Radiation-induced Fibrosis Research Articles

112. Deferoxamine Transdermal Application Decreases Fibrosis and Improves Elasticity in Irradiated Porcine Skin

Purpose: Radiation therapy is notorious for its effectiveness in oncologic treatment but its devastating side effects to the surrounding soft tissue. We have previously demonstrated the efficacy of a transdermal drug delivery system (TDDS) to deliver deferoxamine (DFO) cutaneously to irradiated porcine skin safely without any systemic effects. With this study, we aim to demonstrate the beneficial effects of deferoxamine on irradiated porcine skin. Methods: Six red Duroc pigs (three months old) were irradiated with a single fraction of 30 Gy bilaterally. The pigs were allowed to recover for 12 weeks to allow chronic fibrosis to develop. The pigs were then divided into 4 different treatment conditions: normal, non-irradiated skin, irradiated skin with no patch (IR control), vehicle patch, and 1.0mg DFO TDDS patch. The pigs were treated with daily patch changes for 4 weeks and harvested immediately following the 4 weeks of treatment of after waiting another 4 weeks post-treatment. Histological analysis and suction cutometer analysis were performed. P<0.05 was considered significant on statistical analysis. Results: Dermal thickness was noted to be significantly lower in the DFO cohort than the IR control or vehicle cohort at both 4 and 8 weeks post-treatment. Collagen ultrastructure was significantly different between the DFO treatment groups, more closely resembling the normal skin groups, compared to the no patch or vehicle patch groups. Collagen density was also significantly lower in the DFO cohort compared the vehicle or IR control groups. CD31+ expression was significantly higher in the DFO group compared to all other conditions except the normal skin. DFO also had significantly more elasticity than the IR control or vehicle group. Conclusion: DFO effectively treats radiation-induced fibrosis in a porcine model, allowing the skin to more closely resemble normal skin than irradiated skin.

Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-β (transforming growth factor-β) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

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&amp;lt;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.

Near-infrared Nrf2 activator IR-61 dye alleviates radiation-induced lung injury

Oxidative stress injury and subsequent inflammatory response are considered to play critical roles in radiation-induced lung injury (RILI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates oxidative stress response and represses inflammation, but its therapeutic value in RILI remains elusive. Our previous studies have shown that the near-infrared (NIR) IR-61 dye evokes intracellular antioxidant defense by enhancing Nrf2 signaling and promoting anti-inflammatory effects. We established a model of RILI in mice exposed to whole-thoracic irradiation. The results showed that IR-61 treatment notably improved pulmonary functions by decreasing lung density and diminishing airway resistance. In addition, IR-61 significantly ameliorated radiation-induced inflammatory cell infiltration and proinflammatory cytokine (IL-1β, IL-6, and TNF-α) release, thereby mitigating inflammatory response. Furthermore, IR-61 mitigated radiation-induced lung fibrosis by decreasing the collagen deposition and the levels of fibrogenesis-related factors (collagen I, collagen III, α-SMA, and fibronectin). More importantly, IR-61 was found to accumulate in the mitochondria of macrophages in irradiated lung tissues. Therefore, the functions of IR-61 in macrophages were further studied in irradiated macrophage cell lines, MH-s and RAW 264.7 in vitro. The results indicated that IR-61 upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1) and decreased the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines (IL-1β and IL-6) in macrophages after radiation. In summary, our study suggests that IR-61 effectively mitigates RILI by activating Nrf2 signaling in irradiated lung tissues. In particular, Nrf2-mediated anti-inflammatory and antioxidant effects in irradiated lung tissue macrophages play critical roles in protecting against RILI.

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus

Background & AimsHigh-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis.MethodsUsing a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry.ResultsAt 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype.ConclusionsBased on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype.Lay summaryIrradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors.

Re-Du-Ning injection ameliorates radiation-induced pneumonitis and fibrosis by inhibiting AIM2 inflammasome and epithelial-mesenchymal transition.

BackgroundRadiation-induced lung injury (RILI) is a common side effect in chest radiotherapy patients, and there is no good medicine to treat it. Re-Du-Ning (RDN) injection is a traditional Chinese medicine that is clinically used to treat upper respiratory tract infections and acute bronchitis. RDN has the advantage of high safety and mild side effects. The mechanism of most traditional Chinese medicine preparations is unknown. PurposeTo illustrate the mechanisms of RDN for the treatment of RILI. MethodsFemale C57BL/6 mice were used to establish a RILI model via irradiation, and RDN injection was intraperitoneally administered at doses of 5, 10, and 20 ml/kg. The cytokines were measured by ELISA and qPCR. The data related to Absent in melanoma 2 (AIM2) inflammasome were analyzed via ELISA and a network pharmacological approach. In addition, the data related to epithelial-mesenchymal transition (EMT) were analyzed via immunofluorescence, Western blotting, and a network pharmacological approach. ResultsRDN robustly alleviated RILI. Meanwhile, RDN downregulated inflammatory cells’ infiltration and the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. Next, the potential molecular mechanisms of RDN were predicted through network pharmacology analysis. RDN may ameliorate radiation pneumonitis (RP) by inhibiting AIM2-mediated pyroptosis. Moreover, RDN treatment inhibited EMT and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway. The active compounds from Lonicera japonica Thunb. decreased the phosphorylation of Akt. ConclusionThese findings demonstrate that RDN, as a traditional Chinese medicine preparation, will be a candidate drug for treating RILI.

Inhaled amifostine for the prevention of radiation-induced lung injury

ObjectiveTo alleviate radiation-induced lung injury and prevent the related pneumonitis and pulmonary fibrosis by inhaled amifostine (AMI). Methods15 ​Gy 60Co γ-ray irradiation was performed on the thoracic area of rats once to establish the radiation injury model. AMI was intraperitoneally (i.p.) injected or intratracheally (i.t.) administered to the rats 30 ​min pre-irradiation. The protective effects of the two AMI administration manners were compared in the aspects of hematopoietic system, lung edema, and histopathological examination, and the mechanisms were explored. ResultsCompared to i.p. AMI, i.t. AMI remarkably alleviated radiation-induced lung injury and prevented consequent pneumonitis or pulmonary fibrosis. Specifically, i.t. AMI notably protected white blood cells and platelets, reduced the lung wet/dry weight ratio, and decreased collagen volume fractions compared to the model group (P ​< ​0.05), while i.p. AMI showed no significant difference with the model group (P ​> ​0.05). The high therapeutic efficiency of i.t. AMI was related to its high antioxidation and anti-inflammation effects with downregulation of pro-inflammatory cytokines, the enhanced superoxide dismutase activity, the low levels of malondialdehyde and total proteins. ConclusionInhaled AMI is a promising medicine for preventing radiation-induced lung injury, including pneumonitis and pulmonary fibrosis.

Transcriptional inhibition of miR-486-3p by BCL6 upregulates Snail and induces epithelial\u2013mesenchymal transition during radiation-induced pulmonary fibrosis

BackgroundIonizing radiation (IR) can induce pulmonary fibrosis by causing epithelial mesenchymal transition (EMT), but the exact mechanism has not been elucidated. To investigate the molecular mechanism of how radiation induces pulmonary fibrosis by altering miR-486-3p content and thus inducing EMT.MethodsThe changes of miR-486-3p in cells after irradiation were detected by RT-qPCR. Western blot was used to detect the changes of cellular epithelial marker protein E-cadherin, mesenchymal marker N-cadherin, Vimentin and other proteins. The target gene of miR-486-3p was predicted by bioinformatics method and the binding site was verified by dual luciferase reporter system. In vivo experiments, adeno-associated virus (AAV) was used to carry miR-486-3p mimic to lung. Radiation-induced pulmonary fibrosis (RIPF) model was constructed by 25Gy60Co γ-rays. The structural changes of mouse lung were observed by HE and Masson staining. The expression of relevant proteins in mice was detected by immunohistochemistry.ResultsIR could decrease the miR-486-3p levels in vitro and in vivo, and that effect was closely correlated to the occurrence of RIPF. The expression of Snail, which induces EMT, was shown to be restrained by miR-486-3p. Therefore, knockdown of Snail blocked the EMT process induced by radiation or knockdown of miR-486-3p. In addition, the molecular mechanism underlying the IR-induced miRNA level reduction was explored. The increased in BCL6 could inhibit the formation of pri-miR-486-3p, thereby reducing the levels of miR-486-3p in the alveolar epithelial cells, which would otherwise promote EMT and contribute to RIPF by targeting Snail.ConclusionIR can exacerbate RIPF in mice by activating the transcription factor BCL6, which inhibits the transcription of miR-486-3p and decreases its content, which in turn increases the content of the target gene slug and triggers EMT.

Lymph Node Yield in Gastrointestinal Cancer Surgery With or Without Prior Neoadjuvant Therapy: Protocol for a Systematic Review and Meta-analysis.

BackgroundLymph node yield is the number of lymph nodes retrieved during oncological resection and histopathologically identified in the resection specimen. It is an important surrogate parameter for assessing the oncological radicality of the resection of gastrointestinal carcinomas, as well as a prognostic factor in these diseases. It remains unclear if and to what extent neoadjuvant chemotherapy, radiotherapy, or chemoradiotherapy, which have become established treatments for carcinoma of the esophagus, stomach, and rectum and are increasingly used in pancreatic carcinoma, affect the lymph node yield.ObjectiveThis systematic review with meta-analysis is conducted with the aim of summarizing the available evidence regarding the lymph node yield, an oncological surrogate marker, in patients with gastrointestinal carcinomas undergoing surgery after neoadjuvant therapy compared to those undergoing surgery without neoadjuvant therapy.MethodsRandomized and nonrandomized studies comparing oncological resection of esophageal, stomach, pancreatic, and rectal carcinoma with and without prior neoadjuvant therapy are eligible for inclusion regardless of study design. Publications will be identified with a defined search strategy in 2 electronic databases: PubMed and Cochrane Library. The primary endpoint of the analysis is the number of lymph nodes identified in the resected specimen. Secondary endpoints include the number of harvested metastatic lymph nodes, operation time, postoperative complications, pathological TNM staging, and overall and recurrence-free survival time. Using suitable statistical methods, the endpoints between patients with and without neoadjuvant therapy, as well as in defined subgroups (neoadjuvant chemotherapy, radiotherapy, or chemoradiotherapy; and patients with esophageal, gastric, pancreatic, or rectal cancer), will be compared.ResultsThe literature search and data collection started in October 2021. Results are expected to be published in mid-2022.ConclusionsThis meta-analysis will provide the most up-to-date and complete summary of the evidence on an association between neoadjuvant therapy and lymph node yield in gastrointestinal cancer surgery. The underlying hypothesis is that neoadjuvant therapy decreases the number and size of lymph nodes through lymphocyte depletion and radiation-induced fibrosis, thus leading to a lower possible lymph node yield. The findings of the meta-analysis will show if this hypothesis is supported by evidence.Trial RegistrationPROSPERO CRD218459; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021218459International Registered Report Identifier (IRRID)DERR1-10.2196/35243

Altered DNA Methylation and Gene Expression Profiles in Radiation-Induced Heart Fibrosis of Sprague-Dawley Rats.

Radiation-induced heart disease (RIHD) is a serious side effect of radiotherapy for thoracic tumors. Advanced myocardial fibrosis in the late phase of RIHD can lead to myocardial remodeling, heart function impairing and heart failure, resulting in serious clinical consequences, and its pathogenesis remains vague. DNA methylation is one of the important epigenetic mechanisms which often occurs in response to environmental stimuli and is crucial in regulating gene expression. We hypothesized DNA methylation may contribute to pathogenesis in radiation-induced heart fibrosis (RIHF) and altered DNA methylation patterns probably influenced the genes expression in RIHF. In present study, we found genome-wide differences in DNA methylation status and RNA expression were demonstrated and we screened out 44 genes whose altered expression maybe were regulated by CpG island methylation within the gene promoter in RIHF of Sprague-Dawley rat by employing gene expression arrays and human CpG island microarrays. Gene expression and CpG island methylation levels of several candidate genes were further validated. Our investigation provided a new dimension to reveal the specific mechanisms of RIHF and explore the potential therapeutic targets for it.

Inhibiting the Cytosolic Phospholipase A2–Arachidonic Acid Pathway With Arachidonyl Trifluoromethyl Ketone Attenuates Radiation-Induced Lung Fibrosis

Radiation-induced lung fibrosis (RILF) is a serious late complication of thoracic radiation therapy. Inflammation is crucial in fibroblast activation and RILF, and arachidonic acid (AA) is an important inflammatory mediator released by cytosolic phospholipase A2 (cPLA2) and reduced by arachidonyl trifluoromethyl ketone (ATK)-targeting of cPLA2. Here, we aimed to investigate the roles of the cPLA2/AA pathway in RILF and assess the potential of targeting cPLA2 to prevent RILF. A computed tomography scan was used to obtain the mean lung density, and hematoxylin-eosin, Masson's trichrome, and Sirius Red staining were used to assess the histopathologic conditions in mouse models. AA levels in mouse serum and cell supernatants were tested by enzyme-linked immunosorbent assay. Fibroblast phenotype alterations were examined by a Cell Counting Kit-8, manual cell counting, and a Transwell system. The protein levels were evaluated via Western blotting, immunofluorescence, and immunohistochemistry. AA protected fibroblasts against radiation-induced growth inhibition and promoted fibroblast activation, which was characterized by enhanced α-smooth muscle actin expression and migration capacity. Radiation could activate fibroblasts by upregulating cPLA2 expression and AA production, which could be reversed by ATK. Moreover, inhibiting cPLA2 with ATK significantly attenuated collagen deposition and radiation-induced pulmonary fibrosis in mouse models. We further identified extracellular-signal regulated protein kinase (ERK) as the downstream target of the radiation-AA regulatory axis. Radiation-induced AA increased phosphorylated-ERK levels, promoting cyclinD1, cyclin-dependent kinase 6, and α-smooth muscle actin expression and contributing to fibroblast activation. Inhibiting P-ERK impaired radiation- and AA-induced fibroblast activation. The related molecular mechanisms were verified using specimens from animal models. Our findings uncover the role of the cPLA2/AA-ERK regulatory axis in response to radiation in pulmonary fibroblast activation and recognize cPLA2 as the key regulatory molecule during RILF for the first time. Targeting cPLA2 may be a promising protective strategy against RILF.

P144 a Transforming Growth Factor Beta Inhibitor Peptide, Generates Antifibrogenic Effects in a Radiotherapy Induced Fibrosis Model.

Radiation-induced fibrosis (RIF) is a severe side effect related with soft tissues sarcomas (STS) radiotherapy. RIF is a multicellular process initiated primarily by TGF-β1 that is increased in irradiated tissue, whose signaling leads to intracellular Smad2/3 phosphorylation and further induction of profibrotic target genes. P144 (Disetertide©) is a peptide inhibitor of TGF-β1 and is proposed as a candidate compound for reducing RIF associated wound healing problems and muscle fibrosis in STS. Methods: A treatment and control group of WNZ rabbits were employed to implement a brachytherapy animal model, through catheter implantation at the lower limb. Two days after implantation, animals received 20 Gy isodosis, intended to induce a high RIF grade. The treatment group received intravenous P144 administration following a brachytherapy session, repeated at 24–72 h post-radiation, while the control group received placebo. Four weeks later, affected muscular tissues underwent histological processing for collagen quantification and P-Smad2/3 immunohistochemistry through image analysis. Results: High isodosis Brachytherapy produced remarkable fibrosis in this experimental model. Results showed retained macro and microscopical morphology of muscle in the P144 treated group, with reduced extracellular matrix fibrosis, with a lower area of collagen deposition measured through Masson’s trichrome staining. Intravenous P144 also induced a significant reduction in Smad2/3 phosphorylation levels compared with the placebo group. Conclusions: P144 administration clearly reduces RIF and opens a new potential co-treatment approach to reduce complications in soft tissue sarcoma (STS) radiotherapy. Further studies are required to establish whether the dosage and timing optimization of P144 administration, in different RIF phases, might entirely avoid fibrosis associated with STS brachytherapy.

Mouse mesenchymal stem cell-derived exosomal miR-466f-3p reverses EMT process through inhibiting AKT/GSK3\u03b2 pathway via c-MET in radiation-induced lung injury

BackgroundRadiation-induced lung fibrosis (RILF) is a common complication of thoracic radiotherapy. Alveolar epithelial cells play a crucial role in lung fibrosis via epithelial-mesenchymal transition (EMT). Exosomes derived from mesenchymal stem cells own the beneficial properties to repair and regeneration of damaged tissues, however the underlying mechanisms remain poorly understood.MethodsMouse mesenchymal stem cells-derived exosomes (mMSCs-Exo) were isolated by differential centrifugation, and their protective effects were assessed in vivo and in vitro, respectively. EMT-associated proteins were measured via western blot assay and/or immunofluorescence staining. The miRNA expression was measured by microarray assay and qPCR. Furthermore, bioinformatics prediction with KEGG analysis, luciferase assay, and rescue experiments were performed to explore the molecular mechanism underlying miR-466f-3p.ResultsmMSCs-Exos were efficiently isolated ranging from 90-150 nm with high expression of exosomal markers (CD63, TSG101, and CD9). mMSCs-Exos administration efficiently relieved radiation-induced lung injury with less collagen deposition and lower levels of IL-1β and IL-6. Meanwhile, in vitro results showed mMSCs-Exos treatment obviously reversed EMT process induced by radiation. Among enriched miRNA cargo in exosomes, miR-466f-3p was primarily responsible for the protective effects via inhibition of AKT/GSK3β pathway. Our mechanistic study further demonstrated that c-MET was the direct target of miR-466f-3p, whose restoration partially abrogated mMSCs-Exo-mediated inhibition in both EMT process and AKT/GSK3β signaling activity induced by radiation.ConclusionsOur findings indicated that exosomal miR-466f-3p derived from mMSCs may possess anti-fibrotic properties and prevent radiation-induced EMT through inhibition of AKT/GSK3β via c-MET, providing a promising therapeutic modality for radiation-induced lung fibrosis.

Radiation-induced lung injury - what do we know in the era of modern radiotherapy?

Radiation-induced lung injury (RILI) that is usually divided into an early radiation-induced pneumonitis (RIP) and late chronic radiation-induced lung fibrosis (RILF) remains a clinically significant toxicity in radiation oncology. Thus, a thorough understanding of underlying molecular mechanisms and risk factors is crucial. This review, focused on patients treated with modern radiotherapy (RT) techniques, describes the different clinical presentations of RIP, with most typical imaging findings and usefulness of pulmonary function tests and laboratory assessment in differential diagnosis. The most critical patient- and treatment-related predictors are summarized and discussed — age and sex, comorbidities, tumour characteristics, concomitant treatment, and RT-plan parameters. The conventional grading scales and contemporary approach to quantitative assessment (radiomics, CT density changes) is described as well as treatment methods.

The Effect of Ophiopogonin C in Ameliorating Radiation-Induced Pulmonary Fibrosis in C57BL/6 Mice: An Update Study.

BackgroundThe aim of this study was to assess and update the protective effects and underlying mechanisms of Ophiopogonin C (OP-C), a biologically active component separated and purified from Ophiopogon japonicus, in ameliorating radiation-induced pulmonary fibrosis in C57BL/6 mice administered thoracic radiation.Methods and MaterialsWe randomly divided 75 mice into five groups and administered a dose of 12-Gy whole thoracic radiation to establish a pulmonary fibrosis animal model. Mice were treated with OP-C or dexamethasone combined with or without cephalexin by daily gavage for 4 weeks. All mice were sacrificed after the completion of thoracic irradiation at 28 weeks. Serum levels of interleukin-6 and transforming growth factor-β1 (TGF-β1) were evaluated. Moreover, superoxide dismutase (SOD) levels in lung tissue were measured. The severity of fibrosis was evaluated using the hydroxyproline content of the lung tissue. The pathological changes in the five groups were detected by hematoxylin and eosin and Masson trichrome staining. Smooth muscle actin expression was detected using immunohistochemical staining. Matrix metalloproteinases-2 (MMP-2) and tissue inhibitors of metalloproteases-2 (TIMP-2) were examined by immunohistochemical staining of the lung sections, and semiquantitative analysis was used to calculate the expression of MMP-2 and TIMP-2.ResultsIrradiated mice treated with OP-C or DXE combined with or without cephalexin significantly reduced mortality in mice and fibrosis levels by 1) reducing the deposition of collagen and accumulation of inflammatory cells and fibroblasts, 2) downgrading levels of the promote-fibrosis cytokine TGF-β1, and 3) increasing SOD activity in the lung tissue compared with that of irradiated mice without treatment. However, there were no statistical differences in fibrosis levels among the irradiated mice treated with OP-C or DXE combined with or without cephalexin.ConclusionOP-C significantly ameliorates radiation-induced pulmonary fibrosis and may be a promising therapeutic strategy for this disorder.

Fat Grafts Augmented With Vitamin E Improve Volume Retention and Radiation-Induced Fibrosis.

Treatments for radiation-induced fibrosis range from vitamin E (VE) and pentoxifylline (PTX) systemically to deferoxamine and fat grafting locally. Regarding fat grafting, volume retention hinders its long-term functionality and is affected by 2 factors: inflammation and necrosis secondary to hypovascularity. The authors aimed to simultaneously improve fat graft retention and radiation-induced fibrosis by integrating VE and PTX into fat grafts locally. Forty adult CD-1 nude male mice, 6 weeks old, underwent scalp irradiation and recovered for 4 weeks to allow for development of fibrosis. Mice received 200 μL of donor human fat graft to the scalp. Mice were separated into 4 conditions: no grafting, fat graft without treatment, graft treated with PTX, and graft treated with VE. Fat graft volume retention was monitored in vivo with micro-computed tomography scans at weeks 0, 1, 2, 4, 6, and 8 after grafting. Histological and cytokine analysis of the scalp skin and fat grafts were performed. VE-treated grafts had significant improvement in dermal thickness and collagen density of overlying skin compared with all other groups. VE decreased 8-isoprostane and increased CD31+ staining compared with the other grafted groups. Cytokine analysis revealed decreased inflammatory and increased angiogenic markers in both the fat graft and overlying skin of the VE group. Fat graft volume retention was significantly improved in the VE group starting at 1 week post grafting. Radiation-induced fibrosis and fat graft volume retention are both simultaneously improved with local administration of VE.

Satellite Cells are Activated in a Rat Model of Radiation-Induced Muscle Fibrosis.

Radiation-induced muscle fibrosis is a long-term side effect of radiotherapy that significantly affects the quality of life and even reduces the survival of cancer patients. We have demonstrated that radiation induces satellite cell (SC) activation at the molecular level; however, cellular evidence in a rat model of radiation-induced muscle fibrosis was lacking. In this study, we evaluated SC activation in vivo and investigated whether radiation affects the proliferation and differentiation potential of SCs in vitro. For in vivo studies, Sprague-Dawley rats were randomly divided into six groups (n = 6 per group): non-irradiated controls, 90 Gy/1 week-, 90 Gy/2 weeks-, 90 Gy/4 weeks-, 90 Gy/12 weeks- and 90 Gy/24 weeks-postirradiation groups. Rats received a single dose of radiation in the left groin area and rectus femoris tissues were collected in the indicated weeks. Fibrosis, apoptosis, and autophagy were evaluated by Masson's trichrome staining, TUNEL staining, and electron microscopy, respectively. SC activation and central nuclear muscle fibers were evaluated by immunofluorescence staining and hematoxylin and eosin staining. IL-1β concentrations in serum and irradiated muscle tissue samples were determined by ELISA. For in vitro studies, SCs were isolated from rats with radiation-induced muscle fibrosis and their proliferation and differentiation were evaluated by immunofluorescence staining. In vivo, fibrosis increased over time postirradiation. Apoptosis and autophagy levels, IL-1β concentrations in serum and irradiated skin tissues, and the numbers of SCs and central nuclear muscle fibers were increased in the irradiated groups when compared with the control group. In vitro, cultured SCs from irradiated muscle were positive for the proliferation marker Pax7, and differentiated SCs were positive for the myogenic differentiation marker MyHC. This study provided cellular evidence of SC activation and proliferation in rats with radiation-induced muscle fibrosis.

Semi-Supervised Segmentation of Radiation-Induced Pulmonary Fibrosis From Lung CT Scans With Multi-Scale Guided Dense Attention.

Computed Tomography (CT) plays an important role in monitoring radiation-induced Pulmonary Fibrosis (PF), where accurate segmentation of the PF lesions is highly desired for diagnosis and treatment follow-up. However, the task is challenged by ambiguous boundary, irregular shape, various position and size of the lesions, as well as the difficulty in acquiring a large set of annotated volumetric images for training. To overcome these problems, we propose a novel convolutional neural network called PF-Net and incorporate it into a semi-supervised learning framework based on Iterative Confidence-based Refinement And Weighting of pseudo Labels (I-CRAWL). Our PF-Net combines 2D and 3D convolutions to deal with CT volumes with large inter-slice spacing, and uses multi-scale guided dense attention to segment complex PF lesions. For semi-supervised learning, our I-CRAWL employs pixel-level uncertainty-based confidence-aware refinement to improve the accuracy of pseudo labels of unannotated images, and uses image-level uncertainty for confidence-based image weighting to suppress low-quality pseudo labels in an iterative training process. Extensive experiments with CT scans of Rhesus Macaques with radiation-induced PF showed that: 1) PF-Net achieved higher segmentation accuracy than existing 2D, 3D and 2.5D neural networks, and 2) I-CRAWL outperformed state-of-the-art semi-supervised learning methods for the PF lesion segmentation task. Our method has a potential to improve the diagnosis of PF and clinical assessment of side effects of radiotherapy for lung cancers.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-β-induced tumor-associated fibrosis

AbstractPancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-β-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.Fluvastatin, a cholesterol-lowering drug, radiosensitizes pancreatic cancer (PC) cells partly by inhibiting DNA damage response and/or autophagic flux. Fluvastatin also significantly suppresses intra-tumor radiation-induced fibrosis, as it inhibits radiation/TGF-β-induced activation of pancreatic stellate cells. Together, fluvastatin and radiation co-treatment may be a potential therapeutic approach against PC and warrants further clinical evaluation.

CircTUBD1 Regulates Radiation-induced Liver Fibrosis Response via a circTUBD1/micro-203a-3p/Smad3 Positive Feedback Loop.

Background and AimsRadiation-induced liver fibrosis (RILF), delayed damage to the liver (post-irradiation) remains a major challenge for the radiotherapy of liver malignancies. This study investigated the potential function and mechanism of circTUBD1 in the development of RILF.MethodsBy using a dual luciferase assay, RNA pull-down assays, RNA sequencing, chromatin immunoprecipitation (known as ChIP) assays, and a series of gain- or loss-of-function experiments, it was found that circTUBD1 regulated the activation and fibrosis response of LX-2 cells induced by irradiation via a circTUBD1/micro-203a-3p/Smad3 positive feedback loop in a 3D system.ResultsKnockdown of circTUBD1 not only reduced the expression of α-SMA, as a marker of LX-2 cell activation, but also significantly decreased the levels of hepatic fibrosis molecules, collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), and connective tissue growth factor (CTGF) in a three-dimensional (3D) culture system and RILF model in vivo. Notably, knockdown of circTUBD1 alleviated early liver fibrosis induced by irradiation in mice models.ConclusionsThis study is the first to reveal the mechanism and role of circTUBD1 in RILF via a circTUBD1/micro-203a-3p/Smad3 feedback loop, which provides a novel therapeutic strategy for relieving the progression of RILF.