Candidate Stem Cell Markers Research Articles

Differential Marker Expression between Keratinocyte Stem Cells and Their Progeny Generated from a Single Colony.

The stemness in keratinocyte stem cells (KSCs) is determined by their gene expression patterns. KSCs are crucial in maintaining epidermal homeostasis and wound repair and are widely used candidates for therapeutic applications. Although several studies have reported their positive identifiers, unique biomarkers for KSCs remain elusive. Here, we aim to identify potential candidate stem cell markers. Human epidermal keratinocytes (HEKs) from neonatal foreskin tissues were isolated and cultured. Single-cell clonal analysis identified and characterized three types of cells: KSCs (holoclones), transient amplifying cells (TACs; meroclones), and differentiated cells (DSCs; paraclones). The clonogenic potential of KSCs demonstrated the highest proliferation potential of KSCs, followed by TACs and DSCs, respectively. Whole-transcriptome analysis using microarray technology unraveled the molecular signatures of these cells. These results were validated by quantitative real-time polymerase chain reaction and flow cytometry analysis. A total of 301 signature upregulated and 149 downregulated differentially expressed genes (DEGs) were identified in the KSCs, compared to TACs and DSCs. Furthermore, DEG analyses revealed new sets of genes related to cell proliferation, cell adhesion, surface makers, and regulatory factors. In conclusion, this study provides a useful source of information for the identification of potential SC-specific candidate markers.

Co-expression of transcription factor AP-2beta (TFAP2B) and GATA3 in human mammary epithelial cells with intense, apicobasal immunoreactivity for CK8/18

AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation

BackgroundA hypoxic microenvironment leads to an increase in the invasiveness and the metastatic potential of cancer cells within tumors via the epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. However, hypoxia-induced changes in the expression and function of candidate stem cell markers and their possible molecular mechanism is still not understood.MethodsLung cell lines were analyzed in normoxic or hypoxic conditions. For screening among the stem cell markers, a transcriptome analysis using next-generation sequencing was performed. For validation, the EMT and stem cell characteristics were analyzed. To determine whether an epigenetic mechanism was involved, the cell lines were treated with a DNA methyltransferase inhibitor (AZA), and methylation-specific PCR and bisulfite sequencing were performed.ResultsNext-generation sequencing revealed that the CXCR4 expression was significantly higher after the hypoxic condition, which functionally resulted in the EMT and cancer stemness acquisition. The acquisition of the EMT and stemness properties was inhibited by treatment with CXCR4 siRNA. The CXCR4 was activated by either the hypoxic condition or treatment with AZA. The methylation-specific PCR and bisulfite sequencing displayed a decreased CXCR4 promoter methylation in the hypoxic condition.ConclusionsThese results suggest that hypoxia-induced acquisition of cancer stem cell characteristics was associated with CXCR4 activation by its aberrant promoter demethylation.

Expression of p63 and CD44 in oral squamous cell carcinoma and correlation with clinicopathological parameters

Squamous cell carcinoma (SCC) is the sixth most frequent malignant tumor of the head and neck region. Despite advances in therapeutic options over the last decades, the rate of mortality and morbidity has not been improved markedly. A small subset of cells, cancer stem cells (CSCs), with self-renewal properties have become a major focus of current cancer research. CD44 and p63 are identified as candidate stem cell markers in normal squamous epithelium and SCC. The role of these markers in oral squamous cell carcinoma (OSCC) is still debatable. The aim of this study was to evaluate immunohistochemical expression of these markers in OSCC samples and also correlates the expression of these markers with some clinicopathological parameters of prognostic significance including histological grading, TNM staging, overall survival (OS) rate as well as patients’ age, gender, and tumor location. CD44 and p63 were expressed in all studied lesions with different degrees. Statistically significant difference was observed between CD44 and p63 expression with tumor grade and stage with higher expression in high grade and advanced OSCCs. No significant relationship was detected between markers immunoreactivity and patients age, gender, tumor location as well as OS. These markers can possibly advance our understanding of the initiating mechanisms and pathogenesis of OSCC and also result in novel therapeutic target in cancer treatment.

Label retention and stem cell marker expression in the developing and adult prostate identifies basal and luminal epithelial stem cell subpopulations

BackgroundProstate cancer is the second most frequent cancer among males worldwide, and most patients with metastatic disease eventually develop therapy-resistant disease. Recent research has suggested the existence of cancer stem-like cells, and that such cells are behind the therapy resistance and progression.MethodsHere, we have taken advantage of the relatively quiescent nature of stem cells to identify the slow-cycling label-retaining stem cell (LRC) populations of the prostate gland. Mice were pulsed with bromodeoxyuridine (BrdU) during prostate organogenesis, and the LRC populations were then identified and characterized in 5-day-old and in 6-month-old adult animals using immunohistochemistry and immunofluorescence.ResultsQuantification of LRCs in the adult mouse prostate showed that epithelial LRCs were significantly more numerous in prostatic ducts (3.7 ± 0.47% SD) when compared to the proximal (1.4 ± 0.83%) and distal epithelium (0.48 ± 0.08%) of the secretory lobes. LRCs were identified in both the basal and epithelial cell layers of the prostate, and LRCs co-expressed several candidate stem cell markers in a developmental and duct/acini-specific manner, including Sca-1, TROP-2, CD133, CD44, c-kit, and the novel prostate progenitor marker cytokeratin-7. Importantly, a significant proportion of LRCs were localized in the luminal cell layer, the majority in ducts and the proximal prostate, that co-expressed high levels of androgen receptor in the adult prostate.ConclusionsOur results suggest that there are separate basal and luminal stem cell populations in the prostate, and they open up the possibility that androgen receptor-expressing luminal stem-like cells could function as cancer-initiating and relapse-responsible cells in prostate cancer.

Clinical Relevance of a Candidate Stem Cell Marker, p75 Neurotrophin Receptor (p75NTR) Expression in Circulating Tumor Cells.

Despite advances in its diagnosis and multimodal therapies, the prognosis of esophageal squamous cell carcinoma (ESCC) patients remains poor, because of high incidences of metastasis . Recent reports suggested that circulating tumor stem cells (CTSCs), rather than circulating tumor cells (CTCs), were more accurate diagnostic marker for metastasis, because tumor stem cells or cancer stem cells (CSCs) are more responsible for metastasis through processes such as epithelial mesenchymal transition (EMT) and tumor initiation. A neurotrophin receptor p75 (p75NTR) is expressed in a candidate CSC s in ESCC, which possess enhanced tumorigenicity along with strong expression of EMT-related genes. Our recent report using two-color flow cytometry demonstrated that CTC counts based on a combined expression of epithelial cell adhesion molecule (EpCAM) and p75NTR was significantly higher in peripheral blood samples of ESCC patients than healthy controls. In addition, EpCAM + p75NTR+, but not EpCAM + p75NTR- CTC counts, correlated with clinically diagnosed distant metastasis and pathological venous invasion in surgically resected primary ESCC tumors. Malignant cytology of the isolated EpCAM + p75NTR+ cells was microscopically confirmed as well. These results demonstrated that EpCAM + p75NTR+ CTC count was a more accurate diagnostic marker than EpCAM+ CTC count, suggesting the highly metastatic potential of CTCs with p75NTR expression.Investigation using the isolated EpCAM + p75NTR+ CTCs to assess their stem cell properties may shed light on their roles in tumor metastasis in ESCC.Further investigations based on large-scale prospective studies with long term follow up may provide us with evidences for its clinical use.

Identification of stem-like cells and clinical significance of candidate stem cell markers in gastric cancer.

The existence of gastric cancer stem cells (CSCs) has not been definitively proven and specific cell surface markers for identifying gastric CSCs have largely not been identified. Our research aimed to isolate potential gastric CSCs and clarify their clinical significance, while defining markers for GCSC identification and verification. Here, we report that spheroid cells possess stem cell-like properties, and overexpress certain stem cell markers. CD133 or CD44-positive cells also exhibit properties of CSCs. The expression of Oct4, Sox2, Gli1, CD44, CD133, p-AKT, and p-ERK was significantly higher in metastatic lesions compared to that in primary lesions. Elevated expression of some of these proteins was correlated with a more aggressive phenotype and poorer prognosis, including Oct4, Sox2, Gli1, CD44, and p-ERK. Multivariate Cox proportional hazards model analysis showed that only CD44 is an independent factor. Knockdown of CD44 down-regulated the stem cell-like properties, which was accompanied by the down-regulation of p-ERK and Oct4. Oct4 overexpression could reverse the decreased CSCs properties induced by CD44 knockdown. Taken together, our research revealed that spheroid cell culture, and CD133 or CD44-labeled FACS methods can be used to isolate gastric CSCs. Some CSC markers have clinical significance in predicting the prognosis. CD44 is an independent prognostic factor and maintains the properties of CSCs in CD44-p-ERK-Oct4 positive feedback loop.

Inflammation-Related DNA Damage and Cancer Stem Cell Markers in Nasopharyngeal Carcinoma.

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. To investigate the involvement of stem cells in Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC), we used double immunofluorescence staining to examine several cancer stem/progenitor cell markers (CD44v6, CD24, and ALDH1A1) in NPC tissues and NPC cell lines. We also measured 8-nitroguanine formation as an indicator of inflammation-related DNA lesions. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in the stroma of NPC tissues than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. Similarly, more intense staining of CD44v6 and ALDH1A1 was detected in an NPC cell line than in an immortalized nasopharyngeal epithelial cell line. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. 8-Nitroguanine was detected in both CD44v6- and ALDH1A1-positive stem cells in NPC tissues. In conclusion, CD44v6 and ALDH1A1 are candidate stem cell markers for NPC, and the increased formation of DNA lesions by inflammation may result in the mutation of stem cells, leading to tumor development in NPC.

Models of Breast Morphogenesis Based on Localization of Stem Cells in the Developing Mammary Lobule

SummaryCharacterization of normal breast stem cells is important for understanding their role in breast development and in breast cancer. However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known. In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals. Comparing predictions of these models with immunohistochemical analysis of tissue sections for candidate stem cell markers, we defined distinct areas where stem cells reside in the mammary lobule. An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of nulliparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

Characterization of sphere-forming cells with stem-like properties from the gastric cancer cell lines MKN45 and SGC7901

Traditionally, it was presumed that gastric cancer was derived from tumor cells with stem‑like properties. In the present study, stem‑like cells from the gastric cancer cell lines MKN45 and SGC7901 were enriched by growing them as spheres in a defined serum‑free medium. Following enrichment for stem‑like cells, cluster of differentiation (CD)24 and CD44 were applied as candidate stem cell markers to examine the expression profile. It was revealed that the sphere‑derived cells contained a higher proportion of cells expressing the stem cell surface markers CD24 and CD44 when compared with the parental cells. It was also identified that the expression of cytokeratin 18 in sphere‑derived cells was decreased and the expression of vimentin and aldehyde dehydrogenase 1 (ALDH1) was increased compared with the parental cells. This finding supports the existence of a population of tumor sphere‑forming cells with stem cell properties in the MKN45 and SGC7901 cell lines. Furthermore, the stem cell population was enriched in cells expressing CD24, CD44, vimentin and ALDH1 cell surface markers. These results support the existence of gastric cancer stem cells and provide an alternative approach to the diagnosis and treatment of gastric cancer.

Integrin alphavbeta5 identifies human corneal epithelial stem cells

AbstractPurpose Stem cell (SC) therapy is the main treatment modality to restore vision in patients with limbal stem cell deficiency. If corneolimbal epithelial stem cells (LESC) can be readily identified, isolated and maintained ex vivo, better quality grafts can be generated for patients with this debilitating disease. With prior knowledge that the extracellular matrix protein vitronectin (VN) is present within the LESC niche and that it supports SC in vitro, we postulated that the VN receptor αvβ5 is expressed by, and can be used to identify and isolate SC within the cornea.Methods Epithelial cells were isolate and expand from cadaveric human corneas and αvβ5 expression determined by flow cytometry and immunofluorescence. Integrin expressing cells were isolated by magnetic‐activated cell sorting then assessed by immunocytology, colony forming efficiency, RT‐PCR and microarray analysis.Results Integrin αvβ5+ cells co‐localized to N‐cadherin+/CK‐15+ putative LESC. αvβ5 was restricted to 3% of the total epithelial cells, which expressed more SC markers and formed more colonies compared to αvβ5‐ cells. Transcriptional profiling of αvβ5+/‐ cells by global gene array identified several interferon‐inducible genes, including the chemokine IP‐10, which localized to LESC. The role of IP‐10 in these cells is yet to be elucidated, but may be involved in immunoprotection by attracting mesenchymal SC into the corneal stroma.Conclusion Integrin αvβ5 is a candidate SC marker since its expression is restricted and αvβ5+ limbal epithelial cells have properties of LESC. Knowledge of the niche’s composition and the genes expressed by its SC will facilitate isolation and maintenance of these cells in culture for therapeutic purposes.

The CD44+ALDH+ Population of Human Keratinocytes Is Enriched for Epidermal Stem Cells with Long-Term Repopulating Ability

Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH(+) CD44(+) population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6(hi) CD71(lo) population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44(+) ALDH(+) keratinocytes exhibited other stem cell properties. CD44(+) ALDH(+) keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44(+) ALDH(+) cells, and holoclone formation in vitro. CD44(+) ALDH(+) cells were multipotent, producing greater numbers of hair follicle-like structures than CD44(-) ALDH(-) cells. Furthermore, 58% ± 7% of CD44(+) ALDH(+) cells exhibited label-retention. In vitro, CD44(+) ALDH(+) cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44(+) ALDH(+) population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs.

Leucine‐rich repeat‐containing G protein‐coupled receptor 5 expression in ductular reactions after chemotherapy for metastatic colorectal cancer

Oval cells with ductular reactions (DR) in damaged liver are referred to as "intermediate hepatobiliary cells". In a preliminary study, we had found expression of the leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) known as a potential marker for stem cells in the small intestine and colon in DR in liver damaged by chemotherapy for metastatic colorectal cancer (CRC). The aim of this study was to confirm LGR expression in DR in damaged liver after chemotherapy. A total of 68 liver specimens obtained after surgical resection were stained with monoclonal antibodies for cytokeratin (CK)7, neural cell adhesion molecule (NCAM; a bile ductular and liver progenitor cell marker), CD133 (a candidate stem cell marker of hepatocellular carcinoma as well as CRC) and LGR5. Additionally, these mRNA levels were investigated according to the location in damaged liver after chemotherapy using microdissected specimens. We observed that LGR5 was expressed in DR with CD133, CK7 and NCAM expression. By contrast, LGR5, CD133 and NCAM were not expressed in mature bile ducts with CK7. In transcriptional analysis, LGR5 mRNA levels in fibrotic tissue including DR were higher compared with that in adjacent normal liver without significant difference. These findings suggest that LGR5 may be involved in maintaining DR in damaged liver.

Characterization of sphere-forming cells with stem-like properties from the small cell lung cancer cell line H446

A relatively novel paradigm in tumor biology hypothesizes that cancer growth is driven by tumor cells with stem-like properties. However, direct proof of a population of stem cells in small cell lung cancer (SCLC) remains elusive. In this study, we enriched for stem-like cells from the SCLC cell line H446 by growing them as spheres in a defined serum-free medium. Sphere-derived cells have increased in vitro clonogenic and in vivo tumorigenic potentials as well as drug-resistant properties. After enrichment for stem-like cells, we used multiple candidate stem cell markers to examine the expression profile and found that the sphere-derived cells contained a higher proportion of cells expressing the stem cell surface markers uPAR and CD133 when compared with parental cells. To identify a selectable marker for the sphere-forming cells, we evaluated the sphere-forming abilities of uPAR+ and uPAR− cells as well as the sphere-forming abilities of CD133+ and CD133− cells. Both CD133+ and CD133− cell fractions were capable of forming spheres, and no statistically significant difference was observed in the sphere-forming efficiency between these two populations. In contrast, cells derived from the uPAR+ fraction were capable of forming spheres, whereas cells derived from the uPAR− fraction remained as single cells. Moreover, uPAR+ cells efficiently formed transplantable tumors, whereas uPAR− cells were unable to initiate tumors when transplanted at equivalent cell numbers. In addition, uPAR+ cells could differentiate into CD56+cells, CK+ cells, and uPAR− cells. These data support the existence of a population of tumor sphere-forming cells with stem cell properties in the H446 SCLC cell line. Furthermore, the stem cell population may be enriched in cells expressing the uPAR cell surface marker.

Analysis of immunoexpression of common cancer stem cell markers in ameloblastoma

Recent studies have established that, in benign tumors, a large number of cancer stem cells are present, which have great implications in tumor development. However, in ameloblastoma, a highly aggressive, locally invasive tumor with a high recurrence rate, whether or not cancer stem-like cells are present remains undetermined. Therefore, in this study we analyzed the protein expression of three candidate stem cell markers in ameloblastoma. Immunohistochemical staining for cancer stem cell (CSC) markers (CD133, CD44 and ABCG2) and for the proliferation marker Ki-67 was performed using 23 ameloblastoma cases. In all 23 samples, CD133, CD44 and ABCG2 were expressed. Nine (39.13%) cases showed high expression and 14 cases (60.87%) showed low expression for CD133. Twelve of the 23 cases (52.17%) showed high expression and 11 cases (47.83%) showed low expression for both CD44 and ABCG2, respectively. Ki-67 was mainly expressed in peripheral ameloblast-like cells, suggesting that these cells have a higher degree of differentiation and, therefore, are less likely to contain cancer stem-like cells. On the other hand, cells positive for CSC markers situated at the close proximity to peripheral cells were devoid of Ki-67 and may have the potential to be cancer stem-like cells. After analyzing the correlation between expression of three CSC markers with clinicopathological factors and Ki-67 expression, only CD44 expression was correlated with tumor recurrence (P=0.0391). In conclusion, this study showed various expression patterns of different types of cancer stem cell markers and the presence of candidate CSC-like cells in ameloblastoma, which are possibly involved in cell proliferation, tumor progression and recurrence.

CD133+ niches and single cells in glioblastoma have different phenotypes

Putative CD133(+) brain tumor stem cells have been shown to be located in niches and as single cells. This is the first study providing insight into the different phenotypes of CD133(+) cells in glioblastoma according to localization. Paraffin sections were stained by double immunofluorescence with CD133 and the candidate stem cell markers Sox2, Bmi-1, EGFR, podoplanin and nestin, the proliferation marker Ki67 and the endothelial cell markers CD31, CD34, and VWF. Cell counting showed that the CD133(+) cells in the niches had a significantly higher expression of Sox2, EGFR and nestin compared to CD133(+) single cells, but only a 3% Ki67 labeling index versus 14% found for CD133(+) single cells. Only low endothelial cell marker expression was found in the niches or the CD133(-) tumor areas, while 43% CD133(+)/CD31(+) and 25% CD133(+)/CD34(+) single cells were found. CD133(+) blood vessels within CD133(+) niches were less proliferative and more often Bmi-1(+) than CD133(+) blood vessels outside niches. In conclusion, different CD133(+) cell phenotypes exist according to the in situ localization, and also the phenotype of CD133(+) blood vessels vary according to the localization. CD133(+) niches contain stem-like cells with a lower proliferation index than CD133(+) single cells, which have an endothelial differentiation profile suggesting a role in angiogenesis.

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA-expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU-incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell-lineage-specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell-lineage-specific markers during archeocyte differentiation into other cell types. Choanocytes are food-entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell-like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.

Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1, where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study, RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies, flow cytometry, and chromatin immunoprecipitation identified two candidate stem cell marker genes, CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1), to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors, we detected a relatively abundant CD133(high)-ASCL1(high)-ALDH1(high) subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133(high) subpopulation depended on continued ASCL1 expression. Whereas CD133(high) cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor, CD133(low) cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity, including variation in key regulatory factors such as ASCL1, can modulate tumorigenicity in SCLC.

Independent prognostic value of the basal‐like phenotype of breast cancer and associations with EGFR and candidate stem cell marker BMI‐1

To study the relationship between basal-like breast cancers, epidermal growth factor receptor (EGFR) and candidate stem cell markers (BMI-1, EZH2, Oct-4) in a population-based setting. Immunohistochemistry was evaluated in a series of 190 breast cancers. Basal-like phenotype (BLP) 1-5 was found in 4.3-14.3% of cases. EGFR was expressed in 9% of cases and associated with cytokeratin (CK) 5 and P-cadherin positivity, but not with survival; 28% of CK5+ cases were EGFR+. On multivariate analysis, basal-like differentiation and lymph node status were independent prognostic factors of comparable strength. BMI-1 positivity (42.6%) was associated with absence of basal-like features, oestrogen receptor positivity and low Ki67, but not related to survival. BMI was not associated with EZH2 expression, and these markers tended to show opposite associations with other variables, suggesting different roles in breast cancer. Oct-4 expression was not detected in this series. Basal-like features and lymph node status were strong and independent prognostic factors in this population-based series of breast cancer. Neither EGFR nor BMI-1 had significant prognostic impact, whereas EZH2 expression was associated with decreased survival. BMI-1 was inversely related to basal-like factors, and a stem cell phenotype of the basal-like subgroup could not be verified by this marker.

Prostatic Stem Cell Marker Identified by cDNA Microarray in Mouse

Identifying prostatic stem cells is important to elucidate the mechanisms by which the prostate develops and control prostate cancer. We recently reported that the proximal region of the mouse prostate contains a population of stem cells. However, to our knowledge the specific marker of stem cells in the proximal region remains unknown. We performed cDNA microarray analysis of cells obtained from the proximal region and from the remaining regions in dorsal prostates to identify several candidate stem cell markers. After we focused on 1 candidate among them we confirmed the expression of this candidate gene by reverse transcriptase-polymerase chain reaction analysis and immunohistochemistry. We also investigated the relation between positive cells for this marker and those for telomerase reverse transcriptase. Finally, we investigated the functional potential of prominin positive cells in 3-dimensional culture. Seven of 4,800 genes analyzed showed proximal/remaining ratios greater than 20. Of these genes we focused on prominin because it is a cell surface marker widely used to identify and isolate stem cells from various organs. We found a prominin positive cell population enriched in the basal cell layer in the proximal region, and the coincidence of prominin and telomerase reverse transcriptase immunostaining. We also found that prominin positive cells gave rise to numerous and large-branched ducts, whereas prominin negative cells formed far fewer such structures in 3-dimensional culture. A small population of prominin positive cells in the mouse prostate basal layer of the proximal region represents a stem cell population.