receptor-alpha (cd123) is overexpressed on cd34+cd38- leukemic stem cells (lscs) in acute myelogenous leukemia (aml) but not on normal cd34+cd38- bone marrow cells. treating aml-engrafted nod/scid mice with a cd123-specific monoclonal antibody impaired lscs homing to the bone marrow and reduced overall aml cell repopulation including the proportion of lscs in secondary mouse recipients. a 2015 study packaged nanoparticles with mir-34a and ammonium bicarbonate and delivered them to prostate cscs in a mouse model. then they irradiated the area with near-infrared laser light. this caused the nanoparticles to swell three times or more in size bursting the endosomes and dispersing the rna in the cell. mir-34a can lower the levels of cd44.