Abstract
As breast cancer migrates into the brain, it acquires distinct genomic, immunologic and phenotypic changes through interactions with the local microenvironment. We sought to determine how targetable gene expression changes mediate breast cancer extravasation into and proliferation within the brain. We created a brain metastatic cell line (“brain-seeking”) via serial intracardiac injection of mCherry+ 4T1 breast cancer cells in Balb/c mice and flow cytometry isolation of micrometastases. We conducted in vitro and in-vivo studies to uncover immunological and genetic differences between primary breast cancer and brain metastases. In culture, we observed significant upregulation in adhesion (p=0.01) and invasion (p=0.0004) along with upregulation of adhesion-promoting genes such as Sdc3, Serpine2 or ECM1 in the brain-seeking cells. We observed significant differences in survival of Balb/c mice injected intracranially with brain-seeking cells compared to the 4T1 breast cancer cells (p=0.04). In the mammary fat pad (MFP), parental 4T1 cells caused increased mortality compared to brain-seeking cells (p=0.003). This rejection of brain-seeking 4T1 cells in the MFP could be due to their prior adaptation to the brain microenvironment, while rejection of parental 4T1 cells in the brain microenvironment could be due to them lacking those adaptations. Consistent with this finding, of 20,382 analyzed genes in both cell lines, 108 related to immune regulation were significantly upregulated in brain-seeking cells including Cxcl1, Dusp1, C3. Flow cytometry showed increased cytotoxic and helper T cells in intracranially injected parental 4T1 cells vs. brain-seeking cells (p <0.001). In the MFP, however, 4T1 parental cells displayed CD3+ T cell depletion and an increased number of neutrophils (p <0.05) while the “brain-seeking cells” showed increased numbers of CD3+ and CD4+ cells (p <0.01) and macrophages (p<0.05). These findings suggest that brain metastases evolve from their primary tumor through adhesive and invasive adaptations promoting extravasation, along with unique immunological adaptations that allow them to proliferate free of immune surveillance in the brain microenvironment.