ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma

Background

Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.

Methodology/Principal Findings

We have isolated populations of cells with high ALDH activity (ALDH^high) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDH^high and ALDH^low cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice.

Conclusions/Significance

We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133⁺ and ALDH^low cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH^high cell populations. ALDH^high/CD44⁺/CD24⁺ or ALDH^low/CD44⁺/CD24⁺ phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.     

Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+), yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) > CD133(+)ALDH(-) > CD133(-)ALDH(-). ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population.     

Uncoupling Warburg effect and stemness in CD133<Superscript>+ve</Superscript> cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133^+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca²⁺ signaling and cell cycle analysis. CD133^+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133^+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133^+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133^+ve cells. In summary, both CD133^+ve/?ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133^+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.     

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APC ^{Min +/-} mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APC ^{Min +/-} mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.     

Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4

Endometrial cancer (EC) is the most common familiar gynecologic malignant tumor identified in the female reproductive system and has been increasing yearly. In this study, we will identify the surface markers and stem cell markers related with cancer stem cells (CSCs) of EC. Tissue samples were obtained from endometrial cancer patients during surgical procedures. Single cells were isolated from the tissues for culturing, transfection into nude mice, and histopathology analysis. RT-PCR demonstrated that the cultured cells strongly expressed stemness-related genes, such as c-Myc, Sox-2, Nanog, Oct 4A, ABCG2, BMI-1, CK-18, Nestin and β-actin. The expression of surface markers CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3 and SSEA4, CD24, and CD133 and chemokine markers such as CXCR4 were measured by flow cytometry. Then the double percentage of CD133+CXCR4+ cells constituted 7.2% and 9.3% in EC cells originated from two different patients, respectively. The CD133+CXCR4+ primary endometrial cancer cells grew faster, exhibited high expression of mRNA of stemness-related genes, produced more spheres, and had higher clonogenic ability than other subpopulations. They are also more resistant to anti-cancer drugs than other subpopulations. These findings indicate that CD133+CXCR4+ cells may possess some characteristics of CSCs in primary endometrial cancer. These cell surface markers may be useful for the development of drugs against CSC molecular targets or as a predictive marker for poor prognosis in primary endometrial cancer.     

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny¹. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44⁺CD24^low/-)². Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues^3-5.Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD133^6-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH⁺ and CD44⁺CD24⁺ pancreatic CSCs are similarly tumorigenic, but ALDH⁺ cells are relatively more invasive⁸. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts⁹. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH¹⁰. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.     

New emerging roles of CD133 in cancer stem cell: Signaling pathway and miRNA regulation

Cancer stem cells (CSC) are rare immortal cells within a tumor that are able to initiate tumor progression, development, and resistance. Advances studies show that, like normal stem cells, CSCs can be both self-renewed and given rise to many cell types, therefore form tumors. A number of cell surface markers, such as CD44, CD24, and CD133 are frequently used to identify CSCs. CD133, a transmembrane glycoprotein, either alone or in collaboration with other markers, has been mainly considered to identify CSCs from different solid tumors. However, the exactness of CD133 as a cancer stem cell biomarker has not been approved yet. The clinical importance of CD133 is as a CSC marker in many cancers. Also, it contributes to shorter survival, tumor progression, and tumor recurrence. The expression of CD133 is controlled by many extracellular or intracellular factors, such as tumor microenvironment, epigenetic factors, signaling pathways, and miRNAs. In this study, it was attempted to determine: 1) CD133 function; 2) the role of CD133 in cancer; 3) CD133 regulation; 4) the therapeutic role of CD133 in cancers.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133^high/CD44^high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133^low/CD44^low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133^high/CD44^high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.     

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44^high/EGFR^low, CD44^high/EGFR^high and CD44^low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44^high/EGFR^low population showed a spindle-shaped EMT-like morphology, while the CD44^low population was dominated by cobblestone-shaped cells. The CD44^high/EGFR^low population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44^high/EGFR^low cells in comparison to CD44^low cells. Moreover, CD44^high/EGFR^low cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44^high/EGFR^high and CD44^low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

One of the theories regarding oral carcinogenesis is that the tumor growth is initiated from cancer stem cells (CSCs) that self-renew and give rise to differentiated tumor cells, like stem cells do in normal tissues. The most common methods of CSC identification are based on CSC marker expression in carcinogenesis. This study examined the expression of CD133 and CD44, the most commonly used CSC biomarkers in oral squamous cell sarcoma (SCC), with the goal of identifying molecular biomarkers whose expression is associated with the multistep oral carcinogenesis. The expression of CD133, CD44, proliferating cell nuclear antigen (PCNA), and Cytokeratin (CK) was examined by Western blot analysis and confirmed by immunohistochemistry in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model. Also, the expression of aldehyde dehydrogenase 1 (ALDH1), OCT-4 and Nanog were investigated for alteration of cancer cell stemness by Western blot. Along with the progress of multistep carcinogenesis, there were slight increases of CD133 and CD44 expression in the dysplasia group compared with normal rats. However, CD133 protein level was significantly overexpressed in SCC. The expression of PCNA and CK were low in normal group, but sequentially increased in SCC. ALDH1, Nanog and OCT-4 expression were significantly increased according to SCC grade during carcinogenesis. The findings indicate that CD133 is useful in identifying oral CSCs, which suggests that CD133 may serve as a predictor to identify CSCs with a high risk of oral cancer development.     

CD98 marks a subpopulation of head and neck squamous cell carcinoma cells with stem cell properties

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98^high cells, in contrast to CD98^low cells, are able to generate tumors in immunodeficient mice, indicating that CD98^high cells have stem cell characteristics. Furthermore, the CD98^high subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98^low fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.     

Aldehyde dehydrogenase 1, a potential marker for cancer stem cells in human sarcoma

Tumors contain a small population of cancer stem cells (CSC) proposed to be responsible for tumor maintenance and relapse. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in different cancer types. This study used the Aldefluor® assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1^high cells from five human sarcoma cell lines and one primary chordoma cell line. ALDH1^high cells range from 0.3% (MUG-Chor1) to 4.1% (SW-1353) of gated cells. Immunohistochemical staining, analysis of the clone formation efficiency, and xCELLigence microelectronic sensor technology revealed that ALDH1^high cells from all sarcoma cell lines have an increased proliferation rate compared to ALDH1^low cells. By investigating of important regulators of stem cell biology, real-time RT-PCR data showed an increased expression of c-Myc, β-catenin, and SOX-2 in the ALDH1^high population and a significant higher level of ABCG2. Statistical analysis of data demonstrated that ALDH1^high cells of SW-982 and SW-1353 showed higher resistance to commonly used chemotherapeutic agents like doxorubicin, epirubicin, and cisplatin than ALDH1^low cells. This study demonstrates that in different sarcoma cell lines, high ALDH1 activity can be used to identify a subpopulation of cells characterized by a significantly higher proliferation rate, increased colony forming, increased expression of ABC transporter genes and stemness markers compared to control cells. In addition, enhanced drug resistance was demonstrated.     

Expression of Androgen Receptor and Cancer Stem Cell Markers (CD44+/CD24− and ALDH1+): Prognostic Implications in Invasive Breast Cancer

BACKGROUND: Androgen receptor (AR) has emerged as a significant prognostic marker in early breast cancer (BCa). Association of AR with cancer stem cell (CSC) markers in BCa is unknown. Aim of the present study was to evaluate the immunohistochemical expression of AR, CD44, CD24 and ALDH1 in a cohort of Pakistani patients diagnosed with invasive BCa and to correlate the expression with 5- year disease free survival. PATIENTS AND METHODS: We evaluated immunohistochemical expression AR, CD44, CD24 and ALDH1 in formalin fixed paraffin embedded archival blocks of 166 cases of primary invasive BCa (stage I-III) and correlated the expression with clinicopathological variables and outcome using univariable and multivariable analysis. Survival data was computed by Kaplan Meier curves. RESULTS: Expression of AR was observed in 62.7% tumors whereas CD44, CD24 and ALDH1 were expressed in 61.4%, 44% and 30.1% tumors, respectively. AR expression was significantly associated with T1-T2 tumors, lower grade, estrogen and progesterone receptor expression (P < .05) and remained an independent prognostic indicator in multivariable analysis (adjusted HR 0.33, 95% CI 0.13–0.81; P = .016). Significant association was observed between concordant expression of AR and CD24 (P = .001) with a favorable impact on survival (P = .007) whereas expression of CSC phenotypes (CD44⁺, CD44⁺/CD24^? and ALDH1⁺) did not correlate with adverse outcome (P > .05). However, AR expression retained the association with better prognosis even in patients whose tumors exhibited a CSC phenotype. CONCLUSIONS: Expression of AR and CD24 in stage I-III invasive BCa correlates with favorable clinicopathological features and delineates a subgroup of patients with better disease-free survival.     

Side Population in Human Non-Muscle Invasive Bladder Cancer Enriches for Cancer Stem Cells That Are Maintained by MAPK Signalling

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2^hi) relative to the non-SP (NSP) fraction (ABCG2^low). ABCG2^hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2^low NSP cells. In vivo, ABCG2^hi SP cells enriched for tumour growth compared with ABCG2^low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2^hi SP cells and MEK inhibition also inhibited the ABCG2^hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2^hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

Aldehyde Dehydrogenase 1 (ALDH1) Is a Potential Marker for Cancer Stem Cells in Embryonal Rhabdomyosarcoma

Cancer stem cells (CSCs) are defined as a small population of cancer cells with the properties of high self-renewal, differentiation, and tumor-initiating functions. Recent studies have demonstrated that aldehyde dehydrogenase 1 (ALDH1) is a marker for CSCs in adult cancers. Although CSCs have been identified in some different types of pediatric solid tumors, there have been no studies regarding the efficacy of ALDH1 as a marker for CSCs. Therefore, in order to elucidate whether ALDH1 can be used as a marker for CSCs of pediatric sarcoma, we examined the characteristics of a population of cells with a high ALDH1 activity (ALDH1^high cells) in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. We used the human embryonal RMS (eRMS) cell lines RD and KYM-1, and sorted the cells into two subpopulations of ALDH1^high cells and cells with a low ALDH1 activity (ALDH1^low cells). Consequently, we found that the ALDH1^high cells comprised 3.9% and 8.2% of the total cell population, respectively, and showed a higher capacity for self-renewal and tumor formation than the ALDH1^low cells. With regard to chemoresistance, the survival rate of the ALDH1^high cells was found to be higher than that of the ALDH1^low cells following treatment with chemotherapeutic agents for RMS. Furthermore, the ALDH1^high cells exhibited a higher degree of pluripotency and gene expression of Sox2, which is one of the stem cell markers. Taken together, the ALDH1^high cells possessed characteristics of CSCs, including colony formation, chemoresistance, differentiation and tumor initiation abilities. These results suggest that ALDH1 is a potentially useful marker of CSCs in eRMS.     

A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem‐like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24‐ cell populations) and the mature luminal cells (CD49f‐EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label‐free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value <0.05, 305, 322 and 98 proteins were identified as significantly different in three pairwise comparisons of ALDH+ versus CD44+CD24‐, ALDH+ versus CD49f‐EpCAM+ and CD44+CD24‐ versus CD49f‐EpCAM+, respectively. Pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti‐CSC therapeutics.