RNA-seq expression profiling of metastasis-associated endothelial cells from murine bone marrow

Breast tumour cells were found to remodel the murine bone marrow vascular microenvironment to support metastatic expansion. To gain insight into the host vasculature in response to tumor colonization, cells from the isogenic murine mammary tumour cell line 4T1.2 were intracardially injected into immunocompetent BALB/c mice. We analysed the transcriptional profiles of endothelial cells extracted from heavily infiltrated (Metastatic) bone marrow stroma and the adjacent tumor-free bone marrow stroma (Non-metastatic), as well as from healthy bones (Control). Methods: Immunocompetent BALB/c mice were intracardically injected with 4T1.2 tumor cells. The bones of these tumor-burdened mice were examined under fluorescence stereomicroscope to microdissect heavily-infiltrated (Metastatic) and the adjacent tumour-free (Non-metastatic) areas. Bones from healthy mice were used as controls. The dissected bone material from both burdened and control mice was processed to isolate EMCNhi/CD31hi endothelial cells using flow cytometry. RNA was extracted for 75bp paired-end RNA-seq profiling using an Illumina NextSeq 500 sequencer. Overall design: Two biological independent mouse pools were formed of healthy control mice and four pools of tumor-burdened mice. Each pool comprised between 1 and 4 mice in order to achieve sufficient material for RNA sequencing. Two control libraries, six non-metastatic libraries and three metastatic libraries were prepared for sequencing.