cell line: JIMT1 cell type: breast cancer cells culture method: polyHEMA anchorage independent 3D culture for four days
Treatment protocol
For gene expression studies JIMT1 cell were grown in 6 well plates in 2D (7d), PH (4 and 7 days) or MG (4 and 7 days) models. Matrigel was dissolved prior to RNA isolation using Dispase (BD Biosciences). To obtain xenograft tumors of JIMT1 cells, the fat pads of BALB/C-nude mice were injected with 1*10-6 JIMT1 cells in 25 ml of medium and 25 ml of Matrigel. The tumors were collected 43 days after injection at 99 - 158 mm2. Tumor size was calculated from palpation results using (lenght/2 * width/2) * π. PolyHEMA plates were coated prior to use with 2.3 ml/well of PolyHEMA (poly (2-hydroxyethyl methacrylate), Polysciences Inc.), ethanol (>99 %) and sterile water in a 4:90:6 ratios. The plates were left to dry for 7 days at 37oC (or until the wells were dry).The Matrigel membrane was created by pipetting of ice-cold Matrigel dilution (1/2 Matrigel (Basement Membrane Matrix Growth Factor Reduced, BD Biosciences) and 1/2 Opti-MEM® (Life technologies)) to 6-well plates. The membrane was left to polymerize to cell culture inbubator for 20 minutes, after which the cells in cell culture medium were added on top.
Growth protocol
JIMT1 cells were cultivated in a 1:1 mixture of Ham’s F-12 + glutamax medium (Gibco Invitrogen, USA) and Dulbecco’s modified Eagles medium (DMEM, glucose 4.5 g/l; Sigma Aldrich), with 10 % FBS, 2 mM L-glutamine, 0.01 mg/ml insulin, and 1 % penicillin/streptomycin. JIMT1 (DSMZ GmbH, Germany) is a HER2+, ER-, PR-, epithelial-like cell line established from a pleural effusion of a 62-year-old female with trastuzumab-resistant ductal breast cancer (Tanner et al. 2004).
Extracted molecule
total RNA
Extraction protocol
RNA isolation was done using mirVana™ (Life technologies) kit according to manufacturer’s instructions. Quality control was performed with Agilent Bioanalyser. Gene expression was analyzed using illumina HumanHT-12 v 4.0 Expression BeadChip (>47 000 probes). Two biological replicates were used for each sample.
Label
Cy3-streptavidin
Label protocol
standard
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Data processing
The Illumina data was preprocessed by Bioconductor beadarray and lumi packages (Du et al., 2008). The lumiExpresso function was used with default settings, including the variance stabilizing transformation and quantile normalization across samples. (Du P, Kibbe WA, Lin SM. (2008). Lumi: A pipeline for processing illumina microarray. Bioinformatics 24: 1547-1548.)
