Understanding Of Cancer Stem Cells Research Articles

Genome-Wide and Phenotypic Evaluation of Stem Cell Progenitors Derived From Gprc5a-Deficient Murine Lung Adenocarcinoma With Somatic Kras Mutations

Lung adenocarcinomas (LUADs) with somatic mutations in the KRAS oncogene comprise the most common molecular subtype of lung cancer in smokers and present with overall dismal prognosis and resistance to most therapies. Our group recently demonstrated that tobacco carcinogen-exposed mice with knockout of the airway lineage G-protein coupled receptor, Gprc5a, develop LUADs with somatic mutations in Kras. Earlier work has suggested that cancer stem cells (CSCs) play crucial roles in clonal evolution of tumors and in therapy resistance. To date, our understanding of CSCs in LUADs with somatic Kras mutations remains lagging. Here we derived CSCs (as spheres in 3D cultures) with self-renewal properties from a murine Kras-mutant LUAD cell line we previously established from a tobacco carcinogen-exposed Gprc5a−/− mouse. Using syngeneic Gprc5a−/− models, we found that these CSCs, compared to their parental isoforms, exhibited increased tumorigenic potential in vivo, particularly in female animals. Using whole-transcriptome sequencing coupled with pathways analysis and confirmatory PCR, we identified gene features (n = 2,600) differentially expressed in the CSCs compared to parental cells and that were enriched with functional modules associated with an augmented malignant phenotype including stemness, tumor-promoting inflammation and anti-oxidant responses. Further, based on in silico predicted activation of GSK3β in CSCs, we found that tideglusib, an irreversible inhibitor of the kinase, exhibited marked anti-growth effects in the cultured CSCs. Our study underscores molecular cues in the pathogenesis of Kras-mutant LUAD and presents new models to study the evolution, and thus high-potential targets, of this aggressive malignancy.

Current approaches in identification and isolation of cancer stem cells.

Cancer stem cells (CSCs) are tumor cells with initiating ability, self-renewal potential, and intrinsic resistance to conventional therapeutics. Efficient isolation and characterization of CSCs pave the way for more comprehensive knowledge about tumorigenesis, heterogeneity, and chemoresistance. Also a better understanding of CSCs will lead to novel era of both basic and clinical cancer research, reclassification of human tumors, and development of innovative therapeutic strategies. Finding novel diagnostic and effective therapeutic strategies also enhance the success of treatment in cancer patients. There are various methods based on the characteristics of the CSCs to detect and isolate these cells, some of which have recently developed. This review summarized current techniques for effective isolation and characterization of CSCs with a focus on advantages and limitations of each method with clinical applications.

Toward understanding cancer stem cell heterogeneity in the tumor microenvironment

The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation are two paramount processes driving tumor progression, therapy resistance, and cancer metastasis. Recent experiments show that cells with varying EMT and CSC phenotypes are spatially segregated in the primary tumor. The underlying mechanisms generating such spatiotemporal dynamics in the tumor microenvironment, however, remain largely unexplored. Here, we show through a mechanism-based dynamical model that the diffusion of EMT-inducing signals such as TGF-β, together with noncell autonomous control of EMT and CSC decision making via the Notch signaling pathway, can explain experimentally observed disparate localization of subsets of CSCs with varying EMT phenotypes in the tumor. Our simulations show that the more mesenchymal CSCs lie at the invasive edge, while the hybrid epithelial/mesenchymal (E/M) CSCs reside in the tumor interior. Further, motivated by the role of Notch-Jagged signaling in mediating EMT and stemness, we investigated the microenvironmental factors that promote Notch-Jagged signaling. We show that many inflammatory cytokines such as IL-6 that can promote Notch-Jagged signaling can (i) stabilize a hybrid E/M phenotype, (ii) increase the likelihood of spatial proximity of hybrid E/M cells, and (iii) expand the fraction of CSCs. To validate the predicted connection between Notch-Jagged signaling and stemness, we knocked down JAG1 in hybrid E/M SUM149 human breast cancer cells in vitro. JAG1 knockdown significantly restricted tumor organoid formation, confirming the key role that Notch-Jagged signaling can play in tumor progression. Together, our integrated computational-experimental framework reveals the underlying principles of spatiotemporal dynamics of EMT and CSCs.

Understanding Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: A Critical Update in Development of New Approaches

Understanding Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: A Critical Update in Development of New Approaches

A Feasible Method of Angiogenesis Assessment in Gastric Cancer Using 3D Microvessel Density.

Background Cancer stem cell (CSC) promotes angiogenesis which plays an important role in tumor occurrence, growth, and metastasis. Accurately, quantifying the tumor vasculature can help understanding CSC characteristics and improve cancer diagnosis, therapy planning, and evaluation. The objective of this study is to present a method for improved angiogenesis assessment. Methods We proposed a three-dimensional microvessel density (3D MVD) to evaluate tumor angiogenesis and tested it in animal models. Six male Balb/c nude mice were divided into normal group and tumor group. The mice in tumor group were orthotopically implanted human gastric cancer, cell line BGC-823. The phase-contrast images were collected at Shanghai Synchrotron Radiation Facility BL13W beamline, which has much higher soft-tissue contrast and spatial resolution than conventional X-ray. After volume reconstruction and vessel extraction, the 3D models of the angiogenesis were established for MVD calculation. Results The results showed that the proposed 3D MVD is positively correlated with the pathological changes of the microvessels. It took the advantage of high resolution of the phase-contrast imaging and added three-dimensional information to the existing MVD measure. Conclusions Our study presents a feasible approach for better understanding of tumor angiogenesis. It may provide doctors and scientists a better tool for cancer investigation and improving medical outcomes.

Transforming growth factor-β in liver cancer stem cells and regeneration.

Cancer stem cells have established mechanisms that contribute to tumor heterogeneity as well as resistance to therapy. Over 40% of hepatocellular carcinomas (HCCs) are considered to be clonal and arise from a stem‐like/cancer stem cell. Moreover, HCC is the second leading cause of cancer death worldwide, and an improved understanding of cancer stem cells and targeting these in this cancer are urgently needed. Multiple studies have revealed etiological patterns and multiple genes/pathways signifying initiation and progression of HCC; however, unlike the transforming growth factor β (TGF‐β) pathway, loss of p53 and/or activation of β‐catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF‐β signaling in gastrointestinal cancers, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support effective targeted therapies for these common and lethal cancers. Moreover, pathways involved in stem cell transformation into gastrointestinal cancers remain largely undefined. Identifying the key mechanisms and developing models that reflect the human disease can lead to effective new treatment strategies. In this review, we dissect the evidence obtained from mouse and human liver regeneration, and mouse genetics, to provide insight into the role of TGF‐β in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477–493)

Current understanding of cancer stem cells: Review of their radiobiology and role in head and neck cancers.

Evidence of cancer cells that bear attributes analogous to those of normal stem cells has developed a hierarchical model of cancer's architecture and progression. This subset of cancer stem cells (CSCs) drives the progression and therapy resistance of cancers. Research to identify the phenotypes of these CSCs presents evidence of a subpopulation that is more resistant to therapy and may proliferate in response. Literature shows that CSCs typically represent around 1%-10% of cell populations in head and neck cancer but this proportion may increase in response to a therapeutic radiation dose. This is shown to be not just as a result of preferential killing, but also their capacity to alter divisional dynamics and enlist the support of a complicit microenvironment in therapy resistance and proliferation. The CSCs represent the apex of a hierarchy in the heterogeneity of cancer cells and may be seen as the agents of treatment failure, metastasis, and tumor recurrence, the principal cause of mortality in head and neck cancers. Greater than 90% of head and neck cancers are squamous cell carcinomas (HNSCCs), and among these an increasing incidence of the involvement of the human papillomavirus (HPV) is reported. Chemoradiotherapy along with surgical resection are the interventions of choice for control and cure of HNSCC, but given CSCs therapy resistance and proliferative responses to radiation, the identification and understanding of the radiobiology of this subpopulation is critical to their targeted elimination. This article reviews the current evidence on CSC generally and in HNSCC specifically to identify their phenotype, evaluate their responses to radiotherapy, and evaluate the defensive mechanisms used to resist therapeutic control.

Smad inhibitor induces CSC differentiation for effective chemosensitization in cyclin D1- and TGF-β/Smad-regulated liver cancer stem cell-like cells.

Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-dependent activation of Smad2/3 and Smad4 in hepatocellular carcinoma (HCC) CSCs and in HCC primary tumors. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing stemness gene expression, and increasing chemoresistance. Cyclin D1 interacts with and activates Smad2/3 and Smad4 to result in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. The cyclin D1-dependent activation of Smad2/3 and Smad4 is also found in HCC patients and predicts disease progression. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-mediated activation of Smad2/3 and Smad4 is an important regulatory mechanism in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequently significant chemosensitization, which could be an effective strategy to target CSCs.

Abstract 1955: Computational correlation among microRNAs and mRNAs specifically expressed in pancreatic cancer stem cells

Abstract The objective of this study was to analyze and identify pancreatic cancer stem cell-specific microRNAs (miRNAs) and messenger RNAs (mRNAs) and investigate their correlations to cancer stem cell biology. More, pancreatic cancer stem cell-specific miRNAs in cancer patients’ serum were analyzed to determine as a diagnostic application. We used sphere cultivation methods to enrich the stem cell population from the pancreatic cancer cell line, Capan-1 with the normal pancreatic cell line, YGIC-6, and analyzed the overall miRNA and mRNA expressions using with microarray analysis. Total RNA from normal and patient's serum was extracted and the expression individual miRNAs in serum was analyzed by real-time PCR. Fifty two miRNAs including mir-26a, miR-99a, miR-106a, mir-125b, miR-192 and mir-429 were differentially expressed in the pancreatic cancer stem cells. From the miRNA analysis, 170 miRNAs were differentially expressed in the normal cell line YGIC-6, 126 miRNAs in the cancer cell line Capan-1 and 12 miRNAs in both the normal and cancer cell line originated stem cell populations. Examining both the miRNA and mRNA profiles, 52 miRNAs and 111 stem cell-associated mRNAs were highly correlated (both p values<0.01) that were differentially expressed in the pancreatic cancer stem cells. These miRNAs and mRNAs were further investigated with cross-correlation analysis, which showed a number of highly correlated miRNAs and mRNAs that are either directly or indirectly linked based on the target prediction software. The correlation degree is shown on the heatmap, and individual miRNAs with their highly correlated individual mRNAs were analyzed to predict possible mechanisms for miRNAs in pancreatic cancer stem cells. To examine miRNAs level in serum, total RNA was extracted from normal and cancer patient's serum and the expression level of individual miRNAs were analyzed. The expression level (Ct value) of mir-106a, mir-125b and mir-429 was differentially expressed in cancer patient's serum than normal and mir-99a was differentially expressed in cancer patients with poor prognosis. Differentially expressed miRNAs in pancreatic cancer stem cells provide insights into possible linkages between individual miRNAs and stem cell-associated target mRNAs in cancer stem cells and have broad implications in our understanding of cancer stem cells and cancer stem cell-targeted cancer therapy. Expression level of miRNAs in serum was differed in normal and cancer patient suggests miRNAs as a diagnostic and prognostic marker. Citation Format: Dawoon E. Jung, Hyungseok Choi, Jee Yeon Heo, Hee Seung Lee, Kyong Joo Lee, Si Young Song. Computational correlation among microRNAs and mRNAs specifically expressed in pancreatic cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1955.

Abstract 1729: Withaferin A blocks formation of IFN-γ-induced metastatic cancer stem cells through inhibition of the CXCR4/CXCL12 pathway in the UP-LN1 carcinoma cell model

Abstract As our understanding of cancer stem cell (CSC) biology improves, there is an urgent need to search for inhibitory agents of CSCs and metastatic CSCs (mCSCs) positive for CXCR4. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera, has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or non-CSCs of a gastrointestinal (GI) carcinoma tumor remains unclear. Side population analysis, flow cytometric phenotyping and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation. Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44high/CD24low grape-like floating (F) and CD44low/CD24high adherent (A) cell subsets, this study demonstrated that, as compared with parental UP-LN1 cells or A cells, WA preferentially reduced F-cell proliferation, tumor sphere formation, and side population cells in vitro in greater efficiencies by apoptosis. This action was mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/IL-6 pathways, both of which are instrumental in the acquisition of metastatic ability. Attenuation of IFN-γ-induced CXCR4 expression in F cells by knockdown with siRNA or blocking with anti-CXCR4 antibody, followed by Western blot analysis, showed significantly reduced metastatic potential in vitro. The extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was significantly greater than on the F cells without WA treatment, or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation. We conclude that WA could efficiently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective therapeutic agent for GI malignancies exhibiting grape-like tumor sphere. Citation Format: Andy Shau-Bin Chou, Li-Lei Ting, Chin-Hsuan Hsieh, See-Tong Pang, Shuen-Kuei Liao. Withaferin A blocks formation of IFN-γ-induced metastatic cancer stem cells through inhibition of the CXCR4/CXCL12 pathway in the UP-LN1 carcinoma cell model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1729.

Use of Cancer Stem Cells to Investigate the Pathogenesis of Colitis-associated Cancer.

Colitis-associated cancer (CAC) can develop in patients with inflammatory bowel disease with long-term uncontrolled inflammation. The mutational history and tumor microenvironment observed in CAC patients is distinct from that observed in sporadic colon cancer and suggests a different etiology. Recently, much attention has been focused on understanding the cellular origin of cancer and the cancer stem cells, which is key to growth and progression. Cancer stem cells are often chemo-resistant making them attractive targets for improving patient outcomes. New techniques have rapidly been evolving allowing for a better understanding of the normal intestinal stem cell function and behavior in the niche. Use of these new technologies will be crucial to understanding cancer stem cells in both sporadic and CAC. In this review, we will explore emerging methods related to the study of normal and cancer stem cells in the intestine, and examine potential avenues of investigation and application to understanding the pathogenesis of CAC.

Mathematical modeling of bone marrow – peripheral blood dynamics in the disease state based on current emerging paradigms, part I

Stemming from current emerging paradigms related to the cancer stem cell hypothesis, an existing mathematical model is expanded and used to study cell interaction dynamics in the bone marrow and peripheral blood. The proposed mathematical model is described by a system of nonlinear differential equations with delay, to quantify the dynamics in abnormal hematopoiesis. The steady states of the model are analytically and numerically obtained. Some conditions for the local asymptotic stability of such states are investigated. Model analyses suggest that malignancy may be irreversible once it evolves from a nonmalignant state into a malignant one and no intervention takes place. This leads to the proposition that a great deal of emphasis be placed on cancer prevention. Nevertheless, should malignancy arise, treatment programs for its containment or curtailment may have to include a maximum and extensive level of effort to protect normal cells from eventual destruction. Further model analyses and simulations predict that in the untreated disease state, there is an evolution towards a situation in which malignant cells dominate the entire bone marrow - peripheral blood system. Arguments are then advanced regarding requirements for quantitatively understanding cancer stem cell behavior. Among the suggested requirements are, mathematical frameworks for describing the dynamics of cancer initiation and progression, the response to treatment, the evolution of resistance, and malignancy prevention dynamics within the bone marrow – peripheral blood architecture.

Understanding cancer stem cells in head and neck cancer: An insight from oral medicine point of view

Cancer stem cells are altered stem cells that give rise to tumors. Head and neck cancer accounts for the significant amount of cancers that occur in the world. There is pointing evidence that cancer stem cells contribute to the origin of head and neck cancers. Understanding the cancer stem cell biology, identifying them and targeting these cells are highly important to effectively manage head and neck cancers. The present article discusses the importance of the role of cancer stem cells in head and neck cancers from an oral medicine point of view.

Abstract B45: A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells

Abstract Many tumors consist of a hierarchy of cells with different proliferative and developmental potential. A small number of cancer stem cells (CSCs) give rise to a larger population of highly proliferative, committed progenitor cells, which may then undergo limited differentiation. Importantly, CSCs are uniquely capable of initiating and sustaining tumorigenesis, and they have been implicated in driving disease recurrence after cancer therapy. Thus understanding CSC biology will be critical to the development of more effective therapies. CSCs are most commonly identified by FACS analysis but the optimal marker combinations are very dependent on the tissue and specific cell-of-origin of the tumor, and they cannot be used to monitor the CSCs in situ, with all the microenvironmental cues intact. Such markers cannot readily be used for real-time assessment of stem cell behavior at a single cell rather than a population level. To address this problem, we have developed and validated a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of the cells with CSC properties. The construct consists of a tandemly repeated composite Sox2-Oct4 response element (SORE6) driving expression of a destabilized green fluorescent protein reporter. The reporter responds to the presence of the core stem cell transcription factors Oct4 and Sox2, with further stem cell selectivity and kinetic resolution coming from the use of a proteosome-targeting degron on the fluorescent protein. Using the human MCF10CA1h breast cancer cell line, we have shown that SORE6-GFP+ cells within the cell population are undifferentiated and enriched for stem cell markers. These cells can self-renew and regenerate GFP- cells, show enhanced asymmetric division, and are enriched for tumorsphere formation in vitro. Most importantly the SORE6-GFP+ cells are enriched for tumor-initiating and metastasis-initiating ability in vivo and they are relatively resistant to chemotherapeutics both in vitro and in vivo. Thus by a number of criteria, the reporter is marking a cell population that is substantially enriched for CSCs. The reporter works in primary human breast cancer cultures and patient-derived xenografts in addition to established cell line models. Our novel imaging approach opens up the possibility of assessing the spatial distribution of CSCs and temporal changes in CSC properties in experimental settings that retain the complex microenvironmental and structural cues of the tumor bed. Citation Format: Binwu Tang, Asaf Raviv, Dominic Esposito, Catherine Daniel, Bao Tram Nghiem, Susan Garfield, Langston Lim, Poonam Mannan, Ana Robles, William Smith, Joshua Zimmerberg, Rea Ravin, Lalage Wakefield. A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B45. doi:10.1158/1538-7445.CHTME14-B45

Cancer Stem Cells of the Digestive System

Stem cells of the digestive system are ideal in many ways for research, given they are abundant, highly proliferative and have a uniform structural arrangement. This in turn has enormously aided the research of cancer stem cells of the digestive system, which is now shaping our understanding of cancer stem cells. In this review, the recent advances in the understanding of cancer stem cells of the digestive system have been summarized, including aspects such as their identification, origin, cell-cycle dormancy, relationship with epithelial-mesenchymal transition, cellular metabolism and the underlying molecular mechanisms. Newly acquired knowledge concerning cancer stem cells have led to the development of novel cancer therapeutics with provisional yet encouraging results.

MicroRNA-29b-1 impairs in vitro cell proliferation, self‑renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells.

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR-29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.

Sanofi-Cell Research outstanding paper award of 2012

In this November issue of Cell Research, we are proud to announce the winners of the 4th Sanofi-Cell Research Outstanding Paper Award, which were selected from papers published in the 2012 print issues of Cell Research. The 2012 Sanofi-Cell Research Outstanding Review Article Award goes to Dr Dean Tang, for his review paper entitled “Understanding cancer stem cell heterogeneity and plasticity”1. The winners of the 2012 Sanofi-Cell Research Outstanding Research Article Award are Drs Chen-Yu Zhang, Ke Zen and Junfeng Zhang, for their paper entitled “Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA”2; and Drs Yigong Shi and Li Yu, for their paper entitled “Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein”3. The award consists of a prize of € 3000 for the Outstanding Review Article Award and € 5000 for the Outstanding Research Article Award sponsored by Sanofi. The three award-winning papers are selected based on the voting by members of the Sanofi-Shanghai Institutes for Biological Sciences Steering Committee. The award-winning review by Dr Tang was published in the March issue of 2012. The review provides an expert analysis on our current understanding of cancer stem cells, with a focus on discussing their heterogeneity and plasticity. In the first award-winning research article, published in the January 2012 issue, Dr Chen-Yu Zhang and his colleagues report the discovery that a plant microRNA that is abundantly found in rice, could be taken up by animals through food intake, and then acts to regulate the expression of a mammalian gene that is involved in host metabolism, revealing a remarkable example of cross-kingdom regulation by small RNAs. In the second award-winning research article, published in the March 2012 issue, Drs Shi and Yu report the discovery of a novel function of Beclin 1, which is known to play an essential role in autophagy and to be a haplo-insufficient tumor suppressor. Through an elegant combination of structural and biochemical studies, they show that Beclin 1 possesses a novel membrane binding activity, thus providing important molecular insight into how this protein functions. Please join us to congratulate the winners on their winning of the 2012 Sanofi-Cell Research Outstanding Paper Award. We hope that this yearly award program will continue to encourage our fellow scientists to submit their best work to Cell Research.

Abstract 3774: Regulation of the pro-invasive properties of neuroblastoma-stem cells by membrane type-1 matrix metalloproteinase.

Abstract Neuroblastoma (NB) is an aggressive childhood cancer that represents the leading cause of cancer deaths in children. Despite aggressive therapy, more than half of the children with advanced NB usually die because of uncontrolled metastatic disease. In order to develop new therapeutic strategies to limit NB's metastatic potential, it is crucial to identify key molecular targets governing the invasive process. The study of Cancer stem cells (CSCs) may be of interest in this regard because after chemotherapy, CSCs persist in tumors and cause relapse and metastasis. We had recently demonstrated that CD133 allowed to detect CSCs in NB. Membrane type-1 matrix metalloproteinase (MT1-MMP) is important in the metastatic process and its expression was correlated with unfavourable outcome in NBs. Our preliminary data had indicated that MT1-MMP was highly expressed in CD133high NB. The purpose of this study is to characterize the interaction between CD133 and MT1-MMP in NB and determinate the role of MT1-MMP in pro-invasive properties of CSCs. We constructed paraffin-embedded blocks of tissue microarrays (TMA) from 235 patients. In vitro experiments were performed on four established NB cell lines (SK-N-DZ, SK-N-FI and SK-N-SH and SJNB-10). We performed immunohistochemical studies on paraffin-embedded TMA sections with two antibodies (CD133, MT1-MMP). To verify correlation of expression of MT1-MMP and CD133, we realized western blot and immunofluorescence (IF) of both proteins in non treated versus treated NB cell lines. CD133high NB cells were isolated by flow cytometry. CD133high and CD133low NB cells were grown within a 3D collagen matrix and the cell migration assay was tested in collagen-coated transwells. All experiments were performed with or without an anti-MT1-MMP neutralizing antibody or GM6001, a broad-spectrum MMP inhibitor. To assess the interaction between CD133 and MT1-MMP, lysates were subjected to immunoprecipitation (IP) using an anti-CD133 antibody, followed by immunodetection with an anti-MT1-MMP antibody. Finally, we determined which domain of MT1-MMP is involved in its interaction with CD133. Different dominant negative mutants of MT1-MMP (catalytically inactive E240A, cytoplasmic domain-deleted CΔ20 and non-phosphorylatable Y573F) were transfected into NB cells followed by an IP. There is a correlation between MT1-MMP and CD133 expressions in tumors of patients and in cell lines. Cells selected after chemotherapy express both CD133 and MT1-MMP. CD133high cells presented higher migration and invasion properties than CD133low which were MT1-MMP dependent. IF and IP showed a colocalization and interaction between CD133 and MT1-MMP. The cytoplasmic domain of MT1-MMP seems to be responsible for the interaction with CD133. These results contribute to a better understanding CSCs properties in NB and may be of great interest to improve new therapeutic strategies. Citation Format: Assila Belounis, Carine Nyalendo, Sonia Cournoyer, Sarah Hadj-Mimoune, Alexandre Benoit, Elliot Lasalle, Jonathan Girard, Mona Beaunoyer, Pierre Teira, Hervé Sartelet. Regulation of the pro-invasive properties of neuroblastoma-stem cells by membrane type-1 matrix metalloproteinase. [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 3774. doi:10.1158/1538-7445.AM2013-3774

Enrichment of colorectal cancer stem cells through epithelial-mesenchymal transition via CDH1 knockdown

Cancer stem cells (CSCs) are considered to be the origin of cancer relapse and metastasis. The better understanding of CSCs, including CSCs in human colorectal cancer (CRC), may facilitate prevention and treatment. This study aimedto establish a CSC enrichment model via the induction of epithelial-mesenchymal transition (EMT) in CRC cells. We established an EMT model using the SW480 CRC cell line by CDH1 knockdown using shRNA interference. CD24+CD44+ cells were enriched in the CDH1 knockdown cells. The cells exhibited mesenchymal morphology and expressed high levels of EMT-related proteins, which confirmed that these cells had undergone EMT. Our results further showed that the proliferation rate of the transfected cells was reduced, whereas their colony-forming capacity and tumorigenesis invivo was significantly enhanced compared to the control cells. In conclusion, these cells were highly enriched CSCs (compared to normal CSCs) and may be used as a stable model for cancer research and anticancer drug screening.

Abstract 5353: Characterization of breast cancer stem cells (BCSCs) in Egyptian breast cancer patients through genetic profiling using the RT-PCR and quantitative real time microarray

Abstract Background: Stem cells (SC) are cells that can differentiate along distinct lineages through systemic differentiation steps. The concept of self renewal is crucial to understand cancer stem cells (CSC) and the mechanisms by which current therapies might evade the available treatment modalities. The aim of the present study was to characterize breast cancer stem cells (BCSC) by genetic profiling of properly isolated BCSCs at different stages using the recently developed quantitative real time microarray technology to understand the origin and contribution BCSCs to breast cancer development and progression. Material and Methods: 60 cases of BC were subjected to enzymatic digestion to obtain single cell suspension for: 1) flowcytometric characterization of cells, 2) mammosphere culture 3) magnetic separation of different subsets of BCSCs (CD44 and CD24), and 4) RNA extraction for RT-PCR and microarray using the SABiosciences's QC PCR profiler Arrays with SC panel of 84 genes (SC specific markers, SC differentiation markers, and signaling pathway markers for SC). Samples for the QC PCR profiler Arrays were categorized into groups: 1) normal breast tissue, 2) BC cells obtained from tumor samples, 3) cells from mammosphere cultures, 4)CD44+/CD24low/EpCAM- cells, 5) CD44+/ CD24low cells. ER, PR and Her-2 assessment was also performed. Results: Discrete mammospheres of different sizes were obtained from 10 cases and IHC of these samples confirmed the SC lineage. By FCM, cells expressing CD44+/CD24low ranged from 0.1% - 79%. MYC, MYST2, DHH, BTRC AND CXCL12 genes showed over-expression in the studied groups in comparison to normal breast tissue. Comparing all studied groups, we identified 5 candidate genes DVL1 and EP300 (Notch pathway), BTRC (Wnt Pathway), DHH (Chro-Chro Modulation), MYST2 (self Renewal), NCAM1 (Cell Adhesion Molecules) which could be used for characterization and molecular target therapy for breast cancerl. Conclusions: Our QC PCR profiler Arrays showed significant differences between the studied groups shedding light on the nature and helping in proper characterization of BCSCs. Correlation between prognostic factors of BC and the number of BCSCs in tumor biopsies together with the probability of mammoshpere formation in culture denotes the contribution of CSCS to the progression of BC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5353. doi:1538-7445.AM2012-5353