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Project description:The survival of isolated metastatic cells and expansion into macroscopic tumour has been recognized as a limiting step for metastasis formation in several cancer types yet the determinants of this process remain largely uncharacterized. In colorectal cancer (CRC), we identify a transcriptional programme in tumour-associated stromal cells, which is intimately linked to a high risk of developing recurrent disease after therapy. A large proportion of CRCs display mutational inactivation of the TGF-beta pathway but paradoxically they are characterized by high TGF-beta production. In these tumours, TGF-beta instructs a transcriptional programme in stromal cells, which confers a high risk of developing metastatic disease. We purified by FACS CD31(+), CD45(+), FAP(+) and Epcam(+) populations from fresh CRC samples and assessed their gene expression profiles Freshly obtained tumours from 6 CRC patients treated at Hospital del Mar or Hospital Clinic (Barcelona, Spain) were minced and incubated with Collagenase IV (100 U mL-1). Enzymatic reaction was stopped by adding 10% FBS, single cells were collected by sequential filtering and resuspended in ammonium chloride (0.15M) to lyse erythrocytes. Cells were stained with FAP unconjugated rabbit antibody. After two washes with HBSS, cells were stained with an APC conjugated anti-rabbit antibody; anti-hEPCAM/TROP1-FITC, anti-CD31-PE and anti-CD45-PE-Cy7 conjugated antibody. Dead cells were labelled with Propidium Iodide. Fluorescence Activated Cell Sorting (FACS) was used to separate 2000 cells from each population; [CD45(+), EPCAM(-), CD31(-), FAP(-)], [CD45(-) EPCAM(+), CD31(-), FAP(-)], [CD45(-), EPCAM(-), CD31(+),FAP(-)] and [CD45(-), EPCAM(-), CD31(-), FAP(+)].
2013-01-17 | E-GEOD-39396 | biostudies-arrayexpress
Project description:Human mucosa was collected from two different individuals undergoing colectomy. After treatment with EDTA, colonic crypts were isolated and further dis-aggregated using Dispase. Next, cells were stained using antibodies directed against the extracellular domain of EpCAM, PTK7, CD11, CD31, and CD45. Cells were analyzed and sorted via flow cytometry (BD Aria). After excluding non-epithelial cells (Epcam. CD11, CD31, and CD45 negative), the EpCAM positive fraction was divided into fractions expressing either high, medium, low, or negative levels of PTK7. Sorted cells were transferred to Trizol for RNA extraction and RNA was purified using the Qiagen RNA Mini Kit.
2016-01-12 | GSE68340 | GEO
Project description:We analysed the signature of sorted CD45-CD31-Ter119-EpCAM+ lung epithelial cells and CD45-CD31-Ter119-EpCAM- lung mesenchymal cells from the control vs. irradiated lungs of BALB/c mice.
2021-12-01 | GSE180823 | GEO
Project description:Human mucosa was collected from two different individuals undergoing colectomy. After treatment with EDTA, colonic crypts were isolated and further dis-aggregated using Dispase. Next, cells were stained using antibodies directed against the extracellular domain of EpCAM, PTK7, CD11, CD31, and CD45. Cells were analyzed and sorted via flow cytometry (BD Aria). After excluding non-epithelial cells (Epcam. CD11, CD31, and CD45 negative), the EpCAM positive fraction was divided into fractions expressing either high, medium, low, or negative levels of PTK7. Sorted cells were transferred to Trizol for RNA extraction and RNA was purified using the Qiagen RNA Mini Kit. Colonic crypts were isolated from normal human mucosa derived from individuals undergoing colectomy. Single cell fractions from these crypts were sorted and isolated RNA processed and hybridized to Affymetrix PrimeView Arrays
2016-01-12 | E-GEOD-68340 | biostudies-arrayexpress
Project description:We are interested in whether fibroblasts (CD45-CD31-Epcam-PDPN+PDGFRA+) cells exhibit unique transcriptional profiles. To this end, we digested tissues and FACS sorted CD45-CD31-Epcam-PDPN+PDGFRA+ fibroblasts CD45-CD31-Epcam-PDPN+PDGFRA- mesothelial cells and compared the transcriptional profiles of these cells. PRJ0014647
2019-07-16 | GSE130813 | GEO
Project description:Epithelial (CD31-CD45-EpCAM+) and stromal (CD31-CD45-EpCAM-) lung cells were derived by FACS from ShhCre;Ezh2fl/fl and control ShhCre;Ezh2fl/+ mouse embryos at day E16.5 (3 biological replicates per genotype-tissue combination). Total RNA extracted from the samples was subjected to NGS library preparation using TruSeq Stranded Total RNA with Ribo-Zero (Illumina). Completed libraries from different samples were sequenced on HiSeq 2000 TruSeq with SBS Kit v3 - HS reagents (Illumina) as 100 bp single end reads at the Australian Genome Research Facility.
2015-05-11 | GSE57391 | GEO
Project description:The survival of isolated metastatic cells and expansion into macroscopic tumour has been recognized as a limiting step for metastasis formation in several cancer types yet the determinants of this process remain largely uncharacterized. In colorectal cancer (CRC), we identify a transcriptional programme in tumour-associated stromal cells, which is intimately linked to a high risk of developing recurrent disease after therapy. A large proportion of CRCs display mutational inactivation of the TGF-beta pathway but paradoxically they are characterized by high TGF-beta production. In these tumours, TGF-beta instructs a transcriptional programme in stromal cells, which confers a high risk of developing metastatic disease. We purified by FACS CD31(+), CD45(+), FAP(+) and Epcam(+) populations from fresh CRC samples and assessed their gene expression profiles
2013-01-17 | GSE39396 | GEO
Project description:To identify genes encoding cell surface proteins that are preferentially expressed in NSCLC cells, CD45-CD31-EpCAM+ cells were purified by FACS from either tumor regions or unaffected regions in resected lung tissues from patients with NSCLC, then subjected to RNA sequencing analysis.
2024-08-05 | GSE226773 | GEO
Project description:To identify genes encoding cell surface proteins that are preferentially expressed in NSCLC cells, CD45-CD31-EpCAM+ cells were purified by FACS from either tumor regions or unaffected regions in resected lung tissues from patients with NSCLC, then subjected to RNA sequencing analysis.
2024-08-05 | GSE226772 | GEO
Project description:We identified an endothelial progenitor/stem like population in the endothelial fraction of preexsiting blood vessels. And we used microarray to see the difference of gene expression profiles between these two populations. Hindlimb muscle of wild type C57BL/6 were digested to single cells and stained with anti-CD31 antibody, anti- CD45 antibody, Propidium iodide and hoechst 33342. CD31+CD45- side population cells and CD31+CD45- main population cells were sorted by flow cytometry for RNA extraction and hybridized on Affimetrix microarrays.
2012-03-28 | E-GEOD-28240 | biostudies-arrayexpress