Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Single Cell Transcriptomic Analysis of Prostate Cancer Cells

ABSTRACT: The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). We wanted to determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell. We clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, we analyzed 10 individual DTC isolated from each of 2 patients with metastatic PCa. We have shown that a transcriptomic profile can be obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled cell samples, but this method can be used to obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa. Custom Agilent 44K whole human genome expression oligonucleotide microarrays were used to profile clonally selected and cultured single passage of cell cycle synchronized C4-2B PCa cells isolated using a micromanipulator under direct visualization with an inverted microscope into ten sets of single, 5-, or 10-cells. Single disseminated tumor cells were isolated from bone marrow (BM) samples of two advanced prostate cancer patients. Essentially, a two-step selection process was employed, in which anti-CD45 and anti-CD61 conjugated to immunomagnetic beads were used for negative selection, and anti-HEA was used for positive selection. Cells were then fluorescently stained for BerEP4, counter stained with RPE anti-CD45, and individually selected (10 single cells each per patient) under fluorescent light using a micropipette system for further analysis. RNA was amplified using the WT-Oviation one-direct system and hybridization against a common reference pool of prostate tumor cell lines. Data from C42B cell data and data from single cells isolated from the bone marrow of patients were normalized and analyzed separately.

ORGANISM(S): Homo sapiens

SUBMITTER: Ilsa Coleman

PROVIDER: E-GEOD-38416 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Injury of the CA1 subregion induced by a single injection of kainic acid (1M-CM-^WKA) is attenuated when juvenile animals (P20) have a history of two sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early life conditioning seizures, we profiled and compared the transcriptomes of CA1 subregions from control, 1M-CM-^WKA, and 3M-CM-^WKA treated animals. More genes were regulated following 3M-CM-^WKA (9.6%) than after 1M-CM-^WKA (7.1%). Following 1M-CM-^WKA, genes supporting oxidative stress, growth, development, inflammation, and neurotransmission were upregulated (e.g., Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12, Grik1). After 3M-CM-^WKA, protective genes were differentially over-expressed (e.g., Cat, Gpx7, GAD1, Hspa12A, Foxn1, adenosine A1 receptor, Ca2+ adaptor and homeostatic proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp, suppressor of cytokine signaling (Socs3)). Distinct anti-inflammatory interleukins not observed in adult tissues (e.g., IL6 transducer, IL23 and IL33 or their receptors (ILF2)) were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1M-CM-^WKA but reduced after 3M-CM-^WKA; pro-inflammatory gene cox1 was either upregulated or unchanged after 1M-CM-^WKA but reduced by ~70% after 3M-CM-^WKA. Enhanced GFAP immunostaining following 1M-CM-^WKA was selectively attenuated in the CA1 subregion after 3M-CM-^WKA. The observed differential transcriptional responses may contribute to early life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca2+ permeability of the hippocampus and redirecting inflammatory and apoptotic pathways which could lead to new genetic therapies for epilepsy. The transcriptomes of the hippocampal CA1 region of Sprague Dawley 23-day-old male rats after 1 or 3 seizures induced by kainic acid injection were compared to the corresponding controls (injected with PBS) using Duke 27k oligonucleotide arrays.

Project description:We identified genes expressed in mouse liver that are regulated by Cux2, a highly female-specific liver transcription factor whose expression is regulated by sex-dependent plasma GH patterns. Using adenovirus to overexpress Cux2 (Adeno-Cux2) in male liver, we show that Cux2 represses ~35% of male-biased genes and induces/de-represses ~35% of female-biased genes. Adeno-CMV was used as a control for adenoviral infection. (Published in Molec Cell Biology, TL Conforto et al, 2012) Liver RNA isolated from the following eight groups of mice was used in the present study: (1) 8 wk old untreated male (M) mice (n = 10; 5 per each pool); (2) 8 wk old untreated female mice (F) mice (n = 11; 5 or 6 per each pool); (3) 8 wk old male mice treated with Adeno-Cux2 and euthanized 5 days later (n = 12; 6 per each pool); (4) 8 wk old female mice treated with Adeno-Cux2 and euthanized 5 days later (n = 8; 4 per each pool); (5) 8 wk old male mice treated with Adeno-CMV and euthanized 5 days later (n = 13; 6 or 7 per each pool); (6) 8 wk old female mice treated with Adeno-CMV and euthanized 5 days later (n = 7; 3 or 4 per each pool); (7) 8 wk old male mice treated with Adeno-Cux2 and euthanized 3 days later (n=11; 5 or 6 per each pool); (8) 8 wk old male mice treated with Adeno-CMV and euthanized 3 days later (n=11; 5 or 6 per pool). These RNA pools were used in four separate sets of competitive hybridization experiments: 1) 8 wk untreated M vs. 8 wk untreated F; 2) 8 wk M + Ad-Cux2 (5 day) vs. 8 wk M + Ad-CMV (5 day); 3) 8 wk F + Ad-Cux2 (5 day) vs. 8 wk F + Ad-CMV (5 day); 4) 8 wk M + Ad-Cux2 (3 day) vs. 8 wk M + Ad-CMV (3 day). Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) RNA samples to Agilent Mouse Gene Expression 4x44k v1 microarrays (Agilent Technology, Palo Alto, CA; catalog # G4122F-014868) were carried out, with dye swapping for each of the three hybridization experiments to eliminate dye bias. Two microarrays, one for each mixed cDNA sample, were hybridized for each of the four fluorescent reverse pairs, giving a total of 8 microarrays.

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Single Cell Transcriptomic Analysis of Prostate Cancer Cells

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