biostudies-arrayexpress00260Michal RajskiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-29035As a model for investigating changes in gene expression in response to epithelial-osteoblast interactions in bone metastases of breast carcinomas, cells that represented malignant epithelial cell compartments (T47D) and cells that represented skeletal compartments (Normal Human (NH) Osteoblasts) were examined in an in vitro mixed co-culture setting. These two types of cells were co-cultivated for 48 h in a low-serum medium [0.2% fetal bovine serum (FBS)] to allow reciprocal signal exchange with minimal background from undefined molecular signals that are inherent in fetal bovine serum. We examined the effects of co-cultivation on each cell pairing in two independent biological replicates. The gene expression profiles of the co-cultures were compared to the expression profiles of the corresponding cells that were kept in monoculture using HEEBO microarrays. After mono or co-culturing, total RNA was extracted and amplified using a modified Eberwine procedure. The amplified RNA was labeled with the fluorescent dye Cy5 and pooled with Cy3 labeled reference RNA, and then the pooled RNA was hybridized onto HEEBO microarrays. After hybridization and washing, arrays were scanned on a fluorescent microscope scanner. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.biostudies-arrayexpressNucleic Acid Extraction - RNeasy; Total RNA was isolated using the RNeasy mini kit (QIAGEN), according to the manufacturer's instructions.; Protocol Type = Extract preparation; Performer: Michal,,Rajski MessageAmp II; RNA was amplified using the Message AmpTM II aRNA Kit (Ambion, Austin, TX) according to the manufacturer's instructionsNucleic Acid Extraction - MessageAmpII; Total RNA was amplified using the Message AmpTM II aRNA Kit (Ambion, Austin, TX), according to the manufacturer's instructionsLabeling - StratageneRef; RNA for the reference was pooled from 11 cell lines - obtained from Stratagene Ambion MessageAmpII Amino Allyl and Cy Dye Coupling for Oligo Arrays CH1; RNA was processed using the Message AmpTM II aRNA Kit (Ambion, Austin, TX) according to the protocol of Kate Rubins/Brown Lab - 04/14/2006 (update 05/25/2006)Labeling - Ambion MessageAmpII Amino Allyl and Cy Dye Coupling for Oligo Arrays CH2; RNA was processed using the Message AmpTM II aRNA Kit (Ambion, Austin, TX) according to the protocol of Kate Rubins/Brown Lab - 04/14/2006 (update 05/25/2006)Growth Protocol - osteoblast co-culture; Human mammary epithelial cells (HMEC) (Cambrex Bio Science Walkersville, Walkersville, MD) were expanded in mammary epithelial basal medium that was supplemented with bovine pituitary extract, human EGF, insulin and antibiotics (Clonetics, Cambrex Bio Science Walkersville, Walkersville, MD). MCF-7, T47D, MDA-MB231, SKBR-3, Hs578T, (ATCC, Atlanta, GA) were propagated in DMEM with 4.5% glucose (Gibco, Grand Island, NY) that was supplemented with 10% FBS (HyClone, Logan, UT), glutamine, 100 U/ml penicillin and 100 ??g/ml streptomycin (Gibco, Grand Island, NY). Normal human Osteoblasts (NHOST) (Lonza Walkersville, Walkersville, MD) were expanded in Clonetics Osteoblast Basal Medium (OBM???) (Lonza Walkersville, Walkersville, MD) supplemented with osteoblast growth medium (OGM TM) SingleQuots?? (Lonza Walkersville, Walkersville, MD) with 10 % FBS. For the co-culture experiments, the cells were cultivated for 48 hours at equal density of 30???000 cells/cm2 (15???000 tumor cells /cm2 and 15???000 normal human Osteoblast cells /cm2) in Clonetics Osteoblast Basal Medium (OBM???) supplemented with 0.2% FBS without any further additives. This medium served as a good universal media for all the cells in the studyHybridization - not providedMIAME ScoreOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsFeature Extraction - VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)Assay Data Transformation - ID_REF = ID_REF<br>CH1I_MEAN = Mean feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale<br>CH2I_MEAN = Mean feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale<br>CH1B_MEDIAN = The median feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel; Background<br>CH2B_MEDIAN = The median feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background<br>CH1D_MEAN = The mean feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel<br>CH2D_MEAN = .The mean feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy5 channel<br>CH1I_MEDIAN = Median feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale<br>CH2I_MEDIAN = Median feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale<br>CH1B_MEAN = The mean feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Background<br>CH2B_MEAN = The mean feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Background<br>CH1D_MEDIAN = The median feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale<br>CH2D_MEDIAN = The median feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale<br>CH1_PER_SAT = The percentage of feature pixels at wavelength 532 nm that are saturated.; Type: integer; Scale: linear_scale<br>CH2_PER_SAT = The percentage of feature pixels at wavelength 635 nm that are saturated.; Type: integer; Scale: linear_scale<br>CH1I_SD = The standard deviation of the feature intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel<br>CH2I_SD = The standard deviation of the feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel<br>CH1B_SD = The standard deviation of the feature background intensity at wavelength 532 nm.; Type: float; Scale: linear_scale; Channel: Cy3 Channel; Background<br>CH2B_SD = The standard deviation of the feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background<br>PERGTBCH1I_1SD = The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale<br>PERGTBCH2I_1SD = The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm.; Type: integer; Scale: linear_scale<br>PERGTBCH1I_2SD = The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale<br>PERGTBCH2I_2SD = The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale<br>SUM_MEAN = The sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale<br>SUM_MEDIAN = The sum of the median intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale<br>RAT1_MEAN = Ratio of the arithmetic mean intensities of each spot for each wavelength, with the median background subtracted. Channel 1/Channel 2 ratio, (CH1I_MEAN - CH1B_MEDIAN)/(CH2I_MEAN - CH2B_MEDIAN) or Green/Red ratio.; Type: float; Scale: linear_scale<br>RAT2_MEAN = The ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale<br>RAT2_MEDIAN = The ratio of the median intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale<br>PIX_RAT2_MEAN = The geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale<br>PIX_RAT2_MEDIAN = The median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale<br>RAT2_SD = The geometric standard deviation of the pixel intensity ratios.; Type: float; Scale: linear_scale<br>TOT_SPIX = The total number of feature pixels.; Type: integer; Scale: linear_scale<br>TOT_BPIX = The total number of background pixels.; Type: integer; Scale: linear_scale<br>REGR = The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.; Type: float; Scale: linear_scale<br>CORR = The correlation between channel1 (Cy3) & Channel 2 (Cy5) pixels within the spot, and is a useful quality control parameter. Generally, high values imply better fit & good spot quality.; Type: float; Scale: linear_scale<br>DIAMETER = The diameter in um of the feature-indicator.; Type: integer; Scale: linear_scale<br>X_COORD = X-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale<br>Y_COORD = Y-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale<br>TOP = Box top: int(((centerX - radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale<br>BOT = Box bottom: int(((centerX + radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale<br>LEFT = Box left: int(((centerY - radius) - yoffset) / pixelSize).; Type: integer; Scale: linear_scale<br>RIGHT = Box right: int(((centerY + radius) - yoffset) / pixelSize); Type: integer; Scale: linear_scale<br>FLAG = The type of flag associated with a feature: -100 = user-flagged null spot; -50 = software-flagged null spot; 0 = spot valid.; Type: integer; Scale: linear_scale<br>CH2IN_MEAN = Normalized value of mean Channel 2 (usually 635 nm) intensity (CH2I_MEAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel<br>CH2BN_MEDIAN = Normalized value of median Channel 2 (usually 635 nm) background (CH2B_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background<br>CH2DN_MEAN = Normalized value of mean Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale; Channel: Cy5 channel<br>RAT2N_MEAN = Type: float; Scale: linear_scale<br>CH2IN_MEDIAN = Normalized value of median Channel 2 (usually 635 nm) intensity (CH2I_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale<br>CH2DN_MEDIAN = Normalized value of median Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEDIAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale<br>RAT1N_MEAN = Ratio of the means of Channel 1 (usually 532 nm) intensity to normalized Channel 2 (usually 635 nm) intensity with median background subtracted (CH1D_MEAN/CH2DN_MEAN). Channel 1/Channel 2 ratio normalized or Green/Red ratio normalized.; Type: float; Scale: linear_scale<br>RAT2N_MEDIAN = Channel 2/Channel 1 ratio normalized, RAT2_MEDIAN/Normalization factor or Red/Green median ratio normalized.; Type: float; Scale: linear_scale<br>LOG_RAT2N_MEAN = Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEAN)].; Type: float; Scale: log_base_2<br>VALUE = Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEDIAN)].; Type: float; Scale: log_base_2Image Adquisition - Scanner Model: GenePix 4100A 01UnknownTranscriptomicsGenomicsProteomics<h4>Background</h4>Bone metastasis is a main cause of morbidity in breast cancer. Since breast cancer is a heterogeneous disease, the interactions of cancer cells with the skeletal host cells might also be diverse. We hypothesized that gene expression signatures induced by heterotypic interaction of breast cancer cells and osteoblasts might be of clinical relevance.<h4>Methodology/principal findings</h4>We established an ex vivo co-culture model using benign breast epithelial cells or a panel of 5 malignant breast epithelial cells in combination with primary human osteoblasts and determined associated gene expression changes with HEEBO microarrays. Pretreatment gene expression profiles of 295 early stage breast cancers published from the Netherlands Cancer Institute with a median follow up of 12.6 years allowed evaluating in vitro effects in the in vivo situation.The effects of the interaction between osteoblasts and breast cancer cell lines of different origin were very heterogeneous. Hs578T cells started to proliferate in co-culture with osteoblasts, SKBR-3 induced a TGF-β response and MDA-MB231 cells showed two distinct sets of up-regulated genes: A set of interferon response genes associated with an up-regulation of STAT1 was in vivo remarkably coherent providing a basis for segregation of tumors into two groups. In a uni-variate analysis, early stage tumors with high expression levels (n = 136) of this gene set had a significantly lower overall survival rate (p = 0.005) (63% at 10 years) than tumors with low expression levels (n = 159) (overall survival: 77% at 10 years). The second gene set was associated with IL-6 and did not significantly change the overall survival rate (p = 0.165), but was significantly associated with a shorter time to bone metastasis (p = 0.049; 74% vs. 83% at 10 years).<h4>Conclusion/significance</h4>An IL-6 gene expression pattern induced by heterotypic interaction of breast cancer cells with osteoblasts in vitro is associated with a higher rate of bone metastasis in vivo.transcription profiling by arrayHomo sapiensGlobal gene expression analysis of the interaction between cancer cells and osteoblasts to predict bone metastasis in breast cancer.Rajski M, Vogel B, Baty F, Rochlitz C, Buess MTatiparthi ReddyMichal Rajski26falseNormal human osteoblasts and T47D mono and cocultureAs a model for investigating changes in gene expression in response to epithelial-osteoblast interactions in bone metastases of breast carcinomas, cells that represented malignant epithelial cell compartments (T47D) and cells that represented skeletal compartments (Normal Human (NH) Osteoblasts) were examined in an in vitro mixed co-culture setting. These two types of cells were co-cultivated for 48 h in a low-serum medium [0.2% fetal bovine serum (FBS)] to allow reciprocal signal exchange with minimal background from undefined molecular signals that are inherent in fetal bovine serum. We examined the effects of co-cultivation on each cell pairing in two independent biological replicates. The gene expression profiles of the co-cultures were compared to the expression profiles of the corresponding cells that were kept in monoculture using HEEBO microarrays. After mono or co-culturing, total RNA was extracted and amplified using a modified Eberwine procedure. The amplified RNA was labeled with the fluorescent dye Cy5 and pooled with Cy3 labeled reference RNA, and then the pooled RNA was hybridized onto HEEBO microarrays. After hybridization and washing, arrays were scanned on a fluorescent microscope scanner. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.E-GEOD-29035GSE2903522235336EFO_000276810.1371/journal.pone.0029743