Unknown,Transcriptomics,Genomics,Proteomics

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CD133+ cells contain cancer stem cell-like properties in Malignant Mixed Mullerian Tumor (Carcinosarcoma) of the Uterus

ABSTRACT: Cancer stem cells (CSCs) that display tumor-initiating properties have recently been identified. We herein identify and characterize CSCs in human uterine carcinosarcoma, a highly aggressive and therapy-resistant gynecologic malignancy, which is considered to be of mesodermal origin. FU-MMT-1, a cell-line, which was established by us (Emoto M, Cancer 1992) from a patient with uterine carcinosarcoma, was evaluated. FU-MMT-1 contained a high population of CD133, CD44, CD90, and CD29 positive cells. Using the magnetic bead cell separation method, we isolated CD133+ cells, which predominantly form spheres in culture. These CD133+ cells form transplantable tumors in vivo. A qRT-PCR analysis of the genes implicated in stem cell maintenance revealed that CD133+ cells express significantly higher levels of OCT4, NANOG, and BMI-1 than CD133M-oM-

ORGANISM(S): Homo sapiens

SUBMITTER: Satoshi Kondo

PROVIDER: E-GEOD-27172 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cancer stem cells (CSCs) drive prostate cancer (PCa) progression and metastasis. These cells exhibit remarkable self-renewal, chemoresistant and invasive potentials, and are thought to participate in the changes in cellular architecture that lead to epithelial-to-mesenchymal transition (EMT). Conventional therapies fail to eliminate CSCs, which results in tumor recurrence and progression to castration resistant PCa (CRPC). Recent evidence suggests that castration itself can induce EMT, which could potentiate the â??stemnessâ?? and number of CSCs within the tumor. Hence, there is an urgent need for EMT- and CSC-targeted therapies that could prevent progression to CRPC. 22Rv1, DU145, LNCaP, and PC3 cells were grown under sphere-forming conditions standardized in our lab. We have previously demonstrated that these enriched PCa cell lines exhibit phenotypic characteristics associated with CSCs in vivo. Over-expression of the stem-associated CD133 biomarker was determined via flow cytometric analysis. Total RNA was isolated from CD133+ prostasphere-derived cells. Gene expression profiling was carried out using the nCounter NanoString system and three different CodeSets associated with cancer or stem cells. Functional annotation was performed using the over-representation analysis tool from ConsensusPathDB. Functional annotation analysis of upregulated transcripts suggests that prostasphere-derived cells undergo a transcriptional reprogramming that triggers a major phenotypic shift. These changes are similar to the mesenchymal shift associated with EMT that drives PCa progression to CRPC. Each cell line exhibited distinct expression profiles that are presented and analyzed individually. Furthermore, our analysis revealed over-represented pathways that were common to all cell lines, which are related to the MAPK/ERK, PI3K/AKT, Notch, and Wnt stem cell fate signaling networks. Transcriptional analysis of induced CSCs not only offers insight into their underlying pathophysiology, but can also be used as a platform for the discovery of therapeutic targets for CSC-specific intervention. In this contribution, we present a flexible in vitro platform for the identification of functionally relevant therapeutic targets, using cell samples with low numbers of malignant stem/progenitors that were enriched in vitro. This approach represents a novel and effective strategy that can be used in the development of therapeutic strategies, which could ultimately be tailored to the biology of individual patients. 22Rv1, DU145, LNCaP and PC3 prostasphere cultures were sampled on Day 4 (P 2), Day 8 (P 3) and Day 12 (P 4), and compared relative to parental monolayers on Day 0 of culture (P 1). Three independent measurements were carried out for monolayers at Day 0 using the CancerReference Kit. Day 12 prostaspheres were enzymatically dissociated and sorted via MACS using the Anti-CD133/2 (293C3)-PE Antibody (Miltenyi) into CD133+ and CD133- fractions. Total RNA was extracted from CD133+ cells and analyzed using the nCounter GX Cancer Reference kit, the Stem Cell panel and the custom made ITESM CodeSet from NanoString Technologies. This series contains the Cancer Reference kit data.

Project description:Cancer stem cells (CSCs) drive prostate cancer (PCa) progression and metastasis. These cells exhibit remarkable self-renewal, chemoresistant and invasive potentials, and are thought to participate in the changes in cellular architecture that lead to epithelial-to-mesenchymal transition (EMT). Conventional therapies fail to eliminate CSCs, which results in tumor recurrence and progression to castration resistant PCa (CRPC). Recent evidence suggests that castration itself can induce EMT, which could potentiate the âstemnessâ and number of CSCs within the tumor. Hence, there is an urgent need for EMT- and CSC-targeted therapies that could prevent progression to CRPC. 22Rv1, DU145, LNCaP, and PC3 cells were grown under sphere-forming conditions standardized in our lab. We have previously demonstrated that these enriched PCa cell lines exhibit phenotypic characteristics associated with CSCs in vivo. Over-expression of the stem-associated CD133 biomarker was determined via flow cytometric analysis. Total RNA was isolated from CD133+ prostasphere-derived cells. Gene expression profiling was carried out using the nCounter NanoString system and three different CodeSets associated with cancer or stem cells. Functional annotation was performed using the over-representation analysis tool from ConsensusPathDB. Functional annotation analysis of upregulated transcripts suggests that prostasphere-derived cells undergo a transcriptional reprogramming that triggers a major phenotypic shift. These changes are similar to the mesenchymal shift associated with EMT that drives PCa progression to CRPC. Each cell line exhibited distinct expression profiles that are presented and analyzed individually. Furthermore, our analysis revealed over-represented pathways that were common to all cell lines, which are related to the MAPK/ERK, PI3K/AKT, Notch, and Wnt stem cell fate signaling networks. Transcriptional analysis of induced CSCs not only offers insight into their underlying pathophysiology, but can also be used as a platform for the discovery of therapeutic targets for CSC-specific intervention. In this contribution, we present a flexible in vitro platform for the identification of functionally relevant therapeutic targets, using cell samples with low numbers of malignant stem/progenitors that were enriched in vitro. This approach represents a novel and effective strategy that can be used in the development of therapeutic strategies, which could ultimately be tailored to the biology of individual patients. 22Rv1, DU145, LNCaP and PC3 prostasphere cultures were sampled on Day 4 (P 2), Day 8 (P 3) and Day 12 (P 4), and compared relative to parental monolayers on Day 0 of culture (P 1). Three independent measurements were carried out for monolayers at Day 0 using the CancerReference Kit. were sampled Day 12 prostaspheres were enzymatically dissociated and sorted via MACS using the Anti-CD133/2 (293C3)-PE Antibody (Miltenyi) into CD133+ and CD133- fractions. Total RNA was extracted from CD133+ cells and analyzed using the nCounter GX Cancer Reference kit, the Stem Cell panel and the custom made ITESM CodeSet from NanoString Technologies. This series contains data for the ITESM CodeSet.

Project description:Cancer stem cells (CSCs) drive prostate cancer (PCa) progression and metastasis. These cells exhibit remarkable self-renewal, chemoresistant and invasive potentials, and are thought to participate in the changes in cellular architecture that lead to epithelial-to-mesenchymal transition (EMT). Conventional therapies fail to eliminate CSCs, which results in tumor recurrence and progression to castration resistant PCa (CRPC). Recent evidence suggests that castration itself can induce EMT, which could potentiate the âstemnessâ and number of CSCs within the tumor. Hence, there is an urgent need for EMT- and CSC-targeted therapies that could prevent progression to CRPC. 22Rv1, DU145, LNCaP, and PC3 cells were grown under sphere-forming conditions standardized in our lab. We have previously demonstrated that these enriched PCa cell lines exhibit phenotypic characteristics associated with CSCs in vivo. Over-expression of the stem-associated CD133 biomarker was determined via flow cytometric analysis. Total RNA was isolated from CD133+ prostasphere-derived cells. Gene expression profiling was carried out using the nCounter NanoString system and three different CodeSets associated with cancer or stem cells. Functional annotation was performed using the over-representation analysis tool from ConsensusPathDB. Functional annotation analysis of upregulated transcripts suggests that prostasphere-derived cells undergo a transcriptional reprogramming that triggers a major phenotypic shift. These changes are similar to the mesenchymal shift associated with EMT that drives PCa progression to CRPC. Each cell line exhibited distinct expression profiles that are presented and analyzed individually. Furthermore, our analysis revealed over-represented pathways that were common to all cell lines, which are related to the MAPK/ERK, PI3K/AKT, Notch, and Wnt stem cell fate signaling networks. Transcriptional analysis of induced CSCs not only offers insight into their underlying pathophysiology, but can also be used as a platform for the discovery of therapeutic targets for CSC-specific intervention. In this contribution, we present a flexible in vitro platform for the identification of functionally relevant therapeutic targets, using cell samples with low numbers of malignant stem/progenitors that were enriched in vitro. This approach represents a novel and effective strategy that can be used in the development of therapeutic strategies, which could ultimately be tailored to the biology of individual patients. 22Rv1, DU145, LNCaP and PC3 prostasphere cultures were sampled on Day 4 (P 2), Day 8 (P 3) and Day 12 (P 4), and compared relative to parental monolayers on Day 0 of culture (P 1). Three independent measurements were carried out for monolayers at Day 0 using the CancerReference Kit. Day 12 prostaspheres were enzymatically dissociated and sorted via MACS using the Anti-CD133/2 (293C3)-PE Antibody (Miltenyi) into CD133+ and CD133- fractions. Total RNA was extracted from CD133+ cells and were analyzed using the nCounter GX Cancer Reference kit, the Stem Cell panel and the custom made ITESM CodeSet (14 genes associated with prostate cancer) from NanoString Technologies. This series contains data for the Stem Cell panel.

Project description:BACKGROUND: Several in vitro assays have been used to identify “cancer stem cells” (CSC), including expression of cell surface markers and Hoechst dye efflux properties. However, each of these methods has potential pitfalls that complicate interpretation of the results. Focusing on colon cancers (CC), the CD133 antigen has been proposed as a marker of colon CSC. However, conflicting results have been reported in the literature indicating the need of a systematic analysis of CSC within CC and a complete validation of markers for the isolation of these cells. AIMS: Aim of this study was to confirm that CD133 expression is a valid method for isolating CSC in CC and verify if other antigens can increase the specificity of this marker for isolating CSC in CC. METHODS: CD133+ and CD133- cells were isolated from different human CC lines (CaCo-2, HT29, LOVO, HCT-116) by FACS sorter and the tumor-initiating potential of CD133+ cells was assessed in vitro, by soft-agar colony formation assay, and in vivo, upon transplantation into nude mice. Furthermore, the gene expression profile of CD133+ versus CD133- CaCo-2 cells was compared by the means of microarray analysis. Then, in the effort to identify a common “tumor stem cell” signature for CC, the most relevant transcripts resulting from gene expression profiling on CD133+ cells was assessed by real-time PCR on SP-fraction isolated by FACS sorter from the same CC cell lines. Finally, we deplete CD133 expression in the CaCo-2 cell line by the means of siRNA and verified by Western Blot analysis whether there was a functional correlation between CD133 and the target genes. Moreover, CaCo-2 and HCT116 cells were exposed to sodium butyrate (NaBu) for 72h. Colon cells differentiation was assessed by Alkaline phosphatase activity and expression of CD133 and target genes was tested by western blot. RESULTS: We confirmed that only CD133+ cells have a tumor-initiating potential in vitro and in vivo. Furthermore, microarray analysis of CD133+ versus CD133- CaCo-2 cells revealed a significant overexpression of various transcripts involved in cell proliferation, invasion and stemness in CD133+ cell fraction. Comparison of the transcripts by real-time PCR revealed that the genes of Endothelin-1 (END-1) and NR4A2 are highly expressed in both CD133 + cells and in SP fractions. Finally, when we deplete CD133 expression in Caco-2cells by siRNA, we observed a significant attenuation of END-1 and NR4A2 expression, thus demonstrating that CD133 is involved in the transcriptional regulation of these genes. Interestingly, we also showed that the expression of all three genes was inversely correlated with cell differentiation status as demonstrated by the fact that their expression decreases in a time- and dose-dependent manner after differentiation induced by NaBu. CONCLUSION: Overall, this study confirms the role of CD133antigen as CSC marker and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression, hypothesizing that CD133 is involved in the transcriptional regulation of these gene. Microarray analysis was performed on CD133+ and CD133- sorted CACO-2 cells. For both fractions, cells were sorted three independent times. Sample preparation was performed according to Affymetrix recommendations. A total of 6 arrays were hybridized, including 3 CD133+ replicates and 3 CD133- replicates.