Project description:Comparative analysis of the transcriptome of primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a siRNA against murine SPARC (4T1-C18), primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a scramble sequence (4T1-SCR) or lung metastasis foci from 4T1-SCR tumor-bearing mice (4T1-SCR MTTS). Three experimental conditions, 4T1-C18, 4T1-SCR and 4T1-SCR MTTS. Biological replicates: 4 4T1-C18, 4 4T1-SCR, 4 4T1-SCR MTTS independently grown in different mice. 2 days-old tumors and 30 days old lung foci. One replicate per array. All microarrays were processed the same day
Project description:Comparative analysis of the transcriptome of primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a siRNA against murine SPARC (4T1-C18), primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a scramble sequence (4T1-SCR) or lung metastasis foci from 4T1-SCR tumor-bearing mice (4T1-SCR MTTS).
Project description:Comparative analysis of the transcriptome of 4T1 cells stably transduced with a lentiviral vector expressing a siRNA against murine SPARC (4T1-C18) with 4T1 control cells stably transduced with a lentiviral vector expressing a scramble sequence (4T1-SCR). Two-condition experiment, 4T1-C18 vs. 4T1-SCR cells. Biological replicates: 4 SPARC knock down, 4 control, independently grown in vitro and harvested. One replicate per array. Microarrays were hybridized in three different days.
Project description: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).
Project description:Metabolic rewiring is essential for breast tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. Transcriptional and metabolomic analysis have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic profile characterized by increased HIF-1α activity and dependence on glycolysis. Stable isotope tracing analysis (SITA) performed in vivo confirmed that the glycolytic nature of liver-metastatic breast cancer cells is retained when these cells are grown as primary tumors or as liver metastases. However, our data also reveal that unique metabolic adaptations are specifically induced by the liver microenvironment. Indeed, liver metastases display elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to primary tumors. Moreover, breast cancer liver metastases rely strongly on glucose and glutamine-derived carbons to support de novo GSH synthesis. Glutathione is a tripeptide that acts as a major scavenger for reactive oxygen species (ROS). Liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine, confirming their increased capacity to buffer ROS. We demonstrated the dependence of liver metastases formation on these distinct metabolic adaptations by depleting glutamate-cysteine ligase (Gcl), the rate-limiting enzyme in glutathione biosynthesis. Gcl depletion, and decreased GSH levels, strongly reduced the capacity of liver-metastatic cells to form distant metastasis within the liver. We performed gene expression analysis of liver metastasis and primary tumors from liver-metastatic breast cancer cells derived from 4T1 cells. 4T1-2776 (76) and 4T1-2792 (92) cells were injected in the mammary fat pads of BALB/c and grown as primary tumors or were grown as liver metastasis after splenic injection. Tissues were harvested from primary tumors and liver metastasis and submitted to laser-capture microdissection (LCM). For liver metastasis, tumour tissue was harvested at 10 days, 2 weeks and 3 weeks after splenic injection. For each time point, tissue was isolated from the tumor centre (core), a peripheral area of the tumor (margin), a region of the liver proximal to the tumor (adjacent) and a region of the liver far from the tumor (distant). For primary tumors from 4T1-2776 cells (1034, 1036, 1043) and 4T1-2792 (MFP2L, MFP3L, MFP5L) tissue was collected from the tumor core and margin, at the experimental endpoint.
Project description:Metastasis is the most important cause of cancer-related death and it has been the most important issue both in basic and clinical cancer researches. Through in vivo spontaneous metastasis animal model, we successfully cultivated and expanded the metastasis lineage cells for genomic, methylomic, and transcriptomic analyses based on the high efficiency and excellent viability of a modified CTC-capturing system together with primary and metastasis tumors as well as parental 4T1-hHER2 breast cancer cell line.
Project description:We utilized 3D-organotypic cultures whose physical properties were altered by inclusiong of type I collagen to create biomechanically rigid microenvironments that approximated those typically observed in primary mammary tumors. Compliant 3D-organotypic cultures were also generated to recapitulate the biomechanical properties of pulmonary microenvironments typically encountered by disseminated breast carcioma cells. The murine 4T1 progression series represents an established model of triple negative breast cancer development and metastasis and consists of isogenically-derived nonmetastatic 67NR, systemically invasive 4TO7, and highly metastatic 4T1 cells that were propagated for 6 days in the absense or presense of TGF-beta in either rigid or compliant 3D-cultures. Afterward, total RNA was extracted and subjected to miRNA profiling. two replicates of each growth and treatment condition for each cell line.