Radiation-induced Fibrosis Research Articles

Avb6 Integrin-Targeted PET Imaging to Monitor Radiation-Induced Pulmonary Fibrosis In Vivo

<h3>Purpose/Objective(s)</h3> Radiation-induced pulmonary fibrosis (RIPF) is a common, debilitating late complication from radiation therapy, with poor prognosis and limited treatment options. RIPF is a particularly challenging dose-limiting normal tissue toxicity in pediatric hematological malignancies after total body irradiation, lung cancers, breast cancers, and thoracic lymphomas. Currently, clinical monitoring of RIPF progression remains challenging given the lack of imaging tools that can reliably detect RIPF at an early stage and the difficulty distinguishing RIPF from tumor recurrence. In the lung, α_vβ₆ integrin plays a central role in the pathogenesis of RIPF, by serving as a major TGF-beta activator after radiation injury. The objective of this study is to investigate the use of α_vβ₆ integrin-targeted PET imaging (using ⁶⁸Ga-A20FMDV2) to monitor radiation-induced pulmonary fibrosis in a murine model. <h3>Materials/Methods</h3> Using the small animal radiation research platform, the right lungs of C57BL/6J female mice (aged 8-10 weeks) were irradiated focally at 70 Gy using a 3 × 3 mm beam collimator, while the contralateral lung served as control. In addition, sham-treated animals served as control. This high dose focal irradiation model was used as prior studies demonstrated fibrosis in this model starting at 4-6 weeks post-RT. At 6 weeks post-RT, animals were administered 7.4 MBq of ⁶⁸Ga-A20FMDV2 through IV injection and imaged 1 hour later using a small animal micro-PET/CT system. Micro-PET/CT fusion images were reconstructed and maximum intensity projection images were generated. <h3>Results</h3> At 6 weeks post-RT, there was significantly increased uptake of ⁶⁸Ga-A20FMDV2 focally in the irradiated region within the right lung, compared to sham-treated control animals and the unirradiated left lung. <h3>Conclusion</h3> α_vβ₆ integrin-targeted PET imaging using ⁶⁸Ga-A20FMDV2 can serve as a powerful tool to study the evolution of radiation-induced pulmonary fibrosis <i>in vivo</i>, with significant potential for clinical translation.

MRNA Alteration Mapping of Radiation-Induced Pulmonary Fibrosis in C57BL/6 Mice

<h3>Purpose/Objective(s)</h3> Radiation-induced lung injury, including radiation-induced pneumonitis and pulmonary fibrosis, is a common serious dose-limiting toxicity of thoracic radiotherapy, and can be life-threatening. There is no definite conclusion of the etiology of radiation-induced pulmonary fibrosis (RIPF), and the effective interventions are still lacking. In this study, based on RIPF mouse model, we aim to identify genes responsive to irradiation, compare the difference in genome expression between normal lung tissues and the irradiated ones, and to provide the mRNA alteration mapping that can make the predictive model and potential interventions of RIPF. <h3>Materials/Methods</h3> Thirty C57BL/6 mice were randomized into 3 groups: control group (sham radiation), 16 Gy thoracic radiation group and 20 Gy radiation group to establish the RIPF mouse model. Lung tissues were harvested at 3 and 6 months after irradiation. The global gene expression of lung tissues was assessed by RNA sequencing. Differentially expressed genes were identified and subjected to function and pathway enrichment analysis. Infiltration of immune cells was evaluated by CIBERSORT. <h3>Results</h3> RIPF mouse model was successfully established in both 16 Gy and 20 Gy thoracic radiation groups. At 3 months after radiation, 317 mRNAs were significantly upregulated and 254 downregulated in the 16 Gy thoracic radiation group. 203 mRNAs were upregulated and 149 downregulated significantly in the 20 Gy group. At 6 months after radiation, 651 mRNAs were significantly upregulated and 131 downregulated in the 16 Gy group. 106 mRNAs were significantly upregulated and 4 downregulated in the 20 Gy group. Several functions and pathways including angiogenesis, epithelial cell proliferation, extracellular matrix, complement and coagulation cascades, TNF signaling pathway, NOD-like receptor signaling pathway, and HIF-1 signaling pathway were significantly enriched based on the differentially expressed genes in irradiation groups at 3 months after thoracic radiation. Cellular senescence, myeloid leukocyte activation, regulation of lymphocyte activation, mononuclear cell proliferation, immunoglobulin binding, and some other immune system associated pathways were also significantly enriched in irradiation groups at 6 months after radiation. We confirmed a dramatic increase of macrophage, neutrophil, dentric cell and natural killer cell, and a decrease of B cell in the two irradiation groups at 3 months. At 6 months, the infiltration of B cell in the 20 Gy group grew and became higher than the control group. <h3>Conclusion</h3> By RNA-seq, the irradiation responsive genes were identified. The differentially expressed genes were mainly associated with cell injury, cellular metabolism, epithelial and endothelial cell proliferation, and immune cell activation and regulation. Our results provided comprehensive clues on the mechanism of RIPF, which hopefully, may turn into the potential treatment target in preventing and reversing RIPF.

Multi-Omics Classifier of Tumor Recurrence vs. Radiation-Induced Lung Fibrosis in NSCLC Patients Treated with SBRT

<h3>Purpose/Objective(s)</h3> Differentiation between radiation-induced lung fibrosis (RILF) and tumor local recurrence (LR) remains challenging in SBRT treated NSCLC patients. We aimed to integrate spatially and time-resolved 4D radiomics with dosiomics biomarkers to develop a novel robust multi-omics classifier of LR vs RILF. <h3>Materials/Methods</h3> 210 NSCLC patients (101/48% T1-2N0M0, 109/52% T1-3N1/xM0/x) treated with SBRT (median dose 60 Gy/8 fractions) between 2009 and 2019 were identified. Image-based quantitative features were extracted from the SBRT planning and follow-up (FU) CTs at the time point of diagnosis of LR/RILF or a matched time point for CTs without LR/RILF (radiomics) and from dose distributions (dosiomics). The region of interest was the planning target volume plus a 10mm margin (PTV_+10mm). Time-dependent alterations of the radiomics features in FU CTs were integrated (delta-radiomics). To identify relevant features associated with RILF or LR, resampling (iterations = 1000) of feature selection methods were applied. An ensemble classifier comprising random forests, neural networks, and logistic regression was then trained. The area under the ROC curve (AUC) with a 70%/30% training/testing split and a 5-fold cross-validated AUC on the entire cohort were calculated for performance assessment at CT FU. <h3>Results</h3> Out of the 210, 36 patients (17%) were reported with LR and 44 (21%) with RILF. The median FU time was 19 months (range, 5-87). 20 LR and 23 RILF FU CTs, both with median time after SBRT of 15 months, were identified. 53 patients with matching clinical characteristics without LR/RILF (none), at matched FU time points (median time after SBRT: 17 months), were additionally selected, resulting in a cohort of 96 patients (20/21% LR, 23/24% RILF, 53/55% none). Discrimination of RILF versus LC using 4D radiomics features was achieved with a testing and 5-fold AUC of 0.82 [95%CI 0.79 0.86] and 0.85 [0.83 0.88]. The addition of a dosiomics feature improved performance to 0.85 [0.82 0.87] and 0.88 [0.85 0.91]. The PTV_+10mm-based classifier includes 4 textural features (2 delta-radiomics, 1 radiomics from the FU CT and 1 dosiomics). A non-significant correlation was found between the significant features and tumor volume before RT (Spearman's r <0.1, p<0.05). <h3>Conclusion</h3> Our data indicate that integrative omics by combining radiotherapy volume and dose constraints with spatially and time-resolved radiomics may provide a novel mean for better discrimination of tumor recurrence vs RILF after SBRT.

Selectively Targeting Activated Myofibroblasts by ABT-263 Reverses Radiation-Induced Intestinal Fibrosis in Mice

<h3>Purpose/Objective(s)</h3> Radiation induced intestinal fibrosis (RIF) is an irreversible and severe late effect of abdominal and pelvic radiation therapy. The goal of this study was to determine whether clearance of activated fibroblasts with ABT-263, a BCL2/xl inhibitor that can selectively kill myofibroblasts, can reverse RIF. <h3>Materials/Methods</h3> C57BL/6J mice were exposed to a single dose of 12 Gy irradiation on the whole abdomen. The intestinal wall thickness of mice was monitored by MRI at 0, 8^th, 12^th and 18^th week. Eighteen weeks after IR, mice were given 2 cycles of vehicle or ABT-263 treatment by gavage (50 mg/kg per day, 5 days per cycle). Two weeks after last treatment, intestinal sections were harvested for evaluation the effect of ABT-263 on irradiation induced activation of fibroblasts and extracellular matrix deposition by immunohistochemistry and Masson staining. The expression of TGF-β1, α-SMA, BCL-2/BCL-w proteins and phosphorylation of STAT3 in intestinal homogenate were analyzed by western blot analysis. Human and murine fibroblasts were cultured to evaluate the effects of ABT-263 on irradiation or classical TGF-β1-induced (5 ng/ml) activation of fibroblasts. <h3>Results</h3> At 18 weeks after 12Gy abdominal irradiation, C57BL/6J mice developed a significant thickness of the irradiated intestinal wall, with increased number of a-SMA⁺ fibroblasts (activated fibroblasts). Treatment of the RIF mice with ABT-263 after developed RIF could reverse the disease by specific clearance of activated fibroblasts. In vitro, inhibition of Bcl-2 and Bcl-xL by the small molecule BH3 mimetic, ABT-263, or Bcl-xL-specific small interfering RNA induced apoptosis in TGFb1 or irradiation-activated fibroblasts, but not in quiescent fibroblasts. <h3>Conclusion</h3> To our knowledge, this is the first study to demonstrate that RIF can be reversed by a specific myofibroblasts depletion drug ABT-263. ABT-263 has the potential to be developed as a new treatment for RIF.

Fibroblast Subpopulations Are Modified with Fat Grafting to Treat Radiation-Induced Fibrosis

Fibroblast Subpopulations Are Modified with Fat Grafting to Treat Radiation-Induced Fibrosis

Preclinical models of radiation-induced cardiac toxicity: Potential mechanisms and biomarkers.

Radiation therapy (RT) is an important modality in cancer treatment with >50% of cancer patients undergoing RT for curative or palliative intent. In patients with breast, lung, and esophageal cancer, as well as mediastinal malignancies, incidental RT dose to heart or vascular structures has been linked to the development of Radiation-Induced Heart Disease (RIHD) which manifests as ischemic heart disease, cardiomyopathy, cardiac dysfunction, and heart failure. Despite the remarkable progress in the delivery of radiotherapy treatment, off-target cardiac toxicities are unavoidable. One of the best-studied pathological consequences of incidental exposure of the heart to RT is collagen deposition and fibrosis, leading to the development of radiation-induced myocardial fibrosis (RIMF). However, the pathogenesis of RIMF is still largely unknown. Moreover, there are no available clinical approaches to reverse RIMF once it occurs and it continues to impair the quality of life of long-term cancer survivors. Hence, there is an increasing need for more clinically relevant preclinical models to elucidate the molecular and cellular mechanisms involved in the development of RIMF. This review offers an insight into the existing preclinical models to study RIHD and the suggested mechanisms of RIMF, as well as available multi-modality treatments and outcomes. Moreover, we summarize the valuable detection methods of RIHD/RIMF, and the clinical use of sensitive radiographic and circulating biomarkers.

Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes

Abstract Background Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate. Purpose We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation. Methods Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system. Results Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered. Conclusion Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy. Funding Acknowledgement Type of funding sources: None.

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.

Pencil beam scanning proton FLASH maintains tumor control while normal tissue damage is reduced in a mouse model.

Preclinical studies indicate a normal tissue sparing effect when ultra-high dose rate (FLASH) radiation is used, while tumor response is maintained. This differential response has promising perspectives for improved clinical outcome. This study investigates tumor control and normal tissue toxicity of pencil beam scanning (PBS) proton FLASH in a mouse model. Tumor bearing hind limbs of non-anaesthetized CDF1 mice were irradiated in a single fraction with a PBS proton beam using either conventional (CONV) dose rate (0.33-0.63Gy/s field dose rate, 244MeV) or FLASH (71-89Gy/s field dose rate, 250MeV). 162 mice with a C3H mouse mammary carcinoma subcutaneously implanted in the foot were irradiated with physical doses of 40-60Gy (8-14 mice per dose point). The endpoints were tumor control (TC) assessed as no recurrent tumor at 90days after treatment, the level of acute moist desquamation (MD) to the skin of the foot within 25days post irradiation, and radiation induced fibrosis (RIF) within 24weeks post irradiation. TCD50 (dose for 50% tumor control) was similar for CONV and FLASH with values (and 95% confidence intervals) of 49.1 (47.0-51.4) Gy for CONV and 51.3 (48.6-54.2) Gy for FLASH. RIF analysis was restricted to mice with tumor control. Both endpoints showed distinct normal tissue sparing effect of proton FLASH with MDD50 (dose for 50% of mice displaying moist desquamation) of <40.1Gy for CONV and 52.3 (50.0-54.6) Gy for FLASH, (dose modifying factor at least 1.3) and FD50 (dose for 50% of mice displaying fibrosis) of 48.6 (43.2-50.8) Gy for CONV and 55.6 (52.5-60.1) Gy for FLASH (dose modifying factor of 1.14). FLASH had the same tumor control as CONV, but reduced normal tissue damage assessed as acute skin damage and radiation induced fibrosis.

Effect of hyperbaric oxygen treatment on skin elasticity in irradiated patients.

Hyperbaric oxygen treatment (HBOT) is often used in an attempt to reverse/treat late radiation-induced tissue fibrosis (LRITF). This study aimed to quantify the effects on skin elasticity. Skin retraction time was used as a marker of skin elasticity in 13 irradiated breast cancer patients. The measurements were carried out on the affected side as well as the unaffected/healthy side at a mirrored location. Readings were taken at the start and end of HBOT (mean 43 sessions, 80 min at 243 kPa). Patient age ranged from 39-70 years. All patients underwent surgical lumpectomy and radiotherapy prior to undergoing HBOT. The mean time between radiotherapy and HBOT was 70 months. Seven of the 13 patients underwent chemotherapy. Mean irradiated skin retraction time improved from 417 (SD 158) pre-HBOT to 171 (24) msec post-HBOT (P < 0.001). Mean pre-HBOT retraction time in the non-irradiated skin was 143 (20) msec and did not change. This promising pilot study that suggests that HBOT may improve skin elasticity in patients with LRITF.

Natural variation in macrophage polarization and function impact pneumocyte senescence and susceptibility to fibrosis.

Radiation-induced pulmonary fibrosis (RIPF), a late adverse event of radiation therapy, is characterized by infiltration of inflammatory cells, progressive loss of alveolar structure, secondary to the loss of pneumocytes and accumulation of collagenous extracellular matrix, and senescence of alveolar stem cells. Differential susceptibility to lung injury from radiation and other toxic insults across mouse strains is well described but poorly understood. The accumulation of alternatively activated macrophages (M2) has previously been implicated in the progression of lung fibrosis. Using fibrosis prone strain (C57L), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL6/J), we demonstrate that the accumulation of M2 macrophages correlates with the manifestation of fibrosis. A comparison of primary macrophages derived from each strain identified phenotypic and functional differences, including differential expression of NADPH Oxidase 2 and production of superoxide in response to M2 polarization and activation. Further, the sensitivity of primary AECII to senescence after coculture with M2 macrophages was strain dependent and correlated to observations of sensitivity to fibrosis and senescence in vivo. Taken together, these data support that the relative susceptibility of different strains to RIPF is closely related to distinct senescence responses induced through pulmonary M2 macrophages after thoracic irradiation.

Radiotherapy-induced oxidative stress and fibrosis in breast cancer are suppressed by vactosertib, a novel, orally bioavailable TGF-β/ALK5 inhibitor

Radio-resistance resulting from radiotherapy-induced fibrosis is a major clinical obstacle in breast cancer treatment because it typically leads to cancer recurrence, treatment failure, and patient death. Transforming growth factor-β (TGF-β) is a key signal messenger in fibrosis, which plays an important role in radiation-induced fibrosis and cancer stem cell (CSC) development, may be mediated through the generation of oxidative stress. This study was conducted to confirm the efficacy of vactosertib, a TGF-β/ALK5 inhibitor, as a potent inhibitor in radiation-induced oxidative stress generation, fibrosis and CSC development. We used a 4T1-Luc allograft BALB/c syngeneic mouse model and 4T1-Luc and MDA-MB-231 cells for histological analysis, qRT-PCR, western blotting, ROS analysis, mammosphere formation analysis, monolayer fluorescence imaging analysis. Radiotherapy induces TGF-β signaling, oxidative stress markers (4-HNE, NOX2, NOX4, PRDX1, NRF2, HO-1, NQO-1), fibrosis markers (PAI-1, α-SMA, FIBRONECTIN, COL1A1), and CSC properties. However, combination therapy with vactosertib not only inhibits these radiation-induced markers and properties by blocking TGF-β signaling, but also enhances the anticancer effect of radiation by reducing the volume of breast cancer. Therefore, these data suggest that vactosertib can effectively reduce radiation fibrosis and resistance in breast cancer treatment by inhibiting radiation-induced TGF-β signaling and oxidative stress, fibrosis, and CSC.

Association between Submucosal Fibrosis and Endoscopic Submucosal Dissection of Recurrent Esophageal Squamous Cell Cancers after Chemoradiotherapy.

Simple SummaryThe efficacy and safety of endoscopic submucosal dissection for early esophageal cancer after chemoradiotherapy have not been established. In this study, we focused on the fibrosis of the submucosa. As a result, we found that endoscopic submucosal dissection for early esophageal cancer can be performed reliably without adverse events, but the procedure takes longer for lesions with strong fibrosis of the submucosa.Endoscopic resection is a treatment of choice for a metachronous early-stage esophageal squamous cell carcinoma (ESCC) appearing after a radical cure of esophageal cancer by chemoradiotherapy (CRT). However, non-curative resection, and procedural complications including perforation due to radiation-induced submucosal fibrosis, are a concern. This study aimed to evaluate the association between submucosal fibrosis and the usefulness and safety of endoscopic submucosal dissection (ESD) in ESCC after CRT. This study retrospectively analyzed 13 lesions in 11 patients in our institute. Submucosal fibrosis under the lesion (F score) was classified into three levels (F0: none or mild, F1: moderate, and F2: severe) based on endoscopic and histopathologic findings. All lesions were F1 or greater (F1: 8 lesions and F2: 5 lesions). En bloc and R0 resection rates were both 100%. The procedural speed was slower in F2 than in F1 (F1 vs. F2; 15.1 mm2/min vs. 7.1 mm2/min, p = 0.019), without procedure-related adverse events. At a median follow-up of 42 months (range: 14–117 months) after ESD, 7 of 11 (63.6%) patients were alive without recurrence, and without ESCC-related death. ESCC after CRT reliably and safely resected en bloc by ESD but was more difficult in lesions with strong submucosal fibrosis.

The comparison of transforming growth factor beta-1 serum levels in early-stage breast cancer patients treated with external beam whole breast irradiation plus boost versus interstitial brachytherapy accelerated partial breast irradiation

PURPOSETransforming growth factor beta-1 (TGF-β1) is a profibrotic cytokine used as an early biomarker to develop radiation-induced fibrosis (RIF). This study aimed to compare TGF-β1 serum levels in early-stage breast cancer patients treated with whole-breast radiation therapy (WBRT) plus boost versus accelerated partial breast irradiation (APBI) using multicatheter interstitial brachytherapy. METHODS AND MATERIALSThis clinical trial study was conducted on 20 women with early-stage breast cancer after breast-conserving surgery candidate for adjuvant radiotherapy in Golestan hospital, Ahvaz, in 2021. In one group APBI with high-dose-rate brachytherapy (n = 10), the other group WBRT with external beam radiation plus boost (n = 10) was performed. Serum level of TGF-β1 was evaluated before radiotherapy, immediately after the end of radiotherapy and three months after the end of radiotherapy by Enzyme-linked immunosorbent assay technique (ELISA). ResultsMedian serum TGF-β1 level before radiotherapy was not significantly different between the two groups (p = 0.971). In both APBI and WBRT groups, serum TGF-β1 levels significantly decreased immediately after radiotherapy compared to before treatment (p = 0.005 and p = 0.007, respectively); But three months after radiotherapy, serum TGF-β1 levels increased significantly in the WBRT group (40.50 to 77.41 pg/mL; p = 0.017), while no significant change was observed in the APBI group (24.75 to 30.50 pg/mL; p = 0.332). ConclusionSHigher TGF-β1 values in the WBRT group after radiotherapy can be used as an early and vital biomarker in this treatment, and this data may corroborate links between TGF-beta1 and fibrosis and fibrosis rates between APBI and WBRT; It also shows the preference for using the brachytherapy technique in this group of patients. However, due to the small number of samples, definitive conclusions require further prospective studies.

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.

Impact of Radiation-induced Trismus on Patients’ Life Quality

Head and neck cancers (HNC) account for 10–15% of all cancers globally. Curative radiation therapy (RT) and concurrent chemoradiotherapy (CCRT) with or without surgery are widely accepted standard treatment modalities for these patients. Radiation-induced fibrosis is one of the side effects of these treatments, and it causes degeneration, inflammation, pain, and atrophy of the masticatory muscle fibers and the temporomandibular joint, resulting in trismus. Trismus, also known as hypomobility of the mouth, is defined as a restricted opening of the mouth. Radiation-induced trismus (RIT) can occur within the first three months of treatment or up to 12–48 months later during the follow-up period. It has a significant negative influence on daily activities and vital oral functions, including speech problems, difficulty eating or drinking, psychological problems, malnutrition, dehydration, and difficulty with oral hygiene, reducing the quality of life (QoL). Given the underappreciated nature of trismus and its negative consequences in HNC patients receiving RT or C-CRT, the purpose of this review was to provide a concise review of the physical, social, and emotional implications of RIT and their impact on QoL measures, as well as the available evaluation methods for physicians in the related medical fields.

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.

Downregulation of miR-761 ameliorates radiation-induced pulmonary fibrosis by regulating PGC-1α

Background: Radiation-induced pulmonary fibrosis (RIPF) is a serious complication in patients treated with transthoracic irradiation. To date, there are no effective drugs for RIPF treatment. In this study, we attempted to explore the function of miR-761 in RIPF, further investigate its potential mechanism and evaluate its effectiveness in the treatment of RIPF. Methods: qRT-PCR analysis was used to detect miR-761 and peroxisome proliferator-activated receptor gamma (PPARg) coactivator-1 (PGC-1α) expression. Western Blot (WB) assay was applied to verify the regulation of PGC-1α by miR-761 and the expression of fibrosis-related proteins. Gel contraction assay was performed to demonstrate the level of fibroblast activation in vitro. A mouse RIPF model was used to validate the anti-fibrotic effect of Antagomir761. Bioinformatics analysis and dual-luciferase reporter assays were utilized to confirm the regulation relationship between miR-761 and PGC-1α. Results: The results showed that miR-761 was significantly elevated in irradiated mice lungs and fibroblasts. Overexpression of miR-761 in vitro promoted fibroblast activation. Whereas inhibition of miR-761 attenuated the degree of RIPF and inhibited fibroblast activation. Mechanistically, PGC-1α was a direct and functional target of miR-761, overexpression of PGC-1α inhibited irradiation-induced fibroblast activation, and knockdown of PGC-1α caused miR-761 inhibitor loses its anti-activation ability in irradiated cells. Conclusion: Our findings demonstrated that miR-761 regulated RIPF by targeting PGC-1α. Inhibition of miR-761 restored PGC-1α expression and attenuated RIPF damage, and miR-761 was a potential target for preventing the development of RIPF.

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.

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.