Putative Stem Cell Markers CD133 Research Articles

Characterization and Differentiation of Sorted Human Fetal Pancreatic ALDHhi and ALDHhi/CD133+ Cells Toward Insulin-Expressing Cells.

The enzyme aldehyde dehydrogenase (ALDH) is found in developing and multipotent cell populations, and is important for the production and regulation of retinoic acid, which controls β-cell differentiation in the pancreas. The role of ALDH-expressing cells in the formation of endocrine-like cells and co-localization with the putative stem cell marker CD133 has not been examined during human pancreatic development. This study focuses on the co-expression of CD133 on ALDH+ cells from the human fetal pancreas (18-22 weeks of fetal age) with transcription factors (TFs) central to endocrine cell development. Fluorescence-activated cell sorting demonstrated that cells with high ALDH activity (ALDHhi) had increased co-expression of CD133 and endocrine-lineage TFs when compared with cells with low ALDH (ALDHlo) expression. Hormone-expressing (insulin, somatostatin) and ductal cells (CK19) were noted in the ALDHhi population, while mesenchymal (vimentin) and endothelial (CD31) markers were predominantly found in ALDHlo cells. Culture of sorted ALDHhi or ALDHhi/CD133+ cells resulted in loss of endocrine TF, insulin, and CK19 expression. The formation of cell clusters from cultured ALDHhi or ALDHhi/CD133+ cells led to restored CK19 expression and showed endocrine TFs and insulin expression. In summary, pancreatic ALDHhi cells contain a heterogeneous CD133-enriched population with a subset of β-cell associated markers in the developing human pancreas.

A pilot study for cellular detection of circulating tumor cells and disseminated tumor cells of patients with hepatocellular carcinoma.

e15038 Background: Circulating tumor cells (CTC) in peripheral blood (PB) and disseminated tumor cells (DTC) in bone marrow (BM) have been recently focused as predictive and prognostic markers in solid tumors. But hepatocellular carcinoma (HCC) usually lacks epithelial antigens, only a few reports described CTC or DTC at a cellular level. The aim of this study is to establish methodology for detecting CTC and DTC in HCC at a cellular level, and to determine to what degree DTC match the phenotype criterion of putative cancer stem cells. Methods: PB (n-17) and BM samples (n-12) were preoperatively collected from patients with HCC between October 2011 and December 2012. Mononuclear cells were collected by Ficoll gradient separation. CD45+ cell population was deleted through magnetic activated cell sorting system. Subsequently, double immunofluorescence for AFP, Arginase-1 or Hep-Par1 and CD45 was performed. PB samples from 20 healthy volunteers and BM samples from 3 patients with benign disease were used as controls. Our protocol has been approved by the ethical committee of Hokkaido University Hospital and written informed consent was obtained from all enrolled patients. Results: Positive rates of CTC and DTC were 70.5% (12/17) and 75.0% (9/12), respectively. No CTC or DTC was found in the control groups. Poorly differentiated HCC were found in 50.0% of CTC+ patients and 66.6% of DTC+ patients, whereas, 20% in CTC- patients and 0% of DTC- patients. There’re higher risk of recurrence in CTC+ or DTC+ patients, compared with that of CTC- or DTC- patients (33.3%, 55.5% vs 20%, 0%). Over 40% of DTC expressed putative stem cell marker CD133, although the median positive rate of CD133 in primary HCC was reported to be less than 1%. Conclusions: Our protocol might provide highly specific detection of CTC, DTC from HCC patients. Detection of CTC and DTC might closely correlate with histological grade of differentiation and recurrence rate.

Enrichment of Prostate Cancer Stem-Like Cells from Human Prostate Cancer Cell Lines by Culture in Serum-Free Medium and Chemoradiotherapy

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133+/CD44+ cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133+/CD44+ prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133+/CD44+ cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133+/CD44+ PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133+/CD44+ cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133+/CD44+ prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

Analysis of neuroendocrine marker synaptophysin and stem-cell related marker CD133 on circulating tumor cells (CTC) in patients with metastatic non-small cellular lung cancer (NSCLC).

e18010 Background: In NSCLC heterogeneity or switch to neuroendocrine phenotype and the expression of stem-cell related markers have been associated with higher tumor aggressiveness. We evaluated the presence of CTC bearing stem cell-related marker CD133+ and the neuroendocrine marker synaptophysin (SYP) in patients with metastatic non small cellular lung cancer (NSCLC). Methods: Forty-four blood samples (20 mL) were collected after informed consent from 16 consecutive patients (pts) with metastatic NSCLC before and during chemotherapy at our Institution. Blood was enriched for tumor cells by CD45 depletion using a magnetic bead separation technique and potential CTC were identified by flow cytometry. CTC were defined as EpCAM+ CK7/8 + CD45-CD53-. Multiparameter cytometry assessed surface expression of the combinations of CD133 and SYP. Results: CTC phenotype: Twenty-five of all 44 samples (57%) were found positive for presence of at least 1 CTC. The median number of CTC was 4 (range: 1-9) / 10mL blood. Twelve samples (27%) showed to be positive for the cancer putative stem cell marker CD133. The median number of CD133+ CTC was 2 (range: 1-7) / 10mL. Seventeen samples (38%) were positive for SYP with a median number of 2 SYN+ CTC (range: 1-6 ) /10ml. Eleven samples (25%) resulted double positive for CD133 and SYP. Tumor heterogeneity: Twelve of the 16 patients (75%) had at least one sample positive for CTC. In 3 pts (25%) CTC expressed only SYP, in 5 pts (42%) both the markers and in 4 pts (33%) neither CD133 nor SYP were expressed. Conclusions: A higher rate of CD133-SYP-double positive CTC compared to the single positive CTC was observed. This suggests that the switch to the neuroendocrine phenotype might occur concomitantly with a stem-cell like more resistant and aggressive phenotype. It needs to be clarified, whether the presence of stem-cell like and neuroendocrine CTC may be a predictive factor for treatment response and outcome in patients with metastatic NSCLC.

Oncolytic Herpes Simplex Virus Counteracts the Hypoxia-Induced Modulation of Glioblastoma Stem-Like Cells

Glioblastoma (GBM), a fatal malignant brain tumor, contains abundant hypoxic regions that provide a "niche" to promote both the maintenance and enrichment of glioblastoma stem-like cells (GSCs) and confer resistance to chemo- and radiotherapy. Since GSCs, with an ability to resist conventional therapies, may be responsible for tumor recurrence, targeting GSCs located in such a hypoxic environment may be critical to improving the therapeutic outcome for GBM patients. Oncolytic viral therapies have been tested in the clinic as a promising therapeutic approach for GBM. In this study, we analyzed and compared the therapeutic effects of oncolytic herpes simplex virus (oHSV) type 1 G47Δ (γ34.5(-)ICP6(-)LacZ(+)α47(-)) in patient-derived GSCs under normoxia (21% oxygen) and hypoxia (1% oxygen). GSCs cultured in hypoxia showed an increased ability to form neurospheres and expressed higher levels of the putative stem cell marker CD133 compared with GSCs cultured in normoxia. G47Δ exhibited a comparable ability to infect, replicate, and kill GSCs in normoxia and hypoxia in vitro. Importantly, G47Δ could counteract hypoxia-mediated enhancement of the stem-like properties of GSCs, inhibiting their self-renewal and stem cell marker expression. Using orthotopic human GSC xenografts in mice, we demonstrated that intratumoral injection of G47ΔUs11fluc, a newly developed G47Δ derivative that expresses firefly luciferase driven by a true late viral promoter, led to an equivalent frequency of viral infection and replication in hypoxic and nonhypoxic tumor areas. These findings suggest that oHSV G47Δ represents a promising therapeutic strategy to target and kill GSCs, not only in normoxic areas of GBM but also within the hypoxic niche.

Expression of putative stem marker nestin and CD133 in advanced serous ovarian cancer

It is hypothesized that "cancer stem cells" are responsible for the resistance to chemotherapy of cancer cells in ovarian cancers. The objective of the studies was to explore if the stem cell biomarkers could be used to predict the tumor chemotherapy-resistance in serous ovarian cancer patients. Expression of two putative stem cell markers CD133 and nestin, and vascular epithelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) were detected in 123 cases of advanced serous ovarian cancer specimens by immunohistochemistry. To estimate intra-tumoral microvessel density (MVD), CD34 immunostaining was also performed. CD133 and nestin were defined to be positive in 35.0% and 32.5% of the serous ovarian carcinoma tissues, respectively. It was observed that overexpression of nestin but not CD133 was associated with the cisplatin-based chemotherapy resistance and shorter overall survival of the patients, and nestin was found to be an independent prognostic factor. Moreover, positive nestin expression also correlated to increased expression of EGFR and VEGF, and elevated MVD in tumors. The results of this study suggest that serous ovarian cancers with high expression level of nestin represent an aggressive malignant phenotype associated with poor prognosis, and treatment targeted the nestin positive cancer cells might be a promising therapeutic strategy for this subgroups.

Two Domains of Vimentin Are Expressed on the Surface of Lymph Node, Bone and Brain Metastatic Prostate Cancer Lines along with the Putative Stem Cell Marker Proteins CD44 and CD133

Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines (derived from lymph node, bone or brain prostate metastases, respectively) demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells.

Abstract 5205: Selectively high Wnt pathway activity in subpopulations of colon cancer cells and its significance for tumor initiation

Abstract Several cell surface markers that are reported to enrich for tumor-initiating colon cancer cells are presumed targets of the canonical Wnt/beta-catenin pathway. Furthermore, colon cancers typically express the Wnt-effector beta-catenin in a heterogeneous pattern, with strong nuclear expression detected only in a small fraction of tumor cells. We therefore hypothesized that high beta-catenin expression and Wnt pathway activity mark a tumorigenic subpopulation. To label viable tumor cells according to the level of Wnt pathway activity, we constructed lentiviral vectors that express GFP under control of an optimal, beta-catenin-responsive TCF/LEF1 promoter (TOP-GFP). We transduced primary colon cancer xenografts and two well characterized colon cancer cell lines with these vectors, used flow cytometry to isolate cell subpopulations with differential GFP expression, and examined these subpopulations for tumorigenicity and gene expression. High GFP expression accurately marked tumor cells with nuclear beta-catenin immunostaining in Caco2 but not SW1222 cells and in a fraction of primary colon cancer xenografts. Gene expression analysis in the concordant cases was consistent with increased Wnt-pathway activity within the GFP-high cell population, including high expression of the putative stem cell markers CD133, CD44 and LGR5. In SW1222 cells and some primary tumor xenografts, the GFP-high fraction correlated poorly with nuclear beta-catenin localization and GFP flow cytometry did not separate cells with a gene expression profile suggesting Wnt pathway activity. Among these various sources, only Caco2 cells showed increased tumorigenicity of GFP-high cells in immunodeficient mouse xenografts. The utility of TOP-GFP and related reporters to identify tumor-initiating colon cancer cells with high Wnt activity thus varies among primary human colorectal tumors and established cell lines. Furthermore, Wnt pathway activity might fluctuate substantially within individual tumor cells. We conclude that high Wnt activity among tumor cell subpopulations does not necessarily signify tumor-initiating potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2011-5205

Stem Cell Characteristics in Prostate Cancer Cell Lines

BackgroundRecent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. ObjectiveSix established prostate cancer (PCa) cell lines—DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3—were examined for their stem cell properties in vitro. Design, settings, and participantsThe colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, α2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18). Results and limitationsFive out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133+ cells was detected. CD133+ cells; however, like DU145 BCRP+ (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed α2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells. ConclusionsThese results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.

CD133, One of the Markers of Cancer Stem Cells in Hep‐2 Cell Line

In recent years, a growing body of evidence has been reported that a tumor clone is organized as a hierarchy that originates from rare stem cells. CD133, a cell surface antigen, was identified as a stem cell maker for human leukemia, brain tumors, and prostate cancer. The purpose of this study was to detect the expression of CD133, a putative marker of cancer stem cells in the Hep-2 cell line, and isolate CD133 positive cells to observe their proliferation and differentiation ability in vitro. Immunocytochemical staining technology and flow cytometry were used to detect the expression of the putative stem cell marker CD133 in a Hep-2 cell line. The immunomagnetic beads were applied to purify CD133 positive cells. CD133+ tumor cells were cultured in vitro to observe their ability to proliferate and differentiate. Only a small proportion (<5%) of cells in the Hep-2 cell line expressed CD133. CD133+ cells possess a marked capacity for self renewal, extensive proliferation, and mutilineal differentiation potency in vitro. CD133 is one of the markers for cancer stem cells in human laryngeal tumors, the Hep-2 cell line.