Mouse Brain Metastasis

While it is known that initiation and outgrowth of primary tumors is primarily driven by aberrant activation of kinases, their role in promoting the development of metastatic lesions is less well studied. We hypothesized that growth of metastasis can be promoted by specific selection of disseminating cells expressing certain kinases critical for survival in the metastatic environment. We identified that in breast cancer brain metastases, cyclin-dependent kinase 5 (CDK5) is highly up-regulated as compared to matched primary breast tumors. To investigate the role of CDK5 in brain metastasis, we established a knockdown of CDK5 in EO771.Br3 GFP-labeled brain-seeking breast cancer cells. The resultant EO771.Br3.shCtl and EO771.Br3.shCDK5 cells were injected into the left carotid artery of immunocompetent mice to establish experimental brain metastases. Upon reaching terminal morbidity, all mice were euthanized and brains harvested. GFP+ brain lesions were isolated, followed by sorting for live GFP+ cells and live CD45+ infiltrating immune cells. Sorted live cells were submitted for single-cell RNA sequencing analysis. To investigate how CDK5 knockdown affects gene expression of MHC-I-driving transcription factors, we also extracted RNA from EO771.Br3.shCtl (duplicates), EO771.Br3.shCDK5 #1 and EO771.Br3.shCDK5 #2. Cells were cultured until 80% confluent in 10 cm dishes. Bulk RNA was extracted using TRI reagent and quality-checked with use of an Agilent RNA 6000 Pico Kit by Agilent Bioanalyzer 2100. RNA with an integrity number of greater than 7 was used for library preparation.