Transcript profiling of mouse 4T1 tumors grown in preirradiated vs. nonirradiated mammary tissue

The present study was designed to identify genes induced by irradiation in the 4T1 breast cancer model mimicking aggressive local relapse after radiotherapy. For this purpose, we obtained the transcriptomes of 4T1 tumors grown in either preirradiated (IRR+4T1) or non-irradiated (4T1) mammary tissue. Primary tumors were initiated by orthotopic injection of 4T1 (ATCC; CRL-2539) mammary carcinoma cells into BALB/c mice (Charles River Laboratories) as described (1). To investigate the effect of mammary tissue irradiation on gene expression, the fourth mammary gland was either pretreated with a single dose of 20 Gy X-ray irradiation or left untreated before implanting 4T1 tumor cells (1, 2). Tumor tissue samples were collected, flash-frozen and homogenized in RLT buffer using a Dispomix Drive (Medic Tools). Total RNA was isolated using the RNeasy Mini Kit (Qiagen), converted into labeled cDNA and hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST arrays. RMA-normalized expression values were calculated with the affy package from Bioconductor 2.4 (3), and differentially expressed genes were identified using moderated t-statistics calculated with the empirical Bayes method as implemented in the Bioconductor limma package (4). To be considered as differentially expressed between tumors grown in preirradiated (IRR+4T1) and nonirradiated (4T1) mammary tissue, genes had to pass the filters: P-value ≤ 0.05, a log2 fold change ≥ 0.4 and a log2 average expression value ≥ 4.0. References: 1. Kuonen F, Laurent J, Secondini C, Lorusso G, Stehle JC, Rausch T, et al. Inhibition of the Kit ligand/c-Kit axis attenuates metastasis in a mouse model mimicking local breast cancer relapse after radiotherapy. Clin Cancer Res 2012;18:4365-74. 2. Monnier Y, Farmer P, Bieler G, Imaizumi N, Sengstag T, Alghisi GC, et al. CYR61 and αVβ5 integrin cooperate to promote invasion and metastasis of tumors growing in preirradiated stroma. Cancer Res 2008;68:7323-31. 3. Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80. 4. Smyth GK, Speed T. Normalization of cDNA microarray data. Methods 2003;31:265-73.