Potential Cancer Stem Cell Marker Research Articles

MicroRNA and transcriptome analysis in periocular Sebaceous Gland Carcinoma

Sebaceous gland carcinoma (SGC) is a rare, but life-threatening condition with a predilection for the periocular region. Eyelid SGC can be broadly categorised into two subtypes, namely either nodular or pagetoid with the latter being more aggressive and requiring radical excision to save life. We have identified key altered microRNAs (miRNA) involved in SGC shared by both subtypes, hsa-miR-34a-5p and hsa-miR-16-5p. However, their gene targets BCL2 and MYC were differentially expressed with both overexpressed in pagetoid but unchanged in nodular suggesting different modes of action of these two miRNAs on BCL/MYC expression. Hsa-miR-150p is nodular-specifically overexpressed, and its target ZEB1 was significantly downregulated in nodular SGC suggesting a tumour suppressor role. Invasive pagetoid subtype demonstrated specific overexpression of hsa-miR-205 and downregulation of hsa-miR-199a. Correspondingly, miRNA gene targets, EZH2 (by hsa-miR-205) and CD44 (by hsa-miR-199a), were both overexpressed in pagetoid SGC. CD44 has been identified as a potential cancer stem cell marker in head and neck squamous cell carcinoma and its overexpression in pagetoid cells represents a novel treatment target. Aberrant miRNAs and their gene targets have been identified in both SGC subtypes, paving the way for better molecular understanding of these tumours and identifying new treatment targets.

Abstract P6-07-03: Long non-coding RNA H19 promotes cancer stemness and worsen breast cancer survival

Abstract Background: Cancer stem cells (CSC) are good sources of tumor initiation, heterogeneity, progression, and metastasis because of their unique characteristics. Several potential markers for CSCs have been suggested for breast cancer, including CD44+/CD24−/low, aldehyde dehydrogenase 1 (ALDH1), and epithelial cell adhesion molecule/epithelial-specific antigen. We previously reported that ALDH1 gene expression is related to aggressive phenotypes and poor prognosis in breast cancers. In this study, we conducted differential analysis of mRNA expression in ALDH1-positive breast cancer to identify genes associated with CSC. Next, we performed basic and clinical studies of one gene. Methods: Messenger RNA was isolated from ALDH1-positive cells and ALDH1-negative cells in 5 ALDH1-positive breast cancers. Microarray analysis revealed that several genes were significantly associated with the ALDH1 gene. Among them, we examined a long non-coding RNA of H19 in this study. We evaluated the effect of H19 on CSCs using RNA interference and a sphere formation assay using two cell lines, HCC1937 and iCSCL10A cells. We also investigated H19 expression in 192 surgical specimens by in situ hybridization and analyzed the relationship between H19 expression and clinic pathological findings in breast cancer patients. Results: Through in vitro experiments, we confirmed that suppression of H19 reduced sphere formation in both HCC1937 and iCSCL10A cells. Among surgical specimens, 48 samples (25%) expressed H19. We verified thatH19 positivity was significantly higher in ALDH1-positive cases than in ALDH1-negative cases (68% vs 9.7%, p < 0.001). H19 was significantly highly expressed in triple-negative breast cancer (TNBC) (46%) compared with other subtypes: luminal (33%), luminal-HER2 (6%), and HER2-enriched subtype (15%). H19-positive patients showed significantly worse prognosis (5-year disease-free survival 75.8% vs 91.5%, p = 0.001 and 5-year overall survival 88.7% vs 97.7%, p = 0.002). The effect of H19 expression on prognosis was the most significant in TNBC compared to in other subtypes (5-year disease-free survival 63.6% vs 88.9%, p = 0.038). Conclusions: H19 is clearly associated with CSCs and correlated with poor prognosis in breast cancer patients, particularly TNBC. Our future studies will investigate the role of H19 in maintaining the nature of CSCs and protein-coding genes associated with H19. Citation Format: Shima H, Kida K, Yamada A, Sugae S, Narui K, Miyagi Y, Ryo A, Ichikawa Y, Ishikawa T, Endo I. Long non-coding RNA H19 promotes cancer stemness and worsen breast cancer survival [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-03.

Gli1 is a potential cancer stem cell marker and predicts poor prognosis in ductal breast carcinoma

Glioma-associated oncogene homolog 1 (Gli1) maintains the cancer stem cell-like characteristics in various tumors. However, its expression in cancer stem cells (CSC) in ductal breast carcinoma has not been well studied. We aimed to characterize Gli1 as a potential CSC marker and investigate its clinical significance in ductal breast carcinoma. Immunohistochemical staining was used to study the relationship of Gli1 to clinicopathologic features, cell cycle regulation-related genes, and CSC markers. Gli1 was expressed to a greater extent in ductal breast carcinoma than in normal breast tissues (P=.002). Its expression was significantly correlated with tumor grade (P=.044), pT stage (P=.017), and molecular subtype (P=.008). Expression was associated, not only with the expression of HIF-1α (P<.001), but also with greater microvessel density (MVD; P=.012). Kaplan-Meier survival analysis revealed that Gli1 was significantly associated with lower overall survival (OS; P=.02). Univariate Cox regression analysis confirmed that Gli1 was a poor prognostic factor for OS (P=.037) and was associated with the expression of the cell cycle-related genes cyclin D1 (P=.011), p21 (P=.009), and pAkt-Thr308 (P=.038). Moreover, Gli1 expression correlated significantly with the expression of two CSC markers, Sox2 (P=.01) and LSD1 (P=.01). Gli1 could be a stem cell marker and an indicator of poor prognosis in patients with ductal breast carcinoma.

Expression of Cancer Stem Cell Markers OCT4 and CD133 in Transitional Cell Carcinomas.

Treatment failure, recurrence, and metastasis in bladder cancer are attributed to a subset of tumor cells expressing cancer stem cell (CSC) markers. This study aimed to explore the expression levels and the clinical significance of putative CSC markers OCT4 and CD133 in bladder cancer. Tissue microarray-based immunohistochemical analysis was applied to investigate the expression patterns of potential CSC markers OCT4 and CD133 in bladder cancer samples. The correlation between the expressions of each marker and clinicopathologic parameters was then analyzed. There was a significant association between OCT4 expression and the TNM stage of bladder cancer (P<0.001). Our analysis demonstrated a significant association between the intensity of staining and the presence of lamina propria and muscularis propria invasion (P=0.02 and 0.02, respectively), whereas a relative inverse correlation was found between CD133 expression with lamina propria invasion (P=0.051) and muscularis propria invasion (P=0.07). The correlation of OCT4, but not CD133, with the invasiveness of bladder cancer revealed that OCT4 can be considered as a key regulator of tumor progression, aggressive behavior, and metastasis; therefore, OCT4 can be a potential marker for targeted therapy of bladder cancer.

OV6+ cancer stem cells drive esophageal squamous cell carcinoma progression through ATG7-dependent β-catenin stabilization

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that exhibit capacities for tumor initiation and progression. Identifying CSCs and their related pathways is necessary for the development of new therapeutic targets against tumors. However, the molecular mechanism of CSCs in esophageal squamous cell carcinoma (ESCC) remains elusive. This study demonstrated that OV6 expression was closely associated with ESCC patients' clinical outcome and prognosis. OV6+ cells possessed stronger stem-like properties, including self-renewal, stem cell-associated gene expression, tumorigenicity, chemo-resistance, invasion, and metastasis. Autophagy maintained the stem-like properties of OV6+ cells by stabilizing ATG7-dependent β-catenin. Furthermore, a significantly positive correlation between ATG7 and OV6 expression was detected in human ESCC biopsies, and this correlation could be used to predict ESCC patients' prognosis. Taken together, our findings provide a novel potential CSC marker for ESCC. OV6+ cancer stem cells can promote the progression of ESCC through ATG7-dependent β-catenin stabilization. OV6 may serve as a novel prognostic biomarker and therapeutic target for ESCC patients.

Co-Expression of Putative Cancer Stem Cell Markers CD44 and CD133 in Prostate Carcinomas.

Cancer stem cells (CSCs) are the main players of prostate tumorigenesis thus; characterization of CSCs can pave the way for understanding the early detection, drug resistance, metastasis and relapse. The current study was conducted to evaluate the expression level and clinical significance of the potential CSC markers CD44 and CD133 in a series of prostate tissues. One hundred and forty eight prostate tissues composed of prostate cancer (PCa), high-grade prostatic intraepithelial neoplasia (HGPIN), and benign prostate hyperplasia (BPH) were immunostained for the putative CSC markers CD44 and CD133. Subsequently, the correlation between the expression of these markers and the clinicopathological variables was examined. A higher level of CD44 expression was observed in 42% of PCa, 57% of HGPIN, and 42% BPH tissues. In the case of CD133 expression PCa, HGPIN, and BPH samples demonstrated high immunoreactivity in 46%, 43%, and 42% of cells, respectively. Statistical analysis showed an inverse significant correlation between CD44 expression with Gleason score of PCa (P = 0.02), while no significant correlation was observed between CD133 expression and clinicopathological parameters. A significant reciprocal correlation was observed between the expression of two putative CSC markers CD44 and CD133 in PCa specimens while not indicating clinical significance. Further clinical investigation is required to consider these markers as targets of new therapeutic strategies for PCa.

Prostate Cancer Stem Cell Markers Drive Progression, Therapeutic Resistance, and Bone Metastasis.

Metastatic or recurrent tumors are the primary cause of cancer-related death. For prostate cancer, patients diagnosed with local disease have a 99% 5-year survival rate; however, this 5-year survival rate drops to 28% in patients with metastatic disease. This dramatic decline in survival has driven interest in discovering new markers able to identify tumors likely to recur and in developing new methods to prevent metastases from occurring. Biomarker discovery for aggressive tumor cells includes attempts to identify cancer stem cells (CSCs). CSCs are defined as tumor cells capable of self-renewal and regenerating the entire tumor heterogeneity. Thus, it is hypothesized that CSCs may drive primary tumor aggressiveness, metastatic colonization, and therapeutic relapse. The ability to identify these cells in the primary tumor or circulation would provide prognostic information capable of driving prostate cancer treatment decisions. Further, the ability to target these CSCs could prevent tumor metastasis and relapse after therapy allowing for prostate cancer to finally be cured. Here, we will review potential CSC markers and highlight evidence that describes how cells expressing each marker may drive prostate cancer progression, metastatic colonization and growth, tumor recurrence, and resistance to treatment.

The Met tyrosine kinase receptor as a therapeutic target and a potential cancer stem cell factor responsible for therapy resistance (Review).

The MET tyrosine kinase receptor plays an important role during tumor development and progression being responsible for proliferation, morphogenetic transformation, cell motility and invasiveness. High expression of the MET receptor has been shown to correlate with increased tumor growth and metastasis, poor prognosis and resistance to radiotherapy. Moreover, MET expression and activation has been shown to be associated with therapy resistance. The occurrence of resistance to targeted therapy might be related to the presence of cancer stem cells (CSCs). CSCs are a subpopulation of cells in the tumor that possess the ability of self-renewal, clonogenicity, radioresistance and self-sustained protection from apoptosis. Recently, MET has been postulated as an essential factor supporting the functional stem cell phenotype in some tumors and as a CSC factor is believed to be responsible for therapy resistance. This review presents the results from recent studies identifying MET as a potential marker of CSCs and tumor initiating cells, demonstrating pivotal role of MET in supporting stem cell phenotype and indicating the role of MET in acquiring resistance to antitumor therapy.

Neural Stem Cell Marker Nestin As a Novel Unfavorable Prognostic Biomarker for Multiple Myeloma

Background:Neural stem cell marker nestin is a suitable diagnostic and prognostic indicator of malignancy and potential cancer stem cells marker in solid tumors. Currently, nestin has been demonstrated as an epithelial-to-mesenchymal transition related protein associated with tumor metastasis, chemoresistance, and metabolic changes through altering mitochondrial dynamics in solid tumors. However its role in hematological malignancies remains unknown. Our recent work confirmed nestin protein as a tumor-specific marker for CD138+38+ plasma cells (PC) of multiple myeloma (MM) and potential predictor of worse response to conventional and novel therapy but prognostic potential of nestin has never been studied in MM so far.Aim:The aim of this study was to evaluate whether pretreatment levels of nestin can predict overall (OS) and progression-free (PFS) survival of MM patients.Methods: Nestin was detected in CD138+38+PC of 84 newly diagnosed MM patients (36M/48F; median age 70 years) by flow cytometry. Nestin was assessed as the percentage of nestin-positive PC (Nes+PC) and ratio of median fluorescence intensity of nestin (MFI ratio) and isotypic control. Survival cut-off points were established based on time-dependent ROC analysis. P-values below 0.05 were considered as statistically significant in all analyses. Cox proportional hazards models were used to assess the association of prognostic factors with OS and PFS. The independence of the marker was assessed by multivariate Cox regression model. Survival rates were estimated using the Kaplan-Meier method. Differences in survival among subgroups of patients were compared using the log-rank test.Results: ROC analysis divided patients into 2 groups: high (>50) and low (≤50) %Nes+PC subgroup and high (>4) and low (≤4) MFI subgroup. High %Nes+PC subgroup were associated with shorter OS (p=0.042) and PFS (p=0.003). High MFI ratio subgroup (>4.4) were significantly associated with shorter OS (p=0.005); and PFS (p=0.025) (Fig.1). Univariate hazard ratios (HR) showed prognostic impact of %Nes+PC (p=0.003) and of MFI ratio (p=0.017) for OS; %Nes+PC (p=0.005) and of MFI ratio (p=0.028) for PFS (Tab.1). 5 parameters: ISS: stage 3 vs. stage 1, del p53, age (at diagnosis), M protein type: IgG vs. other, LDH (µkat/l) and Beta2 microglobulin (mg/l) were significantly associated with OS after adjustment by nestin. However %Nes+PC or MFI ratio did not achieve independent prognostic status in multivariable model (p=0.223 and p=0.989).Conclusion: These data demonstrate for the first time that nestin protein might have prognostic implication for MM. However, it does not appear to be independently associated with MM outcome when considering standard prognostic factors. The growing importance of nestin in key cell processes responsible for tumorigenesis and metastasis emphasizes the need for further investigation in MM. This pilot study requires to be further validated on a larger cohort of patients.Supported by NT14575, RVO-FNOs/2014/17P, IRP201550, SGS03/LF/2015-2016. [Display omitted] [Display omitted] DisclosuresBesse:Mundipharma-EDO: Other: travel support. Hájek:Takeda: Consultancy; BMS: Honoraria; Janssen: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding.

Integrin α7 is a functional cancer stem cell surface marker in oesophageal squamous cell carcinoma.

Non-CG methylation has been associated with stemness regulation in embryonic stem cells. By comparing differentially expressed genes affected by non-CG methylation between tumour and corresponding non-tumour tissues in oesophageal squamous cell carcinoma (OSCC), we find that Integrin α7 (ITGA7) is characterized as a potential cancer stem cell (CSC) marker. Clinical data show that a high frequency of ITGA7+ cells in OSCC tissues is significantly associated with poor differentiation, lymph node metastasis and worse prognosis. Functional studies demonstrate that both sorted ITGA7+ cells and ITGA7 overexpressing cells display enhanced stemness features, including elevated expression of stemness-associated genes and epithelial–mesenchymal transition features, as well as increased abilities to self-renew, differentiate and resist chemotherapy. Mechanistic studies find that ITGA7 regulates CSC properties through the activation of the FAK-mediated signalling pathways. As knockdown of ITGA7 can effectively reduce the stemness of OSCC cells, ITGA7 could be a potential therapeutic target in OSCC treatment.

Gli1, a potential regulator of esophageal cancer stem cell, is identified as an independent adverse prognostic factor in esophageal squamous cell carcinoma

The hedgehog (Hh) pathway is involved in cancer stem cell (CSC) maintenance in various tumors. Glioma-associated oncogene homolog 1 (Gli1) is a key mediator of the Hh pathway; however, its expression and clinical significance in esophageal squamous cell carcinoma (ESCC) have not been reported. In this study, we aimed to reveal clinical significance of Gli1 expression in ESCC and further investigate the potential of Gli1 as a CSC regulator of ESCC by comparing its expression with expressions of other stemness genes in ESCC. We assessed the expressions of Gli1, Sox9, CD44, Sox2, LSD1, and Oct4 in 127 patients' tissue specimens of ESCC using immunohistochemistry and in ESCC cell lines using Western blotting. The relationship of Gli1 expression with clinic-pathologic parameters as well as cell-cycle-regulating genes was investigated. We also investigated the biological pathways that are activated in Gli1-high ESCC using The Cancer Genome Atlas (TCGA) data. Gli1 expression was observed in 28.3% of ESCC, and its expression was correlated with the expression of stemness genes, Sox9 (P=0.003) and CD44 (P=0.012). And Gli1, CD44, and Sox9 were highly expressed in more poorly differentiated ESCC cell lines such as TE8 and TE1 cells. Notably, Gli1 expression was positively associated with distant metastasis (P=0.011), increased microvessel density (MVD) (P=0.002), and expression of cell cycle regulators such as p21, cyclin D1, cyclin E1, and NF-κB (P<0.05). Sox9 and CD44 expressions in ESCC were also significantly associated with unfavorable clinic-pathologic parameters such as increased MVD, advanced tumor (pT) stage, and higher TNM stage. Moreover, all three potential CSC markers such as Gli1, Sox9, and CD44 were strongly linked to worse clinical outcome and independent poor prognostic factors in overall survival and disease-free survival in ESCC. Gene set enrichment analysis revealed that the Gli1-high-expressing ESCC patients' group was strongly enriched for gene expression signature of Hh signaling pathway, epithelial-mesenchymal transition, and cancer stem cell. Targeting Gli1, a potential diagnostic marker of ESCC stem cells, will have a profound therapeutic and prognostic value.

Evaluation of circulating cellular DCLK1 protein, as the most promising colorectal cancer stem cell marker, using immunoassay based methods.

DCLK1, as the most potential colorectal cancer stem cell (CSC) marker has been the core of many recent investigations. However, no study has been performed to evaluate the circulating cellular DCLK1 protein (CCDP) that might reflect the presences of colorectal CSC in circulation. We aimed to evaluate CCDP in the peripheral blood mononuclear cells (PBMCs) of colorectal cancer (CRC) patients applying immunoassay based methods including PLA, IPCR and ELISA in order to introduce the method of choice for clinical detection of CCDP. PBMCs were extracted from blood samples of 58 CRC patients along with 58 blood samples of tumor free controls. Total protein of PBMC was extracted and the CCDP level was evaluated. The results of three applied immunoassay tests were compared and the best approach for clinical application was introduced, accordingly. In addition, the correlation of CCD Plevel with clincopathologic findings of CRC patients was assessed. The results of three immuneassay methods confirmed each other. Based on our finding, ELISA could be the most judicious method for clinical evaluation of CCDP considering its simplicity for clinical implications. Our results also showed a significant higher amount of CCDP in peripheral blood of CRC patients compared to control group which was also correlated with patients' clinicopathologic finding such as stage, grade and neoadjuvant history. CCDP could be applied for monitoring purposes in CRC patients. However, its application needs to be more elucidated in future investigations implementing larger samples.

CD114: A New Member of the Neural Crest-Derived Cancer Stem Cell Marker Family.

The neural crest is a population of cells in the vertebrate embryo that gives rise to a wide range of tissues and cell types, including components of the peripheral nervous system and the craniofacial skeleton as well as melanocytes and the adrenal medulla. Aberrations in neural crest development can lead to numerous diseases, including cancers such as melanoma and neuroblastoma. Cancer stem cells (CSCs) have been identified in these neural crest-derived tumors, and these CSCs demonstrate resistance to treatment and are likely key contributors to disease relapse. Patients with neural crest-derived tumors often have poor outcomes due to frequent relapses, likely due to the continued presence of residual treatment-resistant CSCs, and therapies directed against these CSCs are likely to improve patient outcomes. CSCs share many of the same genetic and biologic features of primordial neural crest cells, and therefore a better understanding of neural crest development will likely lead to the development of effective therapies directed against these CSCs. Signaling through STAT3 has been shown to be required for neural crest development, and granulocyte colony stimulating factor (GCSF)-mediated activation of STAT3 has been shown to play a role in the pathogenesis of neural crest-derived tumors. Expression of the cell surface marker CD114 (the receptor for GCSF) has been identified as a potential marker for CSCs in neural crest-derived tumors, suggesting that CD114 expression and function may contribute to disease relapse and poor patient outcomes. Here we review the processes of neural crest development and tumorigenesis and we discuss the previously identified markers for CSC subpopulations identified in neural crest tumors and their role in neural crest tumor biology. We also discuss the potential for CD114 and downstream intracellular signaling pathways as potential targets for CSC-directed therapy. J. Cell. Biochem. 118: 221-231, 2017. © 2016 Wiley Periodicals, Inc.

The role of the cancer stem cell marker USP22 in tumors

Ubiquitin specific peptidase 22 (USP22) is one of the 11 known death-from-cancer signature genes. USP22 is also considered a cancer stem cell marker and is highly expressed in a variety of tumor types. USP22 alters the expression of tumor-associated genes (e.g., proto-oncogenes and tumor suppressor genes) and promotes tumor cell proliferation, invasion and metastasis via the modification of cell cycle activity and the activation of a variety of signaling pathways. In patients with tumors, USP22 expression is related to lymphatic metastasis, clinical stage of tumors, survival rate and a number of other factors. In addition, USP22 is considered an indicator of a poor prognosis and thus may be used to evaluate the prognosis of patients with tumors. As a potential cancer stem cell marker, USP22 may serve as a novel target for tumor therapies. Finally, USP22 also plays an important role in tumor development and progression.

Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas.

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment.

CD90 a potential cancer stem cell marker and a therapeutic target.

Cancer Stem Cells (CSCs) have been recently identified and their role in carcinogenesis has been ascertained. CSCs have been correlated with high relapse in certain cancers, multiple drug resistance against chemotherapy and metastasis. Several markers such as CD133, CD24, CD44, EpCAM, and CD26 have been identified to isolate and characterize CSCs. None of these markers or their combinations are universal in nature and can be used to isolate CSCs from all types of cancer. CD90 is one such marker whose expression has been extensively studied in recent years. CD90+ cells have been isolated from several types of tumors and shown to exhibit cardinal properties of CSCs such as proliferation, differentiation, spheroid formation, metastasis and ability to form tumor xenograft in immunodeficient mice. It is also found to be co-expressed with several other CSC markers. CD90 is therefore, suggested as a candidate marker as well as a potential therapeutic target for elimination of CSCs.

Detection of ALDH1 activity in rabbit hepatic VX2 tumors and isolation of ALDH1 positive cancer stem cells.

BackgroundAldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells.Materials and methodsWe used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein.ResultsWe used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver.ConclusionThe overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0785-0) contains supplementary material, which is available to authorized users.

Expression of the potential cancer stem cell markers CD133 and CD44 in medullary thyroid carcinoma: A ten-year follow-up and prognostic analysis.

To investigate the expression profiles of cancer stem cells (CSCs) markers CD133 and CD44 in a cohort of medullary thyroid carcinoma (MTC) patients, and their prognostic values during 10-year follow-up. MTC samples were obtained for H&E and immunohistochemical analysis. Survival analysis was performed using Kaplan-Meier method and log-rank test. Both the CD133 and CD44 positives were higher in MTC than control. High expression of CD133 and CD44 was positively correlated with capsule invasion and each other, and their co-expression was significantly correlated with capsule invasion, tissue invasion, and metastases at surgery. Tumor size, capsular invasion, tissue invasion, metastases at surgery, surgical plan, lymph node metastases, TNM stage, CD133, and CD44 were prognostic factors for overall survival (OS) and/or disease free survival (DFS). Both the CD133 and CD44 were unfavorable prognostic predictors for OS (P = 0.046, P = 0.03), while only CD44 was a significant predictor for DFS (P = 0.017). OS rate in CD133/CD44 co-expression group was significantly lower than that in non-co-expression group (χ(2) = 8.44, P = 0.004). Our study suggested the high expression of CD133 and CD44 in the MTC, and CD133 and CD44 expressions were correlated with capsule invasion and with OS. CD133 and/or CD44 may be prognostic factors for OS and/or DFS in our MTC patients.

Culturing in serum-free culture medium on collagen type-I-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell.

Background:A sub-population of tumor cells termed cancer stem cells (CSCs) has an important role in tumor initiation, progression, and recurrence. Selecting a suitable procedure for isolation and enrichment of CSCs is the biggest challenge in the study of CSCs. In the present study, the role of the combination of stem cell culture medium and collagen type-I was evaluated for successful isolation and enrichment of HT-29 CSCs.Materials and Methods:HT-29 cells were cultured in serum-containing medium (parental culture medium: Medium + 10% fetal bovine serum) and serum-free medium (stem cell culture medium); both on collagen-coated plates. Spheres forming ability and CD133 expression, as a potential marker of colorectal CSCs, were evaluated in two culture mediums.Results:The results show spheroids usually give rise completely within 15 days in the stem cell culture medium on the collagen-coated plate. CD133 expression in spheroid cells (84%) is extensively higher than in parental cells (25%). Moreover, relative to parental cells, spheroid cells were more radioresistance.Conclusion:Finding of this study suggested that CSCs derived from colon cancer cell line (HT-29) can be propagated and form colonospheres in serum-free culture medium on collagen type-I. According to maintenance of their original phenotype in these conditions, it seems serum-free culture medium on collagen type-I is a suitable way to drug screening of HT-29 CSCs.

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.