Extended Data Fig. 8: RSV penetrates the blood-brain barrier when administered in vivo and does not cause major toxicities, while inhibiting the growth of breast cancer BrM.
A. 4T1.Br3 cells were treated with 7.5 μM RSV, harvested at different timepoints, and probed for indicated proteins. B. 4T1.Br3 and T11.Br1 cells were treated with RSV or vehicle as indicated, harvested, and probed for indicated proteins. C. 4T1.Br3 cells were pre-treated with 7.5 μM RSV or vehicle for 10 days, followed by the addition of the activated CD8+ T cells (E:T = 10:1). Bars indicate a lactate dehydrogenase activity indicative of cell death, in supernatant samples collected 48 h after addition of T cells. Each dot indicates a biological replicate. Two-sided T-test. D. Mass spectrometry analysis of RSV standard and BrM sample isolated from mice bearing EO771.Br3 BrM. The table shows RSV concentration in BrM lesions. N = 3 biological replicates. E. Dynamics of mouse weight changes upon twice daily oral gavage of RSV (100 mg/kg) or vehicle. Two-way ANOVA. F. Top: Representative H&E images of livers of EO771.Br3 BrM-bearing mice after 2 weeks of twice daily oral gavage treatment with vehicle or RSV. Scale bar = 100 μm. Bottom: Serum concentration of AST, ALT and LDH in EO771.Br3 BrM-bearing mice after 2 weeks of twice daily oral gavage treatment with vehicle or RSV. Two-sided T-test. G. Representative H&E staining of brains from mice ICA-injected using 4T1.Br3 or T11 cells and treated with vehicle or RSV. BrMs are outlined in yellow. Scale bar = 3 mm. H. Model illustrating the role of Cdk5 in breast cancer BrMs. Communication between BrM cells and BrM-educated astrocytes leads to the secretion of Emilin-1 by astrocytes, which in turn induces overexpression of Cdk5 in BrMs. Through phosphorylation of Irf2bp1, Cdk5 restricts phosphorylation and expression of Stat1 and Nlrc5, leading to reduced MHC-I expression on the surface of BrM cells, which eventually leads to immune evasion followed by metastatic outgrowth in the brain. Targeting Cdk5 by small molecule inhibitor roscovitine may control the outgrowth of breast cancer BrM. For C, results are shown as means ± SEM. N.S – not significant, RSV – roscovitine, Veh – vehicle, ICA – internal carotid artery, BrM – brain metastasis, LDH – lactate dehydrogenase, AST – aspartate transaminase, ALT – alanine transaminase.
