Administration of TGF-ß Inhibitor Mitigates Radiation-induced Fibrosis in a Mouse Model

Abstract

Background: Radiation-induced fibrosis is a long-term adverse effect of external beam radiation therapy for cancer treatment that can cause pain, loss of function, and decreased quality of life. Transforming growth factor beta (TGF-β) is believed to be critical to the development of radiation-induced fibrosis, and TGF-β inhibition decreases the development of fibrosis. However, no treatment exists to prevent radiation-induced fibrosis. Therefore, we aimed to mitigate the development of radiation-induced fibrosis in a mouse model by inhibiting TGF-β.

Question/purposes: Does TGF-β inhibition decrease the development of muscle fibrosis induced by external beam radiation in a mouse model?

Methods: Twenty-eight 12-week-old male C57BL/6 mice were assigned randomly to three groups: irradiated mice treated with TGF-βi, irradiated mice treated with placebo, and control mice that received neither irradiation nor treatment. The irradiated mice received one 50-Gy fraction of radiation to the right hindlimb before treatment initiation. Mice treated with TGF-c (n = 10) received daily intraperitoneal injections of a small-molecule inhibitor of TGF-β (1 mg/kg) in a dimethyl sulfoxide vehicle for 8 weeks (seven survived to histologic analysis). Mice treated with placebo (n = 10) received daily intraperitoneal injections of only a dimethyl sulfoxide vehicle for 8 weeks (10 survived to histologic analysis). Control mice (n = 8) received neither radiation nor TGF-β treatment. Control mice were euthanized at 3 months because they were not expected to exhibit any changes related to treatment. Mice in the two treatment groups were euthanized 9 months after radiation, and the quadriceps of each thigh was sampled. Masson's trichome stain was used to assess muscle fibrosis. Slides were viewed at 10 × magnification using bright-field microscopy, and in a blinded fashion, five representative images per mouse were used to quantify fibrosis. The mean ± SD fibrosis pixel densities in the TGF-βi and radiation-only groups were compared using Mann-Whitney U tests. The ratio of fibrosis to muscle was calculated using the mean fibrosis per slide in the TGF-βi group to standardize measurements. Alpha was set at 0.05.

Results: The mean (± SD) percentage of fibrosis per slide was greater in the radiation-only group (1.2% ± 0.42%) than in the TGF-βi group (0.14% ± 0.09%) (odds ratio 0.12 [95% CI 0.07 to 0.20]; p < 0.001). Among control mice, mean fibrosis was 0.05% ± 0.02% per slide. Mice in the radiation-only group had 9.1 times the density of fibrosis as did mice in the TGF-βi group.

Conclusion: Our study provides preliminary evidence that the fibrosis associated with radiation therapy to a quadriceps muscle can be reduced by treatment with a TGF-β inhibitor in a mouse model.

Clinical relevance: If these observations are substantiated by further investigation into the role of TGF-β inhibition on the development of radiation-induced fibrosis in larger animal models and humans, our results may aid in the development of novel therapies to mitigate this complication of radiation treatment.

Conflict of interest statement

Each author certifies that neither he or she, nor any member of his or her immediate family, has funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request.

Copyright © 2020 by the Association of Bone and Joint Surgeons.

Figures

Fig. 1

This flow chart shows mouse allocation to each of the groups. Twenty-eight mice were assigned to three groups: a control group, a TGF-ßi, group (irradiated and treated with intraperitoneal injections of a small-molecule inhibitor of TGF-β in a dimethyl sulfoxide vehicle), and a radiation-only group (irradiated and injected with vehicle only); WT = wild type.

Fig. 2

This histologic image (at 10 × magnification) is of the quadriceps muscle of a mouse 9 months after irradiation, treated with the dimethyl sulfoxide vehicle only (no TGF-ß inhibitor). Fresh-frozen tissue was stained with Masson’s trichrome (red indicates muscle; blue indicates fibrosis).

Fig. 3

This histologic image (at 10 × magnification) is of the quadriceps muscle of a mouse 9 months after irradiation, treated with the TGF-ß inhibitor (1 mg/kg daily for 8 weeks) after radiation. Fresh-frozen tissue was stained with Masson’s trichrome (red indicates muscle; blue indicates fibrosis).

Fig. 4

This histologic image (at 10 × magnification) is of the quadriceps muscle of a control mouse, neither irradiated nor treated with a TGF-ß inhibitor. Fresh-frozen tissue was stained with Masson’s trichrome (red indicates muscle; blue indicates fibrosis).