Liposarcoma cells with aldefluor and CD133 activity have a cancer stem cell potential

Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0, 1-1, 7% of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells

Molecular and cellular characterization of ABCG2 in the prostate

BACKGROUND: Identification and characterization of the prostate stem cell is important for understanding normal prostate development and carcinogenesis. The flow cytometry-based side population (SP) technique has been developed to isolate putative adult stem cells in several human tissue types including the prostate. This phenotype is mainly mediated by the ATP-binding cassette membrane transporter ABCG2. METHODS: Immunolocalization of ABCG2 was performed on normal prostate tissue obtained from radical prostatectomies. Normal human prostate SP cells and ABCG2(+ )cells were isolated and gene expression was determined with DNA array analysis and RT-PCR. Endothelial cells were removed by pre-sorting with CD31. RESULTS: ABCG2 positive cells were localized to the prostate basal epithelium and endothelium. ABCG2(+ )cells in the basal epithelium constituted less than 1% of the total basal cell population. SP cells constituted 0.5–3% of the total epithelial fraction. The SP transcriptome was essentially the same as ABCG2(+ )and both populations expressed genes indicative of a stem cell phenotype, however, the cells also expressed many genes in common with endothelial cells. CONCLUSION: These results provide gene expression profiles for the prostate SP and ABCG2(+ )cells that will be critical for studying normal development and carcinogenesis, in particular as related to the cancer stem cell concept

CD133 expression in chemo-resistant Ewing sarcoma cells

<p>Abstract</p> <p>Background</p> <p>Some human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT) are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance.</p> <p>Methods</p> <p>Expression of the CD133-encoding <it>PROM1 </it>gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and <it>in vivo </it>tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays.</p> <p>Results</p> <p><it>PROM1 </it>expression was either absent or extremely low in most tumors. However, <it>PROM1 </it>was highly over-expressed in 4 of 48 cases. Two of the 4 patients with <it>PROM1 </it>over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of <it>PROM1 </it>in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny.</p> <p>Conclusions</p> <p>Up to 10% of ESFT express high levels of <it>PROM1</it>. In some tumors and cell lines the CD133+ fraction is relatively more drug-resistant, while in others there is no apparent difference between CD133+ and CD133- cells. These studies reveal heterogeneity in <it>PROM1</it>/CD133 expression in ESFT tumors and cell lines and confirm that high levels of <it>PROM1 </it>expression are, in at least some cases, associated with chemo-resistant disease. Further studies are required to elucidate the contribution of <it>PROM1/</it>CD133 expressing cells to therapeutic resistance in a large, prospective cohort of primary ESFT.</p

Notch signaling contributes to the maintenance of both normal neural stem cells and patient-derived glioma stem cells

<p>Abstract</p> <p>Background</p> <p>Cancer stem cells (CSCs) play an important role in the development and recurrence of malignant tumors including glioma. Notch signaling, an evolutionarily conserved pathway mediating direct cell-cell interaction, has been shown to regulate neural stem cells (NSCs) and glioma stem cells (GSCs) in normal neurogenesis and pathological carcinogenesis, respectively. However, how Notch signaling regulates the proliferation and differentiation of GSCs has not been well elucidated.</p> <p>Methods</p> <p>We isolated and cultivate human GSCs from glioma patient specimens. Then on parallel comparison with NSCs, we inhibited Notch signaling using γ-secretase inhibitors (GSI) and assessed the potential functions of Notch signaling in human GSCs.</p> <p>Results</p> <p>Similar to the GSI-treated NSCs, the number of the primary and secondary tumor spheres from GSI-treated GSCs decreased significantly, suggesting that the proliferation and self-renewal ability of GSI-treated GSCs were attenuated. GSI-treated GSCs showed increased differentiation into mature neural cell types in differentiation medium, similar to GSI-treated NSCs. Next, we found that GSI-treated tumor spheres were composed of more intermediate progenitors instead of CSCs, compared with the controls. Interestingly, although inhibition of Notch signaling decreased the ratio of proliferating NSCs in long term culture, we found that the ratio of G2+M phase-GSCs were almost undisturbed on GSI treatment within 72 h.</p> <p>Conclusions</p> <p>These data indicate that like NSCs, Notch signaling maintains the patient-derived GSCs by promoting their self-renewal and inhibiting their differentiation, and support that Notch signal inhibitor GSI might be a prosperous candidate of the treatment targeting CSCs for gliomas, however, with GSI-resistance at the early stage of GSCs cell cycle.</p

CD133+ Anaplastic Thyroid Cancer Cells Initiate Tumors in Immunodeficient Mice and Are Regulated by Thyrotropin

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important.In prior studies of ATC cell lines, CD133(+) cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133(+) cells, but not CD133(-) cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133(+) increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133(+) cell pool can be further separated by flow cytometry into two distinct populations: CD133(+/high) and CD133(+/low). Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133(+/high) cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133(+) cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells.To our knowledge, this is the first report indicating that CD133(+) ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133(+) cells and the activated TSH signaling pathway may be useful targets for future ATC therapies

The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features

Identification of a New Rhoptry Neck Complex RON9/RON10 in the Apicomplexa Parasite Toxoplasma gondii

Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as "mammospheres" in three-dimensional cultures.We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy

CD133-positive hepatocellular carcinoma in an area endemic for hepatitis B virus infection

<p>Abstract</p> <p>Background</p> <p>CD133 was detected in several types of cancers including hepatocellular carcinoma (HCC), which raised the possibility of stem cell origin in a subset of cancers. However, reappearance of embryonic markers in de-differentiated malignant cells was commonly observed. It remained to be elucidated whether CD133-positive HCCs were indeed of stem cell origin or they were just a group of poorly differentiated cells acquiring an embryonic marker. The aim of this study was to investigate the significance of CD133 expression in HCC in an area endemic for hepatitis B virus (HBV) infection to gain insights on this issue.</p> <p>Methods</p> <p>154 HCC patients receiving total removal of HCCs were included. 104 of them (67.5%) were positive for HBV infection. The cancerous and adjacent non-cancerous liver tissues were subjected for Western blot and immunohistochemistry analysis for CD133 expression. The data were correlated with clinical parameters, patient survivals, and p53 expression.</p> <p>Results</p> <p>Of 154 patients, 24 (15.6%) had CD133 expression in HCC. Univariate and multivariate logistic regression analysis revealed that CD133 expression was negatively correlated with the presence of hepatitis B surface antigen (HBsAg). The unadjusted and adjusted odds ratios were 0.337 (95%CI 0.126 - 0.890) and 0.084 (95%CI 0.010 - 0.707), respectively. On the other hand, p53 expression was positively associated with the presence of HBsAg in univariate analysis. The unadjusted odds ratio was 4.203 (95%CI 1.110 - 18.673). Survival analysis indicated that both CD133 and p53 expression in HCC predicted poor disease-free survival (P = 0.009 and 0.001, respectively), whereas only CD133 expression predicted poor overall survival (P = 0.001). Cox proportional hazard model showed that p53 and CD133 expression were two independent predictors for disease-free survival. The hazard ratios were 1.697 (95% CI 1.318 - 2.185) and 2.559 (95% CI 1.519 - 4.313), respectively (P < 0.001 for both).</p> <p>Conclusion</p> <p>In area where HBV infection accounts for the major attributive risk of HCC, CD133 expression in HCC was negatively associated with the presence of HBsAg, implicating a non-viral origin of CD133-positive HCC. Additionally, CD133 expression predicted poor disease-free survival independently of p53 expression, arguing for two distinguishable hepatocarcinogenesis pathways.</p