Gene expression analysis of murine 4T1 breast cancer cells derived from primary tumor, lung metastasis and brain metastasis

We recently established an orthotopic breast cancer model of brain metastasis based on the injection of murine breast cancer cell lines into the mammary fat pad. This model is based on the use of 4T1 murine breast carcinoma cells. 4T1-derived tumors recapitulate the main steps of human breast cancer progression, including epithelial-to-mesenchymal transition and metastases to lung and lymph nodes. Bioluminescence imaging revealed the appearance of secondary lesions to the lung and lymph nodes and sporadically to the brain. Brain metastases were confirmed by macroscopic and microscopic evaluation of the brains at necropsy. We then isolated brain metastatic cells, re-injected them orthotopically in syngeneic (BALB/c) mice and isolated again cell lines from brain metastatic lesions for two rounds of selection. We obtained a cell line metastasizing to the brain with 100% penetrance (named 4T1-BM2 for Brain Metastasis, 2nd generation). In parallel we derived after two rounds of in vivo growth tumor cell lines from primary tumors (4T1-T2) and from lung metastases (4T1-LM2). Primary, lung and brain metastasis tumors from 4T1 murine breast carcinoma cells were re-derived from nine independent mice (three mice per cell line). The cell lines were expanded and frozen as independent lines. For RNA extraction, the nine cell lines were thawed at the same time and cultured in parallel, under identical conditions. Samples were also processed at the same time by RNAeasy mini kit (Qiagen) and subsequently RNA content and quality were determined by NanoDrop spectrophotometer.