Fig. 7

Combined treatment with PPP and ASP3026 suppresses NPM-ALK⁺ T cell lymphoma cell tumor growth in vivo. a C.B-17 SCID mice were randomized into four treatment groups at week 3 after i.v. injection of Karpas 299 cells permanently expressing firefly luciferase. Thereafter, mice were treated as illustrated. b Mice developed lymphoma tumors within approximately 3 weeks after injection of Karpas 299 cells, and were monitored weekly using bioluminescence imaging. The intensity of the signal is indicated by color (blue: low; green: intermediate; red: high tumor burden). Control mice treated only with vehicle showed aggressive tumor growth. Mice treated with a PPP (20 mg/kg) or a low dose of ASP3026 (5 mg/kg) alone showed reduced tumor burden compared to vehicle-treated mice. Importantly, mice that received the combination regimen showed almost complete absence of tumor growth after 4 weeks from the injection. c Kaplan–Meier survival curves show that mice treated with PPP or low dose of ASP3026 alone had superior overall survival compared with control mice treated with vehicle. However, mice that received a combination treatment of PPP and ASP3026 showed more improved survival than control mice. Notably, mice that received the combination treatment demonstrated statistically superior overall survival compared to mice treated with PPP or ASP3026 alone. d At the end of the experiments, mice were scarified and, then, tumors were collected, processed, and examined microscopically. H&E staining shows that tumors from control mice as well as tumors from mice treated with PPP or a low dose of ASP3026 alone demonstrate sheets of large anaplastic cells with numerous atypical mitotic forms. In contrast, tumors from mice treated simultaneously with PPP and a low dose of ASP3026 showed extended areas of necrosis (right side of H&E-stained section from the PPP + ASP group) in between residual tumor cells. All tumors were positive for NPM-ALK. PPP or a low dose of ASP3026 alone modestly decreased the proliferation of NPM-ALK⁺ T cell lymphoma cells, as illustrated by the Ki-67 stain. In a clear contrast, combined treatment with PPP and ASP3026 was associated with a remarkable decrease in the proliferation of these lymphoma cells with a complete absence of cell proliferation in the extended necrotic areas (right side of Ki-67-stained section from the PPP + ASP group). Similarly, whereas a slight decrease was noted in phosphorylated IGF-IR and STAT3 after treatment with PPP or a low concentration of ASP3026 alone, a pronounced reduction in the levels of these survival-promoting proteins was observed after combined treatment. Notable PPP or ASP3026 alone slightly decreased the frequency of expression of phosphorylated STAT3 in the nucleus. The effects on phosphorylated STAT3 localization in the nucleus became more evident when combined treatment with PPP and ASP3026 was employed. (Original magnification for all photomicrographs: × 400)