{"paper_id":"eef3b227-4c6b-4615-8cde-c13169bdc61d","body_text":"ARTICLE OPEN\nEndometrial stromal cell ferroptosis promotes angiogenesis\nin endometriosis\nGuojing Li 1,2,3, Yu Lin 1,2,3, Yili Zhang 1,2,3, Nihao Gu 1,2, Bingxin Yang 1,2, Shan Shan 1,2, Na Liu 1,2, Jing Ouyang 1, Yisai Yang 1,\nFeng Sun 1 ✉ and Hong Xu 1,2 ✉\n© The Author(s) 2022\nEndometriosis, a chronic disorder characterised by the presence of endometrial-like tissue outside the uterus, is associated with iron\noverload and oxidative stress in the lesion. Although it is well established that iron overload can trigger ferroptosis, the results of\nprevious studies on ferroptosis resistance and ferroptosis in endometriotic lesions are paradoxical. Here, we found that some\nstromal cells of the cyst walls that were in contact with the cyst fluid underwent ferroptosis. Surprisingly, endometrial stromal cell\nferroptosis triggered the production of angiogenic, in flammatory and growth cytokines. In particular, angiogenic cytokines, such as\nvascular endothelial growth factor A (VEGFA) and interleukin 8 (IL8), promoted human umbilical vein endothelial cell (HUVEC)\nvascular formation in vitro. Moreover, we found that inhibition of p38 mitogen-activated protein kinase/signal transducer and\nactivator of transcription 6 (p38 MAPK/STAT6) signalling represses VEGFA and IL8 expression when endometrial stromal cells\nundergo ferroptosis. Notably, VEGFA and IL8 showed localised expression and were signi ficantly upregulated in ectopic lesions\ncompared to control and eutopic endometrium samples from patients with endometriosis. Thus, our study reveals that endometrial\nstromal cell ferroptosis in the ovarian endometrioma may trigger cytokine secretion and promote angiogenesis of adjacent lesions\nvia paracrine actions to drive the development of endometriosis, providing a rationale for translation into clinical practice and\ndeveloping drugs for endometriosis.\nCell Death Discovery            (2022) 8:29 ; https://doi.org/10.1038/s41420-022-00821-z\nINTRODUCTION\nEndometriosis is an oestrogen-dependent disease, characterised\nby the presence of endometrial glands and stroma outside the\nuterine cavity. It is a chronic in flammatory disorder affecting\napproximately 10% of women of reproductive age, with an\nestimated 200 million affected individuals worldwide [ 1]. Among\npatients with endometriosis, about 40 –50% have fertility problems\nand 50% suffer major pelvic pain, affecting the health and quality\nof the life of patients and causing a major economic burden [ 2, 3].\nAlthough it has been generally accepted that the development of\nendometriosis is closely associated with hormones, in flammation,\ndysfunctional immunity, oxidative stress, genetic and epigenetic\nfactors as well as environmental factors, the pathogenesis of\nendometriosis has not been completely elucidated [ 4].\nFerroptosis, a new type of regulated cell death, is triggered by\nthe iron-catalysed process of lipid peroxidation initiated via\nnonenzymatic (Fenton reactions) and enzymatic mechanisms\n(lipoxygenases) [ 5]. It is characterised by small dysmorphic\nmitochondria with decreased cristae and condensed and ruptured\nouter membranes and is closely related to iron, polyunsaturated\nfatty acid, and amino acid metabolism, and glutathione, phos-\npholipid, coenzyme Q10, and NADPH biosynthesis [ 6, 7]. Ferrop-\ntosis is modulated by intracellular iron overload. However, in\nendometriosis, endometrial cells are not destroyed but instead\nsurvive, implant, and grow in the ectopic lesions that contain high\nlevels of iron as a result of repeated bleeding episodes and the\ngradual accumulation of menstrual debris and antiquated blood in\nthe cyst fluid [ 8]. Thus, it was hypothesised that endometrial cells\ncan resist iron-mediated ferroptosis [ 9]. In addition, a recent study\nreported that ectopic endometrial stromal cells (ESCs) were more\nsensitive to erastin-induced ferroptosis [ 10]. Therefore, ferroptosis\nhas drawn immense attention as a promising target for new\ntherapeutic strategies.\nRecently, a wide range of angiogenic, in flammatory, and\ngrowth-stimulating cytokines have been detected in the serum,\nperitoneal fluid, and endometrium of patients with endometriosis,\nsuggesting the potential role of in flammation in the progression\nof this disorder [ 11]. It has been reported that elevated\nexpressions of vascular endothelial growth factor (VEGF) and\ninterleukin 8 (IL8) induce the production of vascular endothelial\ncells in ectopic endometrial lesions and that anti-VEGF/VEGF\nreceptor treatments suppress the development of endometriosis\nin animal models [ 12, 13]. Interleukin ‐1β (IL‐1β) enhances\nendometriotic cell proliferation, decreases apoptosis, and induces\nthe secretion of IL6 and IL8 in endometriotic tissues, leading to\nincreased proliferation in endometriosis [ 14,\n15]. Ferroptosis\ninduction has been reported to be associated with increased IL6\nand IL8 expression [ 16]. To reduce iron overload, injection of\nReceived: 22 September 2021 Revised: 9 December 2021 Accepted: 23 December 2021\n1International Peace Maternity & Child Health Hospital, Shanghai Municipal Key Clinical Speciality, Institute of Embryo-Fetal Original Adult Dise ase, School of Medicine, Shanghai\nJiao Tong University, Shanghai 200030, China. 2Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200030, China. 3These authors contributed equally: Guojing Li, Yu\nLin, Yili Zhang ✉email: sunfeng0711@126.com; xuhong1558@sjtu.edu.cn\nwww.nature.com/cddiscovery\nOfficial journal of CDDpress\n1234567890();,:\n\ndeferoxamine, an iron chelator, into a murine endometriosis\nmodel decreased in flammation and limited lesion proliferation\n[17]. Further work demonstrated that treatment with the\nferroptosis inhibitor, N-acetylcysteine (NAC), reduced the volume\nand weight of endometriotic lesions induced in rodents compared\nto the controls [ 18]. In addition, an observational cohort study\nshowed the effectiveness of NAC for endometriosis treatment\nwithout side effects [ 19]. These studies reveal a contrasting\nferroptosis mechanism underlying endometriosis, suggesting that\nferroptosis may be a double-edged sword in endometriosis.\nAlthough it is a promising treatment target, it may also be closely\ncorrelated with in flammation and the progression of endome-\ntriotic lesions.\nThe role of ferroptosis in endometriosis has not yet been\nsystematically examined. In this study, we observed ferroptosis in\nectopic lesions. We found that ferroptosis of ESCs induced VEGFA\nand IL8 secretion, and explored its potential effects on angiogen-\nesis of adjacent lesions during the development of endometriosis.\nOur results provide new insights into ferroptosis in endometriosis,\nwhich can be translated into clinical practice.\nRESULTS\nFerroptosis detection in endometriotic cyst\nEndometriotic cysts exhibit localised iron overload and persistent\noxidative stress [ 20], which may trigger ferroptosis. However, it is\nnot clear whether ferroptosis occurs in endometriotic lesions. As\nf e r r o p t o s i si ss p e c ifically characterised by mitochondria that appear\nsmaller than normal with increased membrane density, this feature\ncan be used to distinguish ferroptosis from apoptotic or necrotic\ndeath, or autophagy [ 7]. Using transmission electron microscopy\n(TEM), we could observe shrunken mitochondria with increased\nmembrane density and reduced mitochondrial cristae on the inner\nsurface of the cyst walls (Fig. 1Aa). However, the same mitochon-\ndrial changes were not observed on the outer cyst walls (Fig 1Ab).\nIn addition, lipid reactive oxygen species (ROS) accumulation —\nwhich plays a central role in the ferroptosis pathway in eukaryotic\norganisms [ 21], was measured among eutopic endometrium and\nlesion cyst cells by flow cytometry using the fluorescent probe C11-\nBODIPY. The results demonstrated that the endometriotic cyst\nshowed increased lipid peroxidation compared to eutopic endo-\nmetrium (39.80 ± 1.95% vs. 52.64 ± 2.04%, P < 0.01) (Fig. 1B). More-\nover, we investigated the expression of important ferroptosis-\nrelated genes, such as SAT1, PEBP1 and DPP4— which drive\nferroptosis, and GPX4 and DJ-1— which suppresses it, upon their\nactivation [22–24]. We found that the level of DPP4 was higher in\nectopic lesions, whereas that of DJ-1 was lower in lesions than in\neutopic endometrium (Fig. 1C). It has been reported that DPP4\ncontrols lipid metabolism, while DJ-1 is associated with glutathione\nmetabolism during ferroptosis [ 22, 24]. However, no signi ficant\ndifferences in GPX4, SAT1 and PEBP1 expression levels were\ndetected between the two groups.\nFurthermore, to simulate the microenvironment of ectopic\nendometrial cells, we co-cultured primary ESCs with diluted cyst\nfluid (1:1) for 12 h. Surprisingly, we detected an obvious elevation\nof lipid ROS levels (22.33 ± 4.02% vs. 66.03 ± 2.77%, P < 0.001)\n(Fig. 1D) and observed smaller mitochondria with increased\nmembrane density (Fig. 1E), implying that the contents of\nendometrioma might trigger ferroptosis in the ectopic endome-\ntrium. Altogether, these results suggested that ferroptosis does\noccur in endometriotic cysts.\nTranscription pro files of ESCs following erastin-induced\nferroptosis in vitro\nTo explore why endometrial cells of ectopic lesions survive and\ngrow in spite of stromal cell ferroptosis and also if ESC ferroptosis\nbenefits the progression of ectopic lesions, we investigated\nerastin-induced ESC ferroptosis. First, to explore the susceptibility\nof ESCs to erastin-induced ferroptosis, we treated primary ESCs\nwith erastin at different concentrations (10, 20, 30, 50, and\n100 µM) under a time gradient (0, 3, 6, 9 and 12 h). Obvious ESC\nmorphological changes were observed after treatment with\n30 µM erastin for 12 h (Supplementary Fig. 1). We additionally\nfound markedly elevated lipid ROS accumulations (16.47 ± 3.21%\nvs. 57.20 ± 3.38%, P < 0.001) and notable mitochondrial changes in\nthis culture condition (Fig. 2A, B), indicating that erastin caused\nferroptotic cell death in ESCs. To con firm the concrete effect of\nferroptosis, ESCs were treated with either DMSO or 30 µM erastin,\nand total RNA derived from these cells was subjected to RNA\nsequencing. Bioinformatic analysis indicated that following erastin\ntreatment, 352 transcripts were signi ficantly upregulated or\ndownregulated ( ≥2‐fold, P < 0.05) in ESCs, and multiple secretory\nfactors known to be associated with angiogenesis, in flammation,\nand growth were observed in the list of upregulated entities\nincluding VEGFA, IL8, angiopoietin-like protein 4 (ANGPTK4),\nadrenomedullin (ADM), IL1A, IL2, IL11, cardiotrophin-like cytokine\nfactor 1 (CLCF1), and amphiregulin (AREG) (Fig. 2C, G), consistent\nwith previous studies showing that ferroptosis accelerates\ninflammation [25]. We performed gene ontology (GO) enrichment\nanalysis of differentially expressed genes (DEGs). In addition to\nenrichment in neuronal death, stress response, and apoptotic\nprocesses, DEGs were also enriched in vasculature development\nand cell differentiation (Fig. 2D and E). In addition, Kyoto\nEncyclopaedia of Genes and Genomes (KEGG) pathway enrich-\nment analysis demonstrated that these DEGs were mainly\ninvolved in MAPK signalling (Fig. 2F). We veri fied the upregulation\nby examining the levels of a few secretory factors, which also\nshowed signi ficantly higher expression after treatment with\nerastin (Fig. 2G). Thus, we can conclude that erastin-induced ESC\nferroptosis in turn induced the secretion of angiogenic, in flam-\nmatory, and growth factors, which may be associated with\nangiogenesis and the progression of ectopic lesions.\nESC ferroptosis triggers angiogenesis in vitro\nVEGFA and IL8 are pivotal angiogenic factors [ 26, 27] playing an\nessential role in the pathological progression of endometriosis\n[12]. To con firm that ESC ferroptosis induced angiogenic cytokine\nexpression (VEGFA and IL8), the primary ESCs were first treated\nwith erastin at different concentrations (10, 20, 30, 50 and 100 µM)\nand for different time periods (0, 3, 6, 9, and 12 h). Compared to\nthe control, the expression of VEGFA and IL8 increased\nsignifi\ncantly upon erastin treatment from 30 to 50 µM and in a\ntime-dependent manner from 0 to 12 h (Fig. 2H, I). Various\nferroptosis inducers, such as (1S,3R)-RSL3, TBHP, and the\nendometriotic cyst fluid were used to treat ESCs. (1S,3R)-RSL3 is\nanother potential ferroptosis agonist that directly inhibits GPX4,\nand TBHP was used to simulate oxidative stress conditions, which\nultimately resulted in considerable lipid peroxidation [ 6, 28]. As\nshown in Fig. 3A, B, the mRNA and protein levels of VEGFA and IL8\nwere signi ficantly upregulated under the three conditions. In\naddition, as a precursor of intracellular antioxidant glutathione\n[29], NAC rescued cell death morphologically (Supplemental Fig.\n2A) and inhibited VEGFA and IL8 induction by erastin at the\nmRNA, intracellular, and secretory protein levels (Fig. 3C–E). These\nfindings suggest that ESC ferroptosis induces VEGFA and IL8\nproduction. Investigations in several cell lines including 293T, ISK,\nand KGN also revealed that VEGFA and IL8 levels were only\nupregulated in primary ESCs (Fig. 3F), implying a unique\nmechanism of ESC ferroptosis. We next examined the effect of\nstromal VEGFA and IL8 on HUVEC vascular formation using\nconditioned media derived from erastin-treated ESCs. The Matrigel\ntube formation assay showed that HUVEC tube-like structure\nformation (measured based on the total number of branches) was\nconsiderably enhanced in cells cultured with the medium from\nerastin-induced ESCs (erastin-treated ESCs group vs DMSO-treated\nESCs group, 39 ± 2.51 vs. 24 ± 1.76, P < 0.01; erastin-treated ESC\nG. Li et al.\n2\nCell Death Discovery            (2022) 8:29 \n\ngroup vs. erastin with basic medium group, 39 ± 2.51 vs. 22.8 ±\n1.59, P < 0.001) (Fig. 3G, H). However, the angiogenesis-promoting\neffects of ferroptosis were obviously abrogated in the presence of\nNAC (NAC- and erastin-treated ESCs group vs. erastin-treated ESCs\ngroup, 19.8 ± 3.06 vs. 39 ± 2.51, P < 0.0001) (Fig. 3G, H). These data\nindicate that ESC ferroptosis might promote angiogenesis of\nsurrounding tissues through the release of VEGFA and IL8, thus\ncontributing to primary lesion survival.\nThe p38 MAPK/STAT6 pathway is involved in ferroptosis-\ninduced VEGFA and IL8 induction\nWe further investigated the mechanism of ferroptosis-induced\nVEGFA and IL8 upregulation in ESCs. As KEGG pathway enrich-\nment analysis demonstrated that the DEGs of erastin-treated ESCs\nwere mainly involved in the MAPK signalling pathway (Fig. 2F), we\nfirst examined the activation of the p38 MAPK signalling pathway\nusing western blot. Upon treatment of ESCs with erastin, the\nphosphorylation levels of P38 were elevated but attenuated by\nthe addition of NAC (Fig. 4A). We then treated the cells with\nSB203580, a speci fic inhibitor of p38 MAPK, and noted that the\np38 inhibition signi ficantly decreased erastin ‐induced VEGFA and\nIL8 expression (Fig. 4B, C). Furthermore, analysis of the sequences\nwithin 5 kb from the transcription start sites (TSSs) of VEGFA and\nIL8 using RcisTarget [ 30] identi fied enriched motifs for DNA-\nbinding activators (cisbp _ M3992 motif-binding STAT6) both in\nthe TSSs of VEGFA and IL8 (Fig. 4D). To address this, we selected\nthe region 2000 bp upstream of VEGFA and IL8 TSSs or mutated\nthe M3992 motif, fused these sequences to a reporter gene\nexpressing firefly luciferase, and then co-transfected 293T cells\nwith them and the STAT6 expression plasmid. The results showed\nFig. 1 Ferroptosis detection in endometriosis. A Representative transmission electron microscopy images of the ultrastructure of the inner\nand outer walls of mitochondria of endometrioma. B Single stromal cells were isolated from the paired eutopic and ectopic endometria and\ntheir lipid ROS accumulation was assayed using flow cytometry and C11-BODIPY. Representative data and statistical analyses from five\nindependent experiments are shown. C The relative mRNA expression levels of three upregulated (SAT1, PEBP1, and DPP4) and two\ndownregulated (GPX4 and DJ-1) ferroptosis-related genes were compared between the paired eutopic and ectopic endometria ( n = 24).\nD, E ESCs were cocultured with diluted cyst fluid (1:1) for 12 h in vitro. The lipid ROS levels were examined and transmission electron\nmicroscopy of mitochondria ultrastructure was analysed. Arrowheads indicate deformed mitochondrial structures, while arrows point to\nnormal mitochondrial structures. Comparisons were made using the two-tailed, Student ’ s t test ( B– D). ** P < 0.01, *** P < 0.001, **** P < 0.0001.\nns not statistically signi ficant, EU eutopic endometrium from patients with endometriosis, EC ectopic lesions from patients with\nendometriosis, GPX4 glutathione peroxidase 4, SAT1 spermidine/spermine N1-acetyltransferase 1, PEBP1 phosphatidylethanolamine-binding\nprotein 1, DPP4 dipeptidyl-peptidase-4, ESCs endometrial stromal cells, ROS reactive oxygen species.\nG. Li et al.\n3\nCell Death Discovery            (2022) 8:29 \n\nthat STAT6 speci fically activated the firefly luciferase of the wild-\ntype promoter, but had no effect on the reporter plasmid (Fig. 4D).\nThen, we detected STAT6 activation upon erastin and NAC\ntreatment (Fig. 4A), and using siRNA we knocked down STAT6 in\nerastin-treated ESCs. This partially suppressed VEGFA and IL8\nexpression, suggesting that STAT6 contributes, at least in part, to\nferroptosis-mediated VEGFA and IL8 production (Fig. 4E, F).\nCollectively, the above results indicate that the p38 MAPK/\nSTAT6 signalling pathway contributes to ferroptosis-mediated\nVEGFA and IL8 expression.\nVerification of VEGFA and IL8 expression in biospecimens of\npatients with endometriosis\nWe further investigated the location and expression of VEGFA and\nIL8 in endometriosis biospecimens. Surprisingly, we found that\nVEGFA and IL8 were both highly expressed on the inner surface of\novarian cysts using IF, while no localised speci fic expression was\nobserved in the control and eutopic endometria (Fig. 5A).\nMoreover, the expression of VEGFA and IL8 in lesions was\nsignificantly higher than that in the eutopic and control samples,\nas detected using RT-qPCR and IHC (Fig. 5B, C), indicating that\nFig. 2 Transcription profiles and verification of erastin-treated ESCs. Primary ESCs were treated with erastin (30 µM) or DMSO for 12 h.A Lipid\nROS levels were assessed usingflow cytometry and C11-BODIPY . Representative data and statistical analyses from three independent experiments\nare shown. B Transmission electron microscopy analysis of mitochondria ultrastructure in ESCs under erastin treatment. C Heatmap displaying a\nsubset of DEGs in ESCs treated with 30 µM erastin for 12 h ( ≥2‐fold, P <0 . 0 5 ) . G O (D) and KEGG pathway enrichment ( F) analyses on DEGs in\nresponse to erastin-induced ferroptosis. E Heatmap of angiogenesis-related DEGs. G RT-qPCR analysis was used to validate the DEGs, which\nincluded angiogenic cytokines (VEGF A, IL8, ANGPTK4, ADM, and IL1A), inflammatory (IL2 and IL11) and growth factors (CLCF1 and AREG) ( n = 3,\ncompared with DMSO). H, I The ESCs were treated with erastin at different concentrations (10, 20, 30, 50, and 100 µM) for 12 h and for different\ntime periods (0, 3, 6, 9, and 12 h) using 30 µM erastin. VEGF A and IL8 mRNA expressions were detected using RT-qPCR and statistical analysis from\nthree independent experiments. * Compared with DMSO, # compared with 0 h. Arrowheads indicate deformed mitochondrial structures, while\narrows point to normal mitochondrial structures. Comparisons were made using the two-tailed, Student ’ s t test (A, G), one-way ANOVA (H)a n d\ntwo-way ANOVA (I). *P <0 . 0 5 , * *P < 0.01, ***P < 0.001, ****P < 0.0001,\n#P <0 . 0 5 ,##P < 0.01. ns not statistically significant, ESCs endometrial stromal\ncells, DMSO dimethyl sulphoxide, ROS reactive oxygen species, GO Gene Ontology, KEGG Kyoto Encyclopaedia of Genes and Genomes, DEGs\ndifferentially expressed genes, VEGFA vascular endothelial growth factor A, IL8 interleukin 8, ANGPTK4 angiopoietin-like protein 4, ADM\nadrenomedullin, IL1A interleukin 1A, CLCF1 cardiotrophin-like cytokine factor 1, AREG amphiregulin.\nG. Li et al.\n4\nCell Death Discovery            (2022) 8:29 \n\nVEGFA and IL8 expression might be closely related to the\npathogenesis of endometriosis.\nDISCUSSION\nThe term “ferroptosis”,d efined as a distinct form of regulated cell\ndeath characterised by iron-catalysed lethal lipid peroxidation,\nwas first coined by Dixon et al. [ 7]. Iron accumulation serves as an\ninitial element in ferroptotic cell death [ 31]. Ovarian endome-\ntrioma is an ovarian cyst lined with endometrial tissue histologi-\ncally and functionally resembling eutopic endometrium, which is\ngenerally considered to be filled with menstrual debris and\nantiquated blood [ 8]. Inside the cyst, the concentrations of free\niron, ROS, and lipid peroxide are ten to hundreds of times higher\nthan those in peripheral blood or other types of benign cysts,\nproviding an iron overload and oxidative microenvironment for\nG. Li et al.\n5\nCell Death Discovery            (2022) 8:29 \n\nlesion growth [ 20, 32]. There is a point of view that the implanted\nectopic endometrium can resist ferroptosis and survive in a\nmicroenvironment with iron overload due to dysregulated iron\nhomoeostasis [ 33]. The latest meta-analysis of online datasets\ndemonstrated that the ferroptosis pathway presents a down-\nregulation trend among the ectopic, eutopic, and control\nendometria [ 34]. In contrast, according to previous studies,\nectopic endometriotic lesions had signi ficantly higher levels of\nROS, hydrogen peroxide, and oxidative stress activity [ 35, 36].\nWhether ferroptosis exists and has potential effects on the\ndevelopment of ectopic lesions has not yet been proven. Given\nthat there is no gold standard to detect ferroptosis, we observed\nthe ultrastructure of endometriotic cysts using TEM and found for\nthe first time obvious mitochondrial morphological changes on its\ninner surface, consistent with the morphological features of\nferroptosis. Moreover, we treated primary ESCs with cyst fluid\nin vitro and found a markedly elevated level of lipid ROS and\nshrunk mitochondria with increased membrane density, indicating\nthat ESC ferroptosis was induced by the chocolate cyst fluid.\nSurprisingly, we found that erastin-induced ESC ferroptosis\ncould trigger the production of angiogenic, in flammatory, and\ngrowth cytokines, which may provide an original thinking to the\npositive effect of ferroptosis on the development of endome-\ntriosis. The small molecule erastin has been applied in many\ndiseases as a ferroptosis trigger to explore the mechanism of this\nnewly discovered non-apoptotic cell death in vitro [ 7, 31, 37].\nAlthough a recent study showed that ectopic ESCs were more\nsensitive to erastin-induced ferroptosis than normal ESCs,\nsuggesting that erastin may be a novel therapy for endometriosis\n[10], we show here that higher concentrations of erastin could\ntrigger both eutopic and ectopic ESC ferroptosis and also\nsubsequently induce the secretion of cytokines, such as VEGFA\nand IL8 (Supplemental Fig. 2A –C). VEGFA is generally regarded as\na vital angiogenic factor, which attaches to the vascular\nendothelium to initiate cell proliferation and endothelial angio-\ngenesis and increases vascular permeability [ 11]. Furthermore,\nIL8 signalling increases cell proliferation and survival to promote\nangiogenic responses in endothelial cells [ 38]. According to a\nstudy by Sun et al., IL6 and IL8 secretion was elevated in erastin-\ntreated retinal pigment epithelial cells as senescence-associated\nfactors [16]. Nevertheless, few other studies have investigated the\npotential role of ferroptosis in vascular formation. Our study is the\nfirst to demonstrate that ESCs in the process of ferroptosis,\nstimulate VEGFA and IL8 secretion, which may contribute to\nendometriotic lesion angiogenesis. Hence, ferroptosis might act as\na double-edged sword in the progression of endometriosis. On\nthe one hand, the agonist erastin might be a promising agent for\ntriggering ferroptosis in lesions. On the other hand, some stromal\ncells undergoing ferroptosis secrete a series of cytokines to\npromote vascular system formation of surrounding tissues via\nparacrine actions, which may enhance benign cell proliferation\nand accelerate the progression of this disorder. Thus, we aimed to\nidentify an inhibitor to attenuate cytokine secretion. Unfortu-\nnately, we have not investigated other cytokines, such as growth\nfactors (ADM and AREG) and in flammatory factors (IL2), induced\nby ferroptosis. Further studies are also needed to explore more\npotential effects of ferroptosis on endometriosis.\nAccording to our study, NAC could serve as an ef ficacious agent\nfor suppressing ferroptosis-induced cytokine secretion. NAC, the\nacetylated precursor of\nL-cysteine, is generally considered as an\nanti-inflammatory or anti-oxidative agent. However, recent studies\nshowed that NAC could also reverse lipid ROS levels and act as an\ninhibitor against ferroptosis [ 39, 40]. Its curative effect has been\nstudied in a variety of diseases, such as Alzheimer ’ s disease,\nnephropathy, and heavy-metal toxicity [ 29, 41]. In endometriosis,\nanimal experiments have already demonstrated its antioxidative\nfunctions in reducing the weight and size of ectopic lesions [ 19].\nMoreover, in an observational cohort study, NAC was proposed as\na promising treatment for endometriosis by decreasing the size\nand number of cysts, reducing dysmenorrhoea symptoms, and\nincreasing chances of conception without side effects, but its\nmechanism has not been elucidated [ 42]. In this study, we\nincubated NAC with erastin-treated ESCs and surprisingly found\nthat NAC rescued cell death morphologically and suppressed the\nsecretion of VEGFA and IL8. Thus, it counteracted the stimulatory\neffect of ferroptosis on angiogenesis, providing a novel insight\ninto mechanisms underlying the therapeutic effects of NAC in\nendometriosis.\nOur study showed that the p38 MAPK/STAT6 signalling is one\nof the main downstream regulator s of erastin-induced ferrop-\ntosis and that it mediates the secretion of VEGFA and IL8 in ESCs.\nA recent report revealed that ferroptosis inducers, such as RSL3\nand erastin, induced the ASK1-p38 MAPK pathway activation\ndownstream of lipid ROS in A549 cells [ 43]. JNK and p38, except\nfor the ERK MAPK pathway, were also responsible for erastin-\ninduced ferroptosis in an acute myeloid leukaemia cell line [ 44].\nFurthermore, VEGFA expressi on was found to be regulated by\nactivation of the p38 MAPK pathway, playing an important role\nin angiogenesis [ 45, 46]. We found increased phosphorylation of\np38 MAPK in erastin-treated ESCs. The addition of a p38\ninhibitor signi ficantly repressed VEGFA and IL8 production.\nOther studies have shown that the coordinated modulation of\nSTAT6 and p38 MAPK exhibited potential effects on the\ninduction of IL4 and IL13 and that p38 MAPK could directly\nregulate the activity of the transactivation domain of STAT6\n[47–49]. In erastin-induced ESCs, we also detected STAT6\nactivation, and transfection of STAT6 siRNA attenuated the\nexpression of VEGFA and IL8. More over, the luciferase reporter\nassay revealed a direct connection between STAT6 and both\nVEGFA and IL8. These data imply a critical link between the p38\nMAPK/STAT6 pathway and ferrop tosis-mediated VEGFA and IL8\nexpression in ESCs.\nWe also observed localised speci fic expression of VEGFA and IL8\nin ectopic cysts and detected higher expression levels of VEGFA\nand IL8 in ectopic lesions, than in eutopic and control endometria\nin patients with endometriosis. Restricted by sample size, we failed\nto explore alterations in the expression levels and functions of\nVEGFA and IL8 during menstrual cycles. According to previous\nFig. 3 ESC ferroptosis induced VEGFA and IL8 production and promoted angiogenesis. A , B ESCs were incubated with several ferroptosis\ninducers, such as (1S,3R)-RSL3 (10 µM), TBHP (20 µM), and the diluted cystic fluid (1:1), for 12 h. VEGFA and IL8 expression levels were detected\nusing RT-qPCR and western blot. Representative data and statistical analysis from three independent experiments are shown. C– E ESCs were\ntreated with 30 µM erastin in the presence of the bioactive inhibitor NAC (10 µM) for 12 h. VEGFA and IL8 expressions were measured using\nRT-qPCR, IF , and ELISA. All the statistical analysis was from three independent experiments. F Other erastin-treated cell lines (293T, ISK, and\nKGN) and VEGFA and IL8 mRNA expression levels were detected using RT-qPCR ( n = 3, compared with DMSO). G The angiogenic ability of\nHUVECs was tested by coculturing with the supernatant from erastin- and NAC-treated ESCs or with a basic medium containing erastin and\nNAC. Representative micrograph images are shown. H The number of branches was analysed ( n = 5). Comparisons were made using the two-\ntailed, Student’ s t test (F) and one-way ANOVA ( A– C, E, and H). *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. ns not statistically signi ficant,\nESC endometrial stromal cell, DMSO dimethyl sulphoxide, VEGFA vascular endothelial growth factor A, IL8 interleukin 8, NAC N-acetylcysteine,\nIF immuno fluorescence, HEK293T human embryonic kidney cell line, ISK human endometrial cancer cell line Ishikawa, KGN human ovarian\ngranulosa cell tumour cells, HUVECs human umbilical vein endothelial cells, TBHP tert-Butyl hydroperoxide solution.\nG. Li et al.\n6\nCell Death Discovery            (2022) 8:29 \n\nstudies, the endometrial stromal cells present no differences in\nVEGF and IL8 expression throughout the menstrual phase [ 50, 51].\nIn conclusion, we detected ferroptosis caused by an iron\noverload on the inner surface of endometriotic cysts. Erastin-\ninduced primary ESC ferroptosis stimulated VEGFA and\nIL8 secretion through the p38 MAPK/STAT6 pathway and\npromoted angiogenesis in vitro. Thus, ferroptosis may play a\ncritical role in the progression of endometriosis by resulting in\nangiogenic effects via paracrine VEGFA and IL8 action on the\nadjacent lesions. NAC, which serves as a potential anti-ferroptosis\nagent, is expected to contribute signi ficantly to the treatment of\nendometriosis.\nFig. 4 p38 MAPK/STAT6 pathway is involved in ferroptosis-induced VEGFA and IL8 upregulation. A ESCs were incubated with erastin and\nNAC, and the activation of p38 and STAT6 was examined using western blot. B, C The p38 inhibitor SB203580 (10 µM) was added to erastin-\ntreated ESCs and the activation of p38 was measured using western blot. VEGFA and IL8 expression and secretion were measured using RT-\nqPCR, western blot ( n = 3). D The prediction model of enriched motifs for DNA-binding activators of STAT6 bind both the TSSs of VEGFA and\nCXCL8 and is validated using luciferase reporter assays. E, F ESCs were transfected with STAT6 siRNAs for 48 h and then incubated with erastin\nfor 12 h. The transfection ef ficiency and expression of VEGFA and IL8 were detected using RT-qPCR and western blot ( n = 3). Comparisons\nwere made using one-way ANOVA ( A– F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns not statistically signi ficant, p38 MAPK/STAT6 p38\nmitogen-activated protein kinase/signal transducer and activator of transcription 6, ESCs endometrial stromal cells, VEGFA vascular\nendothelial growth factor A, IL8 interleukin 8, NAC N-acetylcysteine, TSSs transcription start sites, CXCL8 C-X-C motif chemokine ligand 8.\nG. Li et al.\n7\nCell Death Discovery            (2022) 8:29 \n\nFig. 5 VEGFA and IL8 expression levels in patients with endometriosis. A Immunofluorescence images of control, eutopic endometrium, and\nectopic lesion samples were co-stained for VEGF A or IL8 (red) and vimentin (green). Nuclear DNA was counterstained with DAPI (blue).B, C VEGF A\nand IL8 expression levels of control, eutopic and ectopic endometrium samples detected using RT-qPCR (CON,n = 12; EU and EC, n = 16) and IHC\n(n = 10). D The mechanism of ferroptosis in endometriotic stromal cells. The iron-overload in cyst fluid triggered ferroptosis in cells of the inner\nsurface of endometrioma and induced cytokines like VEGFA and IL8 secretion. The paracrine cytokines further promoted lesion angiogenesis to\nadvance the development of endometriosis. Comparisons were made using one-way ANOVA ( B, C). * P <0 . 0 5 , * *P < 0.01, ***P < 0.001, ****P <\n0.0001. ns not statistically signi ficant, CON the control endometrium, EU eutopic endometrium from patients with endometriosis, EC ectopic\nlesions from patients with endometriosis, VEGF A vascular endothelial growth factor A, IL8 interleukin 8, IHC immunohistochemistry.\nG. Li et al.\n8\nCell Death Discovery            (2022) 8:29 \n\nMETHODS\nPatients and sample collection\nThirty-six women of reproductive age who underwent laparoscopic and\nhysteroscopic procedures at the International Peace Maternity and Child\nHealth Hospital (IPMCH), Shanghai Jiao Tong University School of\nMedicine, were recruited into this study. All enrolled women had regular\nmenstrual cycles and did not receive hormonal therapy or use\nintrauterine contraception for at least 6 months prior to surgery.\nPatients with metabolic diseases, hypertension, in flammatory disease,\nautoimmune disorders, and cancer were excluded from the study.\nTwelve women with teratoma diagnosis who underwent combined\nhysteroscopy and laparoscopy according to their surgery options\nwithout macroscopic lesions in the uterine cavity served as the control\ngroup. Normal endometrial samples were obtained using an endometrial\ncurette. Eutopic and ectopic endometrial tissues were collected from 24\npatients with ovarian endometrioma using curette and laparoscopy,\nrespectively. The demographic and baseline characteristics of patients in\nterms of age, body mass index, and parity had no signi ficant difference\nbetween the control and endometriosis groups (Supplemental Table 1).\nWe dissected the samples using surgical scissors and tweezers and\ndivided them into three groups. Group 1 samples were transported to the\nlaboratory in phosphate-buffered saline (PBS) (Gibco, New York, USA) on\nice for cell isolation. Group 2 samples were immediately fixed in 4%\nparaformaldehyde and then embedded in paraf fin for immunohistochem-\nical analysis. Group 3 samples were maintained in cryotubes and stored in\nliquid nitrogen for further RNA and protein extraction. The cyst fluid was\naspirated by a 50 ml syringe and was then put in sterile 15 ml centrifugal\ntubes and stored at −80 °C until use.\nThe study protocol was approved by the ethics review committee of\nIPMCH and was conducted according to the principles of the Declaration\nof Helsinki. Written informed consent was obtained from all the\nparticipants.\nIsolation and culture of primary ESCs\nPrimary ESCs were isolated from the eutopic or ectopic endometrium of\nwomen with endometriosis. The tissues were cut into pieces and digested\nwith type I collagenase (1 mg/ml, Gibco, New York, USA) for 0.5 –1h a t\n37 °C. After removing debris and epithelial cells using 100 and 40 μm cell\nstrainers, respectively, ESCs were resuspended in DMEM/F12 containing\n10% foetal bovine serum (FBS) (Gibco, New York, USA) and 1%\npenicillin–streptomycin (Gibco, New York, USA) and cultured in 5% CO\n2\nat 37 °C. The culture medium was replaced after the stromal cells had\nattached, to remove blood cells and debris. After reaching 80 –90%\nconfluency in 2 –3 days, cells were seeded into 12-well plates for in vitro\nexperiments.\nESC purity was detected using immuno fluorescence for vimentin\nand cytokeratin 7 as markers of stromal and epithelial cells,\nrespectively. The number of vimentin-positive cells was greater than\n95% (Supplemental Fig. 3).\nCell cultures and treatment\nThe human embryonic kidney cell line HEK293T, human endometrial\ncancer cell line Ishikawa (ISK), human ovarian granulosa cell tumour cells\n(KGN), and human umbilical vein endothelial cells (HUVECs) were\npreserved in the Shanghai Key Laboratory of Embryo Original Diseases.\nHEK293T cells were cultured in DMEM High (Gibco, New York, USA)\ncontaining 10% FBS. ISK and KGN cells were grown in DMEM/F12 (Gibco,\nNew York, USA) containing 10% FBS. HUVECs were incubated in an\nendothelial cell growth medium (PromoCell, Heidelberg, Germany)\ncontaining an endothelial cell growth supplement. All media were\nsupplemented with 1% penicillin –streptomycin and cells were cultured\nin 5% CO\n2 at 37 °C. The medium was replaced every 2 days until 90%\nconfluency was reached. All experiments were performed using cell lines\nfrom the fifth to tenth passage.\nESCs were treated with different erastin concentrations (10, 20, 30, 50,\nand 100 µM) for 12 h, and for different time periods (0, 3, 6, 9, and 12 h)\nwith 30 µM erastin. Cell morphology was observed using an inverted\nmicroscope (Leica, Germany). To identify the speci fic effects of ferroptosis\non ESCs, primary ESCs were treated with several ferroptosis inducers, such\nas erastin (30 µM), (1S,3R)-RSL3 (10 µM), tert-butyl hydroperoxide solution\n(TBHP, 20 µM), diluted cyst fluid (1:1 dilution with complete medium), and\nwith inhibitor NAC (10 µM) for 12 h. Furthermore, HEK293T, ISK and KGN\nwere treated with 30 µM erastin for 12 h. To explore the role of p38 MAPK\nin ferroptosis-induced VEGFA and IL8 induction, ESCs were treated with\nerastin in the absence or presence of the p38 inhibitor, SB203580 (10 µM)\nfor 12 h. All reagents were purchased from Sigma-Aldrich (USA).\nTransmission electron microscopy (TEM)\nEctopic cyst walls were cut into 1 cm 3 piece and fixed with 2.5%\nglutaraldehyde at 4 °C overnight immediately after they were obtained\nfrom the operating table. Primary ESCs were treated with or without\nthe cyst fluid (1:1 diluted with complete medium) or dimethyl\nsulphoxide (DMSO) or 30 μM erastin for 12 h and then were washed\nthrice with PBS and fixed with 2.5% glutaraldehyde at 4 °C overnight.\nAfter washing in PBS twice for 10 min, the ectopic cyst wall pieces were\nfixed with 1% osmic acid at 4 °C for 2 h. Subsequently, the samples\nwere dehydrated with an ethanol g radient and 100% acetone solution\nfor 15 min and then embedded in epoxy resin. Ultrathin (70 nm)\nsections, obtained usi ng an ultramicrotome, were stained with lead\ncitrate and uranyl acetate for evaluation. Cell and mitochondrial\nmorphology was captured using a t ransmission electron microscope\n(Hitachi H-7650, Japan).\nAnalysis of lipid ROS accumulation\nDMSO- or erastin (30 µM)- or cyst fluid-treated cells or cells from the ESC\nisolation procedures were incubated with C11-BODIPY (581/591) (Invitro-\ngen, California, USA) for 30 min at 37 °C. Cells were subsequently\nresuspended in 1 ml of fresh PBS and strained through a 40 μm cell\nstrainer for flow cytometry analysis. Lipid ROS levels were measured using\nan FFACScan (BD, New York, USA) through the FL1 channel (527 nm).\nApproximately 10,000 cells were analysed per sample. Data analysis was\nperformed using the FlowJo version 10.0.\nRNA-seq, GO, and Kyoto encyclopaedia of genes and genomes\n(KEGG) analyses\nPrimary ectopic ESCs were seeded in 6 cm plates and treated with 30 µM\nerastin for 12 h. The cells from each group were collected using the\nRNAiso Plus reagent (Takara Bio, Tokyo, Japan) and sent to Annoroad\nGene Technology (Beijing, China) for RNA-seq. The raw data were\nsubmitted to the GEO database (PRJNA783151). The raw RNA-seq data\n(FASTQ files) were filtered using the Perl script. Bowtie2 was used to build\nthe genome index, and the RNA-seq data were then aligned to the\nreference genome using HISAT2. Read counts for each gene were\ncounted using HTSeq, and fragments per kilobase million mapped reads\nwere then calculated to estimate the gene expression level in each\nsample. DEGs were identi fied using the DESeq2 [ 52] package in R. The\nDEG threshold was set at q ≤ 0.05, and |log2_ratio | ≥1. DEG GO ( http://\ngeneontology.org/ ) and KEGG ( http://www.kegg.jp/ ) enrichment were\nperformed using the hypergeometric test, where the p-value was\ncalculated and adjusted as a q-value. GO and KEGG terms with q <0 . 0 5\nwere considered to be signi ficantly enriched.\nDual-luciferase reporter assay\nThe 2000 bp promoter sequence of VEGFA and IL8 was searched on the\nUCSC website ( http://genome.ucsc.edu/cgi-bin/hgGateway). Based on the\nobtained promoter sequence, we predicted the possible signal transducer\nand activator of transcription 6 (STAT6) transcription factor-binding region\nof the VEGFA and IL8 promoter sequences. Surprisingly, we found a\ncommon predicted STAT6 transcription factor-binding region in the VEGFA\nand IL8 2000 bp promoters (in the VEGFA mRNA 5 ′-UTR [GGGAAG,\n1447–1452 nt] and IL8 mRNA 5 ′-UTR [AGGAAG, 1852 –1857 nt)] regions).\nThen, we synthesised a target wild type (WT) sequence (GGGAAG/\nAGGAAG) and a mutant sequence (Mut) (GGGAAG/AGGAAG mutation)\nusing site-directed mutagenesis. The synthesised WT or Mut VEGFA and IL8\npromoters were inserted into the KpnI and xhoI digestion sites of pGL4.22\nvectors using the pEASY\n®-Basic Seamless Cloning and Assembly Kit\n(Transgen, Beijing, China). Recombinant plasmids were validated using\nsequencing. For the luciferase assay, HEK293T cells were seeded in 24-well\nplates until they reached 70% con fluency and, subsequently, allocated into\nfive groups: (1) transfected with GL-vector and Flag, (2) transfected with\nGL-VEGFA-WT and Flag-STAT6, (3) transfected with GL-VEGFA-Mut and\nFlag-STAT6, (4) transfected with GL-IL8-WT and Flag-STAT6, and (5)\ntransfected with GL-IL8-Mut and Flag-STAT6. Cell lysates were harvested\nafter 48 h of transfection and dual-luciferase reporter assays were\nperformed using a Dual-Luciferase Reporter Assay System kit (Promega,\nWisconsin, USA).\nG. Li et al.\n9\nCell Death Discovery            (2022) 8:29 \n\nsiRNA transfection\nSmall interfering RNA (siRNA) targeting STAT6 was designed and synthesised\nby GenePharma (Shanghai, China), which also provided the negative control\nsiRNA. The STAT6 siRNA sense and antisense strand sequences are\npresented in Supplemental Table 2. Primary ESCs were seeded into 12-\nwell plates and cultured until they reached 60–80% confluency, followed by\ntransfection with 20 pmol STAT6 siRNAs per well using the Lipofectamine ®\nRNAiMAX reagent (Invitrogen, California, USA) in Opti-MEM (Gibco, New\nYork, USA) according to the manufacturer ’ s instructions. After 48 h of\ntransfection, the cells were treated with 30 µM erastin or DMSO for 12 h. To\ndetermine the transfection ef ficiency, quantitative real-time PCR (RT-qPCR)\nand western blot were performed 48 h after transfection.\nTotal RNA extraction and RT-qPCR\nTotal RNA was extracted from tissues and cultured cells using the RNAiso\nPlus reagent (Takara Bio, Tokyo, Japan) in accordance with the manufac-\nturer’ s protocol. After RNA quantification using the Nanodrop 2000 (Thermo\nFisher Scientific, USA), 1 µg of total RNA was reverse transcribed in a total\nvolume of 20 μl using the PrimeScript\n™ RT reagent kit (Takara Bio, Tokyo,\nJapan). RT-qPCR was performed using TB Green Premix Ex Taq II (Takara Bio,\nTokyo, Japan) and the QuantStudio 7 Flex Real ‐Time PCR system (Applied\nBiosystems, USA). Primers details are mentioned in Supplemental Table 2.\nThe 2\n−ΔΔ Ct method was used to calculate the relative expression levels of\ntarget genes, which were normalised to those of actin beta mRNA levels.\nProtein extraction and western blot analysis\nCultured cells were lysed in radioimmunoprecipitation assay lysis buffer\n(Beyotime, Shanghai, China) supplemented with phenylmethylsulphonyl\nfluoride and protease inhibitor cocktail (Sigma-Aldrich, USA) on ice for\n10 min and centrifuged at 12,000 g for 10 min at 4 °C. Protein concentra-\ntions were quanti fied using a BCA assay kit (Beyotime, Shanghai, China). A\ntotal of 20 µg protein was separated using 10% or 12.5% sodium dodecyl\nsulphate polyacrylamide gel electrophoresis and blotted onto polyvinyli-\ndene fluoride membranes, which were then blocked with 5% non-fat milk\ndiluted in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room\ntemperature. Blocking was followed by incubation with the primary\nantibodies, namely, anti-phospho-p38 MAPK (1:1000, Cell Signalling, 9211,\nMassachusetts, USA), anti-p38 MAPK (1:1000, Cell Signalling, 9212,\nMassachusetts, USA), anti-phospho-STAT6 (1:1000, Cell Signalling, 56554,\nMassachusetts, USA), anti-STAT6 (1:1000, Cell Signalling, 5397, Massachu-\nsetts, USA), anti-VEGFA (1:1000, Abcam, ab46154, Cambridge, UK), anti-IL8\n(1:1000, Proteintech, 17038-1-AP, Chicago, USA), and anti- β-actin (1:5000,\nProteintech, HRP-60008, Chicago, USA) at 4 °C overnight. The membranes\nwere subsequently incubated with horseradish peroxidase-conjugated\nsecondary antibodies (1:5000, Proteintech, SA00001-2, Chicago, USA) at\nroom temperature for 1 h, and the signals were visualised using an\nenhanced chemiluminescence detection reagent (Sigma-Aldrich, USA).\nEnzyme-linked immunosorbent assay (ELISA)\nThe culture supernatant of ESCs was collected after treatment, centrifuged\nat 3000 g for 5 min, and then stored at −80 °C until testing. VEGF and IL8\nconcentrations were measured using ELISA kits according to the\nmanufacturer’ s protocol (Neobioscience, Shenzhen, China).\nImmunofluorescence (IF)\nESCs were fixed with 4% paraformaldehyde at 4 °C for 10 min and then\npermeabilized with 5% Triton-100 at room temperature for 30 min. After\ndeparaffinization, dehydration, rehydration and antigen retrieval, the\nparaffin sections were subjected to the same procedures. Samples were\nblocked with 5% bovine serum albumin (BSA) at room temperature for 1 h\nand subsequently incubated with primary antibodies against vimentin\n(1:100, Abcam, ab8978, Cambridge, UK), anti-cytokeratin 7 (1:100, Abcam,\nab68459, Cambridge, UK), anti-VEGFA (1:100), and anti-IL8 (1:100) over-\nnight at 4 °C. After washing with PBS, samples were incubated with Alexa\nFluor 488- or 555-conjugated secondary antibodies (Invitrogen, California,\nA-21202/A-31572, USA) for 1 h at room temperature in the dark and\nstained with 4,6-diamidino-2-phenylindole (DAPI). Immuno fluorescence\nwas detected using a confocal microscope (Leica, Germany).\nImmunohistochemistry (IHC)\nFresh human specimens were fixed with 4% paraformaldehyde solution for\n24 h, embedded in paraf fin, and cut into sections (5 μm), which were then\nimmersed in xylene and ethanol for deparaf finization and rehydration,\nrespectively. Antigen retrieval was performed using Tris-EDTA (pH 9.0)\n(Biosharp, Anhui, China) in a microwave oven. The following procedures were\nperformed using a staining kit (Absin, Shanghai, China). Brie fly, the slides\nwere incubated with 3% H\n2O2 to eliminate endogenous peroxidase activity\nand then blocked with 5% BSA followed first by incubation with primary\nantibodies against VEGFA (1:200) and anti-IL8 (1:200) overnight at 4 °C, and\nsecond by incubation with a secondary antibody. Staining was performed\nusing DAB and haematoxylin. The slides were observed and imaged using a\nmicroscope (Leica, Germany). The VEGFA and IL8 protein expression levels\nwere semi-quantitatively evaluated using the H-score system. The staining\nintensity was assessed using the scoring parameters: strong (3×), medium\n(2×), and weak (1×), and the H-score value ranging from 0 to 300 was\ncalculated according to the formula [53, 54]: H-score = 1* (% cells 1×) + 2* (%\ncells 2×) + 3* (% cells 3×). The H-score was independently evaluated by two\ninvestigators at different times, and the average score was used.\nMatrigel tube formation assay\nPrimary ESCs were treated with erastin (30 µM) and/or NAC (10 µM) in\nserum-free DMEM/F12, and the supernatant was collected after 12 h of\nincubation. HUVECs were pre-treated with serum-free medium for 48 h\nbefore the Matrigel tube formation assay was performed. Subsequently,\nHUVECs were diluted with the supernatant or serum-free DMEM/F12 as a\ncontrol and 50 µl of 10,000 cells/ well were added to a u-slide angiogenesis\nplate (Ibidi, Germany) precoated with BD Matrigel. Then, the slides were\nincubated at 37 °C for 6 h. Tube formation was imaged using an inverted\nmicroscope (Leica, Germany). The number of branches, an index of\nangiogenesis, was measured using ImageJ.\nStatistical analysis\nAll experiments were independently performed in at least triplicate. All\nstatistical analyses were performed using GraphPad Prism version 8. 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Hirsch FR, Varella-Garcia M, Bunn PA, Di Maria MV, Veve R, Bremnes RM, et al.\nEpidermal growth factor receptor in non –small-cell lung carcinomas: correlation\nbetween gene copy number and protein expression and impact on prognosis. J\nClin Oncol. 2003;21:3798 –807.\n54. John T, Liu G, Tsao MS. Overview of molecular testing in non-small-cell lung\ncancer: mutational analysis, gene copy number, protein expression and other\nbiomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors.\nOncogene. 2009;28:S14 –S23.\nAUTHOR CONTRIBUTIONS\nH.X., Y.L., G.L., and F.S. designed the study. G.L., Y.L., Y.Z., N.G., S.S., N.L., and B.Y.\nperformed experiments. F.S., J.O., and Y.Y. collected human samples. G.L. and Y.L.\nanalysed data and wrote papers. All authors edited and revised the paper.\nFUNDING\nThis work was supported by the National Key Research and Development\nProgramme of China (No. 2020YFC2002804), Shanghai Municipal Key Clinical\nSpecialty (No. shslczdzk01802), the National Natural Science Foundation of China\n(82071622, 81771551, and 81901536), the Shanghai Chinese Traditional and Western\nMedicine Clinical Collaboration Pilot Construction Project (No. ZXYXZ-201905), the\nInterdisciplinary Key Programme of Shanghai Jiao Tong University (YG2021ZD30), the\nResearch Programme of International Peace Maternal and Child Health Hospital (Nos.\nCR2018WX06 and YN201916).\nG. Li et al.\n11\nCell Death Discovery            (2022) 8:29 \n\nCOMPETING INTERESTS\nThe authors declare no competing interests.\nADDITIONAL INFORMATION\nSupplementary information The online version contains supplementary material\navailable at https://doi.org/10.1038/s41420-022-00821-z.\nCorrespondence and requests for materials should be addressed to Feng Sun or\nHong Xu.\nReprints and permission information is available at http://www.nature.com/\nreprints\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims\nin published maps and institutional af filiations.\nOpen Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing,\nadaptation, distribution and reproduction in any medium or format, as long as you give\nappropriate credit to the original author(s) and the source, provide a link to the Creative\nCommons license, and indicate if changes were made. The images or other third party\nmaterial in this article are included in the article ’ s Creative Commons license, unless\nindicated otherwise in a credit line to the material. If material is not included in the\narticle’ s Creative Commons license and your intended use is not permitted by statutory\nregulation or exceeds the permitted use, you will need to obtain permission directly\nfrom the copyright holder. To view a copy of this license, visit http://creativecommons.\norg/licenses/by/4.0/.\n© The Author(s) 2022\nG. Li et al.\n12\nCell Death Discovery            (2022) 8:29","source_license":"CC0","license_restricted":false}