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RNA-seq of Highly Metastatic and Lowly Metastatic 4T1 Single Clones Treated with Iron Chelator Deferasirox (DFX)

ABSTRACT: Using bulk RNA-seq, the transcriptional profile of two cell lines with high or low metastatic properties derived from single clones of the murine 4T1 cell line treated or untreated with iron chelator DFX was analyzed in order to determine the mechanism underlying the inhibition preference of DFX for highly metastatic 4T1 cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE218133 | GEO | 2022/11/16

REPOSITORIES: GEO

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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:Purpose: Compare the tumor outgrowth and immunology of an immunocompetent 4T1- and Py230-based intraductal model for triple-negative breast cancer (TNBC). Methods: BALB/c-derived 4T1 and C57BL/6-derived Py230 mammary tumor cells were side-by-side intraductally inoculated in lactating and syngeneic mice, 4T1 and Py230 primary tumors were subsequently resected at 1, 3 and 6 weeks (w) post-inoculation (p.i.), and RNA was isolated from the 4T1 and Py230 primary tumors using in house developed protocols. Results: The differentially expressed genes in the 4T1 versus Py230 primary tumor datasets at the 3 different time points were categorized into 28 hallmark gene sets using GSEA. Nine hallmarks could be related to tumor immunology and collectively showed a decreased difference in expression over time, although every immunology-related gene set remained most expressed in 4T1 primary tumors across all time points. Seven hallmarks could be related to cellular mitosis and tumor progression and were significantly upregulated at 1 w p.i. in 4T1 primary tumors, whereas at 3 w p.i. they became significantly upregulated in Py230 primary tumors, indicative for enhanced Py230 tumor proliferation. The hallmark epithelial-mesenchymal transition was significantly upregulated at 3 w p.i. in 4T1 compared to Py230 primary tumors, indicative for metastatic progression. Gene sets involved in adipose/stromal processes within the mammary gland were downregulated or weakly expressed at 1 w p.i., but became upregulated at 3 w p.i. in 4T1 compared to Py230 primary tumors, indicating that at the time 4T1 tumor cells were less proliferative compared to Py230 tumor cells, the surrounding stroma and fat tissue were more active in the 4T1- compared to the Py230-based intraductal model. The hallmark androgen response also showed a significantly upregulated expression in Py230 tumors at 3 and 6 w p.i., indicating that androgen signaling is important in invasive Py230 tumors. Conclusion: Our findings highlight an innovative 4T1- and Py230-based intraductal mouse model for TNBC with a different tumor outgrowth and associated tumor microenvironment. These differential models may broadly represent the clinically observed TNBC diversity and together provide a powerful tool to evaluate therapeutics.

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RNA-seq of Highly Metastatic and Lowly Metastatic 4T1 Single Clones Treated with Iron Chelator Deferasirox (DFX)

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