cell line: SUM159 cell type: breast cancer clinical subtype: basal
Treatment protocol
16 cell lines representing luminal HER2- (MCF7, T47D, ZR751, CAMA-1), luminal-HER2+ (BT474, SKBR3, AU565), HER2+ (HCC1954) and basal (SUM149, SUM159, MDA-MB-231, MCF10A, MCF12A, BT549, HBL100, and DKAT) breast cancer cell lines were evaluated for response to a single dose of 5Gy.
Growth protocol
In order to identify differences in the radiation response gene expression profiles of specific breast cancer subtypes, we exposed 16 biologically-diverse breast tumor cell lines to 0 or 5GY radiation. Microarray analysis was performed on RNA harvested from those cell lines. Microarray analysis was performed on RNA harvested from those cell lines. Samples were run in triplicate. Breast cancer cell lines displaying gene expression patterns consistent with distinct clinical breast subtypes were selected (25, 26) as follows: Luminal - MCF7, T47D, ZR751, CAMA-1, BT474 (Her2+), SKBR3 (Her2+), AU565 (Her2+); Basal - SUM149, SUM159, MDA-MB-231, MCF10A, MCF12A, BT549, HBL100, HCC1954 (Her2+) and DKAT. These cell lines reflect the heterogeneity of human breast cancer, thereby, providing a valid biological model for study of phenotype specific mechanisms. Cell lines growth conditions: MCF7, ZR751, MDAMB231, and SUM159 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, SH30071.03) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). SUM149 cells were grown in Ham's F-12 medium supplemented with 5% fetal bovine serum (Hyclone, SH30071.03), 5ug/ml Insulin (Sigma-Aldrich®, I- 5500), 1ug/ml Hydrocortisone (Sigma-Aldrich®, H-4001) and 1X Antibiotics / Antimycotic (Gibco, 15240-062). T47D cells were grown in MEM-alpha medium (Gibco, 12561) supplemented with 6% of fetal bovine serum (Hyclone, SH30071.03), 12mM Hepes (Gibco15630), 1XMEM NEAA(Gibco 11140), 1mM Sodium Pyruvate (Gibco, 11360), 1ug/ml insulin (Sigma-Aldrich®, I-5500), 1ug/ml Hydrocortisone (Sigma-Aldrich®, H-4001), 12.5ng/ml EGF (Sigma-Aldrich®, E4127) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). AU565 and HCC1965 cells were grown in RPMI1640 medium supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03), 1XSodium Pyruvate (Gibco 11360-070), 10mM Hepes(Gibco, 15630) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). BT474 and SKBR3 cells were grown in RPMI1640 medium supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03) and 1X Antibiotic-Antimycotic(Gibco, 15240-062). BT549 were grown in RPMI1640 medium supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03), 1XSodium Pyruvate (Gibco 11360-070), 10mM Hepes(Gibco, 15630), 8ug/ml Insulin(life technology 12585014) and 1X Antibiotic-Antimycotic(Gibco, 15240-062). CAMA1 cells were grown in MEM (Gibco 11095-080) medium supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03), 1XSodium Pyruvate (Gibco 11360-070), 1XNEAA (Gibco 11140) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). HBL100 cells were grown in McCoy’s 5a medium (Gibco 16600-082) supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03) and1X Antibiotic-Antimycotic (Gibco, 15240-062). MCF10A cells were grown in DMEM/F12 (GIBCO 11330-032) medium supplemented with 5% of Horse Serum (Invitrogen #16050-122), 0.5ug/ml of hydrocortisone(Sigma-Aldrich®,H4001), 10ug/ml of Insulin (life technology 12585014), 20ng/ml of EGF(Sigma-Aldrich® , e9644) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). MCF12A cells were grown in DMEM/F12 (GIBCO11330-032) medium supplemented with 10% of fetal bovine serum (Hyclone, SH30071.03), 1ug/ml of hydrocortisone (Sigma-Aldrich, H-4001), 5ug/ml of Insulin (life technology 12585014) and 1X Antibiotic-Antimycotic (Gibco, 15240-062). DKAT cells were grown in MEGM (Lonza, CC-3150) medium and 1X Antibiotic-Antimycotic (Gibco, 15240-062). All cell lines were maintained in a humidified incubator with 5% CO2 atmosphere at 37o C.
Extracted molecule
total RNA
Extraction protocol
Microarray analysis was performed on RNA harvested from those cell lines. Samples were run in triplicate. Isolation of RNA. Total RNA was collected from approximately 1x10^6 cells in each of the sixteen cell lines. RNA was isolated from the treated cell lines (5Gy) 24 hours after radiation using the RNeasy Mini Isolation Kit (QIAGEN, Valencia, CA). The same protocol was used for control cell lines but with 0Gy radiation. Samples were run in triplicate. RNA was hybridized to Affymetrix U133A2 arrays and processed in the Duke Microarray Core facility.
Label
Biotin
Label protocol
Affymetrix ENZO labeling kit for the cell lines (for the Affymetrix U133A2 arrays).
Hybridization protocol
Hybridization of samples to Affymetrix GeneChip arrays was performed according to Affymetrix manual instructions . RNA from the cell lines was hybridized to Affymetrix U133A2.0 arrays and processed in the Duke Microarray Core facility. Total RNA was collected from approximately 1x10^6 cells in each of the sixteen cell lines. RNA was isolated from the treated cell lines (5Gy) 24 hours after radiation using the RNeasy Mini Isolation Kit (QIAGEN, Valencia, CA). The same protocol was used for control cell lines but with 0Gy radiation. Samples were run in triplicate. RNA was hybridized to Affymetrix U133A2 arrays and processed in the Duke Microarray Core facility.
Scan protocol
Scanning of Affymetrix GeneChip arrays was performed according to the Affmetrix manual instructions
Description
Breast cancer cell lines displaying gene expression patterns consistent with distinct clinical breast subtypes were selected (1, 2) as follows: Luminal - MCF7, T47D, ZR751, CAMA-1, BT474 (Her2+), SKBR3 (Her2+), AU565 (Her2+); Basal - SUM149, SUM159, MDA-MB-231, MCF10A, MCF12A, BT549, HBL100, HCC1954 (Her2+) and DKAT. These cell lines reflect the heterogeneity of human breast cancer, thereby, providing a valid biological model for study of phenotype specific mechanisms. Total RNA was collected from approximately 1x10^6 cells in each of the sixteen cell lines. RNA was isolated from the treated cell lines (5Gy) 24 hours after radiation using the RNeasy Mini Isolation Kit (QIAGEN, Valencia, CA). The same protocol was used for control cell lines but with 0Gy radiation. Samples were run in triplicate. RNA was hybridized to Affymetrix U133A2 arrays and processed in the Duke Microarray Core facility. Cell line s159 - 35049
Data processing
The Affymetrix HG U133 A2.0 Gene Chip array interrogates mRNA expression based on 22,000 probe sets corresponding to 14,500 annotated human genes. All statistical analyses were conducted using the R statistical environment and extension packages from CRAN and the Bioconductor project. Gene expression estimates were obtained from Affymetrix arrays using the RMA algorithm. The quality of the resultant microarrays was assessed using R/Bioconductor. To check for sample outliers and batch effects, principal component analysis of the global gene expression was conducted. Batch effects were corrected by scale-shift normalization prior to analysis.
