Cancer Stem Cell-like Subpopulations Research Articles

Extracellular Vesicle-Mediated Modulation of Stem-like Phenotype in Breast Cancer Cells under Fluid Shear Stress

Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic and stem cell-like. Biophysical stresses such as FSS are also known to cause the production of extracellular vesicles (EVs) that can facilitate cell–cell communication by carrying biomolecular cargos such as microRNAs. Here, we hypothesized that physiological FSS will impact the yield of EV production, and that these EVs will have biomolecules that transform the recipient cells. The EVs were isolated using direct flow filtration with and without FSS from the MDA-MB-231 cancer cell line, and the expression of key stemness-related genes and microRNAs was characterized. There was a significantly increased yield of EVs under FSS. These EVs also contained significantly increased levels of miR-21, which was previously implicated to promote metastatic progression and chemotherapeutic resistance. When these EVs from FSS were introduced to MCF-7 cancer cells, the recipient cells had a significant increase in their stem-like gene expression and CD44+/CD24− cancer stem cell-like subpopulation. There was also a correlated increased proliferation along with an increased ATP production. Together, these findings indicate that the presence of physiological FSS can directly influence the EVs’ production and their contents, and that the EV-mediated transfer of miR-21 can have an important role in FSS-existing contexts, such as in cancer metastasis.

Glioblastoma CD105+ cells define a SOX2− cancer stem cell-like subpopulation in the pre-invasive niche

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. Glioma stem like cells (GSC) represent the highest cellular hierarchy in GBM and have a determining role in tumor growth, recurrence and patient prognosis. However, a better definition of GSC subpopulations, especially at the surgical resection margin, is warranted for improved oncological treatment options. The present study interrogated cells expressing CD105 (CD105+) specifically within the tumor front and the pre-invasive niche as a potential GSC subpopulation. GBM primary cell lines were generated from patients (n = 18) and CD105+ cells were isolated and assessed for stem-like characteristics. In vitro, CD105+ cells proliferated and enriched in serum-containing medium but not in serum-free conditions. CD105+ cells were characterized by Nestin+, Vimentin+ and SOX2−, clearly distinguishing them from SOX2+ GCS. GBM CD105+ cells differentiated into osteocytes and adipocytes but not chondrocytes. Exome sequencing revealed that GBM CD105+ cells matched 83% of somatic mutations in the Cancer cell line encyclopedia, indicating a malignant phenotype and in vivo xenotransplantation assays verified their tumorigenic potential. Cytokine assays showed that immunosuppressive and protumorigenic cytokines such as IL6, IL8, CCL2, CXCL-1 were produced by CD105+ cells. Finally, screening for 88 clinical drugs revealed that GBM CD105+ cells are resistant to most chemotherapeutics except Doxorubicin, Idarubicin, Fludarabine and ABT-751. Our study provides a rationale for targeting tumoral CD105+ cells in order to reshape the tumor microenvironment and block GBM progression.

Tracing tumorigenesis in a solid tumor model at single-cell resolution

Characterizing the complex composition of solid tumors is fundamental for understanding tumor initiation, progression and metastasis. While patient-derived samples provide valuable insight, they are heterogeneous on multiple molecular levels, and often originate from advanced tumor stages. Here, we use single-cell transcriptome and epitope profiling together with pathway and lineage analyses to study tumorigenesis from a developmental perspective in a mouse model of salivary gland squamous cell carcinoma. We provide a comprehensive cell atlas and characterize tumor-specific cells. We find that these cells are connected along a reproducible developmental trajectory: initiated in basal cells exhibiting an epithelial-to-mesenchymal transition signature, tumorigenesis proceeds through Wnt-differential cancer stem cell-like subpopulations before differentiating into luminal-like cells. Our work provides unbiased insights into tumor-specific cellular identities in a whole tissue environment, and emphasizes the power of using defined genetic model systems.

Abstract P5-03-17: Sabutoclax, pan-active BCL-2 protein family antagonist, eliminates cancer stem cells through inhibiting PI3K/Akt pathway in breast cancer

Abstract Introduction/Purpose: Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype often accompanies activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, as well as an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. Methods: MTT assays and drug resistance clonogenic assays were performed to evaluate the cell viability in response to drug treatments. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays and the levels of a series of apoptosis-related proteins by real-time PCR and western blot.The expression levels of AKT and pAKT were assessed by western blot to exam the effect of sabutoclax on the activity of PI3K/AKT pathway. The expression of breast cancer stem cells (BCSC) related markers (CD44, CD24 and ALDH1) were detected by flow cytometry. Mammosphere formation assays and soft agar colony formation assay were performed to test the effect of sabutoclax on the proportion of stem-like cell population. To validate the results showed in breast cancer cell lines, we also tested the ex vivo effect of sabutoclax on nine fresh human breast tumor samples by IHC. The effect of sabutoclax on tumor growth was also studied in mouse xenografts. Results We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Inhibition of BCL-2, MCL-1and BCL-xL leads to caspase-3/7 and caspase-9 activation, sabutoclax modulated Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the PI3K/AKT signaling pathway. A similar effect was observed in a panel of nine breast tumors ex vivo. Conclusions: Our findings indicate that sabutoclax partially overcomes drug resistance of breast cancer cells by reactivation of apoptosis, mediates by the inhibition of several anti-apoptotic BCL-2 family proteins such as BCL-2, MCL-1, BCL-XL and BFL-1, and, by abolition of PI3K/AKT pathway and crosstalk between AKT and BCL-2 family proteins is indispensable for maintaining stemness and chemoresistance in BCSCs. This suggests a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients. Citation Format: Zhang J. Sabutoclax, pan-active BCL-2 protein family antagonist, eliminates cancer stem cells through inhibiting PI3K/Akt pathway in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-17.

Sabutoclax, pan-active BCL-2 protein family antagonist, overcomes drug resistance and eliminates cancer stem cells in breast cancer

Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype is also associated with an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy, and BCL-2 inhibitors are currently being clinically trailed in patients with leukemia, lymphoma or non-small cell lung cancer. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Sabutoclax induced the blockage of BCL-2, MCL-1, BCL-xL and BFL-1, which in turn led to caspase-3/7 and caspase-9 activation and modulation of Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the IL-6/STAT3 signaling pathway. A similar effect was observed in a small panel of nine breast tumors ex vivo. Our findings indicate that sabutoclax partially overcomes the drug resistance phenotype of breast cancer cells by reactivation of apoptosis, mediated by the inhibition of several anti-apoptotic BCL-2 family proteins, and eliminates CSCs by abolition of the IL-6/STAT3 pathway. This offers a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients.

The hedgehog pathway in triple-negative breast cancer.

Treatment of triple‐negative breast cancer (TNBC) remains challenging due to the underlying heterogeneity of this disease coupled with the lack of predictive biomarkers and effective targeted therapies. Intratumoral heterogeneity, particularly enrichment for breast cancer stem cell‐like subpopulations, has emerged as a leading hypothesis for systemic therapy resistance and clinically aggressive course of poor prognosis TNBC. A growing body of literature supports the role of the stem cell renewal Hedgehog (Hh) pathway in breast cancer. Emerging preclinical data also implicate Hh signaling in TNBC pathogenesis. Herein, we review the evidence for a pathophysiologic role of Hh signaling in TNBC and explore mechanisms of crosstalk between the Hh pathway and other key signaling networks as well as their potential implications for Hh‐targeted interventions in TNBC.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features.

Stem Cells Antigen-1 Enriches for a Cancer Stem Cell-Like Subpopulation in Mouse Gastric Cancer.

There is a strong need to identify markers to enrich gastric cancer stem cells (CSCs). However, CSC enrichment markers for mouse gastric cancers have not yet been determined. In our previous study, we generated primary mouse gastric cancer cell line NCC-S1 (S1) established from a Villin-cre;Smad4(F/F) ;Trp53(F/F) ;Cdh1(F/wt) mouse and its metastatic variant cell line NCC-S1M (S1M). Interestingly, S1M cells exhibited CSC-like features, such as increased tumorigenic potential and chemoresistance. By comparing gene expression profiles between S1 and S1M cells, we identified Stem Cells Antigen-1 (Sca-1) as a cell surface marker, which was mostly upregulated in S1M. Sca-1 was upregulated in tumorspheres from S1 cells or after cisplatin treatment in S1 cells. Immunofluorescence (IF) analysis showed that approximately 7% of cancer cells exhibited positivity for Sca-1 in primary mouse gastric cancer tissues. An in vivo-limiting dilution assay showed that Sca-1(high) mouse gastric cancer cells demonstrated increased tumorigenicity compared with Sca-1(negative) cells. The Sca-1 expression was downregulated by TGF-β pathway activation and Wnt pathway inhibition in mouse gastric cancer cells. Sca-1(high) cells showed relatively low TGF-β reporter activity and high TCF/LEF1 reporter activity compared with Sca-1(negative) cells. A chromatin immunoprecipitation analysis demonstrated that Sca-1 was a β-catenin/LEF1 target gene. Sca-1(high) allografts were more resistant to cisplatin/fluorouracil chemotherapy than Sca-1(negative) allografts, and overexpressed Bcl-xL. Eighty-five mouse genes overexpressed in Sca-1(high) S1 cells compared with Sca-1(negative) cells clustered 123 pretreatment gastric cancer patient samples according to survival following chemotherapy. Taken together, Sca-1 is a novel CSC enrichment marker that mediates TGF-β and Wnt/β-catenin signaling in mouse gastric cancer. Stem Cells 2016;34:1177-1187.

Adenoviral Vectors for Prodrug Activation-based Gene Therapy for Cancer

Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient's tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity.

Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24− Subpopulations of Breast Cancer Cells

BackgroundSTAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH+), or cell surface molecule CD44-positive (CD44+) but CD24-negative (CD24−) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells is unknown.Methods and ResultsWe examined STAT3 activation in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH+) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH−) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH+ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH+ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH+ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH+ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH+ cells were further selected for the stem cell markers CD44+ and CD24−.ConclusionThese studies demonstrate an important role for STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24− subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Abstract 5330: Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation.

Abstract Multiple myeloma (MM) remains an incurable clonal plasma cell malignancy. The existence of cancer stem cell-like subpopulation in MM may explain for its unfavourable prognosis and high relapse rate. Studies have shown that CD138neg MM cells were clonogenic and could be serially transplanted, while CD138+ cells could not. We characterized CD138neg cells by checking the presence of such subpopulation and their tumorigenic properties. In concordance with literature, CD138neg cells were present in MM cell lines, and possessed higher clonogenic potential than its non-tumorigenic counterpart as shown in colony formation assay. They were more quiescent, with less than 1% in G2/M phase compared to 5-10% in CD138+ cells. They also demonstrated higher resistance to chemotherapeutic agents, including thalidomide and bortezomib. Since studies in solid tumors have demonstrated that microRNA (miRNA) plays a role in self-renewal, tumorigenicity and chemoresistance in cancer stem cells, we hypothesized that specific miRNAs are the key regulators of the cancer stem cell-like functions in MM clonogenic cells. To search for these candidates, we first identified miRNAs that are differentially expressed among the two subpopulations, CD138+ and CD138neg cells, which were isolated from MM cell lines (NCI-H929, U266 and MM17, our in-house MM cell line) using MACS-immunomagnetic separation system and subjected to Exiqon miRCURY LNA miRNA microarray analysis. Ten candidate miRNAs were identified and selected for further validation by Taqman miRNA assay. By further independent investigation using fifteen MM patient samples, concordantly we found frequent up-regulation of miR-483-5p, miR-1246, miR-1275, miR-1290 and miR-3196 in CD138neg compared to CD138+ cells. MiR-1246 (93.4%, p = 0.006) and miR-1290 (93.4%, p = 0.0015) were chosen for further examination of the potential roles in MM pathogenesis and prognosis because they showed significantly higher expression levels in CD138neg cells than the CD138+ counterparts. Preliminary results showed that inhibiting miR-1246 (p = 0.0478) and miR-1290 (p = 0.0386) reduced cell proliferation in CD138neg cells at 24h or 48h of transfection in unsorted U266 cells, while CD138+ cells were not affected. As predicted using computational algorithms (miRANDA and TargetScan), the downstream targets of the two miRNAs include SEMA6A, STK17A, PHLDA1 and KLF9, which bear functions related to MM pathogenesis or known to be tumor suppressors. Moreover, we also confirmed that their mRNA expressions were down-regulated in U266 CD138neg cells compared with CD138+ cells.Taken together, we suggest that miR-1246 and miR-1290 may play a regulatory role in MM clonogenic cell proliferation. Our findings may provide a novel insight for the microRNA-based therapeutics that specifically targets the clonogenic progenitors, which may prove to be more effective for cure of MM. Citation Format: Coty HY Cheung, Suk-Hang Cheng, Libby Li, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Margaret H.L. Ng. Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2013-5330

Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors

IntroductionProtein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells.MethodsThe impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors.ResultsThe impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent.ConclusionThe ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

Knockdown of the Potential Cancer Stem-Like Cell Marker Rex-1 Improves Chemotherapeutic Effects in Gliomas

In the present study, we show that Rex-1 mRNA and protein are found at high levels in both 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell subpopulations and malignant glioblastoma multiforme (GBM) tissue. We used a combination therapy of small interfering RNA (siRNA) against Rex-1 (siRex-1) and BCNU to target GBM cells. Rex-1 siRNA/BCNU treatment resulted in growth inhibition and a diminished S phase. The treatment efficiently induced P38/JNK and Akt/PI3K/GSK3β signaling and led to apoptosis both in vitro and in vivo. We also show that Rex-1/ABCG2 (ATP binding cassette transporter G2)-coexpressing subpopulations were chemoresistant; however, BCNU was not a substrate for ABCG2. siRex-1 treatment led to cell death in GBM subpopulations by promoting apoptosis. Moreover, siRex-1/BCNU combination therapy targeted both the major population and cancer stem cell-like subpopulations. Our findings are important for the development of clinical applications to treat GBM.

Abstract 2140: Assessment of curcumin-induced apoptosis and detection of cancer stem cell-like subpopulations in human esophageal cancer cell lines

Abstract Curcumin, the major active ingredient of turmeric, has been shown to kill several types of malignant cells while it has little or no effect on normal cells, suggesting that it may be an effective therapeutic agent. Although some studies have investigated curcumin effects on esophageal cancer cell lines, no studies have characterized the curcumin-surviving cells, an important population that might show resistance to curcumin. There is increasing evidence that many cancers contain subpopulations that display stem cell characteristics. Cancer stem cells (CSCs) appear to play a central role in tumorigenesis, recurrence, metastasis, and resistance to cancer therapy. The aims of the current study were to assess curcumin-induced cell death and detect CSC subpopulations in six human esophageal cancer cell lines (TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2). Aldehyde dehydrogenase-1A1 (ALDH1A1) expression was used as one marker for CSCs. To evaluate curcumin effects, the cell lines were exposed to 20-80 µM of curcumin for 30 hrs. In all six lines, cell death increased continuously with increasing dosage of curcumin based on a standard crystal violet assay. No cells survived at 80 µM, and the range of cell death across the six cell lines was 66 to 89% at 60 µM. To characterize the progeny of cells that survived curcumin, 1×105 cells from each line were exposed to 40-60 µM curcumin for 30 hours, and the colonies that formed from surviving cells were counted. The number of colonies present after 60 µM treatment varied between the cell lines (ranging from 8 to more than one hundred). The Yes-2 cell line did not survive at 60 µM, but produced 15 colonies after 40 µM treatment. Staining with an antibody against ALDH1A1 was used to compare CSC-like subpopulations within and among the original cells lines as well as cell lines established from the curcumin-treated lines. All cell lines were heterogeneous with respect to ALDH1A1 staining, revealing two subpopulations within each line. The intensity of staining was at least two times higher in the population considered to be CSCs. Interestingly, the curcumin-surviving cell lines showed a significant loss in the high-staining population compared to the untreated cells (T-test, p<0.05), suggesting that curcumin not only can kill cancer cells but also CSCs. Thus, esophageal CSCs appear to be highly sensitive to curcumin. Therefore, curcumin may be a more effective compound for esophageal cancer therapy than other chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2140. doi:10.1158/1538-7445.AM2011-2140

Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of 'stemness genes' such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition, glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.

The Radiation Response of Cancer Stem Cell-like Subpopulations from Established Human Cell Lines

The Radiation Response of Cancer Stem Cell-like Subpopulations from Established Human Cell Lines