Yostina Wagdy ,Silvia Eid , Raouth Nessim , Christine Ateya , Sara Samer , Magda Hanna , Mikel Melad , Simon Samir , Kerollos Sadek , Samuel Yahia , Kerollos Kamal , Mena Welleam , Mina Zosser .
Keywords Stem cell
A B S T R A C T Stem cells are defined by their self-renewal capacity and the ability to give rise to all differentiated progeny necessary for one specific organ. These two
Cancer stem cell Solid tumor
characteristics are also inherent in cancer stem cells (CSCs), which are thought to be the only subpopulation within a tumor endowed with tumorigenic potential. CSCs combine many features that render cancer one of the leading
Tumor-initiating cells Pancreatic cancer
causes of death in the Western world: metastasis, tumor recurrence, and therapy refractoriness. Although the concept of 'cancer stem cell' was first proposed more then a
Colon cancer
century ago, it has attracted a great deal of attention recently due to advances
Breast cancer
in stem cell biology, leading to the identification of these cells in a wide variety of human cancers
Glioblastoma
Solid tumors represent the major cancer burden, and epithelial cancers arising in
CD133
tissues that include breast, lung, colon, prostate and ovary constitute approximately 80% of all cancers, while other tumor entities such as
CD44
glioblastoma multiforme and pancreatic ductal adenocarcinoma are less
ALDH
frequent, but display extraordinarily high mortality rates {1}
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1- Introduction Human development follows a predetermined plan that turns a fertilized egg into a complex, multicellular organism. The fertilized egg develops into a totipotent ball of cells that further differentiates into the three germ layers: endoderm, ectoderm, and mesoderm . These three primitive cell types develop into all tissues in the adult body. Tissues in which malignancies originate, such as the blood, brain, breast, skin,and gut, are organized as a cellular hierarchy with a small population of tissue-specific stem cells responsible for both development and maintenance of tissues for the human life time {2}. . The cancer stem cell theory proposes that tumors have a cellular hierarchy that is a caricature of their normal tissue counterpart because they reflect the pluripotency of the originally transformed cell that cancer stem cells may be responsible for growing and maintaining tumors {2}.. First, most tumors arise from a single cell, but not all the cells within a tumor are identical.known as tumor heterogeneity In fact, there are many different types of cells in a tumor; some are cancerous, whereas others are infiltrating normal cells that are thought to support the growth of the cancer cells . Cancers originally develop from normal cells that gain the ability to proliferate aberrantly and eventually turn malignant. These cancerous cells then grow clonally into tumors and eventually have the potential to metastasize. These cancer stem cells share many characteristics with normal stem cells, including self-renewal and differentiation. With the growing evidence that cancer stem cells exist in a wide array of tumors,Studies of normal and cancer stem cells from the same tissue have shed light on theontogeny of tumors.Understanding the biology of cancer stem cells will contribute to the identification of molecular targets important for future therapies {2}..
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1-1-Characteristics of CSCs The basic characteristics that distinguish CSCs are: (1) promotion of tumorigenesis when they are transplanted into immunosuppressed mice; (2) possession of specific cell-surface markers that are not expressed by noncancer stem cells; (3) tumors that arise from CSCs include both tumorigenic and nontumorigenic cells (heterogeneity); and (4) capacity for self-renewal in seriated transplants over several generations.{3}{4} These characteristics are derived from the intrinsic properties of CSCs, which reside in their ability
1-2-Origin of CSCs Two main hypotheses exist regarding the origin of CSCs: (1) origin from a somatic tissue cell that undergoes genetic mutations, becomes cancerous, and acquires stem characteristics; and (2) derivation from embryonic stem or adult cells as a result of genetic mutations. The first theory does not exclude the second, because the mode of onset may depend on the location of the origin of the tumor {5}.
1-3-The hierarchical theory The hierarchical theory hypothesizes that the tumor originates from embryonic stem cells or somatic cells (present in all tissues) undergoing mutations. These changed stem cells give rise to stem cells that are further altered. Unlike the previous theory, in the hierarchical model, during cell division, one of the two daughter cells retains the ability to replicate, whereas the other loses this capacity and differentiates. Differentiated CSCs represent the majority of the tumor; further mutations that alter the characteristics of the parent cells may intervene during the process of CSC duplication, giving rise to cells that are functionally different. Unlike the stochastic model, the hierarchical model considers that tumorigenicity resides in a small subpopulation of cells composing the tumor that retain the capacity of stemness (Figure 1) {6} .
Figure 1
CSC Differentiate tumor cell
ccancer
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2- RELATIONSHIP BETWEEN NORMAL AND CANCER STEM stem cell is : a primitive cell defined by its capacity to self-renew ) specialized mitotic cell division in which a stem cell creates one (asymmetric) or two (symmetric) daughter stem cells ) and differentiate : ( the overall process of progenitor cells activating genetic and epigenetic mechanisms to define the specialized characteristics of mature cells) into at least one mature cell type {2} while cancer stem cells (CSCs) are cells that drive tumorigenesis, as well as giving rise to a large population of differentiated progeny that make up the bulk of the tumor, but that lack tumorigenic potential and it is a self-renewing cell within a tumor that has the capacity to regenerate the phenotypic diversity of the original tumor.{2}
3- The Difference Between Liquid and Solid Tumors Solid tumors refer to a solid mass of cancer cells that grow in organ systems and can occur anywhere in the body. “All of the solid systems in the body can have cancer,” Two types of solid tumors are seen in adults: epithelial tumors and sarcomas. Epithelial tumors, which can also be called carcinomas, account for 90% of the solid tumors people have, according to Engstrom, and they occur in the lining (epithelium) that is on the outside or inside of the organ. For instance, the digestive system, which starts in the mouth and ends at the anus, is lined all the way down with an epithelium. An epithelial tumor can occur anywhere along that lining, so a person could have a mouth cancer, throat cancer, esophageal cancer, stomach cancer, bowel cancer, or anal cancer Sarcomas are also called “connective tissue tumors” because they occur in the tissue that keeps the organs together. Connective tissues are the muscles, tendons, fat, nerves and other tissues that connect, support or surround structures and organs in the body. Sarcomas are usually named for the type of tissue where they first occur, according to Morgan. For instance, bone tumors are called osteosarcomas (osteo means bone,sarcoma is the connective tissue tumor) {7}.
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Cancer tumors are initially broken down into solid tumors (organ tumors) and liquid tumors (blood cancers
Tumors of the brain are observed in both children and adults. Children are also at risk for congenital tumors of the adrenal gland
(neuroblastoma), kidney (Wilms tumor), muscle (rhabdomyosarcoma), and retina (retinoblastoma).
Liquid Tumors : Liquid tumors occur in the blood, bone marrow, and lymph nodes. They include leukemia,lymphoma, and myeloma. About 61,000 people are diagnosed with blood cancers each year, and both adults and children can develop a blood cancer. Depending on the type of cancer a person has, the outcomes of blood cancers vary {7}.
4-Methods for CSC identification and isolation in solid tumors The methods used for the identification and isolation of tumor stem cell populations apply the same techniques used to identify normal stem cells from their differentiated progeny. Cancer stem cells can be identified via surface markers, determination of ALDH activity, ability to efflux vital dyes, and ability to form tumor spheres in vitro. The subpopulation identified and isolated using these methods is then subjected to tests that prove their tumorigenic ability: quantitative assays of xenografts (which test tumorigenicity) and methods to assess selfrenewal in vivo (which test self-renewal capacity).
4-1- ALDH activity ALDH is an intracellular enzyme that is present normally in the liver. Its best-known functions are the retinol conversion to retinoic acid and the oxidation of toxic aldehyde metabolites, such as those formed during the alcohol metabolism and certain chemotherapeutic drugs (eg, cyclophosphamide and cisplatin). a small percentage of ALDHhigh tumor cells can produce new head and neck tumors when transplanted into immunosuppressed mice. The majority of isolated cells with ALDHhigh also exhibited high expression of
CD44.{8}{9}{10}
4-2- Side populations Another strategy used to identify highly tumorigenic cellular subpopulations is based on the ability of these cells to efflux a fluorescent dye that binds to DNA. The cell populations isolated using this method are called side populations .Using this technique, side populations have been identified in normal tissues and in solid tumors.{11}
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4-3-Formation of tumor spheres CSCs grown in culture conditions without serum retain an undifferentiated state. The addition of growth factors guides them toward proliferation and formation of cell aggregates that are termed tumor spheres.{12}
4-4-surface markers (antigen) The identification and isolation of CSCs using surface markers is most commonly used for implantation into nonobese diabetic/severe combined immunodeficient mice to grow xenografts. The surface antigens used are the same that are used to identify normal stem cells. The surface antigens involved in solid tumors are CD133, CD44, and CD24.
CD133 is a transmembrane pentaspan glycoprotein localized on the protrusions of the cell membrane; its presence has been reported in various solid tumors, such as brain, prostate, colorectal, and lung cancers.43–46 In head and neck tumors Cells with high expression of CD133 exhibit high clonogenicity, the ability to form spheres, and tumor and tumorigenic capacity in xenograft models compared with cells with low CD133.{13}-{15} CD44 is a surface glycoprotein that is involved in cell migration and adhesion. It is a known receptor of hyaluronic acid and interacts with other “ligands,� such as matrix metalloproteases {16],{17} CD24 is a mucin adhesion molecule expressed by pre-B lymphocytes and neutrophils. Functionally, CD24 promotes metastasis, as it has been identified as a ligand of P-selectin, an adhesion receptor found on activated endothelial cells and platelets. Lim and Oh showed that the cytoplasmic expression of CD24 was associated with adenocarcinoma of the colon, stomach, bladder, and ovaries, whereas there is no evidence of this activity in head and neck cancer. {18}
6
markers have been used for the identification of cancer stem cells in solid tumors
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4-4-1- Colon cancer stem cells Colon cancer is one of the few solid tumors for which the progression from a normal cell to a cancer cell is reasonably well understood. If applied to a (cancer) stem cell setting, one would hypothesize that the disease starts from one of the few stem cells at the base of the crypt to express Lgr5 ( a leucine-rich repeat with G-proteincoupled receptor 5 [20]. This landmark study seems to provide us with the most specific stem cell marker for normal intestine to date, but its role in tumorigenesis still remains to be determined. it has been particularly difficult to develop high-affinity antibodies against Lgr5 so that studies for the prospective isolation of Lgr5 positive cells from human tumor samples are still lacking. CD133 expression in colon cancer may not be restricted to CSC at all [21].They claimed CD133 to be expressed on colon cancer cells regardless of their differentiation state, and demonstrated that isolated CD133− cells were at least as capable of giving rise to tumors in NOD/SCID mice as their CD133 positive counterparts
CD133 was finally shown to actually change its conformation upon differentiation, which could explain the opposing results seen with different antibodies and diverse staining protocols [19]. Importantly, the AC133 epitope decreases upon differentiation, which was not linked to a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, the only observed change concerned CD133 glycosylation suggesting that CD133 is expressed on both CSC and differentiated progenies. Since this change in glycosylation alone did not affect binding of AC133, its lack of binding to differentiated cells is more likely related to differences in protein folding as a consequence of this glycosylation.Therefore, though CD133 is certainly not a perfect marker and data need to be interpreted with great caution, it has been one of the most successful markers for the identification of CSC in various tumor entities to date .
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4-4-2-Colorectal cancer (CRC) Colorectal cancer (CRC) is the third most common cancer in the world. Approximately 1.23 million individuals worldwide will develop CRC and 0.6 million people will die of it annually . Although the reasons for the age-related rise in sporadic CRC are not fully known, sedentary lifestyle, diet rich in red meat and unsaturated fats, high energy intake, alcohol consumption are considered to be the major risk factors [22]. Hereditary CRC are of two types:Familial Adenomatous Polyposis (FAP) including an Attenuated Form of Polyposis (AFAP), due to germline mutations in the APC gene .Some other hereditary CRC are not associated with an identifiable hereditary syndrome; they are collectively known as familial CRC [23].Colorectal cancer arises from polyps which are the localized lesions (benign growth) projecting up the surrounding mucosa. Most colorectal polyps are hyperplastic Adenomatous polyp or adenoma arising from glandular epithelium with dysplastic morphology and altered differentiation of epithelial cells are the precursor of carcinoma [24]. Epigenetic modifications are useful biomarkers for early detections of CRC-(i) especially for asymptomatic tumors; (ii) unlike genetic mutations they can be quantified miRNAs, DNA methylation are now being used for the diagnosis and determination of prognostic responses to chemotherapy in patients with colorectal tumor [25]. Epigenetic modifications are dominating over genetic modifications because of their reversibility and are being used as therapeutic targets. DNMT and HDAC inhibitors are long been in practice as a backup for drug resistance [26]. However, further studies are needed to elucidate genetic and epigenetic changes that occur in CSCs/CSLCs during the progression of CRC Genetic changes in colorectal cancer : The spectrum of somatic mutations contributing to the pathogenesis of CRC is wide. Sixty-nine genes were identified as relevant to the pathogenesis of CRC. Nevertheless individual CRC carries an average of nine mutant genes per tumor. In addition, every tumor shows a distinct mutational gene signatur
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4-4-3- Breast cancer stem cells breast cancer was the first solid malignancy in which CSC were identified and isolated .CSC defined as CD44+CD24−/low or ALDH-1+ also seem to play a pivotal role in breast cancer metastasis [28] . The CXCR4/SDF-1 axis has been demonstrated to be of high relevance for breast cancer metastasis [29], and also to be a prerequisitin pancreatic CSC-mediated metastasis [27]. Interestingly SDF-1 is upregulated in tissues that serve as frequent hosts fo tumor metastases such as liver, lung, lymph nodes, bone marrow and brain. Consistently, the investigation of bone marrow metastases revealed an enrichment for CD44+CD24−/low in breast cancer patients [30]. CSC-targeted therapies are not yet ready for the clinical evaluation in breast cancer patients, first studies have been performed to investigate the potential clinical implications of CSC: Korkaya et al. have demonstrated that HER2 overexpression promotes tumorigenicity as well as growth and invasiveness of breast cancers due to its effects on normal and CSC identified by ALDH-1 expression [31]. HER2, a member of the EGF receptor family, serves as a marker to identify patients with a more aggressive disease, but was also identified as a new target for therapy with the clinical introduction of trastuzumab leading to significantly improved outcome
Li et al. have compared the effects of neoadjuvant chemotherapy versus HER2 inhibitor lapatinib in patients with breast cancer [32]. Chemotherapy alone led to an increase of CD44+CD24−/low cells and sphere formation capacity of patient biopsies, providing evidence for a therapy-resistant CSC population.When inhibiting HER-2, however, a non-significant trend towards a decreased CSC content could be observed.
BRCA1 expression is required for the differentiation of estrogen receptor-negative mammary stem cells as knockdown of BRCA1 in primary cells led to an enrichment of ALDH-1 expressing breast CSC [33].
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CSC Numbers Are Increased in Breast Cancer Cells Induced into an EMT ,We sought to increase the proportion of CSCs in breast cancer cell populations by inducing them to pass through an EMT. To this end, we modified experimentally transformed HMLER breast cancer cells by short hairpin RNA (shRNA)-mediated inhibition of the human CDH1 gene, which encodes E-cadherin. Confirming previous results, an shEcad vector triggered an EMT and resulted in acquisition of a mesenchymal phenotype (Moreover, expression of a murine E-cadherin gene resistant to the introduced human shEcad construct caused reversion of EMT-associated phenotypes, indicating that EMT induction was not due to off-target shRNA effects ( We next examined whether HMLER cancer cell populations
Salinomycin Selectively Kills Breast CSCs We focused our further investigations on the properties of salinomycin.We observed that the sensitivity of breast cancer cell lines to salinomycin correlated with the relative abundance of their CD44high/CD24low CSC enriched subpopulations Accordingly, we sought to assess the specific effects of salinomycin on CSCs that existed naturally as a subpopulation within HMLER breast cancer cells rather than in populations experimentally induced into an EMT. For these and subsequent compound-treatment experiments, we treated cells for a specified time, allowed cells to recover for 4 days, and then conducted subsequent experimental assays in the absence of additional treatment, since this protocol would ensure that any further toxicity in the continued presence of a chemical compound would not confound the results of assays used to measure CSC representation. We first assayed the effects of treatment on the proportion of breast cancer cells with the CD44high/CD24low
antigenic
phenotype
.Salinomycin
treatment
decreased
the
proportion
of
CD44high/CD24low breast cancer cells by 20-fold relative to vehicle-treated controls; in contrast, paclitaxel treatment increased the proportion by 18-fold. The relative size of the CD44high/CD24lowfractionwas therefore360-foldlower after treatment with salinomycin than with paclitaxel .
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In a second experiment with an independent population ofHMLER breast cancer cells that naturally contains a high proportion of CSCs, we observed an _75-fold reduction in the proportion of CSCs after salinomycin treatment compared to control treatment Weobserved comparable results with cells of the SUM159 human breast carcinoma line . As a functional measure of CSC frequency, we also examined the ability of HMLER breast cancer cells to form tumor spheres after treatment with salinomycin, paclitaxel, or dimethyl sulfoxide (DMSO) control. Salinomycin treatment induced an _10-fold decrease in the number of tumorspheres relative to controls, In contrast, paclitaxel treatment did not affect the number of tumorspheres formed, resulting instead in a significant increase in tumorsphere size
5-treatment Currently, only a handful of drugs demonstrated selective high efficacy against CSCs. In one example, a veterinary antibiotic, the polyether ionophore salinomycin, was found to possess strong breast CSC -killing capabilities compared with many other drugs tested in the combinatorial study.[34] Due to the prohibitive drug toxicity in humans, only targeted nanoformulations of salinomycin may be potentially devoid of toxicity and efficient in the treatment of CSCs and drug-resistant tumors. Another example, the common Indian spice curcumin, recently attracted great attention in clinical oncology due to its preventive, anticancer and radiotherapy/chemotherapy-sensibilizing activities in aggressive and recurrent tumors. Novel synthetic curcumin analogs and potential nanotechnology-based curcumin formulations have recently been highlighted in the review by Mimeault et al..[35 ] Destruction of CSCs in Niches Various physicochemical methods are currently being investigated for specific destruction of CSCs and theCSC-supporting environment (niche). Most approaches include local delivery of vectorized NPs (empty or drug loaded) and subsequent body irradiation for thermoablation or photodynamic therapy (PDT). Glazer et al. used the efficient heating rates of gold NPs (AuNPs) under irradiation at a nonionizing radiofrequency (13.56 MHz) in this way.{36} Irradiation of AuNPs increased the local temperature up to 60째C, which could result in the rapid destruction of the AuNP-accumulating cancer cells. A total of 36 h after intraperitoneal treatment of human pancreatic Panc-1 and Capan-1 cancer xenografts with mAb-conjugated AuNPs, the mice were exposed to radiofrequency irradiation. Tumors significantly shrank after 6 weeks of weekly treatments and developed visible necrosis. There was no evidence of injury in bystander organs after the therapy
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Ultrasound
is another physical treatment that can potentially enhance CSC-directed therapy and
destruction of the CSC niche. In the following example, pulsed ultrasound was applied in order to enhance the penetration of drug-loaded polymeric and lipid NPs into breast cancer spheroids (300–350 ¾m diameter).[37] Effective treatment of solid tumors requires homogeneous distribution of anticancer drugs within the entire tumor volume to deliver lethal concentrations to drug-resistant cancer cells and CSCs. However, penetration of drug-loaded polymeric and lipid particles into the hypoxic and necrotic regions of solid tumors with high concentration of CSCs remains a significant challenge (Figure 2).
Figure 2. The schematic drawing shows solid tumor organization with the characteristic acidic front, vascularized circumference, hypoxic region and necrotic core. Resistant cancer cells and cancer stem cells are typically sequestered in the tumor's hypoxic and necrotic regions.
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6-Conclusions The CSC field, particularly in the area of solid tumors, is a very young one. A model of solid tumor stemcell biology is shown in Figure 3 highlighting the properties that are currently viewed as important for tumor growth
Figure 3 :Model of cancer stem cells (CSCs) in solid tumors. CSCs are associated with the stromal components of th e tumor, including fibroblasts and/or blood vessels, which make up the CSC ‘niche’. The niche cells secrete factors that support CSC self renewal. CSCs retain differentiation potential, giving rise to non-self renewing tumor cells that make up the bulk of the tumor. CSCs may be drug and/or radiation resistant, and may express CSC-specific antigens. Current research focuses on identification of the molecular mechanisms regulating these properties of CSCs (highlighted in green), as they represent potential targets for therapy .
and that may represent therapeutic targets.We are still in the very technically challenging stage of identifying the methods that will allow us to prospectively isolate CSCs from various solid tumors, and most importantly, to demonstrate in vivo that the populations we are looking at are truly CSCs. In the cases where CSCs have been identified,early studies have already shown that they possess important biological properties that directly relate to patient outcome. The breast CSC gene expression profile was shown to correlate with patient prognosis. In addition,specific signaling pathways have been shown to play a functional role in CSC self renewal and/or differentiation, and early studies indicate that CSCs are associated with a microenvironmental niche. The mechanisms by which CSCs evade current therapies are being elucidated. These types of studies should lead to a better understanding of the molecular mechanisms controlling selfrenewal and/or differentiation. The advent of new highthroughput technologies and the ability to apply them to small numbers of cells should lead to the identification of CSC-specific targets. The development of methods for the prospective isolation of CSCs is thus the first step, which then opens the door to a variety of approaches that could ultimately lead to CSC-specific therapies for cancer treatment.
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7-Abbreviation CSCs : cancer stem cells ALDH: aldehyde dehydrogenase Lgr5 : a leucine-rich repeat with G-proteincoupled receptor 5 CRC : Colorectal cancer FAP :Familial Adenomatous Polyposis AFAP
: Attenuated Form of Polyposis
HER2, a member of the EGF receptor family, serves as a marker to identify patients with a more aggressive disease
EGF : The Epidermal Growth Factor NPs : Nanoparticles EMT : Epithelial Mesenchymal Transition BRCA1 : Breast Cancer Tyep 1 susceptibilty protein
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