NPM-ALK silencing impairs the growth of human ALK⁺ cells in vivo. (A) TS cells transduced with ALK-A5 do not generate xenograph tumors in immunocompromised animals. TS cells transduced with ALK-A5 or with the control ALK-A6 lentivirus were injected into FoxChase (C.B-17) SCID mice 48 hours after infection. Tumor growth was observed over time. A total of 12 mice (6 for each group) was studied. (B) ALCL tumor mass growth is hindered by the intratumoral injection of lentiviruses carrying the ALK-A5 shRNA. FoxChase (C.B-17) SCID mice injected with TS cells (2 × 10⁶) were treated intratumorally when the tumor masses were about 1 cm³ with 50μL (0.5-1 × 10⁸ TU) of concentrated lentivirus preparations carrying ALK-A5 or control ALK-A6 shRNA, every alternative day, 3 times. Tumor growth was determined over time. (A-B) Error bars indicate SD. (C) ALK-A5 shRNA leads to cell death in vivo. After treatment, tumor cells were isolated and stained with PE–annexin V and evaluated by flow cytometry. Percentages of EGFP⁺ cells are indicated (left panels). Representative histologic sections (hematoxylin and eosin [H&E]) of tumor masses derived from animals treated with LV-A6 or LV-A5 preparations are shown as indicated (second panels from left; objective magnification, 20×). The same paraffin-embedded tissue sections were also stained for DNA breaks with a TUNEL method. Hoechst counterstain identifies the nuclei (third panels from left; objective magnification, 40×). DNA break points are demonstrated by the positive FITC stains (fourth panels from left; objective magnification, 40×). Variable percentages of positive cells were observed in different areas within the tumor cells treated with ALK-A5 lentiviral preparations. These findings are representative of 3 different experiments with a total of 12 mice for each group.