Radiation-induced Skin Fibrosis Research Articles

Hyaluronic acid-based injectable formulation developed to mitigate metastasis and radiation-induced skin fibrosis in breast cancer treatment

The standard treatment for early-stage breast cancer involves breast-conserving surgery followed by adjuvant radiotherapy. However, approximately 20 % of patients experience distant metastasis, and adjuvant radiotherapy often leads to radiation-induced skin fibrosis (RISF). In this study, we develop an on-site injectable formulation composed of selenocystamine (SeCA) and hyaluronic acid (HyA), referred to as SeCA cross-linked HyA (SCH) agent, and investigate its potential to mitigate metastasis and prevent RISF associated with breast cancer therapy. SCH agents are synthesized using the nanoprecipitation method to modulate cell-cell tight junctions and tissue inflammation. The toxicity assessments reveal that SCH agents with a higher Se content (Se payload 17.4 μg/mL) are well tolerated by L929 cells compared to SeCA (Se payload 3.2 μg/mL). In vitro, SCH agents significantly enhance cell–cell tight junctions and effectively mitigate migration and invasion of breast cancer cells (4T1). In vivo, SCH agents mitigate distant lung metastasis. Furthermore, in animal models, SCH agents reduce RISF and promote wound repair. These findings highlight the potential of SCH agents as a novel therapeutic formulation for effectively mitigating metastasis and reducing RISF. This holds great promise for improving clinical outcomes in breast cancer patients undergoing adjuvant radiotherapy.

O166: Safety and Efficacy of Autologous Fat Grafting in the Treatment of Radiation-Induced Fibrosis and Contour Defects in Head and Neck Reconstruction

Abstract Background Multimodal treatment for head and neck cancer results in significant functional and cosmetic morbidity, often through contour deformity and radiation-induced skin fibrosis (RIF), affecting head and neck function and cosmesis. Autologous fat grafting is an emerging treatment for RIF. We conducted a retrospective cohort review of patient outcomes following autologous fat transfer for reconstruction of head and neck contour deformity and RIF. Methods A retrospective analysis was performed of patients treated at 2 UK centres between 2009-2022. Patient data collated included demographics, oncological treatment, reconstruction, operative details, and pre- and post-operative patient reported outcome measures (PROMs) including University of Washington Quality of Life Questionnaire (UW-QOL), Derriford Appearance Scale (DAS24) and Patient Outcomes of Surgery (POS). Patients were stratified by indication for AFG. Results A total of 45 patients were identified. 89% of patients (85% RIF, 92% contour deformity) reported cosmetic or functional improvement. Two patients (4%) reported no improvement, and three (7%) were lost to follow-up. Psychological health scores improved in patients following AFG for both RIF and contour deformity patients (DAS24, P< 0.0001). The UW-QOL scores revealed statistically significant improvements in multiple functional domains including appearance, activity, recreation, mood, and anxiety in both patients with RIF and contour deformities. Conclusion This study demonstrates that AFG is an effective technique for the correction of contour deformity and RIF in patients following treatment for head and neck cancer. Patients suffering from RIF demonstrated significant improvements in both functional and psychological domains.

Deciphering the fibrotic process: mechanism of chronic radiation skin injury fibrosis.

This review explores the mechanisms of chronic radiation-induced skin injury fibrosis, focusing on the transition from acute radiation damage to a chronic fibrotic state. It reviewed the cellular and molecular responses of the skin to radiation, highlighting the role of myofibroblasts and the significant impact of Transforming Growth Factor-beta (TGF-β) in promoting fibroblast-to-myofibroblast transformation. The review delves into the epigenetic regulation of fibrotic gene expression, the contribution of extracellular matrix proteins to the fibrotic microenvironment, and the regulation of the immune system in the context of fibrosis. Additionally, it discusses the potential of biomaterials and artificial intelligence in medical research to advance the understanding and treatment of radiation-induced skin fibrosis, suggesting future directions involving bioinformatics and personalized therapeutic strategies to enhance patient quality of life.

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice

BackgroundRadiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF.MethodsUsing a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy.ResultsProphylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts.ConclusionsOur study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Establishment of a Novel Explant Human Fasciocutaneous Flap Model to Understand the Mechanism of Radiation-Induced Skin Fibrosis

Establishment of a Novel Explant Human Fasciocutaneous Flap Model to Understand the Mechanism of Radiation-Induced Skin Fibrosis

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Autophagy gene Atg7 regulates the development of radiation-induced skin injury and fibrosis of skin.

Radiation-induced skin injury, which may progress to fibrosis, is a severe side effect of radiotherapy in patients with cancer. However, currently, there is a lack of preventive or curative treatments for this injury. Meanwhile, the mechanisms underlying this injury remain poorly understood. Here, we elucidated whether autophagy is essential for the development of radiation-induced skin injury and the potential molecular pathways and mechanisms involved. We used the myofibroblast-specific Atg7 knockout (namely, conditional Atg7 knockout) mice irradiated with a single electron beam irradiation dose of 30Gy. Vaseline-based 0.2% rapamycin ointment was topically applied once daily from the day of irradiation for 30days. On day 30 post irradiation, skin tissues were harvested for further analysis. In vitro, human foreskin fibroblast cells were treated with rapamycin (100nM) for 24h and pretreated with 3-MA (5mM) for 12h. Macroscopic skin manifestations, histological changes, and fibrosis markers at the mRNA and protein expression levels were measured. Post irradiation, the myofibroblast-specific autophagy-deficient (Atg7Flox/Flox Cre+ ) mice had increased fibrosis marker (COL1A1, CTGF, TGF-β1, and α-SMA) levels in the irradiated area and had more severe macroscopic skin manifestations than the control group (Atg7Flox/Flox Cre- ) mice. Treatment with an autophagy agonist rapamycin attenuated macroscopic skin injury scores and skin fibrosis marker levels with decreased epidermal thickness and dermal collagen deposition in Atg7Flox/Flox Cre+ mice compared with the vehicle control. Moreover, in vitro experiment results were consistent with the in vivo results. Together with studies at the molecular level, we found that these changes involved the Akt/mTOR pathway. In addition, this phenomenon might also relate to Nrf2-autophagy signaling pathway under oxidative stress conditions. In conclusion, Atg7 and autophagy-related mechanisms confer radioprotection, and reactivation of the autophagy process can be a novel therapeutic strategy to reduce and prevent the occurrence of radiodermatitis, particularly skin fibrosis, in patients with cancer.

1493 Dynamic changes in fibroblast subpopulations drives development of radiation-induced skin fibrosis through the fra/c-jun pathway

1493 Dynamic changes in fibroblast subpopulations drives development of radiation-induced skin fibrosis through the fra/c-jun pathway

Discussion of: Decellularized Adipose Matrices Can Alleviate Radiation-Induced Skin Fibrosis; Adv Wound Care (New Rochelle)

Discussion of: Decellularized Adipose Matrices Can Alleviate Radiation-Induced Skin Fibrosis; Adv Wound Care (New Rochelle)

Immunohistochemical and histochemical analysis of the rat skin after local electron irradiation.

Skin cancer is the most frequently diagnosed type of cancer among all malignant neoplasms. The decrease in mitotic activity and the death of intact keratinocytes arise due to the constantly renewing epithelium being highly sensitive to ionizing radiation. The aim of the study is immunohistochemical evaluation of the proliferative-apoptotic balance of keratinocytes, the fibrous component of the skin, and the expression of pro-inflammatory and anti-inflammatory cytokines after single or fractional local electron irradiation. Wistar rats (n = 80) were taken from the ITM&B Vivarium (Sechenov University) and divided into groups: I-control, which were injected with saline; and experimental groups, local electron irradiation at doses: II-8 Gy (single), III-40 Gy (single), IV-summary dose 78 Gy (fractional; 13 Gy per day for 6 days). We performed histological analysis, histochemical analysis using Masson, safranine, and picrosirius red staining, immunohistochemical (Ki-67, caspase-3, p53, types I and III collagens, IL-1, IL-6, IL-4, and IL-10) and morphometric analysis of skin fragments of the outer surface of the thigh, irradiated in accordance with the design of the experiment. The early and delayed effects of local electron irradiation at different doses were studied. After local electron irradiation, dose-dependent morphological changes in the skin of the experimental groups were observed: violation of the histoarchitectonics of the skin confirmed by morphological and morphometric analysis, the proliferation of connective tissue according to the results of histochemical and immunohistochemical studies with signs of the radiation-induced skin fibrosis development, an increase in the levels of pro- and anti-inflammatory cytokines. We observed the most pronounced signs of radiation-induced skin damage in the group of fractional irradiation after 3 months. 8 Gy and 40 Gy single local electron irradiation leads to a shift in the proliferative-apoptotic balance of keratinocytes toward their apoptosis, which activity is directly correlated with the dose of ionizing radiation, and 78 Gy in fractions leads to partial desquamation of the epithelium and inflammatory infiltration. In addition, after 3 months a significant increase in the expression of type I and III collagen fibers and the development of radiation-induced skin fibrosis takes place against the background of 78 Gy fractional local electron irradiation.

Erbium:YAG fractional laser ablation improves cutaneous delivery of pentoxifylline from different topical dosage forms

Topical application of pentoxifylline (PTX) would enable targeted treatment of radiation-induced skin fibrosis. However, PTX is hydrophilic with limited partitioning into the stratum corneum. The objective of this study was to investigate whether use of Erbium:YAG fractional laser ablation and different topical dosage forms (solution, hydrogel and patch) could be used to improve PTX cutaneous delivery as opposed to transdermal permeation. Initial results confirmed that fractional laser ablation significantly increased PTX delivery from each dosage form compared to passive controls. Delivery efficiencies of ∼ 30% were achieved with each dosage form but a large proportion of PTX permeated across the skin; thus, fluences were decreased to create shallower micropores, their depth being linearly dependent on fluence. The hydrogel was selected as the optimal formulation and PTX delivery efficiencies were further increased (44%-67%) by reducing the amount of hydrogel applied (better mimicking conditions of use). As this resulted in PTX depletion in the formulation, a loss of dependence of delivery on laser fluence was observed. These findings suggest that fractional laser ablation at moderate fluences enables an effective and targeted cutaneous delivery of PTX from a hydrogel formulation, which can be easily produced without the need for complex equipment.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery. The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

763 Radiation injury upregulates miR-196, increases dermal collagen, and triggers a pro-fibrotic genomic response that spreads in a murine model of radiation-induced skin fibrosis

Radiation-induced skin fibrosis (RISF) is a devastating result of cutaneous injury by irradiation that is delivered as part of clinical care, causing functional impairment and diminished quality of life for patients. To model RISF, we irradiated C57BL/6 mice (n=6) and collected skin from irradiated and non-irradiated sites after 24 weeks. Fibrosis was quantified by measuring dermal thickness and by machine learning ultrastructure analysis of dermal collagen network in tissue sections. Collagen levels were measured by hydroxyproline assay.

QS61. Irradiated Fibroblast Sub-populations Alter Gene Expression to Heal Murine Skin

Introduction: Radiation-induced skin fibrosis is a well-documented and burdensome sequela of radiation therapy, however the exact mechanism behind this effect is not well understood. We aim to better delineate the cellular mechanisms of acute and chronic radiation-induced skin fibrosis. Methods: Thirty adult B6 mice, with equal proportion of males and females underwent 5 Gy of radiation every other day for 12 days, equaling 30 Gy total, of the dorsal skin. At weeks 0, 1, 2, 4, and 8 after radiation, the skin was harvested and analyzed using histology and immunofluorescence. Dorsal skin also underwent single-cell RNA sequencing, data was demultiplexed and underwent dimension reduction to identify transcriptionally distinct subpopulations, cross-referenced with Enrichr to identify relevant genetic pathways. Results: Dermal thickness was noted to progressively, significantly increase from week 0 to 8. Dermal collagen deposition and disorganization also significantly increased throughout the experiment. CD31+ expression increased in the acute phase, but decreased below baseline levels by the chronic phase. Four distinct fibroblast subpopulations were identified, and the genetic expression of these individual subpopulations demonstrated dynamic changes throughout the experiment. Sca, Dlk-1, and CD26+ immunofluorescence staining further confirmed this dynamicity as their expression fluctuated throughout the 8-week course of the experiment. Conclusion: Identification of this dynamic process amongst the various fibroblast subpopulations during the healing process of irradiation wounds in wild-type mice is a novel finding and paramount to better understanding and treatment of radiation-induced skin fibrosis.

109. Vitamin E Treated Fat Grafts Maintain Volume to Improve Grafting Effects on Radiation-induced Fibrosis

Introduction: Fat grafts and vitamin E administration have both previously been described to mitigate radiation-induced skin fibrosis. Fat graft retention continues to hinder its long-term functionality affected mainly by two factors: initial inflammatory response and chronic necrosis secondary to hypovascularity. Thus, we aimed to simultaneously improve fat graft retention along with radiation-induced fibrosis. Methods: Ten adult CD-1 nude male mice at 6-8 weeks of age underwent scalp irradiation with 5 Gy every other day for 12 days, totaling in 30 Gy. Mice recovered for four weeks to allow chronic fibrosis to develop. After recovery, mice received 1000μL of donor human fat graft to the scalp. Mice were separated into 4 conditions: ungrafted irradiated, untreated fat graft, fat graft treated with pentoxifylline (PTX), fat graft treated with vitamin E (VE). Fat graft volume retention was monitored in-vivo using microCT scans with 3-dimensional reconstruction and analysis at weeks 0, 1, 2, 4, 6, and 8 after grafting. Fat graft and overlying skin were harvested and underwent histological and cytokine analysis. Results: Histological analysis demonstrated improvement in radiation-induced fibrosis, CD31+ expression, and 8-isoprostane expression in the vitamin E group. Cytokine analysis revealed decreased cell differentiation markers and inflammatory markers. Fat graft volume retention significantly improved as early as 1 week post-grafting with local vitamin E administration compared to control. Conclusion: Radiation-induced fibrosis and fat graft volume retention are both simultaneously improved with novel, local administration of vitamin E, improving both cosmesis and functionality of fat grafting in chronically irradiated fields.

Abstract 5595: Statin drugs have anti-tumor activity and enhance responses to anti-PD-1 therapy in preclinical models of head and neck cancer

Abstract Background/Rationale: Head and neck squamous cell carcinoma (HNSCC) is a common malignancy with significant morbidity and mortality. Approximately half of patients with HNSCC develop recurrent or metastatic disease. Recently, anti-PD-1 immune checkpoint blockade was FDA-approved for first-line treatment of recurrent or metastatic disease, and although relatively well tolerated, only a minority of patients respond. There is an unmet clinical need for agents that enhance responses to anti-PD-1 therapy without excessive toxicity. HMG-CoA reductase inhibitors, also known as statin drugs, are in wide clinical use for hyperlipidemia and are inexpensive and well tolerated. Statin drugs are associated with superior survival outcomes in retrospective population studies of several cancers, including HNSCC. Moreover, statins may reduce multiple treatment-related toxicities, such as radiation-induced skin fibrosis and cisplatin chemotherapy-induced hearing loss. Emerging data suggest that statins may also enhance some aspects of anti-tumor immunity, but this has not been studied in HNSCC. Further, the pleiotropic effects of different statin drugs are highly variable. We hypothesized that a subset of statin drugs would enhance anti-tumor immunity and delay tumor growth in preclinical models of HNSCC. Methods: Two immunocompetent, syngeneic murine models of oral cancer (MOC1 and MOC22) were used. To identify which of the seven FDA-approved statin drugs are capable of antitumor activity and enhancing T-cell induced killing, we used a high-throughput ex vivo co-culture assay of murine oral cancer cells and tumor infiltrating CD8+ T lymphocytes. Tumor-bearing mice were then treated with anti-PD-1 antibody (twice weekly IP) and/or statin drug (daily by oral gavage) for up to three weeks, and tumor kinetics and survival were tracked. Results: When added at or near physiologic drug concentrations, all 7 statins strongly inhibited proliferation of MOC1 and MOC22 cell lines. Moreover, the combination of CD8+ T cells and a subset of statins mediated nearly two-fold tumor cell killing effect versus either treatment used alone. In mice, tumor growth delay did not reach statistical significance with statins or anti-PD-1 alone. However, combination therapy induced a significant tumor growth delay versus control mice (p<0.05). Conclusions: Our results have significant implications for both biologic mechanisms and therapeutic applications of statin drugs in HNSCC. Additional experiments to investigate the mechanisms by which statins inhibit tumor cell proliferation and enhance anti-tumor immunity are currently underway. Funding: supported by Winship Cancer Institute and the Morningside Center for Innovative and Affordable Medicine. Citation Format: Andre J. Burnham, Vikash Kansal, Brendan L. Kinney, Chrystal M. Paulos, Gregory B. Lesinski, Nabil F. Saba, Nicole C. Schmitt. Statin drugs have anti-tumor activity and enhance responses to anti-PD-1 therapy in preclinical models of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5595.

Topical Deferoxamine Patch Is Superior to Direct Injection for the Treatment of Radiation-Induced Skin Fibrosis

Lavin, Christopher V. MS; Abbas, Darren B. MD; Fahy, Evan J. MD; Lee, Daniel K. MD; Griffin, Michelle MBChB, PhD; Diaz Deleon, Nestor M. BS; Mascharak, Shamik BS; Gurtner, Geoffrey C. MD, FACS; Longaker, Michael T. MD, MBA, FACS; Wan, Derrick C. MD, FACS Author Information

Decellularized Adipose Matrices Can Alleviate Radiation-Induced Skin Fibrosis.

Objective: Radiation therapy is commonplace for cancer treatment but often results in fibrosis and atrophy of surrounding soft tissue. Decellularized adipose matrices (DAMs) have been reported to improve these soft tissue defects through the promotion of adipogenesis. These matrices are decellularized by a combination of physical, chemical, and enzymatic methods to minimize their immunologic effects while promoting their regenerative effects. In this study, we aimed at exploring the regenerative ability of a DAM (renuva®; MTF biologics, Edison, NJ) in radiation-induced soft tissue injury. Approach: Fresh human lipoaspirate or DAM was injected into the irradiated scalp of CD-1 nude mice, and volume retention was monitored radiographically over 8 weeks. Explanted grafts were histologically assessed, and overlying skin was examined histologically and biomechanically. Irradiated human skin was also evaluated from patients after fat grafting or DAM injection. However, integrating data between murine and human skin in all cohorts is limited given the genetic variability between the two species. Results: Volume retention was found to be greater with fat grafts, though DAM retention was, nonetheless, appreciated at irradiated sites. Improvement in both mouse and human irradiated skin overlying fat and DAM grafts was observed in terms of biomechanical stiffness, dermal thickness, collagen density, collagen fiber networks, and skin vascularity. Innovation: This is the first demonstration of the use of DAMs for augmenting the regenerative potential of irradiated mouse and human skin. Conclusions: These findings support the use of DAMs to address soft tissue atrophy after radiation therapy. Morphological characteristics of the irradiated skin can also be improved with DAM grafting.

QS8: Fat Grafting Depletes Profibrotic Prrx1-positive Fibroblasts In Irradiated Skin And Mitigates Radiation-induced Groin Contracture

Purpose:Contracture is a long-term complication of radiotherapy (RT) that results from pathologic fibrosis of soft tissue in the radiation field. Fat grafting can regenerate fibrotic soft tissue by decreasing collagen density and reorganizing collagen fiber networks. Fibroblasts are the predominant cell involved in extracellular matrix (ECM) synthesis, and dermal fibroblasts possess distinct embryonic origins. We recently identified a profibrotic fibroblast in the mouse ventral dermis marked by embryonic expression of paired related homeobox 1 (Prrx1). Here we sought to investigate how fat grafting alters Prrx1-positive subpopulation distribution as well as functional contractures.Methods:Adult Prrx-1Cre;R26mTmG reporter mice (n=9) underwent whole body lethal irradiation with 9 Gy for hematopoietic depletion. Mice were immediately reconstituted with 2 million nucleated bone-derived cells from donor NSG (NOD.CB17-Prkdcsscid/J) mice via retro-orbital sinus injection. The success of reconstitution and immunodepletion was assessed by fluorescence-activated cell sorting (FACS) analysis of peripheral blood. After 4 weeks, 30 Gy was delivered to the right hindlimb in five fractionated doses to generate limb contracture. The irradiated, contracted limb was then grafted with 200 μl fresh human lipoaspirate and limb extension was measured over the subsequent 8 weeks, at which point skin was harvested for assessment of fibroblast subtypes for FACS and immunofluorescence. A group of mice with radiation-induced groin contracture did not undergo fat grafting and served as the control group.Results:FACS analysis indicated successful immunodepletion and engraftment by 3 weeks post bone marrow transplantation. At one month following groin irradiation, mice had developed significant right hind limb contracture with significantly reduced limb extension (****p≤0.0001). Histologically this was paralleled by thickening of the dermis, and substantial expansion of the fibrogenic Prrx-1-positive fibroblast subpopulation. While human fat graft volume retention was reduced over 8 weeks following implantation, this was associated with significantly improved in limb extension. The skin overlying the grafted fat showed reduced collagen density, as indicated by trichrome staining, as well as a reduction in the fibrogenic Prrx-1-positive fibroblast subpopulation by immunofluorescence imaging, as compared to the control mice.Conclusion:Here we show that fat grafting improves the extensibility of irradiated and contracted hind limbs and reverses radiation-induced skin fibrosis by both reducing the collagen content and by altering the composition of dermal fibroblast subpopulations. Specifically, fat grafting results in a depletion of the Prrx-1-positive fibroblast subpopulation. Further elucidating how this profibrotic fibroblast subpopulation is involved in ventral surface soft tissue fibrosis will facilitate development of novel strategies to treat/prevent debilitating late side-effect of radiotherapy.