Radiation-induced Fibrosis Research Articles

Deferoxamine to Minimize Fibrosis During Radiation Therapy.

Significance: By 2030, there will be >4 million radiation-treated cancer survivors living in the United States. Irradiation triggers inflammation, fibroblast activation, and extracellular matrix deposition in addition to reactive oxygen species generation, leading to a chronic inflammatory response. Radiation-induced fibrosis (RIF) is a progressive pathology resulting in skin pigmentation, reduced elasticity, ulceration and dermal thickening, cosmetic deformity, pain, and the need for reconstructive surgery. Recent Advances: Deferoxamine (DFO) is a U.S. Food and Drug Administration (FDA)-approved iron chelator for blood dyscrasia management, which has been found to be proangiogenic, to decrease free radical formation, and reduce cell death. DFO has shown great promise in the treatment and prophylaxis of RIF in preclinical studies. Critical Issues: Systemic DFO has a short half-life and is cumbersome to deliver to patients intravenously. Transdermal DFO delivery is complicated by its high atomic mass and hydrophilicity, preventing stratum corneum penetration. A transdermal drug delivery system was developed to address these challenges, in addition to a strategy for topical administration. Future Directions: DFO has great potential to translate from bench to bedside. An important step in translation of DFO for RIF prophylaxis is to ensure that DFO treatment does not affect the efficacy of radiation therapy. Furthermore, after an initial plethora of studies reporting DFO treatment by intravenous and subcutaneous routes, a significant advantage of recent studies is the success of transdermal and topical delivery. Given the strong foundation of basic scientific research supporting the use of DFO treatment on RIF, clinicians will be closely following the results of the ongoing human studies.

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

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

1: Fat Grafting Improves Dorsal Skin Fibrosis after Radiation in an Engrailed-1 Mouse Model

Purpose:Fat grafting is known to rejuvenate and improve fibrosis in irradiated skin, but the underlying mechanisms behind these effects are poorly understood. We have previously identified the Engrailed-1 fibroblast sub-population as having a role in post-natal dorsal skin fibrosis in mice. Using a radiation model to the scalp of Engrailed-1 mice, we sought to better understand the regenerative effects of fat grafting in irradiated tissue.Methods:Adult (60-day old) En1Cre;R26mTmG transgenic mice (n=5) underwent total body irradiation with 9 Gy for immunodepletion. Mice were then immediately reconstituted with 2 million nucleated bone marrow cells from donor NSG(NOD.CB17-Prkdcsscid/J) mice via retro-orbital injections. Reconstitution was confirmed with fluorescence-activated cell sorting (FACS) 2 weeks after whole body irradiation. Mice scalps were then irradiated with 5 Gy every other day for 12 days (30 Gy total) and allowed to recover for 4 weeks to facilitate fibrotic conversion. Irradiated scalps were grafted with 200μL of fresh human lipoaspirate. Graph retention was measured in-vivo for 8 weeks using Micro-Computed Tomography Scanner (Bruker SkyScan 1726™) and scalp skin was harvest for histology.Results:Two weeks post bone-marrow transplant, >90% of circulating hematopoietic cells (CD45+) cells in En1Cre;R26mTmG reporter mice were non-fluorescent, signifying successful reconstitution. Volumetric analysis of fat grafting in-vivo was performed using 3-dimensional reconstruction. At 8 weeks post-grafting, 4 of the 5 grafts demonstrated >50% graft retention, confirming successful grafting. Histological sections of scalp skin 8 weeks post-grafting demonstrated significantly less En1+GFP cells compared to non-grafted scalp skin, signifying less presence of En1+ fibroblasts. Furthermore, histology demonstrated significantly less dermal thickening, epidermal thinning, and collagen deposition and disorganization in fat grafted irradiated scalp skin compared to non-grafted scalp skin.Conclusion:Fat grafting mitigates radiation-induced fibrosis in scalp skin by decreasing collagen deposition, remodeling collagen formation, decreasing epidermal thinning, and reducing presence of pro-fibrotic fibroblast sub-populations.

Dosimetric Quantities in Neuroendocrine Tumors over Treatment Cycles with 177Lu-DOTATATE.

Tumor dosimetry was performed for 177Lu-DOTATATE with the aims of better understanding the range and variation of the tumor-absorbed doses (ADs), how different dosimetric quantities evolve over the treatment cycles, and whether this evolution differs depending on the tumor grade. Such information is important for radiobiologic interpretation and may inform the design of alternative administration schemes. Methods: The data came from 41 patients with neuroendocrine tumors (NETs) of grade 1 (n = 23) or 2 (n = 18) who had received between 2 and 9 treatment cycles. Dosimetry was performed for 182 individual lesions, giving a total of 880 individual AD assessments across all cycles. Hybrid planar-SPECT/CT imaging was used, including quantitative SPECT reconstruction, voxel-based absorbed-dose-rate calculation, semiautomatic image segmentation, and partial-volume correction. Linear mixed-effect models were used to analyze changes in tumor ADs over cycles, absorbed-dose rates and activity concentrations on day 1, effective half-times, and tumor volumes. Tumors smaller than 8 cm3 were excluded from analyses. Results: Tumor ADs ranged between 2 and 77 Gy per cycle. On average, the AD decreased over the cycles, with significantly different rates (P < 0.05) of 6% and 14% per cycle for grade 1 and 2 NETs, respectively. The absorbed-dose rates and activity concentrations on day 1 decreased by similar amounts. The effective half-times were less variable but shorter for grade 2 than for grade 1 (P < 0.001). For grade 2 NETs, the tumor volumes decreased, with a similar tendency in grade 1. Conclusion: The tumor AD, absorbed-dose rate, and activity uptake decrease, in parallel with tumor volumes, between 177Lu-DOTATATE treatment cycles, particularly for grade 2 NETs. The effective half-times vary less but are lower for grade 2 than grade 1 NETs. These results may indicate the development of radiation-induced fibrosis and could have implications for the design of future treatment and dosimetry protocols.

Clinical Role of Oral Vitamin C and E Therapy in Skin and Hair Disorders

Clinical Role of Oral Vitamin C and E Therapy in Skin and Hair Disorders

Evaluation of aromatase inhibitor on radiation induced pulmonary fibrosis via TGF- β/Smad 3 and TGF- β/PDGF pathways in rats

Radiation-induced pulmonary fibrosis (RIPF) is a known complication in cancer patients after getting thoracic radiotherapy. Aromatase inhibitors (AIs) as anastrozole have been used instead of tamoxifen for adjuvant endocrine treatment of postmenopausal women with hormone sensitive breast cancer. This study is to evaluate the concurrent treatment of anastrozole and RIPF in rats. Twenty four female Wistar rats were distributed into 4 groups: Control (C), Radiation group (R) (total dose 30 Gy in 10 fractions, 5 fractions/week), anastrozole group (A) (0.003 mg/200 g body weight) orally for 14 consecutive days, and Radiation + anastrozole group (R + A). Radiation exposure resulted in a significant increase (p < 0.05) in pulmonary Transforming growth factor-beta 1 (TGF-β), SMAD family member 3 (Smad3), Platelet-derived growth factor (PDGF), malondialdehyde (MDA), Total nitrate/nitrite (NO), interleukin 1β (IL-1β) and interleukin 6 (IL-6) compared to the control group. While, significant decreases (p < 0.05) in superoxide dismutase (SOD) activity, reduced glutathione (GSH) and connective tissue growth factor (CTGF) were observed in lung tissue. These alterations were minimized by anastrozole intervention. Also, anastrozole markedly hindered the lung histopathological changes observed after radiation. Concomitant use of anastrozole with radiation seems to attenuate radiation-induced pulmonary toxicity via TGF-β/Smad 3 and TGF-β/PDGF pathways in rats.

ADAM10 Evens Out the Double-Edged Sword of Radiotherapy in Pancreatic Cancer

Radiotherapy plays an important role in the management of pancreatic ductal adenocarcinoma (PDAC), especially when patients are not surgical candidates. Radiation-induced tumor death provokes an acute inflammation followed by a late-fibrotic response that parallels the fibroinflammatory tumor microenvironment of PDAC, inciting the question of whether radiation-induced fibrosis contributes to PDAC progression. The study published in this issue by Mueller and colleagues presents a potential mechanism linking radiation-induced fibrosis with expression of a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, which may also contribute to tumor progression. The authors show that ablation of ADAM10 decreases radiation-induced fibrosis and improves survival in preclinical models. These data suggest that targeting ADAM10 may help to improve clinical outcomes with radiotherapy, particularly if definitive radiation is not possible. A better understanding of the biology of radiotherapy in pancreatic cancer remains crucial, and Mueller and colleagues offer important insight in this regard.See related article by Mueller et al., p. 3255.

The Value of MR-DWI and T1 Mapping in Indicating Radiation-Induced Soft Tissue Injury

ObjectiveTo explore the value of MR-DWI and T1 mapping in predicting radiation-induced soft tissue fibrosis and its correlation with radiation inflammation.Methods① a total of 30 C57BL/6 mice were randomly divided into a control group (Nor group), irradiation group (IR group) and irradiation plus glycyrrhetinic acid group (GA group). The IR group and GA group were treated with 6MV X-rays to irradiate the right hind limbs of mice for 30 Gy in a single shot. MRI examinations were performed before and on the 7th day after irradiation to measure the apparent diffusion coefficient (ADC) value and the longitudinal relaxation time (T1) value of the hind limb muscles of the mice. On the 90th day after irradiation, the hind limb contracture was measured, and the right hind limb muscle was taken for HE staining, masson staining, immunohistochemical staining and Western blot analysis to detect the expression of a-SMA and Fibronectin. ② The other 30 mice were grouped randomly as above. On the 7th day after irradiation, the right hind limbs of the mice were examined by MRI to measure the ADC value and T1 value of the thigh muscles, and then the right hind thigh muscles were immediately sacrificed to detect IL-1β, IL-6, TNF-a and TGF-β1 expression with ELISA.ResultsOn the 7th day after irradiation, the ADC values ​​of right hind thigh muscles of mice in Nor group, IR group and GA group were (1.35 ± 0.11)*10-3mm2/s, (1.48 ± 0.07) *10-3mm2/s and (1.36 ± 0.13)*10-3mm2/s, respectively, by which the differences between the IR group and Nor group (P=0.008) and that between IR group and GA group (P=0.013) were statistically significant; T1 values ​​were (1369.7 ± 62.7)ms, (1483.7 ± 127.7)ms and (1304.1 ± 82.3)ms, respectively, with which the differences in the T1 value between the IR group and Nor group (P=0.012) and between IR group and GA group (P<0.001) were also statistically significant. On the 90th day after irradiation, the contracture lengths of the right hind limbs of the three groups of mice were (0.00 ± 0.07)cm, (2.08 ± 0.32)cm, and (1.49 ± 0.70) cm, respectively. There were statistically significant differences in the IR group compared with the Nor group (P<0.001) and the GA group (P=0.030). The ADC value (r=0.379, P=0.039) and T1 value (r=0.377, P=0.040) of the mice’s hindlimbs on Day 7 after irradiation were correlated with the degree of contracture on Day 90 after irradiation; the ADC value (r=0.496, P=0.036) and T1 value (r=0.52, P=0.027) were positively correlated with the Masson staining results and with the expression of α-SMA and Fibronectin. While the ADC value was positively correlated with IL-6 (r=0.553, P=0.002), there was no obvious correlation with IL-1β, TNF-a and TGF-β1; the T1 value was positively correlated with IL-1β (r=0.419, P=0.021), IL-6 (r=0.535, P=0.002) and TNF-a (r=0.540, P=0.002) but not significantly related to TGF-β1 (r=0.155, P=0.413).ConclusionThe MR-DWI and T1 mapping values on the 7th day after irradiation can reflect the early condition of tissue inflammation after the soft tissue is irradiated, and the values have a certain correlation with the degree of radiofibrosis of the soft tissue in the later period and may be used as an index to predict radiofibrosis.

Potential role of senescent macrophages in radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation in clinic with poor prognosis and limited therapeutic options. Previous results have shown that senescent cells, such as fibroblast and type II airway epithelial cell, are strongly implicated in pathology of RIPF. However, the role of senescent macrophages in the development RIPF is still unknown. In this study, we report that ionizing radiation (IR) increase cellular senescence with higher expression of senescence-associated β-galactosidase (SA-β-Gal) and senescence-specific genes (p16, p21, Bcl-2, and Bcl-xl) in irradiated bone marrow-derived monocytes/macrophages (BMMs). Besides, there’s a significant increase in the expression of pro-fibrogenic factors (TGF-β1 and Arg-1), senescence-associated secretory phenotype (SASP) proinflammatory factors (Il-1α, Il-6, and Tnf-α), SASP chemokines (Ccl2, Cxcl10, and Ccl17), and SASP matrix metalloproteinases (Mmp2, Mmp9 and Mmp12) in BMMs exposed to 10 Gy IR. In addition, the percentages of SA-β-Gal+ senescent macrophages are significantly increased in the macrophages of murine irradiated lung tissue. Moreover, robustly elevated expression of p16, SASP chemokines (Ccl2, Cxcl10, and Ccl17) and SASP matrix metalloproteinases (Mmp2, Mmp9, and Mmp12) is observed in the macrophages of irradiated lung, which might stimulate a fibrotic phenotype in pulmonary fibroblasts. In summary, irradiation can induce macrophage senescence, and increase the secretion of SASP in senescent macrophages. Our findings provide important evidence that senescent macrophages might be the target for prevention and treatment of RIPF.

GDF15 Promotes Cardiac Fibrosis and Proliferation of Cardiac Fibroblasts via the MAPK/ERK1/2 Pathway after Irradiation in Rats.

Ionizing radiation exposure is associated with a risk of cardiac fibrosis; however, the underlying molecular mechanism remains unclear. Growth/differentiation factor-15 (GDF15), a fibroblast factor, is a divergent member of the transforming growth factor β superfamily. Next-generation sequencing analyses has revealed that Gdf15 is increased in cardiac fibroblasts during radiation-induced fibrosis. However, the role of Gdf15 in cardiac fibrosis remains unclear. In this study, we demonstrated that the upregulated expression of GDF15 in newborn rat cardiac fibroblasts and adult rats after irradiation could induce fibrosis, which was confirmed by the increased cell proliferation rate and the increased expression of fibrosis markers (Col1α and αSMA) in newborn rat cardiac fibroblasts after transfection with Gdf15 in vitro. Conversely, the downregulation of GDF15 inhibited cardiac fibrosis, as confirmed by G2/M-cell cycle arrest, suppression of cell proliferation, and low levels of Col1α and αSMA expression. We also found that suppressing the expression of Gdf15 in cardiac fibroblasts could lead to a decrease in CDK1 and inhibit phosphorylation of ERK1/2. Thus, GDF15 might promote cardiac fibroblast fibrosis through the MAPK/ERK1/2 pathway and thus contribute to the pathogenesis of radiation-induced heart disease.

Epigenetic Modulation of Radiation-Induced Diacylglycerol Kinase Alpha Expression Prevents Pro-Fibrotic Fibroblast Response.

Simple SummaryTo reduce long-term fibrosis risk after radiotherapy, we demonstrated with different experimental approaches that modulation of the epigenetic pattern at the DGKA enhancer can attenuate pro-fibrotic reactions in human fibroblasts. We used (epi)genomic editing of the DGKA enhancer and administration of various epigenetic drugs and were able to modulate radiation-induced expression of DGKA and pro-fibrotic collagens. Based on our results, clinical application of bromodomain inhibitors will open promising ways to epigenetically modulate DGKA expression and might provide novel therapeutic options to prevent or even reverse radiotherapy-induced fibrotic reactions.Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

Genetic Variants of DNA Repair Genes as Predictors of Radiation-Induced Subcutaneous Fibrosis in Oropharyngeal Carcinoma.

PurposeTo investigate the impact of genetic variants of DNA repair and pro-fibrotic pathway genes on the severity of radiation-induced subcutaneous fibrosis in patients of oropharyngeal carcinoma treated with radical radiotherapy.Materials and MethodsPatients of newly diagnosed squamous cell carcinoma of oropharynx being treated with two-dimensional radical radiotherapy were enrolled in the study. Patients who had undergone surgery or were receiving concurrent chemotherapy were excluded. Patients were followed up at 6 weeks post completion of radiotherapy and every 3 months thereafter for a median of 16 months. Subcutaneous fibrosis was graded according to the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC) grading system and the maximum grade was recorded over the length of the patient’s follow-up. Patients with severe fibrosis (≥G3), were compared to patients with minor (≤G2) fibrotic reactions. Eight single nucleotide polymorphisms of 7 DNA repair genes and 2 polymorphisms of a single pro-fibrotic pathway gene were analyzed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism and were correlated with the severity of subcutaneous fibrosis.Results179 patients were included in the analysis. Subcutaneous fibrosis was seen in 168 (93.9%) patients. 36 (20.1%) patients had severe (grade 3) fibrosis. On multivariate logistic regression analysis, Homozygous CC genotype of XRCC3 (722C>T, rs861539) (p=0.013*, OR 2.350, 95% CI 1.089-5.382), Homozygous AA genotype of ERCC4 Ex8 (1244G>A, rs1800067) (p=0.001**, OR 11.626, 95% CI 2.490-275.901) and Homozygous TT genotype of XRCC5 (1401G>T, rs828907) (p=0.020*, OR 2.188, 95% CI 1.652-7.334) were found to be predictive of severe subcutaneous fibrosis. On haplotype analysis, the cumulative risk of developing severe fibrosis was observed in patients carrying both haplotypes of variant Homozygous AA genotype of ERCC4 Ex8 (1244G>A, rs1800067) and Homozygous TT genotype of XRCC5 (1401 G>T, rs828907) (p=0.010*, OR 26.340, 95% CI 4.014-76.568).ConclusionWe demonstrated significant associations between single nucleotide polymorphisms of DNA repair genes and radiation-induced subcutaneous fibrosis in patients of oropharyngeal carcinoma treated with radiotherapy. We propose to incorporate these genetic markers into predictive models for identifying patients genetically predisposed to the development of radiation-induced fibrosis, thus guiding personalized treatment protocols.

Radiation-Induced Immunity and Toxicities: The Versatility of the cGAS-STING Pathway.

In the past decade, radiation therapy (RT) entered the era of personalized medicine, following the striking improvements in radiation delivery and treatment planning optimization, and in the understanding of the cancer response, including the immunological response. The next challenge is to identify the optimal radiation regimen(s) to induce a clinically relevant anti-tumor immunity response. Organs at risks and the tumor microenvironment (e.g. endothelial cells, macrophages and fibroblasts) often limit the radiation regimen effects due to adverse toxicities. Here, we reviewed how RT can modulate the immune response involved in the tumor control and side effects associated with inflammatory processes. Moreover, we discussed the versatile roles of tumor microenvironment components during RT, how the innate immune sensing of RT-induced genotoxicity, through the cGAS-STING pathway, might link the anti-tumor immune response, radiation-induced necrosis and radiation-induced fibrosis, and how a better understanding of the switch between favorable and deleterious events might help to define innovative approaches to increase RT benefits in patients with cancer.

Recommendations for postoperative radiotherapy in head & neck squamous cell carcinoma in the presence of flaps: A GORTEC internationally-reviewed HNCIG-endorsed consensus

IntroductionHead and neck reconstructive surgery using a flap is increasingly common. Best practices and outcomes for postoperative radiotherapy (poRT) with flaps have not been specified. We aimed to provide consensus recommendations to assist clinical decision-making highlighting areas of uncertainty in the presence of flaps. Material and methodsRadiation, medical, and surgical oncologists were assembled from GORTEC and internationally with the Head and Neck Cancer International Group (HNCIG). The consensus-building approach covered 59 topics across four domains: (1) identification of postoperative tissue changes on imaging for flap delineation, (2) understanding of tumor relapse risks and target volume definitions, (3) functional radiation-induced deterioration, (4) feasibility of flap avoidance. ResultsAcross the 4 domains, international consensus (median score ≥ 7/9) was achieved only for functional deterioration (73.3%); other consensus rates were 55.6% for poRT avoidance of flap structures, 41.2% for flap definition and 11.1% for tumor spread patterns. Radiation-induced flap fibrosis or atrophy and their functional impact was well recognized while flap necrosis was not, suggesting dose-volume adaptation for the former. Flap avoidance was recommended to minimize bone flap osteoradionecrosis but not soft-tissue toxicity. The need for identification (CT planning, fiducials, accurate operative report) and targeting of the junction area at risk between native tissues and flap was well recognized. Experts variably considered flaps as prone to tumor dissemination or not. Discrepancies in rating of 11 items among international reviewing participants are shown. ConclusionInternational GORTEC and HNCIG-endorsed recommendations were generated for the management of flaps in head and neck radiotherapy. Considerable knowledge gaps hinder further consensus, in particular with respect to tumor spread patterns.

MicroRNA-26 regulates the expression of CTGF after exposure to ionizing radiation.

Radiation-induced fibrosis (RIF) is a serious complication that occurs after irradiation and which is caused by the deposition of extracellular matrix (ECM) proteins such as collagen. However, the underlying mechanisms, including the expression of the cytokines, that promote the RIF process, are not yet fully understood. MicroRNAs (miRNAs) have recently been suggested to act as post-transcriptional repressors for many genes; however, their role in the process of RIF remains to be elucidated. Our previous study showed that ionizing radiation increased the type I collagen expression through the activation of transforming growth factor (TGF)-β, while miR-29 repressed this increase. This study aimed to investigate the mechanisms by which the expression of connective tissue growth factor (CTGF), a downstream mediator of TGF-β, is controlled by miRNAs post-transcriptionally after exposure to ionizing radiation. The expression of CTGF in NIH-3T3 cells and mouse embryonic fibroblasts was increased by ionizing radiation. However, this increase was suppressed with a specific inhibitor of TGF-β receptor. Among the predictable miRNAs that target the CTGF gene, the expression of miR-26a was downregulated after exposure to ionizing radiation and this regulation was negatively mediated by TGF-β signaling. miR-26a negatively regulated the CTGF expression at the post-transcriptional level; however, ionizing radiation suppressed this negative regulation. In addition, the overexpression of miR-26a inhibited the expression of CTGF and type I collagen after irradiation. In conclusion, miR-26a modulates the expression of CTGF via TGF-β signaling in irradiated fibroblasts. The results suggest the potential application of miR-26a in the treatment of RIF.

Regional and organ-level responses to local lung irradiation in sheep

Lung is a dose-limiting organ in radiotherapy. This may limit tumour control when effort is made in planning to limit the likelihood of radiation-induced lung injury (RILI). Understanding the factors that dictate susceptibility to radiation-induced pulmonary fibrosis will aid in the prevention and management of RILI, and may lead to more effective personalized radiotherapy treatment. As the interaction of regional and organ-level responses may shape the chronic consequences of RILI, we sought to characterise both aspects of the response in an ovine model. A defined volume of left pulmonary parenchyma was prescribed 5 fractions of 6 Gy within 14 days while the contralateral lung dose was constrained. Radiographic changes via computed tomography (CT) were documented to define differences in radio-exposed lung relative to non-exposed lung at d21, d63 and d171 (n = 2), and at d21, d147 and d227 (n = 2). Gross and histologic lung changes were evaluated in samples derived at necropsy examination to define the chronic pulmonary response to radiation. Irradiated lung demonstrated reduced radio-density and increased homogeneity as evidenced from texture based radiomic feature analysis, relative to the control lung. At necropsy, the radiation field was readily defined by pallor on the pleural surface, which was also evident on the cut surface of fixed lung specimens. The degree and homogeneity of pallor reflected the sparse presence of erythrocytes in alveolar septal capillaries of radiation-exposed lung. These changes contrasted with dilated and congested microvasculature in the contralateral control lung. Referencing data to measurements made in control lung volumes of sheep experiencing acute RILI indicated that interstitial collagen continues to deposit in the radio-exposed lung field. Overall lung vascularity increased during the chronic response, as evidenced by increased expression of endothelial cell marker (CD31); however, vascularity was consistently decreased in irradiated lung and was negatively correlated with lung collagen. Other organ-level responses included increased expression of alpha smooth muscle actin (ASMA), increased numbers of proliferating cells (Ki67 positive), and cells expressing the dendritic cell-lysosomal associated membrane protein (DC-LAMP) antigen. The chronic response to RILI in this model is effected at both the whole organ and local lung level. Whilst the long-term consequences of exposure to radiation involved the continued deposition of collagen in the radiation field, organ-level responses also included increased vascularization and increased expression of ASMA, Ki67 and DC-LAMP. Interrupting the interplay between these aspects may influence susceptibility to pulmonary fibrosis after radiotherapy. We advocate for the importance of large animal model systems in pursuing these opportunities to target local, organ-level and systemic mechanisms in parallel within the same subject over time.

Ultrafiltration Extract of Radix Angelica Sinensis and Radix Hedysari Attenuates Risk of Low-Dose X-Ray Radiation-Induced Myocardial Fibrosis In Vitro.

The risk of radiation-induced heart damage (RIHD) is a growing concern since recent advances in radiation therapy (RT) for cancer treatments have significantly improved the number of survivors. Radiation-induced myocardial fibrosis (RIMF) is the final pathological condition of RIHD and main change leading to serious cardiovascular complications following RT. The aim of this study was to investigate the effect of ultrafiltration extract of Radix Angelica Sinensis and Radix Hedysari (RAS-RH) on the proliferation, apoptosis, and reactive oxygen species (ROS) of cardiac fibroblasts after X-irradiation in vitro. The RAS-RH extract was from the Danggui Buxue decoction (DBD) in TCM. Primary cardiac fibroblasts were irradiated with 1 Gy X-ray to evaluate the effect of RAS-RH on the expression levels of cell proliferation, apoptosis, ROS, and fibrotic molecules. Our data demonstrated that X-irradiation at 1 Gy resulted in the proliferation of cardiac fibroblasts; RAS-RH attenuated the myocardial fibrosis. Furthermore, X-ray radiation reduced the apoptosis of cardiac fibroblasts; RAS-RH accelerated the apoptosis of these cells after irradiation. In addition, the damage driven by ROS in primary cardiac fibroblasts after irradiation was weakened by RAS-RH and the expression of TGF-β1, Col1, and α-SMA increased after irradiation; RAS-RH decreased the expression of these makers. Overall, these data indicate that low-dose X-ray irradiation boosts myocardial fibrosis, and the effect of RAS-RH protects against fibrosis via attenuating the proliferation and accelerating the apoptosis of myocardial fibroblasts after X-irradiation.

Principles of treatment of late rectal radiation damage in cancer patients

Malignant neoplasms of the pelvic organs in the structure of cancer incidence make up about 30 % of the adult population of our country. The widespread use of radiation therapy, unfortunately, has also increased the frequency of radiation damage to the pelvic organs, which, as a rule, take a chronic course. Late radiation damage to the pelvic organs is a fairly common complication of radiation therapy for prostate cancer. According to various authors, the frequency of such iatrogenic pathological changes is up to 25 %. In fact, some patients who are cured of a malignant neoplasm acquire a new disease that significantly reduces the quality of life and requires treatment. During radiation therapy of prostate cancer, various anatomical formations can fall into the zone of radiation fields: the bladder, rectum, intra-pelvic tissue, neurovascular bundles and pelvic bones. It should be noted that isolated lesions of one organ are rare and in most cases they are combined. A special place in clinical practice is occupied by cases of local radiation injuries of the rectum, including severe complications of radiation and combined treatment associated with the formation of fistulas against the background of radiation-induced intra-pelvic fibrosis in the absence of a relapse of the underlying disease. In addition, this situation is associated with a violation of the psycho-emotional status of patients, a sharp decrease in the quality of life, difficulties in social adaptation in society and family, pain syndrome, and problems of medical rehabilitation. Unfortunately, conservative measures for such local radiation injuries are not always effective, and the results of surgical interventions are far from ambiguous and require careful study of patients, search for an algorithm of indications and acceptable standardization of surgical manipulations. This paper presents the basic principles of diagnosis, comprehensive treatment and rehabilitation of patients with late radiation rectitis, depending on the severity of the pathological process. Given the growth of malignant neoplasms of the pelvic organs, we can assume a relative increase in the number of patients with complications associated with radiation therapy, which require rehabilitation measures. Such patients come to the attention of oncologists, radiologists, coloproctologists, gastroenterologists and therapists, etc. Currently, our country lacks a network of specialized regional departments that deal with this problem, as well as treatment standards, clinical recommendations, and an algorithm for diagnostic and rehabilitation measures for local radiation injuries. In connection with the above, it became necessary to summarize the data of clinical studies based on their own experience.

Abstract PO-025: Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells

Abstract Acute Radiation Syndrome and the multiorgan failure from delayed effects of acute radiation exposure present challenging consequences of radiation terrorism or a radiological accident. Recent research suggests that radiation-induced cellular senescence plays an important role in radiation-induced pulmonary fibrosis (RIPF), and that clearance of senescent cells (SCs) could be an effective therapeutic strategy. However, the identification and targeted removal of only senescent cells using drugs have been difficult. Here we have established a bone-marrow stromal cell line from a tdTOMp16+ mouse. We show that 5 Gy irradiation induces ~9% cellular senescence in tdTOMp16+ stromal line after 10 days and can be isolated as a pure population of red tdTOMp16+ cells by FACS sorting. To confirm that these irradiated and then sorted red cells are senescent, we cultured them as single cells in 96-well plates and followed for four weeks. None of the 960 irradiated red cells we plated divided after 2 weeks, 137 of these cells remained intact and assumed large and flat cellular morphology. In contrast, 50 of 800 cells that were irradiated but didn’t turn red, divided, as did 104 of 560 nonirradiated control cells (0 Gy) divided. There was significant upregulation of senescent cell markers including SA-ß-gal, p16, and p21 in the irradiated and sorted red cells when compared to irradiated nonred cells. Sorted irradiated red, non-irradiated, or irradiated non-red cells were placed on the top well of transwells. There was a significant induction of fibrotic genes Ctgf, Tgf- ß, collagen 1a, and collagen 3 in C57/B6 stromal target cells in the bottom layer by irradiated red cells, but not the other cell populations irradiated non-red. Thus, radiation-induced biomarkers of fibrosis are induced by senescent cells. Citation Format: Amitava Mukherjee, Michael Epperly, Donna Shields, Wen Hou, Renee Fisher, Diala Hamade, Joel S. Greenberger. Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-025.

Abstract IA-020: Optimizing stem cell niche for post-irradiation regeneration

Abstract Radiotherapy involves unavoidable co-irradiation of the normal tissue, often resulting in side effects that limit the dose to the tumor and/or reduce quality of life of cancer survivors. The response of normal tissues to irradiation is mainly determined by the survival and regenerative potential of the tissue stem cells, and modulated by inflammatory processes, vasculature damage, altered neuronal innervation and fibrosis. Indeed, transplantation of tissue specific stem cells has been shown to restores tissue homeostasis and prevent late radiation effects. However, the radiation-induced senescence, fibrosis, and chronic inflammation may chance the stem cell niche engender an unfavorable environment for the regenerative potential of the tissue’s stem cells. Here changes in the salivary gland stem cell niche will be discussed using irradiated murine submandibular salivary gland tissue and derived organoids. Recently, we have developed methods to culture murine and patient specific tissue resembling salivary gland organoids (SGO). SGO contain all the glandular lineages, are able to extensively self-renew and up on transplantation rescue salivary gland function, allowing the study of radiation responses. The role of stemness factors, senescence and inflammatory processes in stem cell self-renewal and differentiation and post-irradiation regeneration will be discussed. Using SGO formation efficiency (OFE) as a measure of regenerative potential it is shown that stem cell potency after in vivo irradiation, is time dependent reduced. Interestingly, the conditioned medium of irradiated SGO reduced the OFE of unirradiated SGO. The potential role of radiation-induced senescence-associated secretory phenotype, hippo-signaling and inflammation through activation of cytosolic DNA sensing pathways on stem cells function will be addressed. Radiation-induced environmental changes in the stem cells niche have a severe impact on stem cell function. Modulation of these effect may enhance regenerative potential of surviving stem cells and improve engraftment and regeneration after stem cell transplantation. Supported by the Dutch Cancer Society (KWF, grant No. 10650 and 12092) and The Netherlands Organisation for Health Research and Development (ZonMw, Grant nrs. 11.600.1023 and 40-43600-98-14003). Citation Format: Rob P. Coppes. Optimizing stem cell niche for post-irradiation regeneration [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-020.