Stochastic profiling of matrix-attached MCF10A-5E cells in 3D culture
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PROVIDER: PRJNA491420 | ENA |
REPOSITORIES: ENA
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Project description:The objective of this study was to profile the gene-regulatory heterogeneities of breast epithelial cells grown in an ECM-rich environment. The approach was to use stochastic profiling (Janes et al., Nat Methods 7(4):311-7 [2010]) to collect 10-cell pools of ECM-attached cells obtained by laser-capture microdissection and measure fluctuations in the 10-cell-averaged measurements to infer heterogeneous regulatory states.
2018-09-18 | GSE120030 | GEO
Project description:Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. Using a basal-like breast epithelial line, we have identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains TGFBR3, a high-affinity receptor for transforming growth factor β (TGFβ) and other related ligands. The second program contains the JUND transcription factor together with the basal-like marker, KRT5. By disrupting the TGFBR3 and JUND programs individually, we reveal an important circuit for 3D morphogenesis that is wired together by four negative-feedback loops. Computational modeling of this circuit showed that it could exhibit damped, antiphase oscillations when excited with small impulses of TGFβ-like ligand. We directly visualize the circuit's spontaneous dynamics in organotypic cultures by using live-cell imaging with engineered pathway reporters. Importantly, we show that the essence of the JUND-TGFBR3 expression circuit holds true in early basal-like tumors that heterogeneously express KRT5. Correlated JUND-KRT5 expression depends critically on contact with stromal ECM and local expression of tenascin C. This work illustrates how complex tumor heterogeneities can be deconstructed into intrinsic single-cell expression circuits that are modulated by the microenvironment. Gene expression analysis of outer ECM-attached vs. inner cells of MCF10A-5E clones grown in organotypic 3D culture at day 6. Total RNA was isolated from ~50 outer ECM-attached cells and ~50 inner cells, each separately microdissected from 8 micron sections of MCF10A structures at day 6 of morphogenesis. Total RNA was amplified in quadruplicate, and hybridized to HumanRef-8 v2.0 Expression BeadChips (Illumina).
2015-06-05 | E-GEOD-41527 | biostudies-arrayexpress
Project description:Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. Using a basal-like breast epithelial line, we have identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains TGFBR3, a high-affinity receptor for transforming growth factor β (TGFβ) and other related ligands. The second program contains the JUND transcription factor together with the basal-like marker, KRT5. By disrupting the TGFBR3 and JUND programs individually, we reveal an important circuit for 3D morphogenesis that is wired together by four negative-feedback loops. Computational modeling of this circuit showed that it could exhibit damped, antiphase oscillations when excited with small impulses of TGFβ-like ligand. We directly visualize the circuit's spontaneous dynamics in organotypic cultures by using live-cell imaging with engineered pathway reporters. Importantly, we show that the essence of the JUND-TGFBR3 expression circuit holds true in early basal-like tumors that heterogeneously express KRT5. Correlated JUND-KRT5 expression depends critically on contact with stromal ECM and local expression of tenascin C. This work illustrates how complex tumor heterogeneities can be deconstructed into intrinsic single-cell expression circuits that are modulated by the microenvironment. Gene expression analysis of outer ECM-attached vs. inner cells of MCF10A-5E clones grown in organotypic 3D culture at day 6.
2015-06-05 | GSE41527 | GEO
Project description:Identifying Nuclear Matrix-attached DNA across the Genome
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Project description:ErbB2 activation of MCF10A cells gives rise to a multiacinar phenotype that is incompletely penetrant. To identify candidate upstream regulators of the phenotype, an MCF10A-5E clone was subcloned to express chimeric receptors of human EGFR and rat Erbb2, and this B2B1 subclone was stochastically profiled (PMID: 20228812) after 24 hours of 3D culture with or without synthetic dimerization.
2023-03-22 | GSE214455 | GEO
Project description:Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent.
2016-10-06 | GSE87669 | GEO
Project description:Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent.
2016-10-06 | GSE87667 | GEO
Project description:Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent.
2016-10-06 | GSE87662 | GEO
Project description:Herbaspirillum sp. RU 5E strain:RU 5E Genome sequencing and assembly
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Project description:The fibroblast-populated 3D collagen matrix has been used to study the effect of mechanical stress on cell fate; this process is relevant to the fields of wound healing and tissue engineering. Gene array data was generated from mechanically stressed vs. stress-released matrices. The parameters of the collagen matrix model were: collagen type = bovine type I; collagen concentration = 1.5 mg/mL; initial matrix volume = 0.2 mL; initial matrix diameter = 11 mm (cultured in 24-well plates); cell type = human foreskin fibroblast, passage <10; initial matrix cell concentration = 1,000,000 cell/mL (200,000 cell/matrix); culture medium = 5% FBS in DMEM with 1 µg/mL ascorbate. Matrices (n = 6 per experimental group) were incubated for 24 hr in the attached state; the released groups then underwent matrix detachment from the culture plate (defined as t = 0), while the attached groups were left undisturbed. RNA was isolated from attached and released matrices 6 and 24 hr after t = 0. Gene expression in the attached vs. released condition at 6 or 24 hr then was analyzed by hybridizing the anti-sense RNA derived from attached and released matrices at a given time point onto a single chip.
2012-07-20 | GSE39475 | GEO