Macrophage-induced integrin a5 (ITGA5) expression promotes enzalutamide resistance. (A) Correlation of ITGA5 expression with macrophage abundance in indicated bone metastasis datasets. (B) Expression of ITGA5 in bone metastasis and metastases from different organs in indicated patient datasets. (C) Expression of Itga5 in FPKM of MycCaP-Bo cells FACS-purified from indicated in vivo bone metastasis. (D) Representative histogram of ITGA5 expression on MycCaP-Bo cells FACS-purified from indicated in vivo bone metastasis. Number indicates mean fluorescent intensity. FMO, fluorescent minus one; negative control for flow cytometry staining. (E) Schematic showing key elements in the UniSAM vector. (F) Flow histogram of ITGA5 expression in control (Ctrl), and Itga5 overexpressing MycCaP-Bo cells clone 1 (#1 sgRNA, left) and clone 4 (#4 sgRNA, right); number indicating MFI. (G) In vitro response of control (Ctrl) and cells overexpressing Itga5 (#1, #4) to enzalutamide treatment in the presence of FN1 (1 μg/ml). Response was defined by relative growth of indicated cells with enzalutamide (1 μM) over vehicle treatment (n = 3). (H) High expression of Itga5 promotes enzalutamide resistance in vivo shown by relative growth of control (Ctrl) and cells overexpressing Itga5 (#4) with enzalutamide treatment versus vehicle, shown as relative BLI signal of enzalutamide treatment over vehicle treatment on day 18. Data are mean ± SEM in C, G, and H; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. Pearson correlation analysis was used in A, ANOVA was used in B, C, and G, and two-tailed unpaired Student’s t test was used in H.