Tumor EVs are required to program macrophages to a pro-metastatic phenotype in vitro (A) Schematic describing the method by which TEMs were programmed with tumor EVs, followed by analysis of TEM gene expression and effect of TEMs to TNBC invasion. TNBC Cells BM1 and LMB were used for all experiments. P-values were calculated using a two-way t-test (B) Relative invasion of TNBC cells pre-incubated for 24 h with TEM conditioned media (N = 6). (C) qRT-PCR of Ccl7, Tnfr2, Slpi, and Mmp12 in TEMs programmed with TNBC EVs compared to M-CSF alone (Control) with Gapdh as a loading control. P-values were calculated using a two-way ANOVA (N = 3 for LMB; N = 4 for BM1). (D) Relative invasion of TNBC cells pre-incubated for 24 h with TEM conditioned media (N = 3). TEMs were programmed with M-CSF (Control) or EVs from control (Ctrl EV) or sh-Rab27a tumor cells (shRab27a clones: sh3, sh5). P-values were calculated using a one-way ANOVA comparing changes in BM1 and LMB cells separately between groups (E) Relative BM1 invasion (mean +/− SEM) when cells were treated with TEM media programmed by an M-CSF control (control, grey), BM1 media with EVs (CM, red), or BM1 media with EVs removed (CM w/o EV, purple). p-values were calculated using a one-way ANOVA comparing both changes in both BM1 and LMB cells together between groups.