dataesr/scientific-paragraphs-categorization huggingface dataset:
J o u r n a l P r e -p r o o f To address spatial heterogeneity of TME subtype expression signatures, the IvyGAP dataset (N=122 samples) was stratified according to novel TME classifiers (Supplementary Figure  13A ). Spatial interrogation of TME subtype distribution (based on IvyGAP anatomic neighbourhoods), identified differing gene expression patterns among each anatomic region. TME High samples were most enriched within regions defined as cellular tumour (63%). TME High samples also demonstrated slightly elevated proportions of microvascular proliferation samples (12%) compared to other subtypes. The TME Med cohort manifested a moderate proportion of infiltrating tumour samples (10%) and elevated proportion of microvascular proliferation samples (15%) compared to TME Low samples (6%). In contrast, TME Med samples displayed the highest proportion of pseudopalisading cells around necrosis samples (23%).
J o u r n a l P r e -p r o o f Next, to further study the relationship between TME-subtype and response to ICI, we accessed RNA-seq data from the Zhao study (Zhao cohort) which evaluated immune responses and survival of longitudinally profiled patients during standard therapy and following treatment with PD-1 inhibitors (nivolumab or pembrolizumab)  18. Firstly, the GBM-MCP-counter was applied to pre-and post-anti-PD1 treated tumour samples (n=24)(Figure  5A ). 5B ). We subsequently assessed how TME-subtype proportion changes in pre-and post-anti-PD1 treatment samples, and in responders and non-responders (responders defined as those which revealed an inflammatory response, few tumour cells upon sampling and stable or shrinking tumour volume). Following anti-PD1 treatment, the proportion of TME Low tumours remained the same (33%), the proportion of TME Med tumours decreased from 27% to 22%, and the proportion of TME High tumours increased from 40% to 44% (Figure  5C ).
J o u r n a l P r e -p r o o f Finally, we examined whether patient stratification based on TME-subtype could predict response to oncolytic virus therapy. Sequencing and clinical data were accessed from the Desjardins et al  19  2018 phase 1 clinical trial (NCT01491893) which evaluated convectionenhanced, intratumoural delivery of recombinant non-pathogenic polio-rhinovirus chimera (PVSRIPO) in rGBM patients (PVSRIPO cohort). Samples were first assigned to TME-subtypes (Figure  5E ). Tentatively, TME High patients treated with PVSRIPO showed a trend towards improved OS (P=0. 056) when compared with TME Low and TME Med tumours (Figure  5F ).
It will also be important to consider whether therapeutic pressure may truly drive subtype switching  41, 48. Additional analyses of biopsies in primary and recurrent tumours (posttreatment) may further unravel the impact of intra-tumoural heterogeneity on TMEsubtype classification and TME-subtype specific treatment resistance mechanisms  37. Finally, the predictive potential of novel TME-subtypes was retrospectively assessed in interventional immunotherapy clinical trial datasets. Firstly, our analysis of the small Cloughesy trial dataset tentatively suggests that TME High patients who receive neoadjuvant J o u r n a l P r e -p r o o f anti-PD1 might show improved OS compared to patients receiving adjuvant anti-PD1 alone. Nevertheless, we acknowledge that TME stratification prior to neoadjuvant treatment is not without complexity.
This observation also requires further validation. Thirdly, we investigated whether TME-subtypes were predictive of survival within the small PSVRIPO dataset (NCT01491893 A tailored, Phase 2 study employing a rational hypothesis-driven trial design is now required to validate our findings. This trial should mandate for the robust collection of fresh frozen tissue for retrospective molecular analysis. In conclusion, our multi-centre study introduces novel TME-subtypes which may inform optimal precision immunotherapy treatment strategies in the GBM setting. Our data provides convincing evidence that a TME-subtype classification system represents a canonical "terminus a quo" (starting point) to (i) deepen knowledge of GBM TME biology, (ii) support identification of patient subgroups who may benefit from immunotherapy and/or other TME targeting agents and (iii) provide a platform for the identification of new TME-associated contexts of vulnerability.
Recently, it has been suggested that EGFR therapeutic resistance may arise due to extrachromosomal DNA (ecDNA) amplification, rather than classical chromosomal alterations33. Further interrogation of TME Low ecDNA landscape is required to uncover potential resistance mechanisms which may be hallmarks of this subtype. TME Med GBM is associated with an abundance of immune populations, functional orientation markers, immune checkpoint and endothelial cell markers. TME subtype analysis of IvyGAP anatomical samples revealed TME Med patients comprised the highest proportion of samples defined as 'pseudopalisading cells around necrosis'. Interestingly, pseudopalisades are associated with microvascular hyperplasia and angiogenesis, and may serve as predictors of poor prognosis in GBM34. Thus, despite negative outcomes following anti-angiogenic therapy (NCT00884741 and Checkmate-143/NCT02017717), our J o u r n a l P r e -p r o o f data tentatively suggests that patients identified in the 'colder' TME Med subtype might antiangiogenic treatment combined with immunotherapy due to high endothelial cell abundance, vascularity and diverse immune cell population.
Acknowledgements The work presented in this paper is dedicated to the memory of  Mr Paolo Iacovelli. We would like to thank  J. Heffernan ,  L. M Houlihan  and  J. Mythen  ( Beaumont Hospital Neuropathology Dept ) for their help in establishing the GLIOTRAIN biobank in Dublin. The authors gratefully acknowledge all patients who kindly donated tumour tissue and clinical/ genomic data used in this study.
Methods: A refined and validated microenvironment cell population (MCP)-counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition arounds medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified 3 novel patient clusters, uniquely characterised by TME-composition, functional orientation markers and immune checkpoint proteins. Validation was performed in three independent GBM-RNA-seq datasets. Neo-antigen, mutational, and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal GLASS cohort and three immunotherapy clinical trial cohorts (treatment with neoadjuvant/adjuvant anti-PD1 or PSVRIPO) were further interrogated to assess subtype alterations between primary and recurrent tumours, and to assess the utility of TME classifiers as immunotherapy biomarkers.
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by uncontrolled terminal complement activation leading to intravascular hemolysis (IVH), thrombosis, and impairments in quality of life (QoL). The aim of this study was to identify the clinical drivers of improvement in patient-reported outcomes (PROs) in patients with PNH receiving the complement component 5 (C5) inhibitors eculizumab and ravulizumab. This post hoc analysis assessed clinical outcomes and PROs from 246 complement inhibitor-naive patients with PNH enrolled in a phase 3 randomized non-inferiority study that compared the C5 inhibitors ravulizumab and eculizumab (study 301; NCT02946463). The variables of interest were lactate dehydrogenase (LDH) levels, a surrogate measure of IVH, and hemoglobin (Hb) levels. PROs were collected using Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) and European Organisation for Research and Treatment of Cancer, Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) to assess fatigue and QoL, respectively.
Ravulizumab is a second-generation C5 inhibitor, which provides the same clinical benefit as eculizumab with immediate, sustained, and complete terminal complement inhibition throughout a significantly longer intravenous dosing interval (every 8 weeks)  [13] [14] [15] [16]. The comparable efficacy and safety profiles of both treatments have been demonstrated in two phase 3, randomized, open-label, multicenter, international studies of patients with PNH who were complement inhibitor-naive and -experienced (studies 301 [NCT02946463]  [17, 18]  and 302 [NCT03056040]  [19, 20] , respectively). In both studies, ravulizumab was demonstrated to be non-inferior to eculizumab for all primary and key secondary efficacy endpoints, such as the proportion of transfusion-free patients, stabilization of hemoglobin (Hb) levels, change from baseline (ΔBL) in patient-reported outcome (PRO) measures, and normalization of LDH levels  [17] [18] [19] [20].
However, the combined effects of improved Hb level with reduction in blood transfusion and improved Hb level with reduction in LDH to < 1. 5 × ULN were significant predictors for improvement in EORTC QLQ-C30 GH and FACIT-F scores, respectively. These results illustrate the significance of decreased LDH levels in driving QoL and fatigue outcomes in patients with PNH, highlighting the detrimental effect of IVH and the importance of maintaining LDH levels below 1. 5 × ULN for disease control and QoL. There are limitations to this study that must be considered. Although study 301 collected data covering both physical functioning and fatigue subscales of the EORTC QLQ-C30, only the GH subscale was used for this analysis. Future research may benefit from incorporating additional domains to describe the interactions between clinical outcomes and PROs further.
Declarations Ethics approval and consent to participate The protocol for study 301 (NCT02946463) was approved by the institutional review board or independent ethics committee at each participating center, and the study was conducted in accordance with the Declaration of Helsinki and the Council for International Organizations of Medical Sciences International Ethical Guidelines. All patients gave written informed consent. Competing interests Hubert Schrezenmeier has received travel support, fees, and research support (to University of Ulm) from Alexion, AstraZeneca Rare Disease, and Novartis, and fees (to University of Ulm) from Apellis, Roche, and Sanofi. Austin Kulasekararaj has received fees for service, travel support, and consulting fees from Alexion, AstraZeneca Rare Disease. Lindsay Mitchell has received fees from Alexion, AstraZeneca Rare Disease.
Such spatial control of the division is a conserved feature of asymmetric divisions in other stem cell niches 6 and may account for in vivo observations of organization into clusters of blood cells differentiation  43. Stem cell asymmetric division is considered as a binary process leading to the generation of daughter cells of distinct identities  44 45. The early steps of hematopoiesis are now considered to occur like a continuum  46  with the slow and progressive emergence of distinct cell populations  47. Our data support the idea that heterotypic interaction driving HSPC asymmetric division generated siblings with different differentiation potentialities rather that distinct identities. Such non-stereotypical asymmetric divisions are likely to be a key process maintaining the plasticity of early steps of hematopoiesis.
Mold fabrication for microwells The mold design and fabrication were performed as previously described 32 : microwell shape, size, and arrangement were drawn using the software CleWin and etched in the chrome layer onto a quartz photomask (Toppan Photomask). A wafer with microstructures was made on silicon. Silicon wafers were coated with a first 5-μm layer of resin (SU8-3005; MicroChem; CTS) exposed to UV light at 23 mJ/cm2 (UV KUB2; Kloe) for 5 seconds, and with a second layer, 50 μm thick (SU8-3050; MicroChem; CTS) exposed to UV light through the quartz mask at 23 mJ/cm2 for 8 seconds to create microwells. After development (Developer SU8; MicroChem; CTS), only the exposed structures remained, it was baked at 150°C for 2 hours and coated with gas-phase trichloro(perfluorooctyl)silane (Sigma-Aldrich).
Microscopy For the measurement of polarization index and mitotic spindle orientation, images were taken using a Nikon Ti-eclipse microscope equipped with a spinning disk (Yokogawa-CSU-X1) with a 60×, 1. 5× 1. 4-NA oil objective on an electron-multiplying charge-coupled device camera (Photometrics-Evolve512). MethaMorph was used as acquisition software. Z-stacks of 0. 5µm were taken and a binning of 2x2 was used. For live-cell imaging with trans and laser illumination light, an Olympus IX83 microscope equipped with a PECON CellVivo incubation system controlling temperature (37°C) and CO2 concentration (5%) was used. Images were acquired with a 20× 0. 30-NA air objective on an ORCA-Flash4. 0 Lite (Hamamatsu) camera and using MicroManager 1. 4. 21 software. A binning of 2x2 was used during the acquisition.
For detailed live-cell imaging of lysosome polarization and asymmetric inheritance with trans and laser illumination lights (Figure  3F , Figure  4 , Figure  S2B  and C ), a Nikon Ti2 Eclipse equipped with a Prime BSI Express CMOS camera (Photometrics) and using a Nikon CFI Plan Fluor 60x/0. 75 NA oil objective with 1. 5 objective amplification. PECON CellVivo incubation system was used to control temperature (37°C) and CO2 concentration (5%). Images were acquired with an ORCA-Flash4. 0 Lite (Hamamatsu) camera and using MicroManager 1. 4. 21 software. To improve the signal/noise ratio of LysoBrite and reduce laser intensity and exposition time, HSPCs were pre-stimulated for 20h with the cytokines cocktail, then labelled with LysoBrite, and added to the microwells with osteoblast, as previously described.
Immunofluorescence quantification and analysis Polarization Index, Golgi polarization, prophase centrosome positioning, and magnupodium: In the 3D images of the HSPCs, the positions of the centrosome A (Xa, Ya, Za), the point of contact B (Xb, Yb, Zb), and the most distal point on the HSPC membrane from the point of contact C (Xc, Yc, Zc), excluding thin membrane protrusions, were determined. The polarization index was calculated as d/D using ImageJ software. Distance D was defined as the length between points B and C, whereas distance d as the length between B and the projection of A in the D vector, using the following equation: 𝑁𝑢𝑚𝑒𝑟𝑎𝑡𝑜𝑟 = |(𝑋𝑎 -𝑋𝑏) × (𝑋𝑐 -𝑋𝑏)| + +|(𝑌𝑎 -𝑌𝑏) × (𝑌𝑐 -𝑌𝑏)| + |(𝑍𝑎 -𝑍𝑏) × (𝑍𝑐 -𝑍𝑏)| 𝐷𝑒𝑛𝑜𝑚𝑖𝑛𝑎𝑡𝑜𝑟 = 9(𝑋𝑎 -𝑋𝑏) ! + (𝑌𝑎 -𝑌𝑏) ! +(𝑍𝑎 -𝑍𝑏) ! × 9(𝑍𝑐 -𝑍𝑏) ! + (𝑌𝑐 -𝑌𝑏) ! +(𝑍𝑐 -𝑍𝑏) ! 𝑑 = (𝑁𝑢𝑚𝑒𝑟𝑎𝑡𝑜𝑟 ÷ 𝐷𝑒𝑛𝑜𝑚𝑖𝑛𝑎𝑡𝑜𝑟) × 9(𝑋𝑎 -𝑋𝑏) ! + (𝑌𝑎 -𝑌𝑏) ! +(𝑍𝑎 -𝑍𝑏) ! The same method was used for Golgi polarization analysis (taking the closest point of the Golgi to the cell membrane as "B") and for both prophase centrosomes polarization analysis (polarization index ≤ 0.
All statistical analyses were performed with Prism software (GraphPad). Mitotic Spindle Orientation: Mitotic cell images were resliced to visualize the centrosomes and the point of interaction with the bottom of the microwell or the osteoblast/fibroblast. For the mitotic spindle angle, the angle generated between both centrosomes with respect to the image X axis was subtracted to the angle generated for the point of interaction with respect to the image X axis. Graphs were generated with R  49.
Live imaging quantification and analysis Live Golgi and lysosome polarization: Line-scan from the contact site to the more distal point was used to assess the lysosome localization with respect to the Golgi in the Max projected images. Graphs were done with Prism software (GraphPad). Lysosome inheritance ratio: cells were tracked manually until division. To automatize the daughter cell area selection, CellTracker images were processed with Gaussian Blur (sigma=2), Subtract Background (rolling=50), and then thresholding (Otsu dark) to create a mask for the daughter cell area. Background signal was selected manually inside the microwell for each image. Lysosome inheritance ratios were calculated in untreated images using the first time point following cell division, by dividing the brightest daughter cell by its sister, using the sum of LysoBrite pixel fluorescence values (CTCF) normalized to the CellTracker CTCF for each cell.
Ratios were therefore ≥ 1. Ratios <1. 5 and ≥ 1. 5 were respectively selected as indicative of symmetric and asymmetric lysosome inheritance. In the cases where both daughter cells were very close in the first time point after division, the following time point was analyzed. Manual selection of the daughter area was done when the automatized selection failed. Data were analyzed with Fiji and presented as SuperPlots in the same way previously referred. All statistical analyses were performed with Prism software (GraphPad). Lysosome polarization, mitotic spindle orientation, and inheritance ratio: Lysosome polarization of mother cells were analyzed as previously described in the time-lapse before chromosome condensation, visualized by phase images. Metaphase/anaphase was analyzed in the time-lapse that allow the visualization of the chromosome orientation to determine mitotic spindle orientation with respect to the interaction point.
Data were analyzed with Fiji and presented as SuperPlots, or with R 49. In Figure  5 , the corresponding density map was generated using the "ggdensity" package  50. Statistical analyses were performed with Prism software (GraphPad). (C) Superplot of the cell division time of HSPCs in contact with fibronectin, osteoblast, and skin fibroblast. Each color represents a biological replicate (three biological replicates; fibronectin ntot=97, osteoblasts ntot=95, fibroblasts ntot=77 cells). For each replicate, the median value appears as a large disk of the corresponding color. Mean of medians ± SEM shown as black bars. One-way ANOVA test. n. s: non-significant. (D) Gaussian fitted curves of the relative frequency for the first and second cell division (ntot=122, (ntot=141 cells respectively; three biological replicates).
Acknowledgements This work was supported by the  European Research Council  ( Consolidator Grant   771599  ( ICEBERG ) to MT and  Advanced Grant   741773  (AAA) to LB), by the  Bettencourt-Schueller foundation , the  Emergence program   of the Ville de Paris  and the  Schlumberger foundation for education  and research. AC received PhD fellowship from the  Fondation pour la Recherche Médicale  (Grant  FDT20170437071 ). We thank the  Technological Core Facility Research Institute, UMS "Saint-Louis ", US53/UAR2030. The facility is supported by grants from  Université Paris Cité ,  ITMO-Cancer of the French National Science Alliance (Aviesan) , the  Conseil Regional d'Ile-de-France (Canceropôle) , the  National Cancer Research Institute (InCa) , the  Ministère de la Recherche , the  Association Saint-Louis  and the  Association Jean Bernard.
A representative picture of DNA bers is shown. Scale bar is 10 μm. Right panel. Super-plot of fork progression in BJ-RASV12 cells (n=5). The overexpression of RASV12 was induced with doxycycline (+10 μg/ml, + Dox) or not (-Dox) for 8 days. The grey dots are the merged data of all track lengths measured from ve independent experiments. The black and purple dots are the mean of each individual experiment. The horizontal bar is the median of ve independent experiments and its value is also indicated besides each distribution. The p-value was obtained with a paired t-test comparing the means of n experiments. b The overexpression of RASV12 was induced (+ Dox) or not (-Dox) for 8 days and cells were pulse-labeled with 30 μM BrdU for 20 minutes. BrdU incorporation in S-phase is a readout of cell proliferation and was monitored by immuno uorescence (see methods section).
05). Genes signi cantly down-regulated are shown in green (log2 fold change < -1 and FDR < 0. 05). The genes that are not signi cantly altered, belonging or not to the tested gene lists, are shown in grey. Data are from biological duplicates. d The cGAS-STING pathway contributes to cell proliferation arrest induced by RASV12 in BJ broblasts. The overexpression of RASV12 was induced with +10 μg/ml of Dox (+ Dox) or not (-Dox) for 8 days and cells were pulse-labeled with 30 μM BrdU for 20 minutes. Cells were treated during the same period of time with the cGAS inhibitor RU. 521 (1 or 5 μM) or the STING inhibitor H151 (2. 5 μM). DMSO was used as control. BrdU incorporation was monitored by immuno uorescence as in Fig. 1B.
Acknowledgements We thank  Anne Letessier ,  Benjamin Pardo ,  Benoît Le Tallec  and  Benoît Miotto  for their comments and suggestions on the manuscript. We thank the  MRI imaging facility (Biocampus)  for assistance with image acquisition and analysis. H. T has been supported by  AIRC  under the  iCARE-2 fellowship program. Our project has received funding from  AIRC  and from the  European Union 's  Horizon 2020 research and innovation program  under the  Marie Skłodowska-Curie  grant agreement No  800924. Research carried by YL. L is supported by the  Fondation ARC pour la recherche sur le cancer  ( N°PJA 20191209522 ). Work in the Pasero lab is supported by grants from the  Agence Nationale pour la Recherche (ANR) ,  Institut National du Cancer (INCa)  and the  Ligue Nationale Contre le Cancer (équipe labéllisée).
The staining intensity was evaluated using a semiquantitative scoring system (negative, 0; weak, 1þ; moderate, 2þ; and strong, 3þ) where a staining intensity $2þ was considered to be positive. 11 Local complement activation was defined as C3 $2þ and/ or C5b-9 $2þ in patients with at least 2 available stainings. Immunolabeling of HS. Frozen sections were stained for HS (Amsbio, JM403, 370730, 5 mg/ml), then revealed with AF488 goat anti-mouse IgM (Invitrogen, A10680, 2 mg/ml). The whole slides were scanned with a Zeiss Axioscan Z1 (20Â/numerical aperture, 0. 8), and processed using the NIS-Elements AR software 3. 2. 11 (Nikon). Intensity was blindly evaluated by 2 independent operators (TL and VG) using a similar semiquantitative score to the above. The percentage of area fluorescent intensity of JM403-positive glomeruli, normalized to total number of glomeruli, was also quantified.
Clinical data Hemolysis was defined by the concurrent measure of a haptoglobin < 0. 5 g/L and lactate dehydrogenase > 500 IU/L. AKI was defined as a 1. 5-fold increase of serum creatinine compared with the expected norm or the patients' last known value. Malignant hypertension was defined as diastolic arterial pressure >130 mm Hg associated with central nervous system, cardiac, retinal, or kidney injury. 12 Extrarenal events included acute cardiac (at least 2-fold increase of troponin I rate in the first 24 hours following admission), neurologic disorders (impaired alertness or focal deficit with posterior reversible encephalopathy syndrome lesions in magnetic resonance imaging), hepatic cytolysis, and concomitant diarrhea.
Heat-treated NHS (decomplemented-NHS) was used as a negative control. Flow cytometry. Cells were washed, nonenzymatically detached, labeled, and analyzed by flow cytometry (Fortessa X20). A minimum of 10,000 cells per condition was required, and dead cells were excluded using LIVE/DEAD (L23105, ThermoFisher) reagent. The data were assessed using FlowJo software (V10. 6. 1; FlowJo LLC). The following antibodies were diluted in phosphate-buffered saline-bovine serum albumin 1% and incubated for 30 minutes at 4 C: anti-HS (10E4, Amsbio, 370255, 20 mg/ml) (revealed by AF488 goat anti-mouse IgM [Invitrogen, A10680, 2 mg/ml]) and anti-C3cfluorescein isothiocyanate (Dako, F020102-2, 30 mg/ml). Immunofluorescence. Cells were washed and fixed with paraformaldehyde 4%, then labeled with anti-HS (10E4, Amsbio, 370255, 5 mg/ml; 16 hours, 4 C) and anti-C3c-fluorescein isothiocyanate (Dako, F020102-2, 12 mg/ml; 1 hour, RT).