We performed 30–2 standard automated perimetry OCT (3.5‐mm diameter ring scan) and 15°×15° OCT‐A . Based on a nerve fiber trajectory model, each VF test spot was assigned to an OCT‐A wedge and an OCT ring‐sector. Two univariate linear models (vM and tM) using either CD‐based vascular (vM) or RNFL‐T–based thickness information (tM) and one multivariate model using both (v:tM) were compared in their associations with measured focal VFD, which were higher for the multivariate model v:tM (mean ± SD correlation coefficient: 0.710 ± 0.086) than for either nested model (0.627 ± 0.078 for vM and 0.578 ± 0.095 for tM). Using a focal visual field approach, the combination of RNFL‐T and CD showed better structure–function correlations than thickness or vascular information only.We compare the focal structure–function correlation of structural measurements of peripapillary retinal nerve fiber layer thickness (RNFL‐T) using optical coherence tomography (OCT), capillary density (CD) measurements using OCT‐angiography (OCT‐A), or a combination of both, with visual field deviation (VFD) in early to advanced glaucoma. Primary open angle glaucoma patients ( Early diagnosis and adequate progression monitoring of primary open angle glaucoma (POAG) requires assessment of structural and functional parameters. We present focal structure–function correlations for capillary density (CD) and retinal nerve fiber layer thickness (RNFL‐T) with visual field deviation, using both univariate and multivariate modeling where the combined use of CD and RNFL‐T was identified to have overall advantageous properties over vascular or thickness measurements alone, with focal exceptions for the former. Early diagnosis and adequate progression monitoring of primary open angle glaucoma (POAG) requires the assessment of structural and functional parameters.Since the emergence of optical coherence tomography angiography (OCT‐A), the applicability of capillary density (CD) for glaucoma diagnostics has been intensively evaluated.However, both global and sectoral analyses fail to provide focal information on correlation, while results from studies in early glaucoma only may be limited in their transferability to more advanced stages. Thus, we applied a recently introduced focal approach,All included subjects participated in an ocular imaging study  assessing functional, structural, and blood flow–related parameters in glaucoma patients and were recruited between April 2019 and January 2021. This study was conducted in compliance with the Declaration of Helsinki and the Good Clinical Practice guidelines of the European Union and was approved by the Ethics Committee of the Medical University of Vienna. Informed consent was obtained from all subjects before any study‐related procedures were performed.The following examinations were performed in all subjects to assess eligibility: patient medical history and concomitant medication, pregnancy testing in women with childbearing potential, best‐corrected visual acuity, visual field (VF) testing using SAP, and slit‐lamp examination, including dilated funduscopy and applanation tonometry. The inclusion criteria were diagnosis of POAG with glaucomatous optic neuropathy and VF loss compatible with glaucoma. The exclusion criteria were current smoking, exfoliation glaucoma, pigmentary glaucoma or any other secondary cause for glaucoma, history of acute angle closure or angle closure glaucoma, intraocular surgery within 6 months, diabetes, untreated hypertension (SBP >160 mmHg or DBP >95 mmHg), and pregnancy.After inclusion, peripapillary OCT‐A volume and circumpapillary OCT scans were performed in one eye per subject.fVFD) of all test locations were extracted and tabulated.A 30–2 VF test using the SITA‐Standard protocol was performed with a Humphrey perimeter . Only VF tests with sufficient compliance parameters were processed . Analysis protocols of SAP were exported and focal VF deviations (15°×15° OCT‐A volume scans (384 B‐Scans and 384 A‐Scans/B‐Scan) were acquired using the standard OCT‐A algorithm (full spectrum amplitude decorrelation) of the Heidelberg Spectralis OCT . Only scans with sufficient quality were used for analysis. Superficial vascular complex slabs as generated by the built‐in segmentation software of the OCT‐A device were exported to MATLAB  and each image was processed using a previously described customized algorithm Fig. .23 In We acquired 3.5‐mm diameter structural OCT ring scans using the glaucoma module of the Heidelberg Spectralis OCT (Heidelberg Engineering). Circumpapillary RNFL‐T values at the 3.5‐mm diameter ring position (focus‐adjusted by the device) were extracted using the automatic RNFL‐segmentation of the Heidelberg glaucoma module, which was manually controlled for segmentation errors.et al.,As previously described in Wong fCD) calculation as the proportion of white pixels ( = perfusion) in the ROI   Fig.  and F. AfVFD with fCD and fRNFL‐T, representing vascular (v) and thickness (t) information, were modeled for every VF location (n = 73) using two univariate regression models (vM and tM) with fCD  were used to compare the associations with measured fVFDs. Linear models were compared using the Akaike information criterion (AIC) and likelihood‐ratio tests.Pearson correlation coefficients (fRNFL‐T and fCD. The Davies test was used to test for the existence of a significant change in the slope (breakpoint) of the relationship between fVFD and fCD or fRNFL‐T.To mathematically describe the breakpoint and corresponding slopes (point estimates and 95% CI), segmented regression analysis on a linear mixed model, in which the focal parameters were used as the fixed effect and the random effects were used to account for multiple VF data from the same individual, was performed. Separate models were evaluated for Global SF relationships were also evaluated using Eqs.  for globP values below 0.05 were treated as statistically significant.Stata 13.1  and R , comprising the package “segmented,” were used for statistical analysis. 28 17 patients were classified as advanced, 14 as moderate, and 15 as early glaucomatous VF loss. All baseline characteristics are summarized in Table Fifty‐one POAG patients were enrolled for the present study. After we excluded one patient due to high myopia (>6 dpt) and four patients due to poor OCT‐A acquisition quality, 46 patients remained. Regarding their VFD, POAG patients exhibited the following characteristics: mean ± SD: −10.41 ± 6.76 dB, range (min./max.): −25.7/0.4 dB, median: −9.1 dB, and interquartile range: 10.6 dB. According to a VF loss classification system proposed by the 5th Guidelines of the European Glaucoma Society,P < 0.001 for all three), with an rp of 0.536 (RNFL‐T), 0.749 (CD), and 0.772 (RNFL‐T and CD), respectively.We found a significant correlation between global structural parameters  with global VFD  in tM and three VF locations (4%) in vM. All VF locations remained significant in v:tM  and over vM in 61 (84%) VF locations. Visual representation of the latter comparison revealed that VF locations showing no AIC drop for v:tM versus vM tended to be located temporally within 10° of the horizontal meridian  and vM in 49 (67%) VF locations. Fifteen (63%) of the 24 VF locations for which no better performance of v:tM as compared to vM could be observed using likelihood‐ratio tests were found temporally within 10° of the horizontal meridian  was not superiorly associated with fVFD, compared with the nested model vM. This was the case for the temporal VF within 10° of the horizontal meridian.To our knowledge, this is the first study of focal SF correlation at the level of single VF test spots in a POAG cohort using a combination of thickness and vascular information. This method allowed us to identify a multivariate regression model using both et al. showed a pronounced floor‐like effect for the inner 10° of the VF, approximately corresponding to the temporal peripapillary Garway‐Heath sector for fRNFL‐T, which was lacking for fCD.Taking into account the Garway‐Heath model for sectoral division of the peripapillary region, the temporal VF within 10° of the horizontal meridian corresponds to the temporal Garway‐Heath sector. Indeed, using Garway‐Heath sectors for SF analysis, especially temporal peripapillary CD, was previously shown to better correlate with corresponding VF sensitivity than did temporal peripapillary RNFL‐T.v:tM is not surprising, as the two parameters fCD and fRNFL‐T reflect different aspects of RNFL loss and can, therefore, contribute complementarily to a model. While fCD is a measure of local presence of perfused capillaries, fRNFL‐T represents the local axial RNFL dimension and lacks information on the internal structure of the layer composed of neurons, glia, and vasculature. In this context, the lack of further precision added to model v:tM by variable fRNFL‐T in the temporal VF within 10° of the horizontal meridian could be attributed to the physiologically thinner RNFL‐T in the temporal sector compared with inferior and superior sectors, thus reducing the sectoral dynamic range and, subsequently, minimizing information contributable to the model. Finally, thin RNFL regions are prone to a higher inaccuracy regarding thickness measurements, which could also account for lower associations.From an anatomical point of view, the overall superiority of fCD with fVFD in vM were highly significant (P < 0.001) at more VF locations than those of fRNFL‐T in tM, and comparison of the models revealed that combined model v:tM outperformed vM in fewer VF locations than tM, suggesting better focal correlation between fCD and fVFD, compared with fRNFL‐T and fVFD. Better correlation indices between CD and VFD than between RNFL‐T and VFD were reported by studies investigating these relationships in various glaucoma stages under varying degrees of focality.et al.v:tM was not significantly superior to vM, not only in the temporal VF within 10° of the horizontal meridian (as discussed above) but also in nine additional locations of the superior hemisphere when compared using likelihood‐ratio tests, is currently unknown. However, it can be speculated that minimal fVFDs in the superior hemisphere  not evident and significant in a single SAP session play a role. Assuming that associations of fRNFL‐T with fVFD are overall weaker than those of fCD with fVFD, the additional error introduced by minute artifacts at VF spots in the superior periphery produces a higher tendency for rendering the association fRNFL‐T:fVFD insignificant compared with the association fCD:fVFD. Similar results concerning peripheral VF locations were found in a focal SF analysis in early glaucoma.Associations of fCD and fRNFL‐T, the BP occurred at a nominally, yet not significantly, higher fVFD for fCD compared with fRNFL‐T. While this finding points to an advantageous applicability of CD, as compared with RNFL‐T, for follow‐up at later disease stages, comparative longitudinal studies are required to conclusively answer this question.Using SR allowed us to determine BPs at which the respective SF correlation significantly changed. While this was the case for both Compared with previous studies,Our study has several limitations. First, the sample size of 46 patients is relatively small. However, the focal approach and the large number of highly deviated VF spots resulting from the use of a dataset, including one‐third advanced glaucoma patients, can partly compensate for that. Moreover, our study is by design cross‐sectional; therefore, the results are not conceived to be directly applicable to disease progression. As mentioned earlier, longitudinal studies are, therefore, necessary.In addition, the presence of large vessels within the retinal nerve fiber layer may have affected the performance of RNFL‐T. However, their potential contribution could not be evaluated because current methods of analysis do not allow one to distinguish these components in routine RNFL‐T measurements.Finally, for the calculation of BPs and slopes, we did not divide the VF based on peripapillary sectors or VF degrees, as done previously.The main strengths of this study lie in the use of a well‐phenotyped glaucoma cohort evenly distributed in relation to VFD and containing a substantial number (37%) of patients with advanced glaucomatous VF loss who received ophthalmic examinations, OCT, and OCT‐A imaging with the same machine, and SAP at the same clinical setting; these qualities allowed us to apply a VF‐based approach for SF correlation of, at this stage of disease, precise focality.In conclusion, we present focal SF correlations for CD and RNFL‐T with VFD, using both univariate and multivariate modeling, where the combined use of CD and RNFL‐T was identified to have overall advantageous properties over vascular or thickness measurements alone, with focal exceptions for the former. Using segmented regression modeling, we identified significant changes in the slope for focal CD and RNFL‐T in the course of deteriorating VFD, with a BP for focal CD at higher deviations.Conception and design: M.K., B.T., D.S., L.S., G.G., and D.W. Acquisition of data: M.K., N.H., A.S., and D.W. Analysis and interpretation of data: M.K., J.C., B.T., D.S., C.H., O.F., L.S., G.G., and D.W. Drafting of manuscript: M.K. and D.W. Revision of intellectual content of manuscript: N.H., A.S., J.C., B.T., D.S., C.H., O.F., L.S., and G.G. Approval of final version of manuscript: M.K., N.H., A.S., J.C., B.T., D.S., C.H., O.F., L.S., G.G., and D.W. Responsibility for the integrity of the data analysis: D.W.The authors declare no competing interests.https://publons.com/publon/10.1111/nyas.14732.The peer review history for this article is available at


Human herpesvirus‐8 (HHV8) is a lymphotropic virus associated with different lymphoproliferative disorders, including primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD), diffuse large B‐cell lymphomas, not otherwise specified, and the rare entity known as germinotropic lymphoproliferative disorder (GLPD). In PELs and GLPD the neoplastic cells also contain Epstein–Barr virus (EBV). In addition, occasional cases with atypical and overlapping features among these entities have been recognised, suggesting that the spectrum of the HHV8‐related lesions may not be fully characterised.Here, we report two cases of lymphoproliferative disorder associated with HHV8 and EBV that further expand the spectrum of HHV8/EBV‐positive lymphoproliferative disease.Case 1 represented HHV8/EBV‐positive extracavitary nodal PEL followed by pleural PEL. The striking characteristic of this case was the almost focal and intrasinusoidal localisation of the neoplastic cells and the association with Castleman’s disease features. In the second case, we found the entire spectrum of HHV8‐related disorders, i.e. MCD, GLPD, and PEL, coexisting in the same lymph node, underlining the variability, possible overlap and evolution among these entities. Both cases were well analysed with immunohistochemistry, determination of the EBV latency programme, and molecular analysis for clonality of immnoglobulin genes. In both patients, the disease followed an unexpected indolent course, both being still alive after 8 and 12 months, respectively.Our findings represent further evidence of the overlap among HHV8/EBV‐positive lymphoproliferative disorders, and underline a grey zone that requires further study; they further confirm the experimental evidence that lytic EBV replication influences HHV8‐related tumorigenesis. Since the discovery of human herpesvirus‐8 (HHV8) as the causative agent of Kaposi’s sarcoma in 1994,In particular, HHV8 has been identified in multicentric Castleman’s disease (MCD),Except for GLPD, the majority of these disorders arise in human immunodeficiency virus (HIV)‐positive patients,Similarly to what occurs with EBV (human herpesvirus‐4),How HHV8 and EBV cooperate to promote tumorigenesis remains unclear, but recent experimental data suggest that EBV/HHV8 coinfection enhances HHV8 persistence and tumorigenesis.In humans, occasional cases with atypical and overlapping features among HHV8/EBV‐related LPDs have been recognised, such as lesions intermediate between MCD and GLPD in HIV‐positive patients, or GLPD that progresses to high‐grade EBV or HHV8‐positive lymphoma.Here, we report two cases that showed Castleman’s disease features in association with HHV8/EBV‐positive lymphoproliferations, characterised by early sinusal and germinal centre involvement of the lymph node by atypical cells with plasmablastic morphology. Both cases were analysed with morphology, immunohistochemistry, determination of the EBV latency programme, and molecular analysis for clonality of immunoglobulin genes. Our findings expand the clinical and pathological spectrum of HHV8‐related LPDS, underlying a grey zone and possible evolution among them.One lymph node from each patient was excised and, after fixation in neutral 10% buffered formalin, was cut into 2‐mm‐thick section and paraffin‐embedded. Haematoxylin and eosin‐stained sections were examined. Immunohistochemical staining was performed on the Ventana Benchmark autostainer  on 2‐µm‐thick formalin‐fixed paraffin‐embedded sections. Stains for CD19, CD20, CD79a, PAX5, CD10, bcl‐6, bcl‐2, MUM1, CD38, CD138, IgM, IgG, kappa light chain, lambda light chain, CD21, CD2, CD3, CD5, CD30, ALK, granzyme B, ZAP70 and Ki67 were used, according to the manufacturer’s instructions.in‐situ hybridisation (ISH), performed on the Ventana Benchmark autostainer .HHV8 was detected with an antibody against latent nuclear antigen , and the presence of EBV was studied with both immunohistochemistry  and EBV‐encoded small RNA (EBER) IGH–VDJ rearrangements were studied in all of the specimens according to the BIOMED‐2 protocol.FR1‐JH polymerase chain reaction (PCR) products of all the samples were directly sequenced and compared with the germline sequence by application of the GenBank database (http://www.imgt.org/IMGT_vquest/vquest).The antibody clones, dilutions and antigen retrieval protocols are shown in Table Multiple abdominal lymph nodes were detected in a 75‐year‐old male during the follow‐up for a low‐grade papillary urothelial carcinoma diagnosed 3 years before. Clinical conditions, laboratory values and haematological values were unremarkable. Serum HIV testing gave negative results, whereas testing for EBV DNA and HHV8 DNA gave positive results . An incisional biopsy from an external iliac lymph node was performed, and the diagnosis of HHV8/EBV‐positive EC‐PEL was made, with Castleman’s disease features in the remaining lymph node. No serous effusions were detected. Chemotherapy with rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin and prednisolone resulted in a complete response after six cycles.Eighteen months after the first diagnosis, the patient developed pleural effusion; on cytological examination, HHV8/EBV‐positive PEL was diagnosed. No additional therapy was performed; 8 months after the PEL diagnosis, the patient is alive with no evidence of recurrence.A 49‐year‐old HIV‐positive African woman presented with enlargement of the bilateral inguinal and para‐aortic lymph nodes. The CD4 count was 439, and the viral load was undetectable. An inguinal lymph node biopsy was performed, and a diagnosis of GLPD and/or early EC‐PEL associated with Castleman’s disease features in the same lymph node was made. No body cavity effusions on computed tomography scan or extranodal involvement were seen. There was no bone marrow involvement. The patient was treated with cyclophosphamide, doxorubicin, and VP16, and underwent complete remission. She is still well after 12 months of follow‐up.The lymph node architecture was preserved, with B follicles showing regressive germinal centres with hyaline sclerosis, numerous polyclonal plasma cells in the interfollicular areas, and single atypical cells with plasmablastic morphology Figure . In scatImmunohistochemically, the atypical cells in the sinuses were positive for CD3, but lacked CD2, CD5, CD30, ALK, granzyme B and ZAP70 expression. Staining for CD19, CD20, CD79a, CD138 and PAX5 also gave negative results, whereas the atypical cells expressed MUM1/interferon regulatory factor 4 (IRF4), IgM, and kappa light chain. They were also positive for HHV8 Figure  and EBV,Cytological examination of the pleural effusion that developed 18 months later showed pleomorphic anaplastic large cells, which were negative for CD20 and positive for CD38, HHV8, and EBV Figure .JGH clonality. Whereas the cells occurring in the germinal centres showed an oligoclonal pattern, the intrasinusoidal cells were clearly monoclonal, but a clonal relationship could not be detected. Unfortunately, a comparison of clonality between the nodal lymphoma and PEL was not possible, owing to the low amount and poor quality of DNA in the cytological sample.The atypical cells in the sinuses and in the germinal centres were microdissected and analysed with PCR for The lymph node architecture was preserved, with large reactive germinal centres and expansion of interfollicular areas with massive infiltrates of polyclonal plasma cells with occasional hyaline vascular features and radially penetrating vessels Figure ; single These atypical cells were positive for MUM1/IRF4, kappa light chain, and variably positive for IgM, whereas they did not express CD20, PAX5, CD10, bcl‐6, or CD30. Moreover, they were positive for HHV8 Figure  and EBV,JH clonality on the atypical cells occurring in the sinuses and in the germinal centres showed an identical IGH rearrangement with somatic hypermutations.PCR analysis for Individual cases of HHV8‐related LPD showing overlapping features have been reported.IGH clonality, favour reactivation of EBV infection.Case 1 arose in an immunocompetent patient who initially presented with asymptomatic and isolated abdominal lymphadenopathy. The striking characteristic of this case was the almost exclusive intrasinusoidal involvement of the neoplastic cells and the association with Castleman’s disease features. The intrasinusoidal growth pattern and the strong expression of CD3 simulated an anaplastic large‐cell lymphoma,Interestingly, positivity for HHV8 was detected along the FDC cytoplasmic processes. There is experimental evidence that dendritic cells and macrophages can be infected through the DC‐SIGN receptor expressed on their surfaces. However, we were unable to demonstrate a clear nuclear stain giving evidence of HHV8 infecting FDCs.Dendritic cells play an important role in HHV8 infection and pathogenesis. However, it is not yet clear how this might alter the immune response associated with HHV8 and how it influences HHV8‐related disease development.In the second case, we found the entire spectrum of HHV8‐related disorders  coexisting in the same lymph node, underlining the variability, the overlap and the possible evolution among these entities. Cases with clinicopathological features of MCD enriched for plasmablastic aggregates and GLPD have been reported, suggesting an overlap with MCD and GLPD in a subset of cases.HHV8/EBV coinfection may be a key factor in explaining the overlap and evolution among HHV8/EBV‐positive LPDs.BZLF1. This non‐canonical latency programme has been observed in other lymphoma subtypes.EBV has different latency programmes, and both latent and lytic genes may have an oncogenic effect. Interestingly, in our case we observed a heterogeneous EBV latency programme, with expression also of the lytic gene Cases of MCD associated with EBV have been reported,In summary, our cases varied from the usual morphology and clinical scenario of HHV8‐related LPDs, potentially leading to misdiagnosis. The present cases broaden the spectrum of HHV8/EBV lymphoproliferation and underline the grey zone among HHV8‐related LPDs.The authors declare that they have no conflict of interest.GA, SA, UM: provided tumour samples and clinical data; ML, AT, SE: performed immuhistochemestry and cytogenetic analysis; GM, FM, MV, BC, UM, FF, MRL, LL, LS: analysed and interpreted the data; GM, FM, FF, MRL, LL, LS: designed and coordinate the study; GM, FM, FF, LL, LS: interpreted the data and wrote the manuscript.Figure S1. Case 1, pleural effusion.Click here for additional data file.Figure S2. Cartoon illustrating progression of human herpesvirus 8 (HHV)‐positive multicentric Castleman disease to a broad spectrum of lesions with overlapping features.Click here for additional data file.Figure S3. A case of MCD associated with EBV.Click here for additional data file.Table S1. Antibodies used.Click here for additional data file.Table S2. The differential diagnosis of HHV8‐positive lymphoproliferative disorders.Click here for additional data file.Table S3. Summary of the literature review.Click here for additional data file.


The current study examines the influence of word class  and valence  on the processing of emotional words under different virtual reality (VR) emotional contexts. To this end, 115 participants performed a modified affect labeling task after experiencing different VR scenarios. Their galvanic skin responses were also examined to further gauge the different effects of VR contexts. The results demonstrated significant main effect for word valence, indicating more processing of positive words relative to neutral words which are processed more than negative words. The results also demonstrated significant main effect for word class, indicating more processing of nouns in contrast to adjectives. Additionally, the results indicated that both positive and negative VR contexts could stimulate participants to select more positive words though negatively valenced words were processed more under negative VR context relative to positive VR context. However, the amplitude of galvanic skin responses in positive VR was lower than that in negative VR. The results were interpreted in line with the situation-consistency effects, the mood-consistency effects, the specific nature of VR context, and the different features of different word classes in terms of concreteness, imageability, arousal, and valence. Effects of emotional valence  have witnessed burgeoning attention in lexical processing research. While it is generally suggested that valence play a crucial role in lexical processing, the bulk of the previous studies have been prominently featured by discrete emotional stimuli limited to emotional face pictures or sentences  or lexicValence effects can be modulated by word classes because different word classes have different emotional arousal and processing mechanisms for an individual . Nouns, Researches carried out in laboratory settings have well established the role of valence in lexical processing, suggesting that words with different emotional valence could be different in their processing. However, the findings are far from universal. The majority of previous studies have found that negatively valenced words, compared to those with positive or neutral valence, could exert more processing in quantity, intensity, and neural responses e.g., . Using SThe discrete, context-detached stimuli shared by all the previous tasks might explain the rather conflicting findings as revealed by the aforementioned studies because emotion, context and experienced cognition are closed related . In agreOne corollary of the aforementioned relation between emotion and cognition is that linguistic contexts likely impinge on emotional words processing, and that contextual effects may be different for different words. However, what should be noted is that the emotion-cognition relation has been mainly explored within a controlled laboratory setting where emotion invoking stimuli were exposed very briefly. It is still unclear regarding how well stationary laboratory behavior predicts task performance when the stimuli have features more typical of real-world environments . TherefoVirtual reality digital technology can enable its users to be immersed in a 3D virtual environment, and accordingly activate active interaction between the simulated reality and users. Virtual reality can also allow for the use of multiple senses that function simultaneously during information processing . In a VRIn addition to a potentially real emotional context, VR can also create an enhanced self-related context which is a prominent factor in an individual’s emotion information processing . An indiTogether with these predictions for physiological responses, we also hypothesize that VR simulated emotional environments may also influence an individual’s galvanic skin responses because from which we can observe an individual’s stimulated responses under different circumstances . A stresIn sum, the current study constitutes a new attempt to assess the influence of word valence and classes on lexical processing under VR simulated emotional contexts. This attempt represents a significant contribution due to the scarcity of research in this perspective. The current study gains additional significance from a number of factors. First, the current study examines valence and emotional context effects among different word classes, which will help understand any potential influence for different concreteness and imageability. Secondly, the effects of VR simulated emotional contexts on emotional words are largely underrepresented in lexical processing research. Findings from this perspective may prove specifically significant because VR features more typical of real-world environments and can heighten immersive experience . SpecifiAre there any differences between the positive and negative VR contexts in the processing of differently valenced words with different word classes?Are there any differences in an individual’s galvanic skin responses under different VR contexts?One hundred and fifteen participants were recruited from a vocational college. All participants were 2nd graders  whose average age was 20.05 (SD = 0.96). All participants were healthy, reported no history of fear of height, cardiovascular diseases, psychiatric illness, or neurologic disorder. Additionally, all participants had normal or corrected-to-normal vision and were right-handed, and participated in the experiment voluntarily and received a certain reward after the experiment. Consent form were signed by the participants’ representative, and all participants were free to retreat from the experiment whenever they wanted. Ethics approval were also checked and approved by Academy Department of the college.There were 60 emotional word stimuli, including 30 nouns  and an equal number of adjective counterparts . According to M = 7.36, SD = 0.27; M = 5.38, SD = 0.40; M = 2.73, SD = 0.48; F = 465.43, p < 0.001, η2 = 0.981), indicating significantly higher valence of positively nouns relative to neutrally valenced nouns  and negatively nouns , and significantly higher valence of neutrally valenced nouns relative to negatively valenced nouns . Significant differences were also shown between positively, neutrally, and negatively valenced nouns in term of arousal  = 28.91, p < 0.001, η2 = 0.763), indicating significantly higher arousal of positively and negatively valenced nouns relative to neutrally valenced nouns  though insignificant differences were found between negatively and neutrally valenced nouns . However, no significant differences were showed between positively, neutrally, and negatively valenced nouns in terms of the frequency  = 1.27, p = 0.305, η2 = 0.124). Neither were significant differences found between negatively, positively, and neutrally valenced nouns in terms of the familiarity as shown by pilot participants’ self-report  = 0.244, p = 0.785, η2 = 0.008).Results of the repeated measures ANOVA showed significant differences between positively, neutrally, and negatively valenced nouns in term of the valences  = 660.82, p < 0.001, η2 = 0.987), indicating significantly higher valence of positively adjectives relative to neutrally valenced adjectives  and negatively valenced adjectives , and significantly higher valence of neutrally valenced adjectives relative to negatively valenced adjectives . Significant differences were also shown between positively, neutrally, and negatively valenced adjectives in term of arousal  = 12.35, p < 0.001, η2 = 0.579), indicating significantly higher arousal of positively and negatively valenced adjectives relative to neutrally valenced adjectives  though insignificant differences were found between negatively and neutrally valenced adjectives . However, no significant differences were showed between positively, neutrally, and negatively valenced adjectives in terms of the frequency  = 3.02, p = 0.077, η2 = 0.274). Neither were significant differences found between negatively, positively, and neutrally valenced adjectives in terms of the familiarity as shown by pilot participants’ self-report  = 0.121, p = 0.886, η2 = 0.004).Results of the repeated measures ANOVA also showed significant differences between positively, neutrally, and negatively valenced adjectives in term of the valences ; Sensors included SteamVR tracking technology, G-sensor correction, gyroscope gyroscope, proximity sensor, and eye comfort Degree setting (IPD); The control panel was MI PAD4, and the processor was eight-core 2.20 GHz; As to the computer model, the processor was i7-8,700 CPU, 3.20 GHz, and the system model was a 64-bit operating system.T-test showed significant differences between positively and negatively valenced VR scenarios in terms of the valences  = 26.26, p < 0.001) and insignificant differences in terms of the arousal though that of the negative VR context was much higher  = −1.80, p = 0.083).As to the VR context, two types were designed: Positive scenario and negative scenario. In the negative scenario VR elevator design, there were 101 floors in the elevator. Influenced by the differences in sweat glands and body glands of the participants, the elevator was set to accelerate and shake 3–5 times during the ascent to produce a falling effect, which was designed to prompt the participants to show significant changes in their heart rate. The duration of the whole experiment including the preparation time was about 3 min and 20–26 s. In the positive scenario VR elevator design, the elevator did not go up, and around the participants there would appear an endless prairie where elephants and other animals were walking, grazing, or playing leisurely. At the same time, soothing and soft music was played, accompanied by instructions such as “take a deep breath, relax, take a deep breath, and relax,” trying to create a relaxed and peaceful atmosphere. The duration of the whole experiment including the preparation time is also about 3 min 20–26 s. The two VR scenarios’ valence and arousal were classified on a 7-point rating scale by participants from the pilot experiment. Results of the The elevator experiment adopted a 2  × 3  × 2  within-subject design. The independent variables were word class, word valence, and VR context, and the dependent variables were the number of differently valenced nouns and adjectives selected by participants and the galvanic skin responses. For details, see the flowchart .n = 50) were randomly selected to experience the positive VR scenario first and then the negative VR scenario, and the other part of the participants (n = 65) were asked to experience the negative VR scenario first and then the positive VR scenario in order to balance order effects.The experiment was carried out on participants one by one with the help of two colleges whose major was behavioral science and cognitive psychology, respectively. All participants were asked to put on their headset before experiencing the negative and positive VR scenarios, and would be given about 1 min to experience a VR prairie scenario or a snow mountain scenario to relax to slow down their heart rate. The specific time to slow down the heart rate was based on the specific heart rate characteristics of the participants. Using Latin square experimental design, some participants . In order to further test the semantic relatedness between the scenarios and words, a post-experiment norming study was done using a 7-point rating task among 30 participants recruited from the same college . Both results indicated a rather low semantic relatedness between the two.Post-experience conditioned data of participants under different VR scenarios were collected using the adapted affect labeling task, following Data collection of galvanic skin responses was implemented with reference to Before the formal study, 30 students from the same college were recruited and required to pick out the words that could describe the emotion evoked by the scenario experienced from a word list, and then were asked to perform a 7-point rating scale to obtain the information about familiarity level of the emotional words and valences of the two VR scenarios. These students were different from the participants in the formal experiment. After experiencing the 1st VR scenario and selecting words from a given word list, all participants from the pilot and the formal experiment were required to have about 5 min’ rest. Upon task completion, the response sheets provided to participants to perform the task were collected immediately.F = 272.26, p < 0.001, 2η = 0.544), indicating more processing of positively valenced words relative to neutral words  and more processing of neutral words compared to negatively valenced words . The main effect for word class was also significant  = 23.72, p < 0.001, 2η = 0.094), indicating more processing of nouns in contrast to adjectives . Additionally, there were significant two-way interactions between valence and context  = 103.24, p < 0.001, 2η = 0.312), approximately significant two-way interactions between word class and context  = 3.73, p = 0.055, 2η = 0.016), and significant two-way interactions between valence and word class  = 45.65, p < 0.001, 2η = 0.167). Furthermore, there were also significant three-way interactions between context, valence, and word class  = 10.34, p < 0.001, 2η = 0.043).A 2 (context type: positive VR scenario and negative VR scenario) × 3  × 2 (word class: noun and adjective) repeated-measures ANOVA was performed on words selected by participants under different VR scenarios see . The maiThe simple effect analysis indicated significant interference between the three variables in term of word class see .Mpositive nouns = 3.50, SD = 0.23; Mpositive adjectives = 2.41, SD = 0.17; p < 0.001), indicating more processing of positively valenced nouns as compared to adjectives; (2) for negatively valenced words, significant differences were found between nouns and adjectives , indicating more processing of negatively valenced adjectives as compared to nouns; (3) for neutral words, significant differences were also found between nouns and adjectives , indicating more processing of neutral nouns as compared to adjectives. Overall, under positive VR context, the processing of positively and neutrally valenced nouns was significantly more than that of positively and neutrally valenced adjectives, and the processing of negatively valenced adjectives was significantly more than that of negatively valenced nouns.Under positive VR context, (1) for positively valenced words, significant differences were found between nouns and adjectives , indicating processing advantages of positively valenced nouns over adjectives; (2) for negatively valenced words, no significant differences were found between nouns and adjectives , indicating similar processing of negatively valenced adjectives and nouns; (3) for neutral words, significant differences were also found between nouns and adjectives , indicating processing advantages of neutral adjectives over nouns. In a word, under negative VR context, the processing of positively valenced nouns was significantly more than that of positively valenced adjectives, the processing of negatively valenced nouns and adjectives was similar to each other, and the processing of neutral adjectives was significantly more than that of neutral nouns.Under negative VR context, (1) for positively valenced words, significant differences were found between nouns and adjectives  and between neutrally and negatively valenced nouns , whereas no significant differences were found between positively valenced nouns and neutral nouns , indicating more processing of positively and neutrally valenced nouns as compared to negatively nouns; (2) for adjectives, significant differences were found between positively and negatively valenced adjectives  and between neutrally and negatively valenced adjectives , whereas no significant differences were found between positively valenced adjectives and neutral adjectives , indicating more processing of positively and neutrally valenced adjectives as compared to that of negatively valenced adjectives. In sum, under positive VR context, the processing of positively and neutrally valenced nouns and adjectives was significantly more than that of negatively valenced nouns and adjectives while no significant differences were found between positively and neutrally valenced nouns and adjectives.Under positive VR context, (1) for nouns, significant differences were found between positively and negatively valenced nouns  and between positively and neutrally valenced nouns , whereas no significant differences were found between negatively and neutrally valenced nouns ; (2) for adjectives, significant differences were found between positively and negatively valenced adjectives  and between positively and neutrally valenced adjectives , whereas no significant differences were found between negatively and neutrally valenced adjectives . In general, under negative VR context, the processing of positively valenced nouns and adjectives was significantly more than that of negatively and neutrally valenced nouns and adjectives, whereas no significant differences were found between neutrally and negatively valenced nouns and adjectives.Under negative VR context, (1) for nouns, significant differences were found between positively and negatively valenced nouns  = 39.95, p < 0.001, 2η = 0.26), indicating significant different effects of different VR contexts on galvanic skin responses. Post-hoc multiple comparison showed significant differences between positive baseline and galvanic skin responses after positive VR experience (T(114) = −5.94, p < 0.001, Mrelaxed baseline = 1.74, SD = 1.42, Mafter relax = 2.12, SD = 1.67), and between the negative baseline and the galvanic skin response after negative VR experience (T(114) = 0.36, p = 0.72, Mnegative baseline = 2.50, SD = 1.74, Mafter negative experience = 1.95, SD = 0.18). Furthermore, there was a significant two-way interaction of VR context × galvanic skin response  = 59.33, p < 0.001, 2η = 0.34).A 2 (context type: positive VR scenario and negative VR scenario) × 4  repeated-measures ANOVA was performed on galvanic skin responses under different VR scenarios see . The maiMpositive = 1.72, SD = 1.53; Mnegative = 1.75, SD = 1.33; F = 0.01, p = 0.91, 2η = 0.001); (2) For galvanic skin responses after relaxation, no significant differences were found between the positive and negative VR scenario  = 0.91, p = 0.34, 2η = 0.008); (3) For negative baseline, significant differences were found between the positive and negative VR scenarios  = 4.54, p = 0.035, 2η = 0.039); For galvanic skin responses after negative experience, significant differences were found between the positive and negative VR scenarios  = 35.07, p < 0.001, 2η = 0.237).The simple effect analysis also indicated differences in galvanic skin response under different VR contexts. (1) For relaxed baseline, no significant differences were found between the positive and negative VR scenarios in galvanic skin responses , the School-level Graduate Education Reform Project (YJG2022104) and the Introduced Talents Project (1070XJ2322012), Zhejiang Gongshang University, Zhejiang, China, to SZ. SZ was also supported with a grant from Project of Discipline Innovation and Advancement (PODIA) from Foreign Language Education Studies, Beijing Foreign Studies University (grant number: 2020SYLZDXM011), Beijing, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Resistance to gemcitabine is the main challenge of chemotherapy for pancreatic ductal adenocarcinoma (PDAC). Hence, the development of a response signature to gemcitabine is essential for precision therapy of PDAC. However, existing quantitative signatures of gemcitabine are susceptible to batch effects and variations in sequencing platforms. Therefore, based on within‐sample relative expression ordering of pairwise genes, we developed a transcriptome‐based gemcitabine signature consisting of 28 gene pairs (28‐GPS) that could predict response to gemcitabine for PDAC at the individual level. The 28‐GPS was superior to previous quantitative signatures in terms of classification accuracy and prognostic performance. Resistant samples classified by 28‐GPS showed poorer overall survival, higher genomic instability, lower immune infiltration, higher metabolic level and higher‐fidelity DNA damage repair compared with sensitive samples. In addition, we found that gemcitabine combined with phosphoinositide 3‐kinase (PI3K) inhibitor may be an alternative treatment strategy for PDAC. Single‐cell analysis revealed that cancer cells in the same PDAC sample showed both the characteristics of sensitivity and resistance to gemcitabine, and the activation of the TGFβ signalling pathway could promote progression of PDAC. In brief, 28‐GPS could robustly determine whether PDAC is resistant or sensitive to gemcitabine, and may be an auxiliary tool for clinical treatment. Using within‐sample relative expression ordering of pairwise genes, we developed a qualitative gemcitabine signature containing 28 gene pairs, named 28‐GPS. The 28‐GPS outperformed previous signatures in classification accuracy and prognostic performance for pancreatic ductal adenocarcinoma (PDAC). Compared with gemcitabine‐sensitive PDAC samples, gemcitabine‐resistant PDAC samples classified by 28‐GPS showed poorer overall survival, higher genomic instability, lower immune infiltration, higher metabolic level and higher‐fidelity DNA damage repair. The 5‐yA phase III clinical trial showed that the clinical benefit rate of gemcitabine in the treatment of advanced pancreatic cancer reached 23.8%, and gemcitabine was widely used in clinics . As a stKRAS, TP53, SMAD4 or CDKN2A failed to predict whether patients are sensitive or resistant to gemcitabine [et al. [Resistance to gemcitabine in PDAC is a complex biological process, and the underlying mechanism of resistance is not clear. It is well known that PDAC is a quite complex disease characterized by molecular and clinical heterogeneity. Focussing on a single PDAC driver gene such as citabine , 12. Gencitabine  and GemPcitabine . Howevercitabine . Previoucitabine . Cheng e [et al.  have dem [et al. . Thus, oIn this study, based on REO, we developed a qualitative transcriptional signature to predict the response to gemcitabine for PDAC. The prognostic performance of the signature was validated in multiple independent data sets. Finally, we conducted a systematic and comprehensive analysis to further explore the underlying mechanism of gemcitabine resistance.22.1https://dcc.icgc.org/), ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) and Gene Expression Omnibus   and FIREHOSE (http://gdac.broadinstitute.org/). Each chemotherapy drug was accompanied with corresponding response information in the TCGA drug information. We only retained samples with response information to gemcitabine chemotherapy, and for samples with multiple rounds of gemcitabine treatments, we only kept the samples with first response information to gemcitabine. Pancreatic cancer‐CA (PACA‐CA) patients’ chemotherapy information was obtained from the ICGC. In our study, only PDAC samples with survival information were used. Silent mutations were excluded from TCGA mutation data. In PACA‐CA expression data, only tumour‐related specimens were retained. The probe‐level expression values of pancreatic cancer‐AU (PACA‐AU) and GEO data sets were annotated to gene‐level with matched platform information according to the following criteria: if multiple probe‐sets were mapped to the same gene, the gene expression values were averaged, and multiple gene expression values were mapped to the same probe‐set were excluded. In addition, the gene expression data were filtered by the HUGO Gene Nomenclature Committee database to retain the protein‐coding genes.The transcriptome data were downloaded from publicly available databases, including The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium  Table . PDAC sa2.2https://depmap.org/portal/) and corresponding annotation files were obtained from the Cancer Cell Line Encyclopedia  (Table https://www.cancerrxgene.org), the Cancer Therapeutics Response Portal  and the work of Cancer Genome Project (CGP) [Gene expression data of PDAC cell lines were downloaded from the Cancer Dependency Map (e) Table . Only prct (CGP)   was used for data preprocessing and subsequent analysis [Single‐cell data of 24 PDAC tumour samples were obtained from Peng et al. . r packaanalysis . All funanalysis , 22, 23.2.4i and Gj were used to represent the expression value of gene i and gene j, respectively. Fisher's exact test was used to evaluate whether the frequency of a specific REO pattern (Gi > Gj or Gi < Gj) in the resistant group was significantly higher than the frequency in the sensitive group (P < 0.05). A panel of DEG‐related gene pairs was screened out by pairwise comparisons of all DEGs. To improve the accuracy of the prediction, DEG‐related gene pairs were filtered by maintaining the consistency of the REO pattern and the direction of average rank difference. Using univariate Cox proportional hazards model, the DEG‐related gene pairs were further filtered in TCGA, PACA‐CA and PACA‐AU data sets to obtain a set of prognosis‐related gene pairs. Finally, the common gene pairs of the three data sets were defined as the gemcitabine signature.In the TCGA data set, PDAC samples were classified into the resistant and sensitive groups based on their response to gemcitabine, where patients with complete response (CR), partial response (PR) and stable disease (SD) comprised the sensitive group and progressive disease (PD) belonged to the resistant group. Differentially expressed genes (DEGs) were identified between gemcitabine‐resistant and ‐sensitive groups using the Wilcoxon rank‐sum test. For a gene pair composed of DEGs, G2.5r package ‘pROC’. A single sample was classified into the resistant group when the number of 28‐GPS voting for resistance was no less than the classification threshold; otherwise, the sensitive group. Specifically, for a given gene pair in 28‐GPS, if Gi was greater than Gj, it would be scored as 1. Then, the scores of all gene pairs in 28‐GPS were added and recorded as resistance score. The classification threshold should be correspondingly adjusted with the number of matched gene pairs within the sample.The classification threshold was obtained from the receiver operating characteristic (ROC) curve, which was drawn by the 2.6P value was adjusted using the Benjamini–Hochberg (BH) procedure. Tumour mutational burden (TMB) was calculated based on the number of nonsynonymous somatic mutations. Homologous recombination defect score (HRDscore) was derived from the work of Thorsson et al. [Fisher's exact test was used to identify genes with significantly differential mutation frequencies between gemcitabine‐resistant and ‐sensitive groups. The Wilcoxon rank‐sum test was applied to detect DEGs and differentially expressed methylations (DEMs) between the two groups with a false discovery rate (FDR) < 0.05. The n et al. . TMB, HR2.7r package ‘clusterProfiler’ (version 4.0.5) [Functional enrichment analysis of the genes with corresponding deregulation direction of DEGs and DEMs was performed using the n 4.0.5) . Metaboln 4.0.5) , 27. Sub2.8https://cibersortx.stanford.edu/) and TIMER 2.0 (http://timer.cistrome.org/). T‐cell receptors (TCR) richness was obtained from Thorsson et al. [et al. [et al. [The proportion of infiltrating immune cells in the tumour immune microenvironment was estimated using transcriptome‐based algorithms CIBERSORTx , EPIC 2, TIMER , QUANTISn et al. . Immune‐ [et al. . The Wil [et al. . Differe2.9r¦ > 0.4 and P < 0.05, was used to calculate the correlation between DEGs and differentially expressed immune checkpoint genes or differentially expressed DDR genes. The correlations among metabolic pathway, immune pathway and DDR type were also calculated by Spearman rank correlation. cytoscape software  was used to visualize the correlation network.Spearman rank correlation, with ¦2.10The overall survival time curves were estimated by the Kaplan–Meier method, and tested using the log‐rank test.2.11r Package ‘InferCNV’ [CNV analysis was carried out using the nferCNV’ . The CNV2.12r package ‘CellChat’ (v1.1.3) [Cell–cell communication was investigated via the (v1.1.3) . After c2.13r software (v 4.1.1). The significance of the P value is shown in the following way: *P < 0.05; **P < 0.01; ***P < 0.001; ns: No significance.All statistical analyses in this study were performed using 33.1P < 0.05, Wilcoxon rank‐sum test). Among all gene pairs composed of DEGs, we screened out 2516 candidate gene pairs , of which 1028 gene pairs had REO patterns consistent with the direction of average rank difference. Subsequently, 407, 153 and 164 prognosis‐related gene pairs were identified from the TCGA, PACA‐CA and PACA‐AU data sets, respectively . Finally, the common 28 gene pairs (28‐GPS) of the three data sets were defined as gemcitabine signature . As a frequent KRAS mutation type observed in PDAC, we found that p.G12D mutation exhibited significantly higher resistance scores of 28‐GPS than other types of mutation  cells, and immature dendritic cells (DC). In addition, PDAC cell lines with high TNFSF14 expression were accompanied by high AUC or IC50 values of gemcitabine  and metabolism. Using ssGSEA, resistant samples classified by 28‐GPS reached significantly higher DDR and metabolic pathway enrichment scores than sensitive samples  likewise serve as a hot spot for immunotherapy and two CAR‐T drugs Kymriah and Yescarta have been approved by the Food and Drug Administration (FDA) [+ cells with targeting KRAS mutation showed effective treatment against cancer with mutant KRAS G12D [KRAS G12D in our study achieved a higher 28‐GPS resistance score. Therefore, gemcitabine combined with CAR‐T may be a promising approach for PDAC treatment. Genomic analysis in TCGA indicated that resistant samples classified by 28‐GPS displayed higher genomic instability, such as high HRDscore. In addition, as a tolerant DNA damage repair process accompanied by mutagenesis [In this study, the results suggest some possible combination regimens for PDAC. In the TCGA data, resistant samples classified by 28‐GPS showed lower immune infiltration and down‐regulation of the immune checkpoint genes. PDAC cell lines with higher expression of immune activating gene on (FDA) , 45. An RAS G12D . And, PDagenesis , TLS wasagenesis .et al. [Although some immune cells showed inconsistent infiltration proportion between gemcitabine‐resistant and ‐sensitive samples, most of the immune cell infiltration presented no statistical significance. For example, Tregs exhibited high immune infiltration by CIBERSORTx and low immune infiltration by XCELL and QUANTISEQ in gemcitabine‐resistant samples, but the differences in Tregs infiltration showed no significance between resistant and sensitive samples detected by CIBERSORTx and XCELL Fig. . A compret al.  showed tet al. [ERCC1 gene is involved in multiple DDR pathways and overexpression of ERCC1 is well documented in poor gemcitabine responders [et al. [CXCL13 and CCL21) could potentiate antitumour activity of chemotherapy and increase the infiltration of CD8+ T cells. Xiao et al. [+ T‐cell infiltration. Indeed, in our results, the sensitive PDAC samples achieved high enrichment scores in the chemokine signalling pathway and consistently high infiltration proportion in CD8+ T cells.Compared with the gemcitabine‐sensitive PDAC samples, the gemcitabine‐resistant PDAC samples achieved higher enrichment scores in the DDR and metabolic pathways and lower enrichment scores in the immune pathways. As reported by Jain et al. , gemcitaet al. . In addiet al. . For exasponders . Delvecc [et al.  observedo et al.  found thet al. [ANGPTL4 gene and TGFβ signalling pathway, were reflected in cell communication of the resistant group. ANGPTL4 has been found to play an important role in the process of tumour metastasis [in vitro [In single‐cell analysis, cancer cells in the same PDAC sample had both resistant and sensitive cancer cells, which could be used to explain why PDAC patients who responded to gemcitabine at the beginning will develop secondary resistance later. Zou et al.  also protastasis . The actin vitro .Although our work was limited by the independent PDAC data sets with gemcitabine information to investigate the robustness of 28‐GPS, we used PDAC samples with prognostic information and PDAC cell lines with gemcitabine‐used information to validate the 28‐GPS. In addition, resistant samples in our work showed high metabolic levels and high‐fidelity DNA damage repair, which warrants our future detailed biological experiments to validate those discoveries.5In summary, we developed the 28‐GPS for gemcitabine based on transcriptome, which could be applied to predict response to gemcitabine chemotherapy for PDAC. The resistant samples classified by 28‐GPS in TCGA showed multidimensional resistance‐related characteristics compared with the sensitive samples. Collectively, it is worthwhile to further evaluate the clinical applications of 28‐GPS, which may assist clinicians to make a suitable strategy for PDAC patients.The authors declare no conflict of interest.YYG and KDL conceived the research; KDL, YDG and LZW performed the data processing and analysis; HHX, MZ and YWL carried out the data collection; KDL wrote the manuscript; YYG, ZXZ and TTC revised the manuscript; SLW, LS and FYX provided valuable suggestions for this manuscript; All authors have approved the final manuscript.Fig. S1. The workflow of this study.Fig. S2. The genomic landscape between gemcitabine‐resistant and ‐sensitive samples classified by 28‐GPS in TCGA.Fig. S3. The differences in immune infiltration assessed by five algorithms.Fig. S4. Single‐cell annotation and copy number variation.Table S1. The PDAC cell lines used in this study.Table S2. The pharmacological data sets used in this study.Table S3. The composition of 28‐GPS.Click here for additional data file.


BRCA testing is acceptable, cost‐effective and amplifies primary prevention for breast & ovarian cancer. However, data describing lifestyle impact are lacking. We report long‐term results of population‐based BRCA testing on lifestyle behaviour and cancer risk perception.Ashkenazi‐Jewish (AJ) population‐based Two‐arm randomised controlled trials : (a) population‐screening (PS); (b) family history (FH)/clinical criteria testing.North London AJ‐population.BRCA testing or first‐degree relatives of BRCA‐carriers.AJ women/men >18 years. Exclusions: prior BRCA‐mutations: 185delAG(c.68_69delAG), 5382insC(c.5266dupC) and 6174delT(c.5946delT). This was undertaken for all participants in the PS arm and participants fulfilling FH/clinical criteria in the FH arm. Patients filled out customised/validated questionnaires at baseline/1‐year/2‐year/3‐year follow‐ups. Generalised linear‐mixed models adjusted for covariates and appropriate contrast tests were used for between‐group/within‐group analysis of lifestyle and behavioural outcomes along with evaluating factors associated with these outcomes. Outcomes are adjusted for multiple testing (Bonferroni method), with P < 0.0039 considered significant.Participants were recruited through self‐referral. All participants received informed pre‐test genetic counselling. The intervention included genetic testing for three AJ Lifestyle/behavioural outcomes at baseline/1‐year/2‐year/3‐year follow‐ups.n = 530) or FH (n = 504) arms. No significant difference was identified between PS‐ and FH‐based BRCA testing approaches in terms of dietary fruit/vegetable/meat consumption, vitamin intake, alcohol quantity/ frequency, smoking behaviour (frequency/cessation), physical activity/exercise or routine breast mammogram screening behaviour, with outcomes not affected by BRCA test result. Cancer risk perception decreased with time following BRCA testing, with no difference between FH/PS approaches, and the perception of risk was lowest in BRCA‐negative participants. Men consumed fewer fruits/vegetables/vitamins and more meat/alcohol than women (P < 0.001).1034 participants were randomised to PS (BRCA testing have similar long‐term lifestyle impacts on smoking, alcohol, dietary fruit/vegetable/meat/vitamin, exercise, breast screening participation and reduced cancer risk perception.Population‐based and FH‐based AJ  BRCA testing increases vitamin use and has similar lifestyle impact as FH testing on diet, exercise, alcohol, breast screening.Jewish Pop  BRCA testing provides information on cancer risk, which in turn can influence lifestyle habits such as diet, alcohol, smoking and physical activity. While it is hoped that genetic test results will lead to beneficial lifestyle effects to mitigate risk, behaviour change is not easy. Presenting risk information does not necessarily lead to strong sustainable changes in health behaviour.BRCA testing on lifestyle behavioural factors are limited. A small number of studies with short‐term follow‐up have evaluated this in high‐risk populations and shown inconsistent results.BRCA testing through population‐based ascertainment are lacking. It is also unknown whether BRCA testing on a large scale using a population‐based approach  could lead to false reassurance and have a detrimental impact on lifestyle behaviours such as smoking, alcohol consumption, diet, physical exercise and routine mammography screening. Lifestyle factors such as nutrition, physical activity, smoking and alcohol are important because they can affect chronic diseases such as heart disease, stroke, diabetes, lung disease and cancer.BRCA‐associated BC/OC‐risk in PV‐carriers (substantive data on this are lacking), they are of importance for their association with chronic disease, particularly in the majority testing negative. Chronic disease‐related treatment costs are a massive drain on all health systems. These account for 64% outpatient/70% inpatient workload, and 70% of total UK healthcare expenditurePopulation‐based BRCA testing on general lifestyle factors. We have previously reported on primary outcomes of psychosocial well‐being and quality‐of‐life following BRCA testing.BRCA testing (compared with criteria/FH testing) in the Genetic Cancer Prediction through Population Screening (GCaPPS) trial (ISRCTN73338115). We also evaluate the association of demographic and epidemiological factors with these outcomes.Data from population cohort studies show that BC risk perception in non‐carriers decreases over a year and accuracy of risk perception improves with pre‐test counselling in high‐risk women.22.1BRCA pathogenic variants (PVs), called founder mutations: 185delAG(c.68_69delAG), 5382insC(c.5266dupC) and 6174delT(c.5946delT). Individuals with previous BRCA testing, and first‐degree relatives (FDR) of BRCA‐carriers were excluded. Individuals with PVs received post‐test counselling. These details have been described in earlier reports on psychological (primary) outcomesNorth London‐based self‐referred consenting AJ individuals >18 years were randomised (1:1) using a computer‐generated random‐number algorithm to either population‐screening (PS arm) or FH‐based testing (FH arm). All underwent pre‐test genetic counselling.https://siteman.wustl.edu/prevention/ydr/): How many servings of alcohol do you have on a typical day? (0/1/2/3 or more).Standardised items obtained from the UK National Diet & Nutrition Survey (NDNS); Health Survey England (HSE), Harvard Center for Cancer‐Prevention's Harvard Cancer Risk Index (HCRI), and CDC Behavioral Risk‐Factor Surveillance System (BRFSS) were used to assess these outcomes. The NDNS is a rolling programme collecting information on food consumption, nutrient intake and nutritional status of the UK general population funded by Public Health England (PHE) and the UK Food Standards Agency (FSA). HSE is an annual survey monitoring changes in the health and lifestyles of people across the UK. Cancer‐related food consumption was measured with three items adapted from the NDNS: ‘Please indicate how often, on average, you eat each of the following foods: red meat, fruit, vegetables'; with seven response options (ranging from ‘Never’ to ‘More than once a day’), which were categorised for analysis to: <once/week, ≥ once/week and ≥ once/day. Vitamin consumption was assessed with an item adapted from NDNS: ‘Are you taking any vitamin supplements?’ (no/yes). Alcohol consumption was assessed with one item adapted from the NDNS: ‘How often have you had an alcoholic drink during the last 12 months?’ , plus one item adapted from the Harvard Center for Cancer Prevention ‘Your Disease Risk’ website, which is based on the HCRI : ‘During the past month, other than your regular job, did you participate in any physical activities or exercises such as running/golf/gardening/walking for exercise?’ (yes/no); one item from the HCRI: ‘Do you walk (or do other moderate activity) for at least 30 minutes most days, or at least 3 hours/week?’ (yes/no), and one item adapted from the HSE: ‘Thinking about your job in general, would you say that you are very/fairly/not very/not at all physically active’.Physical activity was assessed with one item from the CDC‐BRFSS .Comparative cancer risk was assessed with (a) ‘Compared with other people of your age and sex, do you think your chances of getting cancer at some point in your life are: much lower, lower, about the same, higher, much higher?’ ; and (b) ‘On a scale from 0 to 100, where 0 is no chance at all and 100 is absolutely certain, what do you think are the chances that you will get cancer sometime during your lifetime?’ . Use of a mammogram was assessed with items adapted from the CDC‐BRFSS: Have you ever had a mammogram?’ [yes/no/not sure]; ‘If yes, how long has it been since you had your last mammogram?’ .2.2BRCA founder‐mutation status (positive or negative or unknown), marital status (married/cohabiting versus widowed/divorced/single), income , education , family history (low‐risk versus high‐risk) and age.The responses to the lifestyle questions at ‘baseline’, 1 year, 2 years and 3 years were analysed using generalised linear mixed models, where a random intercept term represented the unexplained heterogeneity corresponding to each subject. Specifically, a logistic mixed‐model was fitted for the binary items and an ordinal logistic regression model fitted for ordered multiple response items. Questions on breast screening (mammogram) were re‐formulated to be a binary response variable that asked whether the subject had the screening/mammogram within the last 12 months and responses were analysed using logistic mixed‐models. The analysis of mammogram outcomes was restricted to women >50 years. Each time‐point was included as a fixed‐effect and interacted with the group term (‘family history’ or ‘population screening’), resulting in all eight group and time mean values being freely estimable. In addition, the model was adjusted for gender (men versus women), P‐values <0.0039 (0.05/13) considered to be significant. Potential group differences over the four time‐points were also explored visually to help interpret the model parameters for group when interacted with time. To visualise any trends, the STATA margin command was used to make predictions over the sample for each of the eight group‐by‐time interactions. Specifically, for the binary items, the marginal prediction was the population averaged  probability of a positive response. For the ordered items, the same predictions were made for each of the possible outcome categories. These marginal predictions with their confidence intervals were then plotted. Statistical analyses used STATA‐17.0 (StataCorp LP).A linear combination of the relevant time and group parameters for each model was used to estimate the accumulated difference between groups over time  in the logit scale, accounting for any baseline difference. A similar linear combination of group‐specific parameters for each model was calculated to estimate the total change from baseline  for each group in the logit = log(odds) scale. This is the cumulative difference between groups over years 1, 2 and 3 (accounting for the baseline difference). As 13 variables were analysed, results were adjusted for multiple testing. We were conservative and used a Bonferroni correction, with 2.2.1 | Core outcome sets (COS)BRCA testing at present.There are no COS for population 2.2.2 | Patient & public involvement (PPI)BRCA testing and the research. It provided community inputs into study protocol development, communication strategy, development of participant/patient‐facing materials, study conduct/delivery. There was PPI/public representation on trial steering and management committees. The extensive PPI exercise was instrumental in generating support, awareness and successful completion of the study.The GCaPPS trial was preceded by a year‐long extensive community engagement process with all denominations in the Jewish community, involving a broad range of stakeholders. This included representatives from the Liberal, Reform, Masorti/Conservative, Orthodox and Unaffiliated community denominations. We engaged with a range of stakeholders including religious leaders, Rabbis, Boards of Deputies, Jewish charities, Jewish Medical Association, cancer charities and patient support groups. This exercise enabled exchanges of ideas and understanding of underlying concerns related to 3BRCA testing and were randomised. In the PS arm, 530 participants and in the FH arm 66 participants underwent BRCA testing. The consort flow‐chart is given in Figure In all, 1034 AJ participants were recruited: 530 were randomised to the PS arm and 504 to the FH arm; 691 women and 343 men consented to P = 0.001) within the population‐testing arm alone. However, this difference  was not statistically significantly different  compared with the intake seen with FH/criteria‐based testing over time  and PS  are given in Table , 2 and 3P = 0.011) groups  and PS‐based (P < 0.001) BRCA testing, but no difference was observed between FH or PS approaches to BRCA testing (P = 0.06) , vegetables  and vitamins  but consume more meat  and alcohol in greater quantities  and more frequently  than women do (Table P = 0.01) and perceive their cancer risk to be lower compared with women (P < 0.0001) (Table P = 0.003) or vegetable intake  and more physically active jobs (P = 0.002) (Table P < 0.0001). BRCA‐carriers perceived their cancer risk to be higher than non‐carriers. However, the effect size was greater for those testing negative  than for those who did not receive a test result . Older participants consumed greater amounts of fruits , vegetables, vitamins  and alcohol  but less meat  and perceived their cancer risks to be lower . In women >50 years of age, mammogram uptake decreased with age  but there was no difference in mammogram screening uptake between the groups.The outputs of the generalised linear mixed‐models showing the association of covariates with the different outcomes are given in Table do Table . Men are1) Table . Higher 2) Table . Partici44.1BRCA testing with FH‐based testing. Our findings are reassuring, as they do not show a significant difference or any detrimental impact on overall lifestyle factors, such as dietary fruit, vegetable or meat intake, alcohol, smoking or physical activity and exercise, with population‐based BRCA testing compared with the standard FH‐based testing. We found an increase in vitamin intake within the population‐based BRCA testing arm. Men eat fewer fruits, vegetables, vitamins and consume more alcohol and meat than women. Fruit, vegetable, alcohol and vitamin consumption increased, whereas meat intake decreased with age. Non‐carriers perceived themselves at lower risk than BRCA‐carriers and overall cancer risk perception decreased with time, but there was no difference between the PS and FH approaches. Routine mammogram screening uptake declined with time for average‐risk women over 50 years, but there was no difference between the FH and PS arms.To the best of our knowledge this is the only RCT study on lifestyle impact comparing population‐based 4.2BRCA testing in the AJ‐population not being directly generalisable to the non‐Jewish general population.Our study has a number of advantages, including population‐based ascertainment, inclusion of men and women, randomised design, adequate questionnaire response rate and long‐term follow‐up. Its limitations include lack of qualitative data and inferences from 4.3BRCA testing is reassuring. Our finding of increased vitamin intake in the population testing arm is contrary to two earlier small non‐randomised cohort studies in high‐risk AJ and general population women that showed no short‐term impact on vitamin intake with BRCA testing.BRCA testing in high‐risk individuals. However, the efficacy or benefit of CAM on cancer risk remains unestablished. According to the UK FSA, half of adults (48%) take food supplements (multivitamins being the commonest form) regularly, with another third having taken them in the past.Although genetic testing can be used to facilitate healthy lifestyle behaviours, a clear cause‐and‐effect association has yet to be demonstrated.The decrease in long‐term job‐related physical activity we found is consistent with the reduction in physical activity linked to increasing age in the population. That this was not accompanied by a significant reduction in physical exercise associated with sport or moderate activities such as walking is reassuring. Our findings of lack of health behavioural impact on diet, physical activity, alcohol and smoking is consistent with other reports from high‐risk women.Some reports indicate a positive impact on lifestyle following ‘direct‐to‐consumer’ (DTC) genetic testing, in up to 23% participants, on factors such as diet, exercise, vitamin intake, alcohol intake and smoking.BRCA testing is consistent with other reports in the literature from high‐risk studies and cancer risk in non‐carriers in population‐based studies.BRCA testing on baseline breast screening behaviours is reassuring. Average risk women in the UK undergo 3‐yearly mammograms through the national breast screening programme. Our findings suggest that most women continue to undergo the routine mammograms and although mammogram uptake decreases with age in women over 50 years, this was not different between women undergoing FH‐ or PS‐based BRCA testing , where a person's attitudes, subjective norms and perceived behavioural control are key to determining an individual's decision to follow a particular behaviour.BRCA testing. The uptake of clinical BRCA testing remains limited and only a small proportion of eligible women undergo BRCA testing. Correspondingly, only a small proportion of at‐risk BRCA‐carriers have been identifiedBRCA‐carriers who can benefit from screening/prevention remain to be identified, missing huge opportunities for precision prevention. Population testing offers the opportunity to maximise carrier identification for primary OC and BC prevention. WeBRCA testing. Israel has recently changed its policy and implemented population BRCA testing. It is important that other health systems, including the NHS, follow suit.Most lifestyle outcomes of population testing are comparable to those of FH/criteria‐based testing. These data coupled with other publications from the UK, Israel and Canada 5BRCA testing in the Jewish population does not adversely affect long‐term lifestyle behaviour or routine mammogram screening for average‐risk women. Overall findings from population testing for most outcomes are comparable to those of FH/clinical criteria‐based testing, supporting the concept of population‐based BRCA testing in the Jewish population. BRCA testing provides the first implementable model for the application of population‐genomics for precision prevention. We call for a change in policy to implement this.Population‐based Conception: RM, IJ. Design & Development: RM, IJ and UM. Questionnaire development: RM, IJ, UM, JW, SS, KL, SG. Data collection: RM, RD, KL. Data analysis: MB, RM. Preparation of tables and figures: RM, MB, FG, MS. Trial management: RM, IJ, UM, KL, RD, JW, SG, LS, HD, YW, CC, IT, UB, AB. Genetic testing: YW. Data collection from Guy’s genetic laboratories: CJ. Initial draft of manuscript: RM, FG, MB, MS. Manuscript writing, review and approval: All authors.Following peer review, the study was funded by ‘The Eve Appeal’ charity (grant number GTCV). The funding body had no role in the study design, data collection, analysis, interpretation or writing of the report or decision to submit for publication. The research team was independent of funders. RM is supported by an NHS Innovation Accelerator (NIA) Fellowship for population testing, MB and UM by MRC core funding (MC_UU_00004/01) and UM by the National Institute for Health Research University College London Hospitals Biomedical Research Centre.The study is supported by researchers at the Barts Cancer Institute Cancer Research UK Centre for Excellence, Queen Mary University of London (C16420/A18066).The funding body  had no role in the study design, data collection, analysis, interpretation or writing of the report or decision to submit for publication. The research team was independent of funders.IJ and UM have a financial interest in Abcodia, Ltd., a company formed to develop academic and commercial development of biomarkers for screening and risk prediction. IJ is a member of the board of Abcodia Ltd, a Director of Women's Health Specialists Ltd and received consultancy from Beckton Dickinson. RM declares research funding from The Eve Appeal and Cancer Research UK into population testing and from Barts & the London Charity, Rose Trees Trust and BGCS outside this work; an honorarium for grant review from Israel National Institute for Health Policy Research; and honorarium for advisory board membership or lectures from AstraZeneca/MSD/GSK/EGL. RM is supported by an NHS Innovation Accelerator (NIA) Fellowship for population testing. The other authors declare no conflicts of interest.http://www.controlled‐trials.com/ISRCTN73338115).The GCaPPS study received full ethics approval from the Institute of Child Health/Great Ormond Street Hospital Research Ethics Committee on 8 June 2008 (REC Reference number 08/H0713/44). The study was registered with the International Standard Randomised Controlled Trial Number Register ‐ ISRCTN 73338115 (All trial volunteers provided written informed consent to participate in the study.Table S1Click here for additional data file.Figure S1.Click here for additional data file.


Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self‐renewal activities, and tumorigenesis in orthotopic xenograft models.ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor‐associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets.ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self‐renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM.We identified ADAMTS3 as a potential therapeutic target for GBM. ADAMTS3 mRNA level is significantly higher in GSCs.Knockdown of ADAMTS3 suppresses GSC's proliferation, self‐renewal activity, and tumor formation in vivo. Despite aggressive treatments such as surgical resection, radiation, and chemotherapy, GBM patients have a median overall survival of 15 months after diagnosis.A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) enzymes are secreted matrix‐associated zinc metalloendopeptidases that play a range of roles in tissue morphogenesis, pathological remodeling, inflammation, and vascular biology. The human family consists of 19 members, which can be divided into sub‐groups based on their known substrates. Control of the extracellular matrix (ECM) function and structure is a central aspect of the ADAMTS domain, as evidenced by the actions of procollagen‐N‐propeptidases in collagen fibril assembly and aggrecanases in the modification or cleavage of ECM proteoglycans.In comparison to other ADAMTS families, the role of ADAMTS3 is not fully known. ADAMTS3 is a procollagen N‐propeptidase that is essential for the maturation of triple‐helical collagen fibrils. It is expressed particularly in cartilage during embryogenesis and adulthood, and it efficiently processes the procollagen II aminopropeptide in vitro.ADAMTS3 expression levels using Gene Expression Omnibus (GEO) (GSE4536) dataset and employed univariate and multivariate analysis using the Gravendeel dataset. Also, we examined the effect of ADAMTS3 knockdown on self‐renewal and tumor formation ability in vitro and in vivo of GSCs isolated from primary GBM tissues.To investigate the link between ADAMTS3 expression and GBM malignancy, we compared the 22.1Normal human astrocytes (NHA)  were maintained in an astrocyte medium containing 10% fetal bovine serum  and 1% penicillin–streptomycin solution . Glioma cells  were maintained in Serum media composing Dulbecco's modified medium  containing 10% Fetal Bovine Serum  and 1% penicillin–streptomycin solution . A Neurobasal Media (NBE) composed of DMEM/F12 medium (Welgene) supplemented with 2% B27 (Gibco), 1% penicillin–streptomycin solution , epidermal growth factor , and basic fibroblast growth factor , were used to maintain glioma stem cells  received from the University of Texas MD Anderson Cancer Center.2.2−ΔΔCt method was used to calculate the cycle threshold (Ct) values from the qPCR results. Primer sequences (5′ to 3′) used for qPCR were as follows: 18 S (loading control): forward (F), CAGCCACCCGAGATTGAGCA and reverse (R), TAGTAGCGACGGGCGGTGTG; ADAMTS3: F, CAGCAGCGAAACTCCCACTTTG and R, GCGTTCCATCATGCACCAGTTG; CD15: F, TTGGGACCTCCTAGTTCCAC and R, TGTAAGGAAGCCACATTGGA; CD133: F, CAGGTAAGAACCCGGATCAA and R, TCAGATCTGTGAACGCCTTG; GFAP: F, GGAACATCGTGGTGAAGACC and R, AGAGGCGGAGCAACTATCCT; SOX2: F, GCTACAGCATGATGCAGGACCA and R, TCTGCGAGCTGGTCATGGAGTT; NES: F, TCAAGATGTCCCTCAGCCTGGA and R, AAGCTGAGGGAAGTCTTGGAGC; OLIG2: F, ATGCACGACCTCAACATCGCCA and R, ACCAGTCGCTTCATCTCCTCCA.Total RNA was extracted using the RiboEx reagent (GeneAll) and purified using the HybridR kit (GeneAll), as directed by the manufacturer. Thermo Scientific RevertAid FirstStrand cDNA Synthesis Kit (Thermo Fisher Scientific) was used to convert 500 ng of total RNA to complementary DNA (cDNA). The quantitative Real‐Time PCR (qPCR) was performed using TB Green Premix Ex Taq (Takara Bio) on a CFX96 real‐time polymerase chain reaction detection system (BioRad Laboratories). The 22.3ADAMTS3 mRNA expression in different glioma stem cell cultures. The gene expressions were normalized using dChip invariant method and PM‐MM difference model was used to obtain the expression values. Also, the Gravendeel dataset obtained from the Gliovis website (http://gliovis.bioinfo.cnio.es/) was used to analyze ADAMTS3 gene expression profiles and correlation analysis with patient survival and other GSC‐related genes.Gene Expression Omnibus (GSE4536) datasets were used to compare the 2.4ADAMTS3  were used to silence ADAMTS3 expression. For packaging the lentivirus, 293FT (Invitrogen) cells were transfected using the CalPhos Mammalian Transfection Kit (Takara Bio), the lentivirus was harvested at 72 h after transfection and concentrated 100‐fold using Lenti‐X concentrator (Takara Bio). Lentivirus infection was performed according to the manufacturer's protocol.Lentiviral vectors expressing nontarget shRNA (shNT), and shRNA construct targeting 2.5http://bioinformatics.sdstate.edu/go/) to determine the genes that are coupregulated with ADAMTS3 (log2 FC ≥1.0 and p ≤ 0.001) in Gravendeel dataset.GO and KEGG pathway analyses were performed using the ShinyGO (version 0.75. 2.6n = 6). After 72 h of incubation, 10 μl of alamarBlue reagent was added to each well and incubated for another 6 h. At the end of the incubation, Fluorescence was measured at a wavelength of 590 nm in a Synergy HTX Multi‐Mode Reader (BioTek Instruments Inc).Cell viability of GSCs after shRNA transduction was assessed using the alamarBlue® cell viability assay . shRNA‐transduced GSCs were seeded onto 96‐well plates at a density of 3000 cells/well  to determine their tumorsphere formation ability. The frequency of tumorsphere formation ability of GSCs was determined using Extreme Limiting Dilution Analysis (ELDA) software available at 2.85 cells) were intracranially injected into the nude mice brains at coordinates: 2 mm right and 1.0 mm anterior of the bregma using a stereotaxic instrument (n = 6 mice per group). Mice that had lost more than 30% of their body weight were sacrificed. The Kaplan–Meier method was used to present overall survival curves. For tissue histology analysis, one mouse was simultaneously sacrificed in the control and the experimental groups after 1 month of GSC injection. All mice experiments were authorized by Chonnam National University's Animal Care Committee following the Republic of Korea government and institutional standards and laws (CNU IACUC‐YB‐2021‐99).For in vivo orthotopic implantation, shNT or shADAMTS3 treated GSCs (5 × 102.92/sr).For imaging procedures, isoflurane at a concentration of 2% was used to maintain the anesthesia of nude mice. Following intraperitoneal injection of 150 mg/kg D‐luciferin, mice were imaged with 10 min of exposure. Total flux values were determined by luminescent regions of interest (ROI) of each mouse and are presented in radiance  between ADAMTS3 and each GSC subtype marker was calculated using GraphPad Prism Ver.8 software.For statistical analyses, Microsoft Excel, SPSS Ver.20, and GraphPad Prism Ver.8.0 were used. Statistical significance between and among groups were assessed by a two‐tailed 33.1ADAMTS3 expression reduced significantly when cells were differentiated in a serum‐containing medium  cultured undifferentiated GSCs had more robust tumorigenic potential, heterogeneous morphology, and indefinite self‐renewal ability.m Figure . These f3.2ADAMTS3 expression levels in our cell lines, ADAMTS3 mRNA expression levels in NHA, glioma cell lines , and patient‐derived GSCs  were examined. ADAMTS3 was highly upregulated in GSCs compared to NHA and glioma cell lines, according to qRT‐PCR analysis  samples than in low‐grade glioma samples   and age (p = 0.001) were found to be factors influencing glioma patients' survival in multivariate analysis.  and Chinese Glioma Genome Atlas (CGGA) datasets  Figure . Moreove. Figure . Based os Figure . We empl. Figure . In addi. Figure . Moreove3.6ADAMTS3 to better understand ADAMTS3's signaling pathways and biological processes. Using the Gravendeel dataset, we found 444 coexpressed genes that were upregulated with ADAMTS3 . Using ShinyGO v0.75, these positively correlated genes were analyzed for GO biological processes and KEGG pathways  is a type of brain tumor that is characterized by tumor infiltration into normal tissues, necrosis, cellular heterogeneity, and angiogenesis.+ glioma stem cells differentiated into lymphatic vessels through histological analysis.A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) enzymes are secreted matrix‐associated zinc metalloendopeptidases that play a variety of roles in tissue morphogenesis and pathological remodeling, inflammation, and vascular biology.ADAMTS3 expression is considerably downregulated during GSC differentiation in serum‐containing media. In orthotopic GBM mouse models, Lee et al. found that serum‐cultured cells lost their tumorigenic capacity.In this study, Recent studies have revealed genes that can be used as prognostic markers in glioblastoma and glioma stem cells.This research was supported by the National Research Foundation (NRF) funded by the Ministry of Science and ICT (MSIT) .The authors declare no conflict of interest.