Possess Stem Cell Characteristics Research Articles

Long non-coding RNAs in breast cancer stem cells.

Breast cancer, one of the most commonly diagnosed cancers worldwide, is a heterogeneous disease with high rates of recurrence and metastasis that contribute to its high mortality rate. Breast cancer stem cells (BCSCs) are a small but significant subset of heterogeneous breast cancer cells that possess stem cell characteristics such as self-renewal and differentiation abilities that may drive metastasis and recurrence. Long non-coding RNAs (lncRNAs) are a class of RNAs that are longer than 200 nucleotides in length and do not possess protein-coding properties. An increasing number of studies have shown that some lncRNAs are abnormally expressed in BCSCs, and have great biological significance in the occurrence, progression, invasion, and metastasis of various cancers. However, the importance of lncRNAs, as well as the molecular mechanisms that regulate and promote the stemness of BCSCs, are still poorly understood. In the current review, we aim to summarize recent studies that highlight the role of lncRNAs in tumor occurrence and progression through BCSCs. In addition, the utility of lncRNAs as biomarkers of breast cancer progression, and their potential use as therapeutic targets for treatment of breast cancer, will be discussed.

MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity.

Simple SummarySolid tumors comprise metabolically hostile areas adjacent to metabolically friendly ones, and their distribution fluctuates in space and time. Selection pressure is maximal in hostile areas where cancer cells develop at least four survival strategies: to adapt themselves, to modify the microenvironment, to hibernate metabolically, and to escape. Escape marks the transition from a localized to an invasive tumor that may ultimately disseminate remotely, forming metastases. Based on the hypothesis that mitochondria are metabolic sensors that control these responses, we previously established that mitochondrial superoxide is a pro-metastatic intracellular signaling agent. Here, we tested MitoQ, a mitochondria-targeted ROS inactivator that already successfully passed Phase I safety clinical trials, as a potential inhibitor of the early steps of the metastatic cascade. Using human breast cancer cells as models in anticipation of preclinical and clinical assays, we report that MitoQ inhibits cancer cell migration, invasion, clonogenicity, sphere formation and spheroid stability.To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-β pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Stem Cell Markers CXCR-4 and CD133 Predict Aggressive Phenotype and Their Double Positivity Indicates Poor Prognosis of Oral Squamous Cell Carcinoma.

Simple SummaryOral cancer is one of the most frequent malignancies in the world, with a poor prognosis. The absence of accurate biomarkers for predicting oral cancer progression is the primary cause of treatment failures. Multiple studies have shown that cancer stem cells play a critical role in tumor growth and chemo resistance. We uncovered, for the first time, the importance of combinatorial expression of stem cell related molecules CXCR-4 and CD-133 as possible biomarkers to predict poor prognosis of oral squamous cell carcinoma. The findings will aid in the identification of high-risk cases in order to provide appropriate therapy.The activation of the SDF-1/CXCR-4 pathway is crucial for the invasion and metastasis of oral cancer cells. The CXCR-4 positive cells possess stem cell characteristics and express the cancer stem cell marker, CD133, in tumors of colon and pancreas. Despite several studies, the co-expression of CXCR-4 and CD133 and its significance is still largely unknown in oral cancer. Therefore, we aimed to investigate the impact of CXCR-4 and CD133 double positivity in the prognosis of oral cancer. The significance of PKC-δ, one of the key signaling molecules that regulates CXCR-4, was also analyzed. Immunohistochemistry and double immunofluorescence was used to investigate the co-localization of CXCR-4, PKC-δ and CD133 in the human tissues and cell lines of oral squamous cell carcinoma. The expression of CXCR-4, PKC-δ and CD133 were found to be higher in poorly differentiated and lymph node metastasis-positive cases. Interestingly, CXCR-4 positive cells showed positive staining for PKC-δ and CD133 in oral cancer tissue and cell lines. Moreover, CXCR-4+/CD133+ and CXCR-4+/PKC-δ+ double positive cases have the worst survival. We discovered, for the first time, that patients with expression of both CXCR-4 and CD133 have a lower survival rate, and CXCR-4+/CD133+, as well as CXCR-4+/PKC-δ+ double positivity, can be utilized to predict poor prognosis. CXCR-4, PKC-δ and CD133 might regulate aggressiveness and invasion of oral cancer cells.

A cocktail of growth factors released from a heparin hyaluronic-acid hydrogel promotes the myogenic potential of human urine-derived stem cells in vivo

Traditional cell therapy technology relies on the maximum expansion of primary stem cells in vitro, through multiple passages and potential differentiation protocols, in order to generate the abundance of cells needed prior to transplantation in vivo. Implantation of in vitro over-expanded and pre-differentiated cells typically results in poor cell survival and reduced regeneration capacity for tissue repair in vivo. We hypothesized that implantation of primary stem cells, after a short time culture in vitro (passage number ≤p3), in combination with controlled release of relevant growth factors would improve in vivo cell viability, engraftment and tissue regeneration. The goal of this study was to determine whether the release of myogenic growth factors from a heparin-hyaluronic acid gel (hp-HA gel) could enhance in vivo cell survival, in-growth and myogenic differentiation of human urine-derived stem cells (USC) with a corresponding enhancement in graft vascularization, innervation and regenerative properties. Human USC were obtained from healthy adult donors (n = 6), expanded and then mixed with a hp-HA gel containing sets of growth factors known to enhance myogenesis (IGF1, HGF, PDGF-BB), neurogenesis (NGF, FGF) and angiogenesis (VEGF), or a cocktail with a combination of growth factors. Primary cultured USC (p3) mixed with the hp-HA gel and the various combinations of growth factors, were subcutaneously injected into athymic mice. In vivo cell survival, engraftment and functional differentiation within the host tissue were assessed. The implanted grafts containing USC and the growth factor cocktail showed the greatest number of surviving cells as well as increased numbers of cells that expressed myogenic and endothelial cell markers as compared to other groups 4 weeks after implantation. Moreover, the graft with USC and the growth factor cocktail showed increased numbers of blood vessels and infiltrating neurons. Thus, growth factors released in a controlled manner from an hp-HA gel containing USC efficiently improved in vivo cell survival and supported vascularization and myogenic differentiation within the grafts. This study provides evidence for the use of primary USC and growth factors in a hydrogel as a novel mode of cell therapy for the promotion of myogenic differentiation for the treatment of injured muscle tissue. Statement of significanceCell therapies are a promising treatment option for neuromuscular dysfunction disorders. However, major limitations in cell retention and engraftment after implantation remain a hindrance to the use of stem cell therapy for the treatment of muscle injuries or diseased tissues. Implanted long-term in vitro cultured cells tend to demonstrate low rates of survival and tissue engraftment, lessened paracrine effects, and poor homing and differentiation. Human USC are an easily obtainable stem cell source that possess stem cell characteristics such as a robust proliferative potential, paracrine effects on neighboring cells, and multi-potential differentiation. In this study, we demonstrated that a combination of primary human USC with a cocktail of growth factors combined in a hyaluronic gel was optimal for cell survival and engraftment, including myogenic differentiation potential of USC, angiogenesis and host nerve fiber recruitment in vivo. The present study also demonstrated that the use of primary urine derived stem cells at early passages, without in vitro pre-differentiation, implanted in a hyaluronic-heparin hydrogel containing a cocktail of growth factors, provided an alternative safe site-specific delivery method for cell therapy.

1182 Melanospheres identify microRNA-519d as a promoter of melanoma progression by modulating EphA4

Non-adherent melanosphere-forming cells have been demonstrated to possess stem cell characteristics and can serve as a cancer model with aggressive behavior. We found that miR-519d was overexpressed in melanospheres and could enhance melanoma progression. EphA4 is the predicted candidate target of miR-519d through computational algorithm prediction. To identify the direct interaction of miR-519d and EphA4 and characterize the biological roles of EphA4 in cutaneous melanoma, we clarified the specific binding sites between miR-519d and EphA4 by luciferase reporter assay. For characterization the roles of EphA4 in melanoma progression, we performed migration, invasion, and adhesion assays in human melanoma cells with or without EphA4 expression. Immunohistochemical study in melanoma surgical specimens and the correlation with patient survival were performed. Our results demonstrated that EphA4 was the direct target of miR-519d. Forced expression of EphA4 could reverse the effects of miR-519d and knockdown of EphA4 phenocopied the effect of miR-519d on increased cell migration, invasion, adhesion as well as metastatic capacities in mice. The immunohistochemical study showed that the expression of EphA4 was decreased in metastatic melanoma compared to its counterpart primary cutaneous melanoma. The EphA4 expression in metastatic melanomas was inversely correlated with patients overall survival. Because melanoma is among the most notoriously aggressive and treatment-resistant human cancers, identifying novel signaling molecules in melanoma progression, such as EphA4 in this study, is helpful to develop tumor biomarkers, to predict patients disease outcome as well as to design individualized therapies.

Differential expression profile analysis of DNA damage repair genes in CD133+/CD133- colorectal cancer cells.

The present study examined differential expression levels of DNA damage repair genes in COLO 205 colorectal cancer cells, with the aim of identifying novel biomarkers for the molecular diagnosis and treatment of colorectal cancer. COLO 205-derived cell spheres were cultured in serum-free medium supplemented with cell factors, and CD133+/CD133- cells were subsequently sorted using an indirect CD133 microbead kit. In vitro differentiation and tumorigenicity assays in BABA/c nude mice were performed to determine whether the CD133+ cells also possessed stem cell characteristics, in addition to the COLO 205 and CD133- cells. RNA sequencing was employed for the analysis of differential gene expression levels at the mRNA level, which was determined using reverse transcription-quantitative polymerase chain reaction. The mRNA expression levels of 43 genes varied in all three types of colon cancer cells (false discovery rate ≤0.05; fold change ≥2). Of these 43 genes, 30 were differentially expressed (8 upregulated and 22 downregulated) in the COLO 205 cells, as compared with the CD133- cells, and 6 genes (all downregulated) were differentially expressed in the COLO 205 cells, as compared with CD133+ cells. A total of 18 genes (10 upregulated and 8 downregulated) were differentially expressed in the CD133- cells, as compared with the CD133+ cells. By contrast, 6 genes were downregulated and none were upregulated in the CD133+ cells compared with the COLO 205 cells. These findings suggest that CD133+ cells may possess the same DNA repair capacity as COLO 205 cells. Heterogeneity in the expression profile of DNA damage repair genes was observed in COLO 205 cells, and COLO 205-derived CD133- cells and CD133+ cells may therefore provide a reference for molecular diagnosis, therapeutic target selection and determination of the treatment and prognosis for colorectal cancer.

Abstract P3-03-17: Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas?

Abstract Cancer stem cells are believed to be responsible for radio- and chemoresistance of malignant tumors. In vitro studies demonstrate that ionizing radiation is capable of reprogramming cancer cells from non-stem state into stem state [1, 2]. Moreover, the embryonic stemness cassette was found to be expressed in tumor cells (including breast cancer cells) after they were polyploidized as a result of genotoxic stress [2, 3], thus prompting us to suppose that the polyploid cells and their descendants released by depolyploidization can possess stem cell characteristics. The aim of the current work is to test whether polyploid cells having stem cell features can be also induced in vivo, namely in locally advanced breast carcinomas as a result of neoadjuvant chemotherapy (NAC), assuming that this process is not autonomous, but rather stipulated by the tumor microenvironment. The study population consisted of 30 breast cancer patients of age ranged from 31 to 75 y. o. diagnosed in the Latvian Oncology Center of the Riga East University Hospital between 2013 and 2015. The tissue specimens were collected after the patients' informed consent was obtained in accordance with the Ethics regulations. The clinico-pathologic information about these patients, including Ki-67 index and the status of ER, PR and HER2 receptors, was obtained from the aforementioned clinics. The majority of patients (n = 28) had locally advanced breast cancer, predominantly Stage III disease. Both diagnostic biopsy and operation material, such as primary tumors surgically removed after NAC using standard doses of paclitaxel and doxorubicin, were subjected to DNA content analysis with image cytometry. Ploidy-related parameters, such as DNA index and the percentage of cells exceeding 4.5c (presumably proliferating and polyploid cells), were determined. Immunofluorescence staining was applied to evaluate expression of such markers/factors as proliferation (Ki-67), stemness (SOX2 and NANOG) and invasiveness (CD44). At the time of diagnosis, 14 patients had primary tumors possessing near-triploid clones, and these cases in comparison with 16 other cases comprised of near-euploid clones had 4.5-fold increase of percentages of cells exceeding the ploidy of 4.5c (p < 0.05) and 1.3-fold increase of percentages of cells positive for Ki-67 (p > 0.05). Of 10 cases diagnosed as “triple-negative”, 6 were near-triploid. Among those cases that showed the resistance to NAC (grades 1 and 2 by Miller-Payne histopathologic scoring), 67% were near-triploid. Polyploidization, which in some resistant cases is gained by NAC, was likely to be attributed to near-triploid clones. Notably, polyploid cells were positive for Ki-67, SOX2, NANOG, and CD44. Thus, these non-quiescent polyploid cells can possess the invasiveness and self-renewal features that were also seen in descendants after depolyploidization. Perhaps, reversible polyploidy plays a definite role in gaining the resistance of tumor cells to chemo- and radiotherapy in vivo. 1. Ghisolfi L. et al. PLoS ONE 2012; 7: e43628. 2. Lagadec C. et al. Stem Cells 2012; 30: 833-44. 3. Salmina K. et al. Exp Cell Res 2010; 316: 2099-112. Citation Format: Gerashchenko BI, Salmina K, Eglitis J, Erenpreisa J. Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-17.

MiR-21 Is Linked to Glioma Angiogenesis: A Co-Localization Study.

MicroRNA-21 (miR-21) is the most consistently over-expressed microRNA (miRNA) in malignant gliomas. We have previously reported that miR-21 is upregulated in glioma vessels and subsets of glioma cells. To better understand the role of miR-21 in glioma angiogenesis and to characterize miR-21-positive tumor cells, we systematically stained consecutive serial sections from ten astrocytomas for miR-21, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), phosphatase and tensin homolog (PTEN), octamer-binding transcription factor 4 (Oct4), sex-determining region Y box 2 (Sox2) and CD133. We developed an image analysis-based co-localization approach allowing global alignment and quantitation of the individual markers, and measured the miR-21 in situ hybridization signal against the immunohistochemical staining of the six different markers. miR-21 significantly co-localized with the hypoxia- and angiogenesis-associated markers HIF-1α (p=0.0020) and VEGF (p=0.0096), whereas the putative miR-21 target, PTEN, was expressed independently of miR-21. Expression of stem cell markers Oct4, Sox2 and CD133 was not associated with miR-21. In six glioblastoma cultures, miR-21 did not correlate with the six markers. These findings suggest that miR-21 is linked to glioma angiogenesis, that miR-21 is unlikely to regulate PTEN, and that miR-21-positive tumor cells do not possess stem cell characteristics.

Extract of fetal membrane would inhibit thrombosis and hemolysis

The innermost layer of fetal membranes is amnion which has anti-adhesive, anti-inflammation and viscoelastic properties, as well as low immunogenicity. Amniotic membrane has been employed in variety of clinical fields as a natural biomaterial. Amniotic epithelial cells possess stem cell characteristics and capability to differentiate into endothelial cells. The basement membrane of amnion is an extracellular matrix enriched scaffold to support adhesion of endothelial cells. The matrix of amniotic membrane contains two kinds of glycosaminoglycans including perlecan (a heparan sulfate proteoglycan) and hyaluronic acid which both inhibit blood coagulation. Moreover, the other ingredients of amniotic membrane such as pigment-epithelium derived factor (PEDF), IL-10, MMP-9 inhibit platelet aggregation. Based on some biochemical and biomechanical evidences, we hypothesized in this paper that amniotic membrane could prevent thrombosis and hemolysis; therefore, has the capability to be applied in blood contacting devices and implants.

B7-H1 enhances proliferation ability of gastric cancer stem-like cells as a receptor.

Cancer stem-like cells (CSCs) are a rare tumorigenic population with the ability to self-renew in numerous cancer types. Their existence is considered a pivotal contributor to tumor recurrence. B7-H1 is a ligand of inhibitory inducible co-stimulator (ICOS) that is broadly expressed on various human cancers. ICOS acts as a ligand of programmed death-1 (PD-1) on T cells, induces the immune escape of cancer cells and also acts as a receptor mediating anti-apoptotic effects on cancer cells. However, the expression and function of B7-H1 on CSCs is not yet clear. In the present study, gastric cancer samples were collected and the B7-H1 expression in gastric cancer CSCs was detected. Ki67, a proliferation marker, was found to be expressed at a higher rate in B7-H1+ CSCs compared with the B7-H1− counterparts. SGC-7901 cells, a gastric cancer cell line, were cultured in serum-free medium to form sphere cells that possessed stem cell characteristics and could express B7-H1 with the stimulation of interferon-γ. The proliferative ability of sphere cells was enhanced following B7-H1 activation with recombinant PD-1 in vivo and in vitro. This effect could be eliminated by neutralizing B7-H1. Overall, B7-H1 can act as a stimulating receptor for CSCs, and induce CSC proliferation. Blocking B7-H1 on CSCs may possess therapeutic potential for treating gastric cancer.

Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application.

Epithelial and mesenchymal cells isolated from the amniotic membrane (AM) possess stem cell characteristics, differentiation potential toward lineages of different germ layers, and immunomodulatory properties. While their expansion and differentiation potential have been well studied and characterized, knowledge about their immunomodulatory properties and the mechanisms involved is still incomplete. These mechanisms have been evaluated on various target cells of the innate and the adaptive system and in animal models of different inflammatory diseases. Some results have evidenced that the immunomodulatory effect of AM-derived cells is dependent on cell-cell contact, but many of them have demonstrated that these properties are mediated through the secretion of suppressive molecules. In this review, we present an update on the described immunomodulatory properties of the derived amniotic cells and some of the proposed involved mechanisms. Furthermore, we describe some assays in animal models of different inflammatory diseases which reveal the potential use of these cells to treat such diseases.

Transplantation of Photoreceptors Derived From Human Müller Glia Restore Rod Function in the P23H Rat

Müller glia possess stem cell characteristics that have been recognized to be responsible for the regeneration of injured retina in fish and amphibians. Although these cells are present in the adult human eye, they are not known to regenerate human retina in vivo. Human Müller glia with stem cell characteristics (hMSCs) can acquire phenotypic and genotypic characteristics of rod photoreceptors in vitro, suggesting that they may have potential for use in transplantation strategies to treat human photoreceptor degenerations. Much work has been undertaken in rodents using various sources of allogeneic stem cells to restore photoreceptor function, but the effect of human Müller glia-derived photoreceptors in the restoration of rod photoreceptor function has not been investigated. This study aimed to differentiate hMSCs into photoreceptor cells by stimulation with growth and differentiation factors in vitro to upregulate gene and protein expression of CRX, NR2E3, and rhodopsin and various phototransduction markers associated with rod photoreceptor development and function and to examine the effect of subretinal transplantation of these cells into the P23H rat, a model of primary photoreceptor degeneration. Following transplantation, hMSC-derived photoreceptor cells migrated and integrated into the outer nuclear layer of the degenerated retinas and led to significant improvement in rod photoreceptor function as shown by an increase in a-wave amplitude and slope using scotopic flash electroretinography. These observations suggest that hMSCs can be regarded as a cell source for development of cell-replacement therapies to treat human photoreceptor degenerations and may also offer potential for the development of autologous transplantation.

Biologische Grundlagen der Limbusstammzellinsuffizienz

Limbal stem cell deficiency results from the loss of tissue regenerating stem and progenitor cells. Corneal epithelial regeneration is maintained by stem and progenitor cells which reside in the schlerocorneal limbus. They possess stem cell characteristics and can be stimulated to proliferate by external signals. The limbus is the stem cell niche for corneal epithelial stem cells and forms a unique microenvironment in which stem cell characteristics are conserved. Regulation of limbal epithelial stem cells is produced by a network of signals within the niche which governs cell fate decisions with regards to proliferation, differentiation or maintenance of a quiescent status.

203 ENDOTHELIAL DIFFERENTIATION OF HUMAN URINE DERIVED STEM CELLS FOR UROLOGICAL TISSUE REGENERATION

voided urine sample may contain malignant cells. In this study, we investigated whether cells isolated from upper urinary tract (UTCs) possess stem cell characteristics and could be an alternative cell source for bladder cancer patients. METHODS: Urine samples were collected from the upper urinary tract of 4 male bladder cancer patients using a ureteral catheter. The samples were centrifuged and the pellets were plated for primary culture. Cells were analyzed for colony forming unit, proliferation rate, cytogenetics, stem cell characterization and tumorigenicity. The results were compared with those of VUC collected from 3 healthy men. RESULTS: We were able to isolate cells possessing stem cell characteristics from urine, which were obtained from upper urinary tract. The UTCs were able to form colonies, had a higher proliferation rate than VUC and had normal karyotype without any chromosomal aberrations. The UTCs possessed stem cell characteristics (expression of CD44 , CD73 , CD90 , CD105 , SSEA4 ) and expressed several markers for urothelial, smooth muscle and endothelial cell lineages. The UTCs did not form teratoma when implanted into the subcapsular space of mouse kidney. CONCLUSIONS: The UTCs possessed stem cell characteristics and can potentially be an alternative cell source for urological tissue regeneration in bladder cancer patients.

Characterization of Urine-derived Cells From Upper Urinary Tract in Patients With Bladder Cancer

To investigate whether cells isolated from the upper urinary tract (UTCs) possess stem cell characteristics and could be an alternative cell source for patients with bladder cancer. Current tissue engineering approaches for urologic tissue regeneration require invasive tissue biopsies to obtain autologous cells, and these procedures are associated with potential complications, such as donor site morbidity. Recently, cells isolated from voided urine (VUCs) have been proposed as an alternative cell source for urologic tissue engineering. However, VUCs should not be used in patients with bladder cancer, because the voided urine sample could contain malignant cells. Urine samples were collected from the upper urinary tract of 4 male patients with bladder cancer using a ureteral catheter. The samples were centrifuged and the pellets plated for primary culture. The cells were analyzed for colony-forming unit, proliferation rate, cytogenetics, stem cell characterization, and tumorigenicity. The results were compared with those of VUCs collected from 3 healthy men. The UTCs were able to form colonies, had a greater proliferation rate than the VUCs, and had a normal karyotype without any chromosomal aberrations. The UTCs possessed stem cell characteristics (expression of CD44+, CD73+, CD90+, CD105+, SSEA4+) and expressed several markers for urothelial, smooth muscle, and endothelial cell lineages. The UTCs did not form teratoma when implanted into the subcapsular space of a mouse kidney. The UTCs possessed stem cell characteristics and can potentially be an alternative cell source for urologic tissue regeneration in patients with bladder cancer.

A subpopulation of CD24+ cells in colon cancer cell lines possess stem cell characteristics

Cancer stem cells (CSCs) have been shown to contribute to the resistance and relapse in a range of cancer types such as breast cancer and glioma. However, colon cancer stem cells remain poorly characterized. Here we reported that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibited cancer stem cell-like characteristics. Using flow cytometry candidate CSCs markers were selected after initial screening of known CSCs markers from other types of cancer on colon cancer cell lines HCT116, SW480 and HT29. CD24 was expressed in the minority of bulk cell population of HCT116 and SW480 cell lines. Moreover, functional tests demonstrated that CD24+ cells exhibited enhanced chemotherapy-resistance, self-renewal and tumorigenic capacity both in vitro and in vivo, compared to CD24- subpopulations. These results suggest that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibits CSCs like characteristics, and represents a nice model to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.

Amnion-derived stem cells: in quest of clinical applications

In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research.

Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-κB signalling

Androgen depletion is a key strategy for treating human prostate cancer, but the presence of hormone-independent cells escaping treatment remains a major therapeutic challenge. Here, we identify a minor subset of stem-like human prostate tumour-initiating cells (TICs) that do not express prostate cancer markers, such as androgen receptor or prostate specific antigen. These TICs possess stem cell characteristics and multipotency as demonstrated by in vitro sphere-formation and in vivo tumour-initiation, respectively. The cells represent an undifferentiated subtype of basal cells and can be purified from prostate tumours based on coexpression of the human pluripotent stem cell marker TRA-1-60 with CD151 and CD166. Such triple-marker-positive TICs recapitulate the original parent tumour heterogeneity in serial xeno-transplantations indicating a tumour cell hierarchy in human prostate cancer development. These TICs exhibit increased nuclear factor-κB activity. These findings are important in understanding the molecular basis of human prostate cancer.

Abstract LB-251: Isolation, expansion, and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer

Abstract The cancer stem cell (CSC) theory suggests that cancer is maintained by a subpopulation of cancer cells which possesses stem cell characteristics, including self renewal, tumor initiation and pluripotent differentiation abilities. A large series of CSC markers has been reported in various cancer types. CD133 is the most frequently used marker and has been applied to isolate cancer stem cells from cancers of the liver, brain, colon and lung, etc. CD44+/CD24-/low were used as CSC markers in breast cancers. CD34 and Sca-1 were used for identification of murine lung stem cells but Sca-1 is not expressed in human tissues. Expression of embryonic genes such as BMI1 and POU5F1 were found in CSC from different tissues and both proteins are key components in maintaining ‘stemness’ of embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells. It still remains unclear whether additional markers are needed to detect lung CSC. To our knowledge, limited studies using CSC markers on a panel of non-small cell lung cancer (NSCLC) cell lines including those raised from Chinese lung cancer patients have been documented. This study aimed to examine the expression profiles of 6 CSC markers (CD24, CD34, CD44, CD133, BMI1 and POU5F1) in a panel of 10 NSCLC cell lines, 163 NSCLC tumor specimens, fetal lung, reactive and regenerating lung tissues. NSCLC cell lines showed expression of CSC markers at various frequencies that ranged from 0-99.7%, suggesting absence of a single universal marker. Six cell lines expressed CD44 while only HCC1833 expressed CD133. Expression of CD44 and CD133 were mutually exclusive. The CD133+ and CD44+ populations showed distinct properties from CD133- and CD44- cells, respectively. Both CD133+ and CD44+ expanded into tumor-spheroids in vitro in non-adherent medium but not CD133- and CD44- cells. CD44+ H1299 cells expressed ‘stemness’ markers including Oct4, Nanog and Sox2 by RT-PCR and immunofluorescence but were lost after induced-differentiation. The clonogenicity of CD44+ cells is significantly higher than CD44- cells and self-renewal ability was maintained after serial passages. CD44+ cells were cisplatin-resistant and initiated tumor formation in nude mice. The xenograft tumors showed poor differentiation that resembled the parental cell line. Freshly sorted tumor cells showed both CD44+ and CD44- populations, suggesting in vivo differentiation. Immunohistochemistry (IHC) results showed CD44 expression in epithelia of developing airways and regenerating lung. In lung cancers, CD44 expression was associated with squamous cell carcinoma (SCC) (P=0.046). When only 96 adenocarcinomas (AD) were included, CD44 expression was associated with non-smokers (P=0.024). Overall, CD44+cells in NSCLC are capable of tumor-initiation and possess stem cell characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-251.