E. J. Chung, A. White, S. Kwon, H. Ahn, and D. E. Citrin; Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
Purpose/Objective(s): Ionizing radiation is a commonly used treatment modality, but for superficial tumors, radiation exposure of overlying skin can result in dermal and subcutaneous fibrosis. Identification of agents that can reverse or mitigate radiation-induced skin injury is an area of unmet need. In this study, we sought to evaluate the effect of ABT-263 on radiation-induced late skin injury using a well-characterized murine model. Materials/
Methods: Ten-week-old female mice (C57L and C3H/HeN) were exposed to 35 Gy hind leg irradiation (IR). ABT-263 (50 mg/kg/day) or vehicle was administered (n>10/group) by oral gavage beginning at 5 weeks after IR (5 consecutive days per 3-week cycle x 3). Passive extension of the irradiated and contralateral unirradiated leg (internal control) was measured at 150 days after IR using a custom jig. Irradiated and contralateral unirradiated skin were collected at 150 days for assessment of skin thickness (H&E, Masson Trichrome Stain) and immunohistochemical assessment of p16, p21, myofibroblast marker (aSMA) and macrophage markers (F4/80, CD206). Gene expression for key senescence and macrophage markers was assessed with quantitative PCR from bulk RNA isolated from skin. The senolytic activity of ABT-263 in irradiated primary fibroblasts and effects of ABT-263 on primary macrophages were evaluated in vitro. Results: Both strains of mice developed progressive contracture of the irradiated hind limb (% contracture vs. unirradiated leg) that was significantly reduced by ABT-263 treatment (p<0.05). Histologically, ABT-263 treatment diminished IR induced collagen accumulation and dermal thickness (C3H/HeN: 201.3 vs. 98.2 µm, p<0.001; C57L: 172.7 vs. 146.0 µm, p=0.028) compared to vehicle treatment. A significant decrease IR-induced p16 mRNA in skin was confirmed in both strains after ABT-263 treatment (C3H/HeN: 6.2-, p=0.002 C57L: 1.8-fold decrease, p=0.005). By immunohistochemistry, the expression of p21 was elevated in unirradiated basal cells, suggesting it is not an optimal IR induced senescence marker in skin, whereas p16 was only highly expressed in dermis after IR. Treatment with ABT-263 markedly decreased IR induced p16 expression in dermis. Most p16 positive cells in irradiated dermis also expressed CD206 and were eliminated by ABT-263 treatment. ABT-263 in vitro treatment effectively eliminated senescent primary fibroblasts isolated from mouse skin (2.5-fold decrease, p<0.001) and was toxic to primary polarized (M2) macrophages (3.4-fold decrease, p=0.03). Conclusion: Treatment with ABT-263 ameliorates radiation-induced skin fibrosis in both C57L and C3H/HeN mice. The reduction in collagen accumulation and skin thickness after ABT-263 treatment was associated with a reduction of CD206+ and p16+ macrophages in irradiated dermis. This study is the first to demonstrate the utility of senolytic therapy in the treatment of radiation induced skin fibrosis and suggests that ABT-263 should be further explored as a therapy in this context.
