Therapeutic Reversal of Radiotherapy Injury to Pro-fibrotic Dysfunctional Fibroblasts In Vitro Using Adipose-derived Stem Cells

Lipi Shukla; Rodney Luwor; Matthew E Ritchie; Shiva Akbarzadeh; Hong-Jian Zhu; Wayne Morrison; Tara Karnezis; Ramin Shayan

doi:10.1097/GOX.0000000000002706

Therapeutic Reversal of Radiotherapy Injury to Pro-fibrotic Dysfunctional Fibroblasts In Vitro Using Adipose-derived Stem Cells

Plast Reconstr Surg Glob Open. 2020 Mar 25;8(3):e2706. doi: 10.1097/GOX.0000000000002706. eCollection 2020 Mar.

Authors

Lipi Shukla^{1

2

3

4}, Rodney Luwor⁵, Matthew E Ritchie⁶, Shiva Akbarzadeh⁴, Hong-Jian Zhu⁵, Wayne Morrison^{1

2

3}, Tara Karnezis^{1

2

3}, Ramin Shayan^{1

2

3}

Affiliations

¹ Regenerative Surgery and Lymphatics Department, O'Brien Institute, St. Vincent's Institute, Fitzroy, Victoria, Australia.
² Department of Plastic Surgery, St. Vincent's Hospital, Fitzroy, Victoria, Australia.
³ Department of Health Science and Exercise Science Bioengineering Lab, Australian Catholic University and O'Brien Institute Tissue Engineering Centre, Regenerative Surgery Group (AORTEC), Fitzroy Street, Fitzroy, Victoria, Australia.
⁴ Skin Bioengineering Laboratory, Victorian Adult Burns Service, The Alfred Hospital, Victoria, Australia.
⁵ Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital, Victoria, Australia.
⁶ Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Abstract

Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes.

Methods: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFβ) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery.

Results: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFβ was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF.

Conclusions: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.