Bilateral diffuse alveolar damage contributes to the fatal toxicity of pre-existing interstitial lung disease mice after partial thoracic irradiation 

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However, the underlying mechanisms of its pathogenesis are still inconclusive. No approved biomarker or medicine is available to prevent pulmonary toxicities in LC-ILD patients. Appropriate management for them remains a challenge for clinicians due to treatment-related complications. Methods To elucidate the histopathological characteristics and molecular mechanisms responsible for this severe toxicity in vivo , C57BL/6J mice were used to develop different lung injury models, including radiation-induced lung injury (RILI), bleomycin-induced pulmonary fibrosis (BIPF), and severe radiation-related lung injury (sRRLI) murine model. Biopsy examination was performed on hematoxylin and eosin (H&E), Masson’s trichrome, and immunohistochemistry-stained lung tissue sections. Changes in lung function were measured. RNA extracted from mouse lung tissues was sequenced on the Illumina Novaseq platform. Results A severe lung injury model after irradiation was built based on pre-existing ILD mice induced by BLM administration. Enhanced lung injury was observed in the sRRLI model, including higher mortality and pulmonary function loss within six months compared to the mono-treatment groups. Autopsy revealed that bilateral diffuse alveolar damage (DAD) with an overlap of exudative, proliferative, and fibrosing patterns was usually presented in the sRRLI model. The histological phenotypes manifested exudative DAD phase in the early phase and proliferating DAD pattern predominated in the late phase. Bioinformatic analysis showed signaling pathways relevant to immune cell migration, epithelial cell development, and extracellular structure organization were commonly activated in the different models. Furthermore, the involvement of epithelial cells and the infiltration of macrophages and CD4 + lymphocytes were validated during extensive lung remodeling in the sRRLI group. They also participated in triggering remarkable abscopal responses in the non-IR contralateral lungs. Conclusions The study provides a preclinical model to better understand radiation-related severe lung injury in pre-existing ILD mice. DAD with progressive inflammation and fibrosis in bilateral lungs contributed to severe or even fatal complications after partial thoracic irradiation. More studies are needed to investigate potential strategies to prevent and rescue severe pulmonary complications. animal model severe lung injury radiation interstitial lung disease diffuse alveolar damage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Patients with interstitial lung disease (ILD), particularly idiopathic pulmonary fibrosis (IPF), are at high risk of developing lung cancer (LC) [ 1 ]. According to a meta-analysis, the prevalence of LC in ILD patients was 15.5% and 11.6% in Asian and European cohorts, respectively [ 2 ]. Similarly, a high proportion (2.4–10.9%) of LC patients was found to be accompanied by ILD, particularly in the Japanese population with a rate of up to 24.3% [ 3 ]. LC patients with comorbid ILD (LC-ILD) have significantly worse survival and poorer quality of life than the general population. Nearly all therapeutic regimens for LC-ILD patients, including surgery, radiotherapy, chemotherapy, and immunotherapy, are associated with increased acute exacerbation (AE) and high mortality risk [ 4 ]. However, the pathogenesis and mechanisms have not been well understood. Appropriate management of LC-ILD patients remains a challenge for clinicians due to treatment-related pulmonary toxicities or even fatal outcomes. For decades, radiotherapy has been a crucial treatment for LC patients in both localized and advanced stages. Stereotactic body radiotherapy (SBRT), as a more sophisticated technique, is preferred over conventional radiotherapy owing to its reduced adverse effects. However, it has been reported that, in early-stage medically inoperable non-small cell lung cancer (NSCLC) patients with previously diagnosed ILD, the median incidence rate of serious radiation pneumonitis (RP) was 11.9%, and the median rate of fatal RP was 6.4% after thoracic SBRT [ 5 ]. In particular, the median survival of LC patients with underlying IPF was significantly worse than that of IPF patients without LC (38.7 vs 63.9 months; HR = 5.0) after definitive concurrent chemoradiotherapy (CCRT) [ 6 ]. The establishment of a best-characterized animal model is necessary to gain insight into the mechanisms underlying treatment-related lung injury. This may help to identify novel therapies and practical biomarkers for predicting and preventing the progression of severe complications [ 7 ]. In this study, for the first time, a severe radiation-related lung injury (sRRLI) model was established using bleomycin (BLM)-induced pulmonary fibrosis (BIPF) mice to disclose the histopathologic characteristics and potential molecular mechanisms responsible for this severe or even fatal toxicity through biopsy examination and bioinformatic analysis. 2. Methods 2.1 Mice and treatments Six-to-eight-week-old male C57BL/6J mice (18–22 g) (GemPharmatech, Jiangsu, China) were randomly divided into four groups: Control (saline), RILI (irradiation (IR) treatment alone), BIPF (BLM treatment alone), and sRRLI group (IR at week 4 after BLM treatment), 6 mice each group. In detail, for IR exposure, a single dose of 16 Gy X-ray (2.0 Gy/min) generated by an X-RAD 320 irradiator (Precision X-Ray Inc., North Branford, CT, USA) was delivered to the right thorax of mice. For BLM treatment, mice were instilled with BLM sulfate (Meilunbio, Liaoning, China; 2 mg/kg body weight) by oropharyngeal administration under anesthesia as previously described [ 8 ]. The same volume of saline was consistently delivered in the Control and RILI groups. General activity and survival of mice were observed and recorded. All animal experimental protocols were authorized by the Animal Ethics Committee of Fudan University and strictly performed according to the International Guiding Principles for Animal Research guidelines. 2.2 Histological staining and analysis Mice lung tissues at 8- and 24-week after IR in different groups were collected and immersed in 4% paraformaldehyde for 24 h. After dehydration and paraffin-embedded, the tissues were sectioned into 4 µm thickness for histopathological examination of hematoxylin and eosin (H&E) (G1005, Servicebio, Wuhan, China) and Masson’s staining (G1006, Servicebio) [ 8 ]. Lung injury was evaluated using the modified Ashcroft scale ranging from 0 to 8 according to the characterization of alveolar septa and lung structure [ 9 ]. The average score was assessed by 0–4 points (4: extremely serious, 3: serious, 2: middle, 1: slight, 0: normal) from five randomly selected microscopic fields using a Leica DFC7000T microscope. 2.3 Immunohistochemical (IHC) staining and analysis The paraffin-embedded mice tissue sections were further stained for IHC analysis following the manufacturer’s instructions. Slides were blocked with BSA (G5001, Servicebio) and then incubated overnight at 4 ℃ with primary antibodies anti-α-SMA (1:2000, BM0002, BOSTER, CA, USA), anti-pro-SP-c (1:2000, ab211326, Abcam, Cambridge, UK), anti-F4/80 (1:5000, Abcam) and anti-CD4 (1:1000, Abcam). Stained images were captured using a microscope. The total density of ten positive microscopic fields per mouse was assessed, and the semi-quantification of protein expression was analyzed using ImageJ software. 2.4 Mouse pulmonary function At 24 weeks post-IR, six mice were randomly selected from each group and pulmonary function was measured. Mice were anesthetized by intraperitoneal injection of 1.5% pentobarbital sodium (Bio-Light Biotech, Guangzhou, China, 80 mg/kg body weight), and the lung functions vital capacity (VC), forced vital capacity (FVC), and dynamic pulmonary compliance (Cdyn) were measured using a small animal lung function analyzer (Data Sciences International, Inc., ST. Paul, MN, USA) [ 10 ]. 2.5 Functional enrichment analysis of RNA-sequencing (RNAseq) Lung tissues at 8 weeks post-IR in different groups were sampled and cryopreserved at − 80 ℃ for RNA sequencing assay described in our previous study [ 8 ]. Briefly, RNA extracted from mouse lung tissue was sequenced on the Illumina Novaseq platform. Clean data filtered by TrimGalore software (v0.6.6) were mapped to the mouse reference genome (mm10) with TopHat software (v2.1.1) [ 11 ] and evaluated using Cufflinks software (v2.2.1). Differentially expressed genes (DEGs) between the indicated groups were filtered with adjusted p 0.585. Functional enrichment analysis, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), was performed using the clusterProfier package (v3.18.1) [ 12 ]. 2.6 Statistical analysis Statistical analysis was performed using GraphPad Prism 8 (San Diego, CA, USA). Experimental data from at least three replicates were presented as mean ± standard error of the mean (S.E.M.). Survival fraction (SF) was calculated with the Kaplan-Meier method. Comparison was analyzed by one-way analysis of variance (ANOVA) and P < 0.05 was considered statistically significant. 3. Results 3.1 Radiation-related severe lung injury in pre-existing ILD mice A sRRLI model was established based on the BIPF mice which was widely used in the field of pulmonary fibrosis [ 13 ]. As shown in Fig. 1 A, mice exhibited hair loss and discoloration within the exposure field post-IR. Lung histopathology displayed thickened interalveolar septa, cellular infiltration, and collagen deposition through 8 to 24 weeks in the RILI group. Moreover, characteristic lung morphology of fibrotic foci was presented in the BIPF group. Significantly, enhanced damage was observed in the sRRLI model, including inflammatory exudation, fibroblast proliferation, and scattered collagenous deposits at week 8 post-IR, and dense remodeling with organized interalveolar fibrosis scars at week 24 post-IR (Fig. 1 B). Semi-quantification of lung lesions confirmed that the injured areas were remarkably increased in the sRRLI model compared to the mono-treatment groups (Fig. 1 C). Although there was no death in the RILI and BIPF groups, lethal toxicity was observed in the sRRLI group, where 23.08% (3/13) of the mice died within six months with acute death occurring in the first week post-IR (Fig. 1 D). Besides, measurement of pulmonary function in the surviving mice showed that VC, FVC, and Cydn in the sRRLI group were declined significantly in comparison to the mono-treatment groups (Fig. 1 E), indicating the ventilation function was severely impaired in the sRRLI group. 3.2 Diffuse alveolar damage (DAD) patterns in the sRRLI model Representative histological phenotypes of the sRRLI group illustrated that, at week 8 post-IR, the aggravated injury was characterized by exudative DAD in the right lung with obvious desquamated pneumocytes, and exudative hyaline membrane (Fig. 2 A a, b). Besides, collagen deposition was distributed along the bronchovascular bundle or subpleural (Fig. 2 A c, d). At week 24 post-IR, proliferative DAD notably predominated in bilateral lung injury with sustained infiltration of inflammatory cells and fibroblasts (Fig. 2 B a, c). Characteristic pneumonia, progressive fibrosis, and extensive remodeling resulted in alveolar duct obstruction, collapse, and interstitial thickness (Fig. 2 B b, d). To disclose the causes underlying the fatal injury, lung biopsy was taken from a case that died at week 9 post-IR in the sRRLI group. Significant DAD with progressive inflammation and fibrosis were observed in bilateral lungs (Fig. 2 C). Histological examination revealed that normal alveolar structure was replaced by obvious collagen deposition and fibrin exudation, including prominent invasive fibroblast infiltration in the right lung (Fig. 2 C a, b), sporadic pulmonary congestion and bleeding in the right upper lobe (Fig. 2 C c), and worsening inflammation in the left lung dominated by infiltration of macrophages, lymphocytes, and giant cells (Fig. 2 C d). These features of overlapping DAD patterns eventually led to irreversible lung function impairment and respiratory failure. 3.3 Mechanisms underlying different treatment-induced lung injury The potential mechanisms of the above lung injury were further investigated using transcriptome sequencing. Bioinformatic analysis showed that the sRRLI mice had undergone more changes than the mono-treatment damage at the transcriptome level (Fig. 3 A, B), and the top 50 common DEGs among different groups were shown in a heatmap (Fig. 3 C). Functional enrichment analysis of 149 crucial common DEGs revealed that the signaling pathways relevant to inflammation, cellular damage, repair responses, such as p53, PI3K-Akt, MAPK, JAK-STAT, HIF-1 pathways were generally involved (Fig. 3 D). Specifically, GO analysis showed that these genes were enriched in epithelial cell development, immune cell migration, and extracellular external stimulus (Fig. 3 E). The protein-protein interaction with most nodes revealed the critical roles of Btg2 , Cdkn1a , Hspa8 , Fosb , and Serpine1 genes (Fig. 3 F). Moreover, the GO pathway analysis by GSEA revealed that the migration and chemotaxis of immune cells (myeloid leukocytes, granulocytes, and neutrophils) were remarkably involved in the lung of sRRLI mice (Fig. 3 G). 3.4 Cellular infiltration during lung remodeling To validate the participation of important cell types during lung remodeling, Fig. 4 A illustrates the cellular morphology and their anatomical localizations in the lung tissues of different groups, including alveolar epithelial cells (AEC II) with pro-SP-c protein expression, myofibroblasts (α-SMA biomarker), macrophages (F4/80 biomarker), and CD4 + lymphocytes. In detail, the intensity of pro-SP-c protein was obviously increased in the sRRLI group compared to the RILI group. AEC II hyperplasia was especially localized in the residual alveoli with normal structures, whereas it was relatively absent in the fibrotic regions (Fig. 4 B). α-SMA was also highly expressed in the sRRLI group (Fig. 4 C), and its positive area extended along the bronchovascular bundle, subpleural area, and broader consolidation area. Furthermore, the infiltration levels of macrophages and CD4 + lymphocytes were notably higher in the sRRLI model than those in the mono-treatment groups (Fig. 4 D, E). The infiltration nests of immune cells were mainly located along the bronchovascular bundle and consolidation regions. Consistent with the bioinformatic analysis, these results indicated that hyperactivation of the immune system was involved in enhanced lung injury of the sRRLI model. 3.5 Radiation-induced abscopal injury in the contralateral lungs Lung injuries were observed not only in the irradiated tissues but also in the non-IR abscopal contralateral lungs. In the RILI group, the injury area in the abscopal lungs increased slightly compared to the control group. In the sRRLI group, however, the degree of lung injury was substantially higher. The histopathological changes showed obvious thickened alveolar walls, inflammatory cell infiltration, and collagen deposition in the contralateral lungs, similar to the phenomenon in the irradiated ipsilateral right lungs (Fig. 5 A, B). Besides, the enhanced AEC II hyperplasia and the increased infiltration of myofibroblasts, macrophages, and CD4 + lymphocytes were also observed in the non-IR abscopal lung tissues, so that the intensities of pro-SP-c, α-SMA, F4/80, and CD4 + lymphocytes in the sRRLI model were significantly higher than those in the mono-treatment groups (Fig. 5 C-F). 3.6 Critical mediators categorized through bioinformatic GOBP/KEGG terms Reliable biomarkers that could accurately monitor the progression of lung injury were further analyzed with bioinformatic GOBP/KEGG pathways in the sRRLI model (Fig. S1). Some signaling factors in the cytokine-cytokine receptor interaction, cell chemotaxis, cytokine-mediated signaling pathways were categorized in Table 1 , including chemokines and chemokine receptors, interleukins (ILs), transforming growth factor-β (TGF-β) family, matrix metalloproteinases (MMPs), and tumor necrosis factor-α (TNF-α), which might contribute to the recruitment and migration of key cells and be associated with the development of inflammatory and fibrotic responses. Table 1 Crucial mediators categorized through bioinformatic GOBP/KEGG terms of the sRRLI model Categories Potential biomarkers Functions Ref. Chemokines CCL2/5/6/7/8/9/12/17/20/22 Chemokines are secreted proteins that control immune cell migration, adhesion, cell-cell interactions during inflammation and immune surveillance through binding to their specific receptors. They contribute to angiogenesis, tumorigenesis, inflammation, and tissue repair and are also associated with pathological disorders of radiation injury. PPBP has been implicated in the early stages of wound healing and is associated with IPF. PPBP was found to be significantly differentially expressed between those with a definite UIP pattern of ILA compared to those without ILA. [ 34 , 37 ] CXCL5/8/9/10/12/13/16 PPBP(CXCL7) Chemokine receptors CCR1/5/7 CXCR1/4/5/7 ACKR1/3/5 Interleukins IL-1/2/4/5/6/8/10/18/33/34 ILs are a series of cytokines expressed by leukocytes and many other cell types. They participate in immunological responses with pro- and anti-inflammatory properties. IL production is involved in pathological processes of radiation-induced cardiopulmonary toxicity. [ 38 , 39 ] TGF-β family TGFB2, BMP6, GDF6/15 TGF-β family members include TGF-β isoforms, activins, nodal, BMPs, and GDFs. They regulate various key events in normal development and physiology. TGF-β is critical in cancer therapeutic resistance, promoting the activation of fibroblasts and ECM synthesis after radiotherapy. [ 40 ] MMPs MMP12/28 MMPs are responsible for collagen and protein degradation in ECM. They are involved in maintaining normal organ and tissue homeostasis and are related to several pathologic conditions, including cancer and fibrosis. MMP2 and MMP9 are relevant to EMT in epithelial cells after radiation. [ 41 ] TNFRSF TNFRSF 1/4/9/11/12/13/18/19/21 TNF and TNFSF/TNFRSF control the coordination of various mechanisms driving co-stimulation and co-inhibition among immune cells. Single-cell sequencing analysis suggested that TNF plays a role in RIPF in T cells and monocytes. [ 42 ] Others IFNGR2 IFNGR2 is a subunit of the interferon-gamma receptor (IFNγR). The (IFNGR1/IFNGR2)2 -IFN-γ dimer activates the cytotoxic activity of innate immune cells via signaling involving Jak1, Jak2, and Stat1. IFN-γ contributed to the enhanced necroptosis in lung epithelial cells. [ 43 ] SAA SAA is a highly conserved family of acute-phase response proteins. SAA level can increase rapidly in emergency conditions such as inflammation, trauma, and viral infection. Endogenous SAA was recently reported as having a protective role in ameliorating lung injury. [ 36 ] PAI-1/Serpine1 PAI-1 is a major TGF-β1/p53 target gene. PAI-1 inhibits the plasmin system by blocking fibrinolysis and ECM degradation. Mature PAI-1 protein could induce lung epithelial cell senescence treated by bleomycin through a p53-independent mechanism. [ 27 ] Discussion To simulate clinical situations, the present study established a novel sRRLI model based on BLM-induced pulmonary fibrosis mice. It was observed that most of the pre-existing ILD mice presented pathological DAD patterns and lung function loss after partial thoracic irradiation. These mice tended to develop severe pneumonitis, progressive fibrosis, and even fatal outcomes within six months post-IR, compared to RILI and BIPF mono-treatment groups. Clinically, a recent retrospective study showed that the incidence of post-treatment AE was higher in LC-ILD patients who received CCRT (54.5%) followed by radiotherapy (16.2%) and chemotherapy (15.6%), compared to non-ILD patients [ 14 ]. Even when relatively low-dose palliative thoracic radiotherapy was delivered, the rate of grade ≥ 3 RP was reported to be 13.7%. Particularly in patients with a higher pulmonary fibrosis score (score 3–5), the incidence of serious RP increased to 37.5% with eventual death [ 15 ]. Autopsy analysis or lung biopsy is scarce after treatment-related severe complications in the clinic. Using an in vivo mouse model, significant pathological changes in lung injury were detailed here. At the early stage, when lung injury in the RILI and BIPF groups gradually settled into relatively stable, the sRRLI mice were still predominated by the exudative DAD with apparent massive hyaline membranes and intra-alveolar fibrin. As time passed, at six months post-IR, the organizing DAD phase became the main pattern, characterized by inter-alveolar fibroblastic proliferation, septal collagen deposition, and fibrotic foci. Severe damage, if irreversible, could eventually result in repeated injury contributing to modality. Autopsy of the lethal mouse revealed overlapping patterns of exudative, proliferative, and fibrotic DAD in bilateral lungs. Such morphological changes may be responsible for the clinical symptoms observed in LC-ILD patients with progressive dyspnea, dry cough, declining lung function, and respiratory failure [ 16 , 17 ]. Common mechanisms underlying different treatment-induced lung injuries were elucidated using transcriptomic analysis here. Signaling pathways relevant to cellular damage and repair, including p53, PI3K-Akt, MAPK, JAK-STAT, HIF-1, and cellular senescence, were universally activated. A recent report using scRNA-seq analysis showed that the activation of PI3K-Akt and p53 pathways in AEC cells participated in the progression of blast-induced lung damage via modulating autophagic and oxidative stress [ 18 ]. Besides, AKT-, MAPK-, or JAK-STAT-relevant inflammatory pathways are potential therapeutic targets for treating acute lung injury (ALI), and novel agents are under investigation [ 19 – 21 ]. Growing evidence suggests that cellular senescence is positively associated with pulmonary fibrosis. Upregulation of the senescence-related proteins p16 INK4a and p21 CIP1 were previously demonstrated in RIPF or BIPF models [ 22 , 23 ]. Senescent cells display senescence-associated secretory phenotype, which is involved in promoting the lung fibroblast proliferation, myofibroblast activation, and ECM production [ 24 , 25 ]. We also found that the common genes Cdkn1a, Fosb , and Serpine1 were among the core positions during the development of lung injury. Our previous study has validated that Cdkn1a , also known as p21 , its expression level was significantly higher in the sRRLI mice and positively correlated with macrophage infiltration via regulating CCL7 secretion in vitro [ 8 ]. Moreover, Fosb proto-oncogene functions in regulating cell proliferation, differentiation, and transformation. It is a subunit gene of the AP-1 transcription factor, which plays a crucial role in lung fibroblasts by promoting macrophage activation and collagen production [ 26 ]. Additionally, PAI-1 (Serpine1) was recently found to be associated with downregulating re-alveolarization in ALI by reducing the AEC II self-renewal [ 27 ], and evidence supported that it was a druggable target for controlling lung cell senescence and fibrosis via inhibiting TGF-β pathway [ 28 ]. Furthermore, our findings suggested a central role of epithelial cell development and immune cell migration in the progression of severe lung injury. The hyperplastic pneumocytes in residual alveoli and the influx of macrophages and CD4 + lymphocytes were presented persistently during disease development. Cumulative evidence showed that M1/M2 macrophages participated in both the acute and chronic phases of lung disease. M2 macrophages mainly dominate the progression of pulmonary fibrosis via TGF-β signaling pathway during the rehabilitation period [ 29 ]. In the sRRLI model, macrophage pools exceeded in both ipsilateral and contralateral lungs so that radiation-induced abscopal lung injury was observed along with the activation of immune responses. This differs from previous observation that focal regions of macrophages existed only in ipsilateral lungs at 26 weeks post-IR [ 30 ]. Consistently, in some clinical cases after thoracic radiotherapy, extensive ground glass abnormalities and focal consolidations were usually observed spreading to bilateral lungs rather than being limited to the irradiated area on CT images [ 31 ]. Proinflammatory and profibrotic factors are considered crucial mediators in causing serious suffering. However, there are no recommendations for reliable biomarkers in the guidelines now due to their relatively low specificity. Experts have advised that patients with higher ILD gender age physiology (ILD-GAP) index scores should be considered when delivering treatments or only observation, as radiological usual interstitial pneumonia (UIP) pattern was significantly associated with thoracic radiotherapy-related life-threatening pneumonitis [ 17 ]. Most noteworthy, a higher level of KL-6 was found to be linked to more severe AE-ILD or treatment-related ILD and thus was proposed as a potential biomarker for poor prognosis [ 32 , 33 ]. Besides, peripheral blood markers such as pretreatment NLR (≥ 3.0), and pretreatment ANC (≥ 5755) were reported to be associated with severe RILI [ 7 ]. We categorized some potential mediators in the early phase of the sRRLI here, including chemokines and chemokine receptors, ILs, TGF-β family, MMPs, and TNF-α. Other factors of growing interest include pro-platelet basic protein (PPBP), growth and differentiation factor 15 (GDF 15), and serum amyloid A (SAA), which have recently been implicated in the development of lung inflammation and fibrosis [ 34 – 36 ]. Conclusions A preclinical in vivo mouse model was established to mimic radiotherapy-related severe pulmonary complications in LC-ILD patients. This study highlighted that the severe or even fatal toxicity was due to DAD with progressive inflammation and fibrosis in bilateral lungs post-IR. Bioinformatics analysis aided in the discovery of critical signaling pathways, and the hyperactivation of inflammatory responses with infiltration of macrophage and CD4 + lymphocyte was further validated. However, further studies are needed to clarify the mechanisms underlying disease progression. Rigorous preclinical and clinical trials are still required to elucidate potential biomarkers and develop effective therapeutic targets for predicting and preventing severe complications in LC-ILD patients following radiotherapy. Abbreviations ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; LC, lung cancer; LC-ILD, LC patients with comorbid ILD; AE, acute exacerbation; SBRT, stereotactic body radiotherapy; NSCLC, non-small cell lung cancer; RP, radiation pneumonitis; CCRT, concurrent chemoradiotherapy; BIPF: bleomycin (BLM)-induced pulmonary fibrosis; ECM: extracellular matrix; UIP: usual interstitial pneumonia; ALI: acute lung injury Declarations Authorship contribution : J.F. and C.S. conceived and designed the experiments; X.L., and Y.Z. performed experiments, data acquisition and analysis; S.Z., L.Z., Y.P. and J.Z. performed experiments and data acquisition; J.F. wrote the original draft of the manuscript; C.S., K.P, and Y.X supervised the work and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgments : We thank for the support of bioinformatic analysis by the Medical Research Data Center of Fudan University. Funding : This study was supported by the National Natural Science Foundation of China (Nos.81903258, 32171235, 12235004, and 12175044) and the Shanghai Pulmonary Hospital Talents Plan (fkyq1904). Ethics approval and consent to participate: All animal studies complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The animal experimental protocol was approved by the Animal Welfare and Ethics Committee of Fudan University. Consent for publication : Not applicable. Conflict of interest: The authors declare that none of them have any conflicts on interest in relation to the present publication. Availability of data and materials: Data presented in this study are available upon request from the corresponding author upon reasonable request. 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Pref-1 induced lung fibroblast differentiation by hypoxia through integrin alpha5beta1/ERK/AP-1 cascade. Eur J Pharmacol. 2021;909:174385. Ali G, Zhang M, Chang J, Zhao R, Jin Y, Zhang J, et al. PAI-1 regulates AT2-mediated re-alveolarization and ion permeability. Stem Cell Res Ther. 2023;14(1):185. Adnot S, Breau M, Houssaini A. PAI-1: A New Target for Controlling Lung-Cell Senescence and Fibrosis? Am J Respir Cell Mol Biol. 2020;62(3):271-2. Lee JW, Chun W, Lee HJ, Min JH, Kim SM, Seo JY, et al. The Role of Macrophages in the Development of Acute and Chronic Inflammatory Lung Diseases. Cells. 2021;10(4). Groves AM, Misra R, Clair G, Hernady E, Olson H, Orton D, et al. Influence of the irradiated pulmonary microenvironment on macrophage and T cell dynamics. Radiother Oncol. 2023;183:109543. Onishi H, Marino K, Yamashita H, Terahara A, Onimaru R, Kokubo M, et al. Case Series of 23 Patients Who Developed Fatal Radiation Pneumonitis After Stereotactic Body Radiotherapy for Lung Cancer. Technol Cancer Res Treat. 2018;17:1533033818801323. Zhang T, Shen P, Duan C, Gao L. KL-6 as an Immunological Biomarker Predicts the Severity, Progression, Acute Exacerbation, and Poor Outcomes of Interstitial Lung Disease: A Systematic Review and Meta-Analysis. Front Immunol. 2021;12:745233. Yamashita H, Kobayashi-Shibata S, Terahara A, Okuma K, Haga A, Wakui R, et al. Prescreening based on the presence of CT-scan abnormalities and biomarkers (KL-6 and SP-D) may reduce severe radiation pneumonitis after stereotactic radiotherapy. Radiat Oncol. 2010;5:32. Menon AA, Lee M, Ke X, Putman RK, Hino T, Rose JA, et al. Bronchial epithelial gene expression and interstitial lung abnormalities. Respir Res. 2023;24(1):245. Takenouchi Y, Kitakaze K, Tsuboi K, Okamoto Y. Growth differentiation factor 15 facilitates lung fibrosis by activating macrophages and fibroblasts. Exp Cell Res. 2020;391(2):112010. Ji A, Trumbauer AC, Noffsinger VP, Meredith LW, Dong B, Wang Q, et al. Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice. Int J Mol Sci. 2023;24(24). Wang L, Jiang J, Chen Y, Jia Q, Chu Q. The roles of CC chemokines in response to radiation. Radiat Oncol. 2022;17(1):63. Liu X, Shao C, Fu J. Promising Biomarkers of Radiation-Induced Lung Injury: A Review. Biomedicines. 2021;9(9). Mukai-Sasaki Y, Liao Z, Yang D, Inoue T. Modulators of radiation-induced cardiopulmonary toxicities for non-small cell lung cancer: Integrated cytokines, single nucleotide variants, and HBP systems imaging. Front Oncol. 2022;12:984364. Wang J, Xu Z, Wang Z, Du G, Lun L. TGF-beta signaling in cancer radiotherapy. Cytokine. 2021;148:155709. Yue H, Hu K, Liu W, Jiang J, Chen Y, Wang R. Role of matrix metalloproteinases in radiation-induced lung injury in alveolar epithelial cells of Bama minipigs. Exp Ther Med. 2015;10(4):1437-44. Sun Z, Lou Y, Hu X, Song F, Zheng X, Hu Y, et al. Single-cell sequencing analysis fibrosis provides insights into the pathobiological cell types and cytokines of radiation-induced pulmonary fibrosis. BMC Pulm Med. 2023;23(1):149. Hao Q, Shetty S, Tucker TA, Idell S, Tang H. Interferon-gamma Preferentially Promotes Necroptosis of Lung Epithelial Cells by Upregulating MLKL. Cells. 2022;11(3). Additional Declarations No competing interests reported. Supplementary Files floatimage6.jpeg Supplementary figure 1 Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2025 Read the published version in Radiation Oncology → Version 1 posted Editorial decision: Revision requested 09 Jan, 2025 Reviews received at journal 20 Oct, 2024 Reviewers agreed at journal 17 Oct, 2024 Reviewers invited by journal 05 Aug, 2024 Editor assigned by journal 29 Jul, 2024 Submission checks completed at journal 29 Jul, 2024 First submitted to journal 28 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4816003","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":338467932,"identity":"afebf705-7685-46b2-9933-4c4b0b1bdfc3","order_by":0,"name":"Jiamei Fu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYBACxgbGBwwfGBgSINwDRGlhNmCcQZIWBgZmA2YekrQwNzCzSduUHc5jYD/8gJnnDHEOY5POOXe4mIEnDWjdDaK08B+Tzm07nNjAkMPAzPOBWFssQVr435CihRGkRQJkC1EOa2Zmtuw5l17MJvHM4OAcYrxv2N7MeONHmXUeP3/ywwdvjhGjpRlEsoERkREpzwDVMgpGwSgYBaMAJwAA23ovt7wZc64AAAAASUVORK5CYII=","orcid":"","institution":"Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Jiamei","middleName":"","lastName":"Fu","suffix":""},{"id":338467933,"identity":"ba3b7184-c48b-4cd4-b2f7-f8f1ea38e35b","order_by":1,"name":"Xinglong Liu","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xinglong","middleName":"","lastName":"Liu","suffix":""},{"id":338467934,"identity":"9fe36185-b809-4886-8219-69560c9dc25c","order_by":2,"name":"Yuchuan Zhou","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuchuan","middleName":"","lastName":"Zhou","suffix":""},{"id":338467935,"identity":"4143b020-6860-4060-8349-ebcadbe8ac0b","order_by":3,"name":"Shengnan Zhao","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Shengnan","middleName":"","lastName":"Zhao","suffix":""},{"id":338467936,"identity":"4ebbbbcc-926d-4202-ae81-71cd2e96d006","order_by":4,"name":"Liang Zeng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zeng","suffix":""},{"id":338467937,"identity":"c9b3358a-e40d-4897-a94d-ac9e3054692b","order_by":5,"name":"Yan Pan","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Pan","suffix":""},{"id":338467938,"identity":"952d472e-805e-48a0-be9a-3938ecb76ff5","order_by":6,"name":"Jianghong Zhang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jianghong","middleName":"","lastName":"Zhang","suffix":""},{"id":338467939,"identity":"254b9d87-3cb2-4cd7-92cc-1253539eae6b","order_by":7,"name":"Kevin M Prise","email":"","orcid":"","institution":"Queen’s University Belfast","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"M","lastName":"Prise","suffix":""},{"id":338467940,"identity":"ce03f8e9-938b-4b70-960f-fbca481b164b","order_by":8,"name":"Chunlin Shao","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Chunlin","middleName":"","lastName":"Shao","suffix":""},{"id":338467941,"identity":"583cdcf8-4f2e-4670-973a-202cd8adec76","order_by":9,"name":"Yaping Xu","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-07-28 09:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4816003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4816003/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13014-025-02596-w","type":"published","date":"2025-02-07T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63464025,"identity":"5b3550cf-acad-4593-8941-806ca72d4b9b","added_by":"auto","created_at":"2024-08-28 12:00:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":377630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiation-related severe lung injury in pre-existing ILD mice. (A)\u003c/strong\u003e The schedule for establishing the severe lung injury model. The right thorax of the mice was exposed to 16 Gy X-ray at week 4 after BLM administration. \u003cstrong\u003e(B)\u003c/strong\u003e Representative H\u0026amp;E and Masson’s staining images of the right lungs at week 8 and week 24 post-IR. Scale bar=50 μm, magnifying scale bar=20 μm. \u003cstrong\u003e(C)\u003c/strong\u003e The score of the right lung injury (n=6). \u003cstrong\u003e(D)\u003c/strong\u003e Survival analysis of different treatment groups (n=13). \u003cstrong\u003e(E)\u003c/strong\u003e Mouse vital capacity (VC), forced vital capacity (FVC), and dynamic pulmonary compliance (Cdyn) were recorded at week 24 post-IR and compared to age-matched controls (n=4–6). * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/6ba1cde083f088a700295785.jpeg"},{"id":63464834,"identity":"b15c129c-bba2-4a4e-9c37-ec7de1fba429","added_by":"auto","created_at":"2024-08-28 12:08:10","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":575563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiffuse alveolar damage (DAD) patterns in the sRRLI model. (A)\u003c/strong\u003e Proliferative DAD in the right lung at week 8 post-IR. (a, b) H\u0026amp;E stained lung sections, bleeding (black arrows), desquamated pneumocytes (green arrows), exudative hyaline membrane (blue arrows) and scattered large protein globules (dark blue arrows), scale bar=20 μm. (c, d) Masson’s stained lung sections, collagenproliferation (black asterisk), scale bar=50 μm (c). collagen deposition and alveolar occlusion, scale bar=200 μm (d). \u003cstrong\u003e(B)\u003c/strong\u003eFibrosing DAD in bilateral lungs at week 24 post-IR. (a) H\u0026amp;Estain, alveolar ducts filled by hyperplastic pneumocytes, fibroblasts and myofibroblasts (black arrows), scale bar=20 μm. (b) H\u0026amp;Estain, septal thickening and inflammatory cells infiltration (green arrows) and cystic change (red asterisk), scale bar=100 μm. (c) Masson’s stain, collagen proliferation and deposition, scale bar=20 μm. (d) Masson’s stain, fibrous deposits along the bronchovascular bundle or subpleural (green asterisk), scale bar=200 μm. \u003cstrong\u003e(C)\u003c/strong\u003eFatal DAD in autopsy. (a) Masson’s stain, collagen proliferation and deposition, scale bar=50 μm. (b) H\u0026amp;E stain, invasive fibroblast infiltration, scale bar=20 μm. (c) H\u0026amp;E stain, congestion and bleeding, scale bar=20 μm. (d) H\u0026amp;E stain, the inflammation and cellular infiltration of macrophages, lymphocytes, giant “foamy” macrophages and large atypical pneumocytes desquamation with extensive hyaline membrane formation, scale bar=50 μm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/a0d359eb54eb40a4a190cd51.jpeg"},{"id":63464030,"identity":"e4288873-c23d-4a4b-a95c-ffa067d4213f","added_by":"auto","created_at":"2024-08-28 12:00:10","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":367288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms underlying different treatment-induced lung injury models\u003c/strong\u003e. (\u003cstrong\u003eA) \u003c/strong\u003eThe gene number of DEGs in RILI, BIPF, and sRRLI models. (\u003cstrong\u003eB)\u003c/strong\u003e Common genes of three lung injury models. (\u003cstrong\u003eC)\u003c/strong\u003e Heatmap of top 50 DEGs.\u003cstrong\u003e (D)\u003c/strong\u003eBarplot of top 15 KEGG pathways enriched by common genes. (\u003cstrong\u003eE)\u003c/strong\u003e Treeplot of GO pathways enriched by common genes. (\u003cstrong\u003eF\u003c/strong\u003e) Protein-protein interaction (PPI) of common genes. (\u003cstrong\u003eG)\u003c/strong\u003e Ridgplot of top 10 GO pathways in GSEA enriched by DEGs of the sRRLI model.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/bf744b7cd8d8ddd93469d30a.jpeg"},{"id":63464027,"identity":"5cfbe490-3ca1-4ad3-ae66-b210cd34d4c0","added_by":"auto","created_at":"2024-08-28 12:00:09","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":963396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe infiltration of different cells during lung remodeling. (A) \u003c/strong\u003eThe protein expressions of pro-SP-c, α-SMA, F4/80 and CD4 were measured using IHC staining in mice lung tissues at week 24 post-IR. Semi-quantification of pro-SP-c \u003cstrong\u003e(B)\u003c/strong\u003e, α-SMA \u003cstrong\u003e(C)\u003c/strong\u003e, F4/80 \u003cstrong\u003e(D)\u003c/strong\u003e and CD4 \u003cstrong\u003e(E)\u003c/strong\u003e IHC intensity was accessed, and data was showed as the means ± SEM in ten fields per mouse in every group. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, ns: no significant. Scale bar=100 μm, magnifying scale bar=20 μm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/96e48e2a72b9182440779175.jpeg"},{"id":63464026,"identity":"54942f9d-5de7-40d6-bad1-e179009db893","added_by":"auto","created_at":"2024-08-28 12:00:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1136710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiation-induced abscopal injury in the contralateral lungs. (A) \u003c/strong\u003eRepresentative H\u0026amp;E staining images and the protein expressions of pro-SP-c, α-SMA, F4/80 and CD4 were measured using IHC staining in mouse left lung tissues at week 24 post-IR. \u003cstrong\u003e(B) \u003c/strong\u003eThe score of the left lung injury. Semi-quantification of pro-SP-c \u003cstrong\u003e(C)\u003c/strong\u003e, α-SMA \u003cstrong\u003e(D)\u003c/strong\u003e, F4/80 \u003cstrong\u003e(E)\u003c/strong\u003eand CD4 \u003cstrong\u003e(F)\u003c/strong\u003e IHC intensity was accessed, and data was showed as the means ± SEM in ten fields per mouse in every group. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, ns: no significant. Scale bar=100 μm, magnifying scale bar=20 μm.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/80730f4e1b6159faa61adfd0.jpeg"},{"id":75930442,"identity":"2a5283b6-95bf-4e66-a534-ea61ba012548","added_by":"auto","created_at":"2025-02-10 16:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4459276,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/f4c667fc-b8e8-4745-a104-ee29911366f2.pdf"},{"id":63464028,"identity":"7b63beb3-6cde-4e15-8793-1ef7e17efec7","added_by":"auto","created_at":"2024-08-28 12:00:09","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":208759,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 1\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4816003/v1/f39d2b00f84189590e7c5d00.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bilateral diffuse alveolar damage contributes to the fatal toxicity of pre-existing interstitial lung disease mice after partial thoracic irradiation ","fulltext":[{"header":"1. Background","content":"\u003cp\u003ePatients with interstitial lung disease (ILD), particularly idiopathic pulmonary fibrosis (IPF), are at high risk of developing lung cancer (LC) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to a meta-analysis, the prevalence of LC in ILD patients was 15.5% and 11.6% in Asian and European cohorts, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Similarly, a high proportion (2.4\u0026ndash;10.9%) of LC patients was found to be accompanied by ILD, particularly in the Japanese population with a rate of up to 24.3% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. LC patients with comorbid ILD (LC-ILD) have significantly worse survival and poorer quality of life than the general population. Nearly all therapeutic regimens for LC-ILD patients, including surgery, radiotherapy, chemotherapy, and immunotherapy, are associated with increased acute exacerbation (AE) and high mortality risk [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the pathogenesis and mechanisms have not been well understood. Appropriate management of LC-ILD patients remains a challenge for clinicians due to treatment-related pulmonary toxicities or even fatal outcomes.\u003c/p\u003e \u003cp\u003eFor decades, radiotherapy has been a crucial treatment for LC patients in both localized and advanced stages. Stereotactic body radiotherapy (SBRT), as a more sophisticated technique, is preferred over conventional radiotherapy owing to its reduced adverse effects. However, it has been reported that, in early-stage medically inoperable non-small cell lung cancer (NSCLC) patients with previously diagnosed ILD, the median incidence rate of serious radiation pneumonitis (RP) was 11.9%, and the median rate of fatal RP was 6.4% after thoracic SBRT [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In particular, the median survival of LC patients with underlying IPF was significantly worse than that of IPF patients without LC (38.7 vs 63.9 months; HR\u0026thinsp;=\u0026thinsp;5.0) after definitive concurrent chemoradiotherapy (CCRT) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The establishment of a best-characterized animal model is necessary to gain insight into the mechanisms underlying treatment-related lung injury. This may help to identify novel therapies and practical biomarkers for predicting and preventing the progression of severe complications [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, for the first time, a severe radiation-related lung injury (sRRLI) model was established using bleomycin (BLM)-induced pulmonary fibrosis (BIPF) mice to disclose the histopathologic characteristics and potential molecular mechanisms responsible for this severe or even fatal toxicity through biopsy examination and bioinformatic analysis.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Mice and treatments\u003c/h2\u003e \u003cp\u003eSix-to-eight-week-old male C57BL/6J mice (18\u0026ndash;22 g) (GemPharmatech, Jiangsu, China) were randomly divided into four groups: Control (saline), RILI (irradiation (IR) treatment alone), BIPF (BLM treatment alone), and sRRLI group (IR at week 4 after BLM treatment), 6 mice each group. In detail, for IR exposure, a single dose of 16 Gy X-ray (2.0 Gy/min) generated by an X-RAD 320 irradiator (Precision X-Ray Inc., North Branford, CT, USA) was delivered to the right thorax of mice. For BLM treatment, mice were instilled with BLM sulfate (Meilunbio, Liaoning, China; 2 mg/kg body weight) by oropharyngeal administration under anesthesia as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The same volume of saline was consistently delivered in the Control and RILI groups. General activity and survival of mice were observed and recorded. All animal experimental protocols were authorized by the Animal Ethics Committee of Fudan University and strictly performed according to the International Guiding Principles for Animal Research guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histological staining and analysis\u003c/h2\u003e \u003cp\u003eMice lung tissues at 8- and 24-week after IR in different groups were collected and immersed in 4% paraformaldehyde for 24 h. After dehydration and paraffin-embedded, the tissues were sectioned into 4 \u0026micro;m thickness for histopathological examination of hematoxylin and eosin (H\u0026amp;E) (G1005, Servicebio, Wuhan, China) and Masson\u0026rsquo;s staining (G1006, Servicebio) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lung injury was evaluated using the modified Ashcroft scale ranging from 0 to 8 according to the characterization of alveolar septa and lung structure [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The average score was assessed by 0\u0026ndash;4 points (4: extremely serious, 3: serious, 2: middle, 1: slight, 0: normal) from five randomly selected microscopic fields using a Leica DFC7000T microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Immunohistochemical (IHC) staining and analysis\u003c/h2\u003e \u003cp\u003eThe paraffin-embedded mice tissue sections were further stained for IHC analysis following the manufacturer\u0026rsquo;s instructions. Slides were blocked with BSA (G5001, Servicebio) and then incubated overnight at 4 ℃ with primary antibodies anti-α-SMA (1:2000, BM0002, BOSTER, CA, USA), anti-pro-SP-c (1:2000, ab211326, Abcam, Cambridge, UK), anti-F4/80 (1:5000, Abcam) and anti-CD4 (1:1000, Abcam). Stained images were captured using a microscope. The total density of ten positive microscopic fields per mouse was assessed, and the semi-quantification of protein expression was analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Mouse pulmonary function\u003c/h2\u003e \u003cp\u003eAt 24 weeks post-IR, six mice were randomly selected from each group and pulmonary function was measured. Mice were anesthetized by intraperitoneal injection of 1.5% pentobarbital sodium (Bio-Light Biotech, Guangzhou, China, 80 mg/kg body weight), and the lung functions vital capacity (VC), forced vital capacity (FVC), and dynamic pulmonary compliance (Cdyn) were measured using a small animal lung function analyzer (Data Sciences International, Inc., ST. Paul, MN, USA) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Functional enrichment analysis of RNA-sequencing (RNAseq)\u003c/h2\u003e \u003cp\u003eLung tissues at 8 weeks post-IR in different groups were sampled and cryopreserved at \u0026minus;\u0026thinsp;80 ℃ for RNA sequencing assay described in our previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Briefly, RNA extracted from mouse lung tissue was sequenced on the Illumina Novaseq platform. Clean data filtered by TrimGalore software (v0.6.6) were mapped to the mouse reference genome (mm10) with TopHat software (v2.1.1) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and evaluated using Cufflinks software (v2.2.1). Differentially expressed genes (DEGs) between the indicated groups were filtered with adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2(fold change)|\u0026gt;0.585. Functional enrichment analysis, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), was performed using the clusterProfier package (v3.18.1) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8 (San Diego, CA, USA). Experimental data from at least three replicates were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M.). Survival fraction (SF) was calculated with the Kaplan-Meier method. Comparison was analyzed by one-way analysis of variance (ANOVA) and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Radiation-related severe lung injury in pre-existing ILD mice\u003c/h2\u003e \u003cp\u003eA sRRLI model was established based on the BIPF mice which was widely used in the field of pulmonary fibrosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, mice exhibited hair loss and discoloration within the exposure field post-IR. Lung histopathology displayed thickened interalveolar septa, cellular infiltration, and collagen deposition through 8 to 24 weeks in the RILI group. Moreover, characteristic lung morphology of fibrotic foci was presented in the BIPF group. Significantly, enhanced damage was observed in the sRRLI model, including inflammatory exudation, fibroblast proliferation, and scattered collagenous deposits at week 8 post-IR, and dense remodeling with organized interalveolar fibrosis scars at week 24 post-IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Semi-quantification of lung lesions confirmed that the injured areas were remarkably increased in the sRRLI model compared to the mono-treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAlthough there was no death in the RILI and BIPF groups, lethal toxicity was observed in the sRRLI group, where 23.08% (3/13) of the mice died within six months with acute death occurring in the first week post-IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Besides, measurement of pulmonary function in the surviving mice showed that VC, FVC, and Cydn in the sRRLI group were declined significantly in comparison to the mono-treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating the ventilation function was severely impaired in the sRRLI group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Diffuse alveolar damage (DAD) patterns in the sRRLI model\u003c/h2\u003e \u003cp\u003eRepresentative histological phenotypes of the sRRLI group illustrated that, at week 8 post-IR, the aggravated injury was characterized by exudative DAD in the right lung with obvious desquamated pneumocytes, and exudative hyaline membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA a, b). Besides, collagen deposition was distributed along the bronchovascular bundle or subpleural (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA c, d). At week 24 post-IR, proliferative DAD notably predominated in bilateral lung injury with sustained infiltration of inflammatory cells and fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB a, c). Characteristic pneumonia, progressive fibrosis, and extensive remodeling resulted in alveolar duct obstruction, collapse, and interstitial thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB b, d).\u003c/p\u003e \u003cp\u003eTo disclose the causes underlying the fatal injury, lung biopsy was taken from a case that died at week 9 post-IR in the sRRLI group. Significant DAD with progressive inflammation and fibrosis were observed in bilateral lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Histological examination revealed that normal alveolar structure was replaced by obvious collagen deposition and fibrin exudation, including prominent invasive fibroblast infiltration in the right lung (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC a, b), sporadic pulmonary congestion and bleeding in the right upper lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC c), and worsening inflammation in the left lung dominated by infiltration of macrophages, lymphocytes, and giant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC d). These features of overlapping DAD patterns eventually led to irreversible lung function impairment and respiratory failure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Mechanisms underlying different treatment-induced lung injury\u003c/h2\u003e \u003cp\u003eThe potential mechanisms of the above lung injury were further investigated using transcriptome sequencing. Bioinformatic analysis showed that the sRRLI mice had undergone more changes than the mono-treatment damage at the transcriptome level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), and the top 50 common DEGs among different groups were shown in a heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Functional enrichment analysis of 149 crucial common DEGs revealed that the signaling pathways relevant to inflammation, cellular damage, repair responses, such as p53, PI3K-Akt, MAPK, JAK-STAT, HIF-1 pathways were generally involved (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Specifically, GO analysis showed that these genes were enriched in epithelial cell development, immune cell migration, and extracellular external stimulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The protein-protein interaction with most nodes revealed the critical roles of \u003cem\u003eBtg2\u003c/em\u003e, \u003cem\u003eCdkn1a\u003c/em\u003e, \u003cem\u003eHspa8\u003c/em\u003e, \u003cem\u003eFosb\u003c/em\u003e, and \u003cem\u003eSerpine1\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Moreover, the GO pathway analysis by GSEA revealed that the migration and chemotaxis of immune cells (myeloid leukocytes, granulocytes, and neutrophils) were remarkably involved in the lung of sRRLI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cellular infiltration during lung remodeling\u003c/h2\u003e \u003cp\u003eTo validate the participation of important cell types during lung remodeling, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA illustrates the cellular morphology and their anatomical localizations in the lung tissues of different groups, including alveolar epithelial cells (AEC II) with pro-SP-c protein expression, myofibroblasts (α-SMA biomarker), macrophages (F4/80 biomarker), and CD4 + lymphocytes. In detail, the intensity of pro-SP-c protein was obviously increased in the sRRLI group compared to the RILI group. AEC II hyperplasia was especially localized in the residual alveoli with normal structures, whereas it was relatively absent in the fibrotic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). α-SMA was also highly expressed in the sRRLI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and its positive area extended along the bronchovascular bundle, subpleural area, and broader consolidation area. Furthermore, the infiltration levels of macrophages and CD4 + lymphocytes were notably higher in the sRRLI model than those in the mono-treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). The infiltration nests of immune cells were mainly located along the bronchovascular bundle and consolidation regions. Consistent with the bioinformatic analysis, these results indicated that hyperactivation of the immune system was involved in enhanced lung injury of the sRRLI model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Radiation-induced abscopal injury in the contralateral lungs\u003c/h2\u003e \u003cp\u003eLung injuries were observed not only in the irradiated tissues but also in the non-IR abscopal contralateral lungs. In the RILI group, the injury area in the abscopal lungs increased slightly compared to the control group. In the sRRLI group, however, the degree of lung injury was substantially higher. The histopathological changes showed obvious thickened alveolar walls, inflammatory cell infiltration, and collagen deposition in the contralateral lungs, similar to the phenomenon in the irradiated ipsilateral right lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Besides, the enhanced AEC II hyperplasia and the increased infiltration of myofibroblasts, macrophages, and CD4 + lymphocytes were also observed in the non-IR abscopal lung tissues, so that the intensities of pro-SP-c, α-SMA, F4/80, and CD4 + lymphocytes in the sRRLI model were significantly higher than those in the mono-treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Critical mediators categorized through bioinformatic GOBP/KEGG terms\u003c/h2\u003e \u003cp\u003eReliable biomarkers that could accurately monitor the progression of lung injury were further analyzed with bioinformatic GOBP/KEGG pathways in the sRRLI model (Fig. S1). Some signaling factors in the cytokine-cytokine receptor interaction, cell chemotaxis, cytokine-mediated signaling pathways were categorized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, including chemokines and chemokine receptors, interleukins (ILs), transforming growth factor-β (TGF-β) family, matrix metalloproteinases (MMPs), and tumor necrosis factor-α (TNF-α), which might contribute to the recruitment and migration of key cells and be associated with the development of inflammatory and fibrotic responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCrucial mediators categorized through bioinformatic GOBP/KEGG terms of the sRRLI model\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotential biomarkers\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFunctions\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eChemokines\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCL2/5/6/7/8/9/12/17/20/22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eChemokines are secreted proteins that control immune cell migration, adhesion, cell-cell interactions during inflammation and immune surveillance through binding to their specific receptors. They contribute to angiogenesis, tumorigenesis, inflammation, and tissue repair and are also associated with pathological disorders of radiation injury. PPBP has been implicated in the early stages of wound healing and is associated with IPF. PPBP was found to be significantly differentially expressed between those with a definite UIP pattern of ILA compared to those without ILA.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCXCL5/8/9/10/12/13/16\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePPBP(CXCL7)\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eChemokine receptors\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCR1/5/7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCXCR1/4/5/7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACKR1/3/5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterleukins\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-1/2/4/5/6/8/10/18/33/34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eILs are a series of cytokines expressed by leukocytes and many other cell types. They participate in immunological responses with pro- and anti-inflammatory properties. IL production is involved in pathological processes of radiation-induced cardiopulmonary toxicity.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGF-β family\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGFB2, BMP6, GDF6/15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGF-β family members include TGF-β isoforms, activins, nodal, BMPs, and GDFs. They regulate various key events in normal development and physiology. TGF-β is critical in cancer therapeutic resistance, promoting the activation of fibroblasts and ECM synthesis after radiotherapy.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMPs\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMMP12/28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMMPs are responsible for collagen and protein degradation in ECM. They are involved in maintaining normal organ and tissue homeostasis and are related to several pathologic conditions, including cancer and fibrosis. MMP2 and MMP9 are relevant to EMT in epithelial cells after radiation.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFRSF\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTNFRSF 1/4/9/11/12/13/18/19/21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF and TNFSF/TNFRSF control the coordination of various mechanisms driving co-stimulation and co-inhibition among immune cells. Single-cell sequencing analysis suggested that TNF plays a role in RIPF in T cells and monocytes.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIFNGR2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIFNGR2 is a subunit of the interferon-gamma receptor (IFNγR). The (IFNGR1/IFNGR2)2 -IFN-γ dimer activates the cytotoxic activity of innate immune cells via signaling involving Jak1, Jak2, and Stat1. IFN-γ contributed to the enhanced necroptosis in lung epithelial cells.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSAA is a highly conserved family of acute-phase response proteins. SAA level can increase rapidly in emergency conditions such as inflammation, trauma, and viral infection. Endogenous SAA was recently reported as having a protective role in ameliorating lung injury.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAI-1/Serpine1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePAI-1 is a major TGF-β1/p53 target gene. PAI-1 inhibits the plasmin system by blocking fibrinolysis and ECM degradation. Mature PAI-1 protein could induce lung epithelial cell senescence treated by bleomycin through a p53-independent mechanism.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo simulate clinical situations, the present study established a novel sRRLI model based on BLM-induced pulmonary fibrosis mice. It was observed that most of the pre-existing ILD mice presented pathological DAD patterns and lung function loss after partial thoracic irradiation. These mice tended to develop severe pneumonitis, progressive fibrosis, and even fatal outcomes within six months post-IR, compared to RILI and BIPF mono-treatment groups. Clinically, a recent retrospective study showed that the incidence of post-treatment AE was higher in LC-ILD patients who received CCRT (54.5%) followed by radiotherapy (16.2%) and chemotherapy (15.6%), compared to non-ILD patients [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Even when relatively low-dose palliative thoracic radiotherapy was delivered, the rate of grade ≥ 3 RP was reported to be 13.7%. Particularly in patients with a higher pulmonary fibrosis score (score 3–5), the incidence of serious RP increased to 37.5% with eventual death [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAutopsy analysis or lung biopsy is scarce after treatment-related severe complications in the clinic. Using an \u003cem\u003ein vivo\u003c/em\u003e mouse model, significant pathological changes in lung injury were detailed here. At the early stage, when lung injury in the RILI and BIPF groups gradually settled into relatively stable, the sRRLI mice were still predominated by the exudative DAD with apparent massive hyaline membranes and intra-alveolar fibrin. As time passed, at six months post-IR, the organizing DAD phase became the main pattern, characterized by inter-alveolar fibroblastic proliferation, septal collagen deposition, and fibrotic foci. Severe damage, if irreversible, could eventually result in repeated injury contributing to modality. Autopsy of the lethal mouse revealed overlapping patterns of exudative, proliferative, and fibrotic DAD in bilateral lungs. Such morphological changes may be responsible for the clinical symptoms observed in LC-ILD patients with progressive dyspnea, dry cough, declining lung function, and respiratory failure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCommon mechanisms underlying different treatment-induced lung injuries were elucidated using transcriptomic analysis here. Signaling pathways relevant to cellular damage and repair, including p53, PI3K-Akt, MAPK, JAK-STAT, HIF-1, and cellular senescence, were universally activated. A recent report using scRNA-seq analysis showed that the activation of PI3K-Akt and p53 pathways in AEC cells participated in the progression of blast-induced lung damage via modulating autophagic and oxidative stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Besides, AKT-, MAPK-, or JAK-STAT-relevant inflammatory pathways are potential therapeutic targets for treating acute lung injury (ALI), and novel agents are under investigation [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Growing evidence suggests that cellular senescence is positively associated with pulmonary fibrosis. Upregulation of the senescence-related proteins p16\u003csup\u003eINK4a\u003c/sup\u003e and p21\u003csup\u003eCIP1\u003c/sup\u003e were previously demonstrated in RIPF or BIPF models [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Senescent cells display senescence-associated secretory phenotype, which is involved in promoting the lung fibroblast proliferation, myofibroblast activation, and ECM production [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe also found that the common genes \u003cem\u003eCdkn1a, Fosb\u003c/em\u003e, and \u003cem\u003eSerpine1\u003c/em\u003e were among the core positions during the development of lung injury. Our previous study has validated that \u003cem\u003eCdkn1a\u003c/em\u003e, also known as \u003cem\u003ep21\u003c/em\u003e, its expression level was significantly higher in the sRRLI mice and positively correlated with macrophage infiltration via regulating CCL7 secretion \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, \u003cem\u003eFosb\u003c/em\u003e proto-oncogene functions in regulating cell proliferation, differentiation, and transformation. It is a subunit gene of the AP-1 transcription factor, which plays a crucial role in lung fibroblasts by promoting macrophage activation and collagen production [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, PAI-1 (Serpine1) was recently found to be associated with downregulating re-alveolarization in ALI by reducing the AEC II self-renewal [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and evidence supported that it was a druggable target for controlling lung cell senescence and fibrosis via inhibiting TGF-β pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, our findings suggested a central role of epithelial cell development and immune cell migration in the progression of severe lung injury. The hyperplastic pneumocytes in residual alveoli and the influx of macrophages and CD4 + lymphocytes were presented persistently during disease development. Cumulative evidence showed that M1/M2 macrophages participated in both the acute and chronic phases of lung disease. M2 macrophages mainly dominate the progression of pulmonary fibrosis via TGF-β signaling pathway during the rehabilitation period [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the sRRLI model, macrophage pools exceeded in both ipsilateral and contralateral lungs so that radiation-induced abscopal lung injury was observed along with the activation of immune responses. This differs from previous observation that focal regions of macrophages existed only in ipsilateral lungs at 26 weeks post-IR [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistently, in some clinical cases after thoracic radiotherapy, extensive ground glass abnormalities and focal consolidations were usually observed spreading to bilateral lungs rather than being limited to the irradiated area on CT images [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProinflammatory and profibrotic factors are considered crucial mediators in causing serious suffering. However, there are no recommendations for reliable biomarkers in the guidelines now due to their relatively low specificity. Experts have advised that patients with higher ILD gender age physiology (ILD-GAP) index scores should be considered when delivering treatments or only observation, as radiological usual interstitial pneumonia (UIP) pattern was significantly associated with thoracic radiotherapy-related life-threatening pneumonitis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Most noteworthy, a higher level of KL-6 was found to be linked to more severe AE-ILD or treatment-related ILD and thus was proposed as a potential biomarker for poor prognosis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Besides, peripheral blood markers such as pretreatment NLR (≥ 3.0), and pretreatment ANC (≥ 5755) were reported to be associated with severe RILI [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We categorized some potential mediators in the early phase of the sRRLI here, including chemokines and chemokine receptors, ILs, TGF-β family, MMPs, and TNF-α. Other factors of growing interest include pro-platelet basic protein (PPBP), growth and differentiation factor 15 (GDF 15), and serum amyloid A (SAA), which have recently been implicated in the development of lung inflammation and fibrosis [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e–\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA preclinical \u003cem\u003ein vivo\u003c/em\u003e mouse model was established to mimic radiotherapy-related severe pulmonary complications in LC-ILD patients. This study highlighted that the severe or even fatal toxicity was due to DAD with progressive inflammation and fibrosis in bilateral lungs post-IR. Bioinformatics analysis aided in the discovery of critical signaling pathways, and the hyperactivation of inflammatory responses with infiltration of macrophage and CD4 + lymphocyte was further validated. However, further studies are needed to clarify the mechanisms underlying disease progression. Rigorous preclinical and clinical trials are still required to elucidate potential biomarkers and develop effective therapeutic targets for predicting and preventing severe complications in LC-ILD patients following radiotherapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; LC, lung cancer; LC-ILD, LC patients with comorbid ILD; AE, acute exacerbation; SBRT, stereotactic body radiotherapy; NSCLC, non-small cell lung cancer; RP, radiation pneumonitis; CCRT, concurrent chemoradiotherapy; BIPF: bleomycin (BLM)-induced pulmonary fibrosis; ECM: extracellular matrix; UIP: usual interstitial pneumonia; ALI: acute lung injury\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u003c/strong\u003e: J.F. and C.S. conceived and designed the experiments; X.L., and Y.Z. performed experiments, data acquisition and analysis; S.Z., L.Z., Y.P. and J.Z. performed experiments and data acquisition; J.F. wrote the original draft of the manuscript; C.S., K.P, and Y.X supervised the work and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eWe thank for the support of bioinformatic analysis by the Medical Research Data Center of Fudan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was supported by the National Natural Science Foundation of China (Nos.81903258, 32171235, 12235004, and 12175044) and the Shanghai Pulmonary Hospital Talents Plan (fkyq1904).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eAll animal studies complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The animal experimental protocol was approved by the Animal Welfare and Ethics Committee of Fudan University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that none of them have any conflicts on interest in relation to the present publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eData presented in this study are available upon request from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTurkkan G, Willems Y, Hendriks LEL, Mostard R, Conemans L, Gietema HA, et al. Idiopathic pulmonary fibrosis: Current knowledge, future perspectives and its importance in radiation oncology. 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PAI-1 regulates AT2-mediated re-alveolarization and ion permeability. Stem Cell Res Ther. 2023;14(1):185.\u003c/li\u003e\n\u003cli\u003eAdnot S, Breau M, Houssaini A. PAI-1: A New Target for Controlling Lung-Cell Senescence and Fibrosis? Am J Respir Cell Mol Biol. 2020;62(3):271-2.\u003c/li\u003e\n\u003cli\u003eLee JW, Chun W, Lee HJ, Min JH, Kim SM, Seo JY, et al. The Role of Macrophages in the Development of Acute and Chronic Inflammatory Lung Diseases. Cells. 2021;10(4).\u003c/li\u003e\n\u003cli\u003eGroves AM, Misra R, Clair G, Hernady E, Olson H, Orton D, et al. Influence of the irradiated pulmonary microenvironment on macrophage and T cell dynamics. Radiother Oncol. 2023;183:109543.\u003c/li\u003e\n\u003cli\u003eOnishi H, Marino K, Yamashita H, Terahara A, Onimaru R, Kokubo M, et al. Case Series of 23 Patients Who Developed Fatal Radiation Pneumonitis After Stereotactic Body Radiotherapy for Lung Cancer. 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Exp Cell Res. 2020;391(2):112010.\u003c/li\u003e\n\u003cli\u003eJi A, Trumbauer AC, Noffsinger VP, Meredith LW, Dong B, Wang Q, et al. Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice. Int J Mol Sci. 2023;24(24).\u003c/li\u003e\n\u003cli\u003eWang L, Jiang J, Chen Y, Jia Q, Chu Q. The roles of CC chemokines in response to radiation. Radiat Oncol. 2022;17(1):63.\u003c/li\u003e\n\u003cli\u003eLiu X, Shao C, Fu J. Promising Biomarkers of Radiation-Induced Lung Injury: A Review. Biomedicines. 2021;9(9).\u003c/li\u003e\n\u003cli\u003eMukai-Sasaki Y, Liao Z, Yang D, Inoue T. Modulators of radiation-induced cardiopulmonary toxicities for non-small cell lung cancer: Integrated cytokines, single nucleotide variants, and HBP systems imaging. Front Oncol. 2022;12:984364.\u003c/li\u003e\n\u003cli\u003eWang J, Xu Z, Wang Z, Du G, Lun L. TGF-beta signaling in cancer radiotherapy. Cytokine. 2021;148:155709.\u003c/li\u003e\n\u003cli\u003eYue H, Hu K, Liu W, Jiang J, Chen Y, Wang R. Role of matrix metalloproteinases in radiation-induced lung injury in alveolar epithelial cells of Bama minipigs. Exp Ther Med. 2015;10(4):1437-44.\u003c/li\u003e\n\u003cli\u003eSun Z, Lou Y, Hu X, Song F, Zheng X, Hu Y, et al. Single-cell sequencing analysis fibrosis provides insights into the pathobiological cell types and cytokines of radiation-induced pulmonary fibrosis. BMC Pulm Med. 2023;23(1):149.\u003c/li\u003e\n\u003cli\u003eHao Q, Shetty S, Tucker TA, Idell S, Tang H. Interferon-gamma Preferentially Promotes Necroptosis of Lung Epithelial Cells by Upregulating MLKL. Cells. 2022;11(3).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"animal model, severe lung injury, radiation, interstitial lung disease, diffuse alveolar damage","lastPublishedDoi":"10.21203/rs.3.rs-4816003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4816003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLung cancer patients with comorbid interstitial lung disease (LC-ILD) have an increased risk of developing severe or even fatal radiation pneumonitis after thoracic radiotherapy. However, the underlying mechanisms of its pathogenesis are still inconclusive. No approved biomarker or medicine is available to prevent pulmonary toxicities in LC-ILD patients. Appropriate management for them remains a challenge for clinicians due to treatment-related complications.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo elucidate the histopathological characteristics and molecular mechanisms responsible for this severe toxicity \u003cem\u003ein vivo\u003c/em\u003e, C57BL/6J mice were used to develop different lung injury models, including radiation-induced lung injury (RILI), bleomycin-induced pulmonary fibrosis (BIPF), and severe radiation-related lung injury (sRRLI) murine model. Biopsy examination was performed on hematoxylin and eosin (H\u0026amp;E), Masson\u0026rsquo;s trichrome, and immunohistochemistry-stained lung tissue sections. Changes in lung function were measured. RNA extracted from mouse lung tissues was sequenced on the Illumina Novaseq platform.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA severe lung injury model after irradiation was built based on pre-existing ILD mice induced by BLM administration. Enhanced lung injury was observed in the sRRLI model, including higher mortality and pulmonary function loss within six months compared to the mono-treatment groups. Autopsy revealed that bilateral diffuse alveolar damage (DAD) with an overlap of exudative, proliferative, and fibrosing patterns was usually presented in the sRRLI model. The histological phenotypes manifested exudative DAD phase in the early phase and proliferating DAD pattern predominated in the late phase. Bioinformatic analysis showed signaling pathways relevant to immune cell migration, epithelial cell development, and extracellular structure organization were commonly activated in the different models. Furthermore, the involvement of epithelial cells and the infiltration of macrophages and CD4\u0026thinsp;+\u0026thinsp;lymphocytes were validated during extensive lung remodeling in the sRRLI group. They also participated in triggering remarkable abscopal responses in the non-IR contralateral lungs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe study provides a preclinical model to better understand radiation-related severe lung injury in pre-existing ILD mice. DAD with progressive inflammation and fibrosis in bilateral lungs contributed to severe or even fatal complications after partial thoracic irradiation. More studies are needed to investigate potential strategies to prevent and rescue severe pulmonary complications.\u003c/p\u003e","manuscriptTitle":"Bilateral diffuse alveolar damage contributes to the fatal toxicity of pre-existing interstitial lung disease mice after partial thoracic irradiation ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 12:00:04","doi":"10.21203/rs.3.rs-4816003/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-09T16:35:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-20T07:45:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64386078750308647471609413234541126208","date":"2024-10-18T00:32:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-05T10:50:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-29T10:09:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-29T05:21:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Radiation Oncology","date":"2024-07-28T09:18:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"radiation-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"raon","sideBox":"Learn more about [Radiation Oncology](http://ro-journal.biomedcentral.com/)","snPcode":"13014","submissionUrl":"https://submission.nature.com/new-submission/13014/3","title":"Radiation Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a38a269e-7fa9-461c-b833-aedabe26f648","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:02:56+00:00","versionOfRecord":{"articleIdentity":"rs-4816003","link":"https://doi.org/10.1186/s13014-025-02596-w","journal":{"identity":"radiation-oncology","isVorOnly":false,"title":"Radiation Oncology"},"publishedOn":"2025-02-07 15:57:46","publishedOnDateReadable":"February 7th, 2025"},"versionCreatedAt":"2024-08-28 12:00:04","video":"","vorDoi":"10.1186/s13014-025-02596-w","vorDoiUrl":"https://doi.org/10.1186/s13014-025-02596-w","workflowStages":[]},"version":"v1","identity":"rs-4816003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4816003","identity":"rs-4816003","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}