miR-181a slows tumor growth rate and prolongs animal survival in the ABC-like DLBCL xenograft model. Xenografts were generated with OCILY19 (GCB-like DLBCL) or OCILY10 (ABC-like DLBCL) cells (5 × 10⁶), induced to express control or miR-181a by supplementing the mice drinking water with DOX (1 mg/mL). (A) Ex vivo assessment of efficient DOX induction in the animals. FACS analyses of RFP+ signal from control and miR-181a–induced OCILY19 and OCILY10 cells extracted from the mice tumors (i), and quantitative real-time–PCR analysis of miR-181a expression (ii) corrected for RNU6B levels, mean ± SE (n = 3; 2 independent experiments), Student t test (*P < .05; **P < .01). (B) Tumor growth curves. The tumor volume was assessed 3 times a week with calipers, 10 mice per cell line (n = 5 control; n = 5 miR-181a). Statistical significance two-way ANOVA tests (****P < .0001) from 2 independent experiments. (C) Kaplan–Meier plots: OCILY19 (n = 8 per group) and OCILY10 (n = 10 per group). Mice where euthanized when the tumor volume reached 1500 mm³ in accordance with the institutional guidelines. Statistical significance by log-rank test from 2 independent experiments. (D) Ex vivo analyses of the tumors. The xenografts were generated as described in panel A but the DOX treatment (8 days) was initiated when the tumors were palpable (∼2 weeks). Shown are representative FACS analyses of PE Texas Red⁺ cells evaluated for cell viability. The cells were stained with Annexin V–FITC and allophycocyanin-Cy7. The graph below depicts cumulative results (n = 4 mice per group), showing the percent difference in live cells between the induced miR-181a and the control cells. Statistical significance by Student t test (**P < .01; ns, not significant) presented as mean ± SD. (E) Immunohistochemistry and TUNEL assay in paraffin embedded xenograft tumors slides. Thin histologic sections were stained with hematoxylin and eosin (H&E), and analyzed for the expression of the B-cell markers CD20 (low expression in OCILY19 cell line⁴³ ) or CD79A. The TUNEL assays were performed with the In Situ Cell Death Detection Fluorescein Kit following the manufacturer’s instructions. Nuclei were labeled with DAPI, and images were acquired on a Zeiss LSM 700 confocal microscope at ×400 magnification. Bar, 40 μm. One representative mouse out of 3 per group is shown. DAPI, 4,6 diamidino-2-phenylindole.