Abstract 980: Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF)
Authors: Ejaz Asim , Epperly Micheal , Fisher Renne , Zhang Xichen , Johngrass Moriah , Schusterman Asher , Kokai Lauren , Greenberger Joel , Rubin Peter 
Published: August 2018

Abstract
Radiation therapy doses above 50Gy to head and neck, breast cancer and soft tissue sarcomas can result in the rare late complication of RIF, which can significantly impair function. Several published case studies suggest injection of fibrotic sites with autologous adipose tissue stem cells can ameliorate RIF. In this study, we elucidated the molecular mechanism in an in vitro co-cultivation system and in vivo using a mouse model of hind limb fibrosis. In vitro Transwell co-cultures contained a bottom layer of irradiated human foreskin fibroblasts (HFFs), mouse or human cell lines derived from clinical fibrosis biopsies, and an upper layer containing freshly prepared ASCs. We quantitated levels of fibrosis -related genes in the lower layer cells and regulatory cytokines in the upper layer cells using quantitative real-time (qRT) PCR. In vivo studies: Female C57BL/6 mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electron beam. The irradiated and contralateral unirradiated flank tissue was monitored for expression of fibrosis related genes at days 1 and 14 post irradiation (PI). Fibrosis was confirmed by histologic staining for collagen and range of limb motion measurements. Irradiated fibrotic sites were injected at day 28 with ASCs from luciferase+ GFP+ mice. RIF was uniformly detected at day 14 and amplified by day 28, and confirmed by histologic staining for collagen by Masson's Trichrome. At day 14 PI there was clear upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), collagen 1 (400 fold), collagen3 (500 fold) and collagen4 (500 fold) compared to nonirradiated contralateral hind limb tissue. Irradiation induced by day 28 a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p &lt; 0.0001) degrees in the contralateral nonirradiated limb. A single ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). To measure possible systemic radiation mitigatory effects of ASCs PI, a single intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (P = 0.047). In transwell co-cultures ASCs demonstrated significant downregulation of profibrotic genes (including collagen 1-4, and TGF β) in both acutely irradiated and irradiation-fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was a key secreted mediator in co-culture models. Addition of human recombinant HGF to irradiated HFFs significantly downregulated profibrotic gene transcripts. Thus, HGF secreted by ASCs is involved in the reduction of RIF in vivo and with in vitro models of radiation fibrosis. Further studies should elucidate the mechanism of HGF and other secreted and cell contact regulators of adipose tissue-based cell therapy of radiation fibrosis.
Citation Format: Asim Ejaz, Micheal Epperly, Renne Fisher, Xichen Zhang, Moriah Johngrass, Asher Schusterman, Lauren Kokai, Joel Greenberger, Peter Rubin. Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 980.