Figure 1.

Novel MDA-IBC3 breast cancer sublines with different propensities for brain metastasis. A) The novel MDA-IBC3 sublines show distinct morphology in cell culture. B) Incidence of brain metastasis in mice injected with MDA-IBC3.1 and MDA-IBC3.2 sublines (P value from 2-sided Fisher’s exact test). C) Representative images of brain metastasis generated after tail-vein injection of MDA-IBC3.1 and MDA-IBC3.2 sublines, with quantification of brain metastasis burden. The horizontal lines indicate means and error bars indicate SD. P value from 2-sided Mann-Whitney test. D) Time to detect brain metastases (BM) was measured by detection of luciferase signal. Kaplan-Meier estimates of brain metastasis-free survival (BrMFS) E) and overall survival (OS) of MDA-IBC3.1 (n = 9) and MDA-IBC3.2 (n = 10) (F). P values from 2-sided log-rank tests and the hazard ratio (HR) with 95% confidence interval are shown for each graph. G) Heatmap from reverse-phase protein array analysis shows top differentially expressed proteins (fold change) in the MDA-IBC3.1 and MDA-IBC3.2 sublines. Data presented as means of 6 replicates for each sample. H) Reverse-phase protein array findings for phosphorylated NDRG1 (NDRG1_pT346). Horizontal lines indicate means and ranges. I) Immunoblotting analysis of expression of total NDRG1 and NDRG1_pT346 between the 2 sublines. GAPDH was used as internal control. Analysis of publicly available datasets for NDRG1 expression in matched primary tumor and brain metastasis (GSE125989, n = 16) J) and in unmatched samples (GSE43837, n = 19) (K). J) P values from 2-sided Wilcoxon tests; K) horizontal lines indicate means and error bars indicate SD. P value from 2-sided Mann-Whitney test. L) Percentage of patients free of brain relapse stratified by NDRG1 expression [GSE2034; NDRG1-low (n = 47); NDRG1-high (n = 60)]. P values from 2-sided log-rank test and the hazard ratio with 95% confidence interval are shown. RPPA = reverse phase protein array.