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    Reply to the reviewers

    The authors wish to thank all three Reviewers for their appreciative comments regarding our ECPT and for very useful suggestions. Response to all points raised are presented below, we hope that the responses and new experiments proposed in the following pages will fully address remaining concerns.

    Reviewer’s comments to the BiorXiv paper by Chesnais et al, 2021

    “High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers”

    https://www.biorxiv.org/content/10.1101/2020.11.17.362277v3


    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The authors highlight the importance of endothelial heterogeneity using endothelial cells from different tissues. They examined aortic and pulmonary endothelium as well as HUVECs. They cultured the cells in identical conditions and also stimulated them with a physiological concentration of vascular endothelial growth factor as well high concentrations as would be found in cancers. They developed a profiling tool that allowed analysis of individual endothelial cells within a monolayer and quantification of inter-endothelial junctions, Notch activation, proliferation and other features.

    **Major comments**

    1. It would be useful to apply this technology one step beyond two-dimensional culture, to use vessels opened up longitudinally so that one can see the monolayer of endothelial cells and assess whether it is relevant in primary material in situ. I think this would be a major utility of the whole approach.

    R: We thank this reviewer for the suggestion. In vivo analysis is not in the objectives of the paper. However, we propose to perform “En face” staining of murine blood vessels following the protocol in the reference below. We will perform stainings for murine CDH5 (VE-Cadherin), NOTCH1 intracellular domain, HES1 and DNA which parallel that used in vitro on human EC. We will then apply our revised ECPT workflow and present data in a new Figure.

    En Face Preparation of Mouse Blood Vessels. Ko KA, Fujiwara K, Krishnan S, Abe JI. J Vis Exp. 2017 May 19;(123):55460. doi: 10.3791/55460. PMID: 28570508

    2. There are some very nice images here but disappointed not see a field that could show staining and markers for several of the target proteins and thus show the heterogeneity and randomness or organisation of the endothelial cells.

    R: We thank the reviewer for the appreciative comment. We propose to include representative microphotographs to illustrate the heterogeneity of different EC monolayers in the revised version of the manuscript. Furthermore, to further illustrate these aspects we will also include spatial correlation maps of cells and features measured with ECPT as explained below.





    3.

    • The Notch signalling is an important aspect of this work, particularly evidence of lateral inhibition would have been of value. For example, one might expect cells adjacent to each other to have alternating high and low NICD. *R: *We thank the reviewers for the suggestion. To address this, we are currently developing a new module to perform spatial autocorrelation analysis based on cell maps built using ECPT. In particular we have developed a new module to export cell maps as spatial objects in R which can be then analysed using the adespatial R package and provide correlation metrics such as the Moran’s autocorrelation index (see reference below). The index works with continuous data, removing the need to establish arbitrary thresholds and thus provides formal metrics to demonstrate heterogeneity in EC monolayers. We have derived this index as an example of such analysis for synthetic data and for one ECPT cell map as shown below.

    Figure 1:* Moran’s spatial autocorrelation analysis using R and adespatial package. Moran’s index has values between –1 and 1. If adjacent cells had a consistent tendency to acquire alternate high and low NICD values, the corresponding bivariate Moran’s index would have an I+ value ~ 0 and an I- value approaching -1. In the example cell map both I+ and I- have relatively small absolute values and large p values which suggest a random cell distribution. The analysis was performed on synthetic data and ECPT derived data (HUVEC at baseline).*

    Community ecology in the age of multivariate multiscale spatial analysis

    S Dray et al, Ecological Monographs, 2012. doi:10.1890/11-1183.1

    • NICD staining alone does score the extent of the signalling because of many factors that can influence the transport of the cleaved NICD. Really a marker of Notch signalling downstream e.g. HES or HEY family, DLL4 fis needed to give more information about this critical aspect. __R): __We thank the reviewer for the suggestion. We are currently performing HES1 staining (with no Pha staining) along with a new NICD mAb (see below). Preliminary qualitative data (Fig 2) show that HES1 staining also reveals single cell heterogeneity of NOTCH activation in the same monolayer. We will include ECPT analysis of HES1 and correlation with NICD and other features as suggested. We will reformat the current Fig 5 to include HES1 analysis and improved metrics of NOTCH pathway activation including spatial analysis (point 3 above).

    Figure 2: HES1 immunostaining on HUVEC (Image enhanced for visualisations). Cell nuclei labelled as 1, 2 and 3 have raw mean grey values of HES1 signal equal to 2271, 11210 and 48261 (C2/C1 and C3/C2 >4 folds).




    I really do not think that in Figure 5 it is justified to have a red line drawn through the cloud of points. The correlation coefficient is so low that this is meaningless. The failure to distinguish a P value from biological relevant is worrying. Much better comparison would have been between NICD staining and a downstream gene regulated by notch.

    R: *We appreciate the reviewer’s concerns and are presenting our analyses of NOTCH activation using new immunostainings (HES1) and robust metrics for NOTCH activation as discussed above. We will therefore remove the mentioned corelation plots from the reviewed version of the manuscript. *

    It is important to know that the antibodies used for staining have be validated by the investigators. They would need to show a single band on Western blots or be able to block staining on immunohistochemistry. We all know the manufacturers can be unreliable and use high concentrations of proteins for Western blots. These should be added as a supplementary figure.

    R: While the paper was under revision the AB8925 (NICD, Abcam) has been retracted from the market. To address this major concern, we have decided to acquire a different antibody targeting the intracellular portion of NOTCH receptor and validated its specificity by western blot. Fig 3 below, show western blots demonstrating a clean band at ~98 Kd as expected for cleaved NOTCH1 intracellular domain (NICD).

    We are currently repeating the whole experiment presented in the current version of the manuscript and the ECPT analysis using the new antibody and including HES1 one of the canonical NOTCH target genes as also suggested by this and other Reviewers. We will provide WB analysis of all antibodies used in the paper in a supplementary figure in the revised manuscript.

    Figure 1, WB analysis (NOTCH1 intracellular domain, AB52627, Abcam). of HUVEC (lanes 2,3), HAoEC (lanes 4,5) and HPMEC (lanes 6,7)

    Reviewer #1 (Significance (Required)):

    This represents a valuable and thorough methodology likely to be highly useful to many groups and show new insights into endothelial biology.

    Wide audience, cancer, cardiology, vascular disease-covid.

    My expertise >100 papers on angiogenis in cancer, basic mechanism, therapy models, bioinformatics IHC, patients, clinical trial. H score 190 Google Scholar

    R: *We thank Reviewer One for their very appreciative comments and we hope that the proposed revisions will fully address remaining concerns. *

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Chesnais et al reports development of workflow for analysis of cultured endothelial cells , which they call Endothelial Cell Profiling tool (ECPT). Using ECPT they analyse several parameters in three different endothelial cell types (HuAEC, HUVEC and HPMEC), such as cell morphology, activation of cytoskeleton, VE-cadherin junctions, cell proliferation and Notch activation, under steady conditions and upon treatment with VEGF. The analysis allows to observe some predicted changes, such as increase in cell cycle and junctional activation in cells treated with VEGF-A, and such changes are highly heterogeneous. Overall, this is a potentially useful albeit not revolutionary tool for batch analysis of cultured endothelial cell phenotypes.

    I have the following comments:

    1. To make their case the authors should provide a comparison with other currently used approaches for EC phenotypic analysis in vitro - what is the advantage of using ECPT? The authors repeatedly use the term "single-cell level of analysis ", but this is in fact the case of any IF based analysis of cultured cells.

    R: We thank the reviewer for the suggestions. Indeed, several tools for imaging based single cell phenotyping are available. However, ECPT represents an improvement under several aspects. First, it allows improved segmentation of difficult-to-segment and heterogeneous cells; second, ECPT allows multi-parametric analysis on large image datasets in a semi-automated and structured way facilitating downstream data analysis; third, ECPT is open source.

    *Furthermore, ECPT is a very flexible workflow including tools which facilitate and automate several tasks such as systematic images re-labelling and grouping. We will now draft a table including a complete list of features and improvements in comparison to other available tools and include it in revised manuscript in appendix1 and include analyses which are not implemented in any currently available software such as spatial autocorrelation. *

    I strongly recommend to stain HPMECs for PROX1, these cells are frequently 100% lymphatic endothelial cells. In this case the authors compare different lineages and not blood endothelial cells from different locations.

    R: *We thank the reviewer for the suggestion. We will address this with a new characterisation as supplementary figure in the revised manuscript. We are currently performing a qRT-PCR screening of several EC marker including arterious, venous and lymphatic markers (e.g., CXCR4, Tie2, CDH5, PROX1, LYVE1 as well as baseline NOTCH1 and Dll4 and downstream genes such as HES1 and HEY2. *

    Please provide evidence for specificity of NICD antibody.

    R: We thank this reviewer for the suggestion. Please see response to Reviewer one point 5.

    Figure 1: HPMEC picture appears out of focus

    R: We thank this reviewer for noticing, we will now include a clearer picture in revised version of the manuscript.

    Figure 3 A - it is not entirely clear what is the difference between activated and stressed phenotype, they look quite similar.


    R: We will clarify the definitions of cell activation in revised version of the manuscript and present this analysis as supplementary material to demonstrate the flexibility of our ECPT rather than in main figures. We have removed Pha staining from the new experiments we are performing to allow HES1 staining and address NOTCH signalling in more details. The assessment of Pha and stress fibres in previous experiments will be moved to supplementary material. The classification is based on PhA staining using CPA classifier which was trained to distinguish among the two by the presence of stress fibres. The general rule to place cells in the stressed category during training of the CPA model was the observation of stress fibres crossing the nucleus while cells with peripheral bundles of actin were placed in the activated category.

    * *

    Figure 5 - what is the difference in NICD localization between "high" and "On" conditions?

    R:

    Since it has been noted by this and other reviewers that this classification might be difficult to interpret and in fact, the established thresholds are somehow arbitrary, we will completely revise the way we present analysis of NOTCH activation data including downstream analysis and more formal metrics of spatial correlation and extent of activation eliminating the need to impose thresholds (also see response to Reviewer one point 3).

    Since the authors make a correlation between Notch activity and junctional stabilization, it would be important to confirm this by other means, such as analysis of Notch target genes.

    R: We thank this reviewer for the comment which resonate with this and other Reviewers’ comments. We will include HES1 analysis in the revised manuscript, please see Response to point 6 and reviewer’s one point 3 above.

    **Technical and minor**

    1. Methods mentions HDMECs (human dermal microvascular endothelial cells) but the authors discuss HPMEC throughout the text 2. Please add scale bars on all microscopy pictures. 3. Please provide the information on what isoform of VEGF-A was used for stimulation and the rationale for selecting the concentration.

    R: We thank this reviewer for flagging these imprecisions and we will fix them in revised version of the manuscript.

    Reviewer #2 (Significance (Required)):

    The authors provide a workflow for the phenotypic analysis of cultured cells. Such tool is potentially useful, although the examples the authors show do not reveal striking examples of why such analysis is better in comparison to existing approaches. My guess is that the analysis may be faster and less tedious, once the training sets are generated, but this is not specified. My speciality is endothelial cells biology.

    R: We thank this reviewer for their very useful and appreciative comments. As mentioned above we will expand appendix 1 to fully explain potential and utility of our ECPT and review the main text to clearly highlight main advantages.* We hope that our plan for revision will fully address remaining concerns.*

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    **SUMMARY:**

    The manuscript by Chesnais et al. presents a novel endothelial cell (EC) profiling tool (ECPT) which provides spatial and phenotypic information from individual ECs, and was tested with a variety of specialized EC subtypes (arterial, venous, microvascular). They present a high throughput immunostaining and imaging-based platform using culture of human ECs on 96-well plates and capture of fixed, stained samples on a Perkin Elmer Operetta CLS system. The authors report the use of this ECPT tool to investigate EC phenotypes from human umbilical vein ECs (HUVEC), human aortic ECs (HAoEC) and human pulmonary microvascular (HPMEC) in relation to 50 ng/mL VEGF stimulation for 48 hours, and the general parameters of proliferation, Notch activation and stress fiber rearrangement (F-actin), and present this as a prospective platform to examine differences in EC phenotypes and responses at a more individualized level.

    **MAJOR COMMENTS:**

    1. Fundamentally, the advantage of single cell technologies is the ability to segregate populations to make novel observations. One area that would be of interest to explore in this manuscript using this ECPT platform would be reporting the results from single cell analysis that is then subsequently pooled within a sub-population, rather than sub-stratifying populations to reflect the multiple phenotypes that may be present within a single "confluent" well. With analysis of EC heterogeneity, it would be of interest to differentiate heterogeneity within EC subtypes at the culture/treatment conditions presented, and heterogeneity between EC subtypes.

    R: *We thank this reviewer for the suggestions, we believe that the new approach to evaluate heterogeneity through spatial autocorrelation can provide a much better and clearer picture of this aspect (see responses to Reviewers One point 3 and Two points 6 and 7. Furthermore, we are currently restructuring the ECPT data structure to a more intuitive layout (list of lists rather than a single huge data frame) without affecting downstream data presentation. We will also update our Shiny App to enable the user to perform analyses on data subsets of interest without any R coding, we will present examples and walkthrough of this approach in appendix. *

    2.

    The term "stable IEJ" is used and refers to 48h after seeding 40,000 cells on a 96-well plate, but it is unclear how the authors defined or demonstrated a "stable" junction. In previous reports, longer-term culturing of EC monolayers well beyond the point of confluence has been shown to result in junctional complex rearrangement (Andriopoulou P et al. Arterioscler Thromb Vasc Biol. 1999; reviewed in Bazzoni G & Dejana E. Physiol Rev. 2004). To this point, the fact that the different EC subtypes investigated had different percentages of "quiescent cells" suggests that the monolayers were not completely quiescent. The statement that the IEJ classification is "an immediate index of EC activation in contrast to quiescence" should be further supported by references or data. The definition of quiescent EC as simply non-proliferating, non-migrating is somewhat reductionist, and oversimplifies EC states. The authors state that HAoEC and HUVEC "...appeared more active...", but it is unclear what "active" means, and whether this may simply reflect that these cells had not yet reached confluence or quiescence in the 48h total culture time. As well, it is unclear how "migratory phenotypes" could occur in confluent monolayers. It would be helpful to see the data for these observations. If leaving ECs longer in culture, are the authors able to achieve a higher percentage of quiescent cells?


    R: *We thank this reviewer for the very insightful comments and for suggesting the references. Indeed, we considered these aspects carefully. Regarding cell culture density and confluency, we previously tested seeding densities of 30000-60000 cell/well of 96 well plates (0.32 cm2, ~95000-190000cells/cm2) and we selected 40000 as the maximum seeding density allowing adhesion of >99% of cells. For HUVEC, a seeding density of 40000 cells/well (125000 cells/cm2) produced a high-density culture immediately after seeding (close to what reported for long-confluent cultures in Andriopoulou P et al, ATVB 1999, 140000 cells/cm2). We allowed further 48h culture aiming to achieve junctional “stabilisation” and “maximal” cell density. For consistency, we also seeded 40000 HAoEC and HPMEC per well in all our experiments, however both cell types are significantly larger than HUVEC (Fig 4). For all cells cultures we used EGM2 medium which has few differences with that reported in Andriopoulou P et al, namely, absence of antibiotics and antimycotics and use of defined cocktail of recombinant growth factors instead of Endothelial Cells Growth Supplement. In the past we compared HUVEC cultured in EGM2 and supplemented M199 medium and in our experience EGM2 promotes higher proliferation rates in sub-confluent cultures but similar morphology upon confluency. Is notable that several other factors (including flow, matrix, perivascular cells) are absent in our culture conditions and therefore the homeostatic balance found in vivo might not be fully achievable under our experimental conditions. However, we argue that the described culture conditions should be sufficient to reach a bona fide relatively quiescent EC phenotype in culture. *

    Save these considerations, we agree with this reviewer that providing examples of longer-term cultures would help substantiating our findings and further validate the ECPT approach. We will perform a supplementary experiment to evaluate this aspect by comparing 48h cultures with longer culture times (72h and 96h). Furthermore, we will expand the methods section with the details discussed above and in relation to the suggested references.

    *Regarding the definition of “stable IEJ” and “active EC”, we used this terminology referring exclusively to our measures of IEJ stability (STB index) and Pha based cell classification where we used the terms of “quiescent”, “active” or “stressed”. Therefore, all statements mentioning more or less “stable IEJ” or “active” EC are relative to the specific context of our experiment (not in absolute terms). *

    Overall, we appreciate that the terminology we employed is a source confusion and might suggest inappropriate over-interpretation of our results. We will correct the text in the manuscript to avoid this confusion and to clarify that our observations are valid within the context of our in vitro conditions. In particular, we will present the data regarding junctions as proportions of different junction per cell, and we will rename cell “activation” categories based on PhA immunostaining using more neutral terms (e.g., No Fibres, Peripheral Bundles, Stress fibres). Finally, we will also attempt to generalise our observations to more physiologic context by performing immunostaining on “en face” preparation of murine blood vessels (cfr response to R1 point 1).

    Fig 4: Cell area density distribution for HUVEC, HAoEC and HPMEC in baseline conditions.

    Could the authors comment on the baseline NICD immunoreactivity in the nuclei in HAoEC and HPMEC compared to HUVEC? Is this a reflection of active NOTCH signaling? Or rather, is it possible contact-inhibition (and downregulation of NOTCH) may not have occurred? Demonstration of EC quiescence would help to ensure similar cell cycle states. The definition of "Notch-positive" and "Notch-negative" cells is a bit misleading, as NICD levels and localization are a better indication of canonical Notch activation, and not the presence or absence of Notch protein(s). Further, NICD activation is also dependent on the levels of Notch ligands, which was not addressed. Are the authors able to confirm "OFF", "Low", "High", and "ON" classifications based on NICD intensity and localization with downstream Notch gene activation at a single-cell level? Or correlation between NICD status and the phase of cell cycle or proliferation status?

    R: *We thank this reviewer for the comment. Overall, NICD either nuclear or cytoplasmic can give a measure of how much a cell is relaying canonical notch signalling in a small timescale (minutes, which is also the timescale affected by lateral inhibition, Sjoqvist M and Andersson ER, Dev Biol, 2019). By evaluating single cells in the context of their population in multiple fields of view and samples we can get an indication of how frequently a particular cell type tends to actively transduce canonical NOTCH (under confluent conditions). As this and other reviewers have pointed out NOTCH signal transduction mediated by NICD can be affected by several factors limiting the potential to infer actual activation of the pathway (i.e., downstream gene transcription. As suggested by this and other reviewers we are including measures of downstream gene activation, in particular we have included HES1 staining in our workflow, and we will include these data in a new analysis (also see response to R1 point 3). We will also provide new metrics of spatial autocorrelation to evaluate the tendency to lateral inhibition (R1 point 3) and correlation between parameters using continuous mesures and therefore we will remove the previous classification based on thresholds. Finally, we are performing a qRT-PCR screening to assess baseline levels of DLL4, NOTCH1 and JAG1 which we will present as supplementary material. *

    Do as I say, Not(ch) as I do: Lateral control of cell fate

    Sjoqvist M and Andersson ER, Dev Biol, 2019

    PMID: 28969930

    The existing workflow/platform is adapted for images obtained from the Operetta CLS system (Perkin Elmer) and Harmony software (proprietary), which may not be available for broader users in the EC field. It would be helpful to include ImageJ macros for the bulk automatic import of TIFF, renaming and upscaling of resolution/bit quality to match the formats that are compatible with the software.

    R: *We thank this reviewer for the comment. We have now included an ImageJ macro (available in the GitHub repository) which in principle can import and elaborate images from any source. We didn’t include a specific option in our current user interface because the relabelling operates by parsing original filenames into fields which are then renamed according to user input and each HT platform adopt different regular expression to encode filename. Any user with a basic literacy in ImageJ macro scripting can achieve relabelling and elaboration of their own file given that their filenames use regular expressions which can be parsed. Also, it is relatively easy (again by modifying the macro) to include user defined pre-processing steps including image scaling. An example of parsing method for Operetta CLS filenames is provided in appendix 1. *

    Could the authors comment on the manpower (hours from start to finish for experiments, staining, imaging, analysis, etc.) and cost of the ECPT pipeline relative to emerging single cell technologies such as single cell-RNA sequencing.

    Further, one major advantage of imaging technologies is the ability to assess live cell dynamics, which are particularly relevant in response to stimuli and agonists. Have the authors utilized the ECPT platform for these approaches, in particular, to assess the differential EC subtype dynamics in proliferative conditions?

    R: In terms of manpower the workflow is not very demanding. Our current dataset is based on images extracted form 4 independent experiments (18 wells each). The process is sequential, therefore a single user trained in cell biology, automated microscopy and in the use of the different ECPT components (ImageJ, CP, CPA and R) could perform the experiment alone. The timing of each experiment will depend on circumstantial factors, however once the ECPT is trained for specific user’s requirements (which can require some trials and errors depending on user’s experience) the whole process from cell fixation and staining, through image acquisition (~2 h acquisition for each experiment on an Operetta CLS system), to dataset build-up can take less than one week. For example, elaborating the current image database (~6000 images for four fluorescence channels) which data are presented throughout, had the following raw elaboration times on a Mac Book Pro 2017 equipped with an intel i7 processor and 16 Gb of RAM:

    *- Image pre-processing and relabelling ~1h *

    - Generation of probability maps for VEC and NICD ~3h

    - CP pipeline run (Cell segmentation, objects measurements and classification) ~16h

    *- Data import (R studio) ~20m *

    We will measure these parameters more precisely in the new experimental run and present timings for each step in a new table in appendix 1.

    After main dataset is created R studio can perform most statistical analyses and data plotting almost instantly.

    *We fully appreciate the value of employing ECPT in live imaging setups and we believe it is one of the most promising future applications. We didn’t address live microscopy experiments in the context of ECPT development and validation presented in the current manuscript therefore we cannot present example data or proof of concept. However, we can confidently comment that time lapse experiment would not endow further layers of complexity in terms of image analysis workflow. Therefore, given appropriate set of live markers (e.g., transgenic fluorescently tagged CDH5 for EC segmentation and junctions analysis) we believe that the current implementation of ECPT is already fully equipped to facilitate elaboration and analysis of imaging data derived from time lapse experiments. *

    The authors should discuss the ability to amend or revise of the ECPT platform to incorporate analysis of additional markers that may be obtained through imaging, and discuss greater implications and utility to specifically tailor the workflow for other researchers in vascular biology, or to other monolayer culture systems. Further, they should better highlight the novel observations obtained with the ECPT compared to traditional methodology.

    R: We thank this reviewer for the comments. We will provide evidence of ECPT flexibility within this manuscript by including, during the time of this review process, a new analysis for downstream NOCTH signalling (HES1). We will move analysis of cell “activation” (i.e., stress fibres analysis) to supplementary information and include a more through discussion of how automated single cell classification could improve content, speed, reliability and robustness of quantification tasks which are currently exposed to long and tedious processing times and conscious/unconscious observer biases.

    **MINOR COMMENTS:**

    We thank this reviewer for the very thorough revision of the manuscript. It is truly invaluable to us to improve it. Below responses to specific technical points, we will fix all stylistic, formatting and typographical issues in revised version of the manuscript.

    There are minor typographical, capitalization and grammatical errors throughout.

    R1:* Thanks, we will fix these in updated version of the manuscript.*

    Why was fibronectin used to coat plates, and what was rationale for using this ECM substrate versus gelatin (most commonly used in EC cultures) or type I collagen?

    R2: We used fibronectin for immunostaining experiments similar to what reported in our previous work (Veschini et al, 2007, 2011, Wiseman et al, 2019) and also in Andriopoulou P et al,1999. In general, in our experience FN gives better cell adhesion in comparison to gelatin when culturing EC on glass or other substrates different from cell culture plastic. FN is the cell culture substrate recommended by Promocell therefore, we also used FN for cell expansion to avoid any phenotypic change which might have been caused by switch in cell culture substrate.

    3. Based on the various box plots present throughout the figures, it appears that some parameters have a large range of values. Is it possible or helpful to set minimum and maximum exclusionary criteria? Further, in the way that these data are presented, it is difficult to appreciate the effects of a treatment such as 48h of VEGF, as the magnitude of STB Index difference, for example, appears small, and it is difficult to understand whether these significant differences are biologically relevant, as assessed.

    R3: *We agree that in absence of exclusion criteria it is difficult to infer biologic meaning out of subtle differences (e.g., the tiny difference in STB index between HAoEC in presence or absence of VEGF). In the current version of the manuscript, we attempted to be agnostic in regards whether some observed small but significant mean differences could endow biologic meaning and discussed larger variation as biologically meaningful, for example the differences in STB index among cell types. We argue that tiny differences in the distribution of some selected parameter across experimental conditions could reflect underlying mechanisms masked by biologic noise, therefore catching a glimpse of these variations via ECPT could inspire novel experiments to specifically address their full biologic significance. *

    *To the interest of better understanding of the current manuscript we will re elaborate our data to provide more immediate metrics and highlight outstanding features. *

    * *

    Use of arrows and further description in Figure 1 would help the reader understand what specific features are different in the various EC subtypes. As well, the representative micrographs for HPMEC appear blurry compared to other panels (Fig. 1).

    In Figure 2, the panels in A, B and C do not correspond horizontally, and it may be cleared to demonstrate "Segmentation & features extraction" overlays from the same representative micrographs shown in panel A. Labeling of the individual panels and software used for panel B would help the readership understand what is being quantified and how. The second panel in "C" appears blurry.

    In Figure 3, labelling the color code for quiescent, activated and stressed categories on graphs and in legend would be helpful to easily identify populations.

    R4-6:* Thanks, we will fix these in updated version of the manuscript.*

    For Figure 4, line separators or more obvious grouping to distinguish discontinuous, linear and stabilized junction types in panel A. What proportion of the different EC subtypes contains discontinuous, linear and stabilized junctions at confluence/quiescence? Is there a correlation between discontinuous junctions and proliferating cells?


    R7: We will perform new analyses to address correlation between proliferation and junctions and proliferation vs HES1. We will restructure data presentation on junctions to display different proportion of junctions per cell or per cell type rather than a unified value (STB index).


    It would be useful to distinguish the effects of published mediators on junctional integrity in intact EC monolayers (i.e. histamine; VEGF) from those shown in this automated quantitation. It appears that 50 ng/mL of VEGF treatment for 48h only slightly increases STB index based on panel C.

    __R7c____: __*We agree that increase of STB index in HAoEC and HPMEC upon VEGF treatment might not be highly biologically meaningful, save consideration in point 3 above. However, difference in HUVEC (+- VEGF) is visually appreciable in images (i.e., VEGF treated HUVEC seem to have more linear junctions) therefore we believe that the ~16 units difference in STB index is biologically meaningful. As discussed in point 7 above, we will restructure data presentation to better clarify these aspects. *

    * *

    Figure 5 panel B should provide legend in graphs/figures or figure legends to highlight the color-coding matching the OFF, Low, High and ON groups. Further, it is unclear the difference between "High" and "ON" groups. The authors state that "thresholds were selected empirically", however, it is unclear whether this was derived through utilization of known Notch activators or inhibitors, and how this relates to the threshold of Notch activity necessary to enhance proliferation or maintain quiescence. In Supplementary Figure 4 (which I believe is mislabelled as Supplementary Figure 5), shows only a weak positive correlation between nuclear NICD intensity and mean STB index. It would be of interest to see the plot from Supplementary Figure 5 for each of the EC subtypes, in the presence and absence of VEGF. As well, for Figure 5, on C and D panels, it would improve clarity to revise "Low" and "High" descriptors with "Low NICD activity" and "High NICD activity".

    R8: *As discussed above we will revise our analyses to remove NOTCH categories and instead show spatial autocorrelation analyses which work on continuous data. *

    In Supplementary Table 1, "Widt/length" should be "Width/length"

    * *

    R9: Thanks, we will fix this in updated version of the manuscript.

    For Supplementary Figure 3, it would be of use to show DNA distribution intensities from proliferating, non-confluent EC subtypes to demonstrate the validity of this methodology to identify cells in G0/G1, S and G2/M phases, as highlighted in panel A. Could the authors comment on the discrepancy between the percentage of cells identified as quiescent by ECPT and the percentage of cells in G0/G1? The comment that "VEGF induced a small detectable increase in proliferation rate in all EC" is curious, as a dose of 50 ng/mL of VEGF should be a relatively strong stimulator of proliferation/migration in ECs.

    R10: We will perform ECPT analysis on sub-confluent or sparse cells to further validate our analysis. Qualitative data on preliminary images seems to confirm that the proliferation rate in sparse cells is very high (>70%). To perform the evaluation we followed and improved a previously published method (Roukos et al, Nat Prot, 2015)

    Regarding the relation between cell in G0/G1 and assessment of “quiescent” phenotype (which nomenclature will be revised as discussed above), it is important to highlight that we reported data on stress fibres analysis (i.e., classification into “quiescent”, “activated” and “stressed” cells) only on the cells in G0/G1 (i.e., we excluded proliferating cells from this analysis as we assumed that all proliferating cells would be “not quiescent” and bias our estimation).

    For Supplementary Figure 5, "Nuclear NOTCH intensity" on the Y-axis should read "Nuclear NICD intensity", as it does not appear that Notch was stained. It would also be of benefit to overlay the ranges for "OFF, Low, High and "ON" to appreciate ranges of activation. Is there any correlation between NICD nuclear intensity and proliferative index?

    R11: We will present correlation between NICD or HES1 and proliferation in revised version of the manuscript.

    Definitions should be provided for many terms. i.e. vascular endothelial-cadherin (VE-CAD; CDH5); HUVEC (human umbilical vein endothelial cell); HAoEC (human aortic endothelial cell); HDMEC (human dermal microvascular endothelial cell); NICD (NOTCH intracellular domain); VEGF (vascular endothelial growth factor); etc. at first appearance.


    R12: We will add this information in revised version of the manuscript.

    For EC subtypes purchased from commercial vendor, it would be of interest to understand how many unique donors these cells/data were derived from, and whether there are any differences in basic donor information such as age, sex, etc. Further, Promocell catalogs proliferative rate for each of their lot numbers, and it would be of interest how this compares to the values determined using the ECPT software analysis package.

    R13: We will add this information in revised version of the manuscript.

    1 In the "Cell culture" section of the methods, HDMEC from Promocell are listed, however, the manuscript and figures show data from HPMEC. Both EC subtypes are available from Promocell, however, HDMEC are from dermal origin.

    1 Vascular endothelial-cadherin should be abbreviated "VE-CAD" or "CDH5" and not "VEC", as this is not a standard or gene notation, and will likely be confused with the more common abbreviations for venous or vascular EC. It seems as though "CDH5" is used most commonly throughout manuscript, so this should be used throughout.

    1 The authors refer to "activated NOTCH" when describing antibodies in the methods, however, it would be clearer to the reader to simply refer to the antibody target (NICD), and mention that this reflects canonical NOTCH downstream activation.

    The sentence in the "Immunostaining" methods "CDH5 is a lineage marker..." should be moved to results/discussion as these details are out of place in methods.

    How were the 3 areas captured per wells designated? Were these locations the automated, and the same for all wells?

    "Appendix - Figure" notation should be revised to "Appendix Figure" for consistency and to avoid confusion.

    R14-19: Thanks, we will fix these in updated version of the manuscript.

    How were artifacts and mis-segmented cell objects excluded?

    R20:* We will add this information in the revised appendix. As general rules, cells containing NaNs values in any of the parameters, cells fragments or merged cells (evaluated using area measurements) and cells with no detectable junctions were all excluded (total cell excluded from analysis were ~ 2.5 % of the initial dataset). *

    In "Statistical analysis" "Tuckey's" should be "Tukey's". "HSD" should be defined "honestly significant difference" or simply removed, as Tukey's is most common name.

    In "Statistical analysis", "significative" should be "significant" or "statistically significant".

    Scale bars should be added to micrographs.

    * *

    R21-23: Thanks, we will fix these in updated version of the manuscript.

    Could the authors comment on the necessity of µclear plates, which substantially increases the cost per plate/experiment.

    R24: mclear plates were used to allow image acquisition with a 40x water immersion objective in the Operetta CLS (impossible with standard 96 well plates). Cell grown on coverslips and mounted on microscopy slides could be used as well with significant increase in acquisition time (Wiseman et al, 2019).

    Were other seeding densities and times investigated?

    R25: We will evaluate sparse cells in revised version of the manuscript as discussed above.

    * *

    More description on potentially novel observations between these three primary EC subtypes would be informative for the readership to appreciate

    The references do not appear in chronological order. Further, consistency of reference formatting should be reviewed, and appropriate journal name abbreviations should be used.

    R26-27:* Thanks, we will fix these in updated version of the manuscript.*

    Reviewer #3 (Significance (Required)):

    • This manuscript presents a conceptual and technical advance, introducing a high throughput imaging platform to assess endothelial phenotypes
    • Within the field of angiogenesis, several tools exist, either proprietary, or leveraging ImageJ software to assist in assessment of cells. The ECPT provides a more complex analysis platform to integrate analysis of multiple endpoints
    • This work would be of interest to vascular biology laboratories to adopt a more comprehensive view of heterogeneous endothelial phenotypes in vitro
    • As a vascular biology researcher, I have had extensive experience with in vitro culture of various endothelial cell subtypes from human and mouse. My field of expertise gives me the perspective of the nuances of the direct handling and phenotyping of ECs, and have worked specifically worked with HUVEC, HAoEC and HPMEC, and assessed the impact of key factors relevant in angiogenesis such as VEGF, Notch and other mediators.

    *R: *We thank the reviewer for the very appreciative comments, and we hope that with the revised version of the manuscript we will be able to fully address remaining concerns.

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    Referee #3

    Evidence, reproducibility and clarity

    SUMMARY:

    The manuscript by Chesnais et al. presents a novel endothelial cell (EC) profiling tool (ECPT) which provides spatial and phenotypic information from individual ECs, and was tested with a variety of specialized EC subtypes (arterial, venous, microvascular). They present a high throughput immunostaining and imaging-based platform using culture of human ECs on 96-well plates and capture of fixed, stained samples on a Perkin Elmer Operetta CLS system. The authors report the use of this ECPT tool to investigate EC phenotypes from human umbilical vein ECs (HUVEC), human aortic ECs (HAoEC) and human pulmonary microvascular (HPMEC) in relation to 50 ng/mL VEGF stimulation for 48 hours, and the general parameters of proliferation, Notch activation and stress fiber rearrangement (F-actin), and present this as a prospective platform to examine differences in EC phenotypes and responses at a more individualized level.

    MAJOR COMMENTS:

    1. Fundamentally, the advantage of single cell technologies is the ability to segregate populations to make novel observations. One area that would be of interest to explore in this manuscript using this ECPT platform would be reporting the results from single cell analysis that is then subsequently pooled within a sub-population, rather than sub-stratifying populations to reflect the multiple phenotypes that may be present within a single "confluent" well. With analysis of EC heterogeneity, it would be of interest to differentiate heterogeneity within EC subtypes at the culture/treatment conditions presented, and heterogeneity between EC subtypes.
    2. The term "stable IEJ" is used and refers to 48h after seeding 40,000 cells on a 96-well plate, but it is unclear how the authors defined or demonstrated a "stable" junction. In previous reports, longer-term culturing of EC monolayers well beyond the point of confluence has been shown to result in junctional complex rearrangement (Andriopoulou P et al. Arterioscler Thromb Vasc Biol. 1999; reviewed in Bazzoni G & Dejana E. Physiol Rev. 2004). To this point, the fact that the different EC subtypes investigated had different percentages of "quiescent cells" suggests that the monolayers were not completely quiescent. The statement that the IEJ classification is "an immediate index of EC activation in contrast to quiescence" should be further supported by references or data. The definition of quiescent EC as simply non-proliferating, non-migrating is somewhat reductionist, and oversimplifies EC states. The authors state that HAoEC and HUVEC "...appeared more active...", but it is unclear what "active" means, and whether this may simply reflect that these cells had not yet reached confluence or quiescence in the 48h total culture time. As well, it is unclear how "migratory phenotypes" could occur in confluent monolayers. It would be helpful to see the data for these observations. If leaving ECs longer in culture, are the authors able to achieve a higher percentage of quiescent cells?
    3. Could the authors comment on the baseline NICD immunoreactivity in the nuclei in HAoEC and HPMEC compared to HUVEC? Is this a reflection of active NOTCH signaling? Or rather, is it possible contact-inhibition (and downregulation of NOTCH) may not have occurred? Demonstration of EC quiescence would help to ensure similar cell cycle states. The definition of "Notch-positive" and "Notch-negative" cells is a bit misleading, as NICD levels and localization are a better indication of canonical Notch activation, and not the presence or absence of Notch protein(s). Further, NICD activation is also dependent on the levels of Notch ligands, which was not addressed. Are the authors able to confirm "OFF", "Low", "High", and "ON" classifications based on NICD intensity and localization with downstream Notch gene activation at a single-cell level? Or correlation between NICD status and the phase of cell cycle or proliferation status?
    4. The existing workflow/platform is adapted for images obtained from the Operetta CLS system (Perkin Elmer) and Harmony software (proprietary), which may not be available for broader users in the EC field. It would be helpful to include ImageJ macros for the bulk automatic import of TIFF, renaming and upscaling of resolution/bit quality to match the formats that are compatible with the software.
    5. Could the authors comment on the manpower (hours from start to finish for experiments, staining, imaging, analysis, etc.) and cost of the ECPT pipeline relative to emerging single cell technologies such as single cell-RNA sequencing. Further, one major advantage of imaging technologies is the ability to assess live cell dynamics, which are particularly relevant in response to stimuli and agonists. Have the authors utilized the ECPT platform for these approaches, in particular, to assess the differential EC subtype dynamics in proliferative conditions?
    6. The authors should discuss the ability to amend or revise of the ECPT platform to incorporate analysis of additional markers that may be obtained through imaging, and discuss greater implications and utility to specifically tailor the workflow for other researchers in vascular biology, or to other monolayer culture systems. Further, they should better highlight the novel observations obtained with the ECPT compared to traditional methodology.

    MINOR COMMENTS:

    1. There are minor typographical, capitalization and grammatical errors throughout.
    2. Why was fibronectin used to coat plates, and what was rationale for using this ECM substrate versus gelatin (most commonly used in EC cultures) or type I collagen?
    3. Based on the various box plots present throughout the figures, it appears that some parameters have a large range of values. Is it possible or helpful to set minimum and maximum exclusionary criteria? Further, in the way that these data are presented, it is difficult to appreciate the effects of a treatment such as 48h of VEGF, as the magnitude of STB Index difference, for example, appears small, and it is difficult to understand whether these significant differences are biologically relevant, as assessed.
    4. Use of arrows and further description in Figure 1 would help the reader understand what specific features are different in the various EC subtypes. As well, the representative micrographs for HPMEC appear blurry compared to other panels (Fig. 1).
    5. In Figure 2, the panels in A, B and C do not correspond horizontally, and it may be cleared to demonstrate "Segmentation & features extraction" overlays from the same representative micrographs shown in panel A. Labeling of the individual panels and software used for panel B would help the readership understand what is being quantified and how. The second panel in "C" appears blurry.
    6. In Figure 3, labelling the color code for quiescent, activated and stressed categories on graphs and in legend would be helpful to easily identify populations.
    7. For Figure 4, line separators or more obvious grouping to distinguish discontinuous, linear and stabilized junction types in panel A. What proportion of the different EC subtypes contains discontinuous, linear and stabilized junctions at confluence/quiescence? Is there a correlation between discontinuous junctions and proliferating cells? It would be useful to distinguish the effects of published mediators on junctional integrity in intact EC monolayers (i.e. histamine; VEGF) from those shown in this automated quantitation. It appears that 50 ng/mL of VEGF treatment for 48h only slightly increases STB index based on panel C.
    8. Figure 5 panel B should provide legend in graphs/figures or figure legends to highlight the color-coding matching the OFF, Low, High and ON groups. Further, it is unclear the difference between "High" and "ON" groups. The authors state that "thresholds were selected empirically", however, it is unclear whether this was derived through utilization of known Notch activators or inhibitors, and how this relates to the threshold of Notch activity necessary to enhance proliferation or maintain quiescence. In Supplementary Figure 4 (which I believe is mislabelled as Supplementary Figure 5), shows only a weak positive correlation between nuclear NICD intensity and mean STB index. It would be of interest to see the plot from Supplementary Figure 5 for each of the EC subtypes, in the presence and absence of VEGF. As well, for Figure 5, on C and D panels, it would improve clarity to revise "Low" and "High" descriptors with "Low NICD activity" and "High NICD activity".
    9. In Supplementary Table 1, "Widt/length" should be "Width/length"
    10. For Supplementary Figure 3, it would be of use to show DNA distribution intensities from proliferating, non-confluent EC subtypes to demonstrate the validity of this methodology to identify cells in G0/G1, S and G2/M phases, as highlighted in panel A. Could the authors comment on the discrepancy between the percentage of cells identified as quiescent by ECPT and the percentage of cells in G0/G1? The comment that "VEGF induced a small detectable increase in proliferation rate in all EC" is curious, as a dose of 50 ng/mL of VEGF should be a relatively strong stimulator of proliferation/migration in ECs.
    11. For Supplementary Figure 5, "Nuclear NOTCH intensity" on the Y-axis should read "Nuclear NICD intensity", as it does not appear that Notch was stained. It would also be of benefit to overlay the ranges for "OFF, Low, High and "ON" to appreciate ranges of activation. Is there any correlation between NICD nuclear intensity and proliferative index?
    12. Definitions should be provided for many terms. i.e. vascular endothelial-cadherin (VE-CAD; CDH5); HUVEC (human umbilical vein endothelial cell); HAoEC (human aortic endothelial cell); HDMEC (human dermal microvascular endothelial cell); NICD (NOTCH intracellular domain); VEGF (vascular endothelial growth factor); etc. at first appearance.
    13. For EC subtypes purchased from commercial vendor, it would be of interest to understand how many unique donors these cells/data were derived from, and whether there are any differences in basic donor information such as age, sex, etc. Further, Promocell catalogs proliferative rate for each of their lot numbers, and it would be of interest how this compares to the values determined using the ECPT software analysis package.
    14. In the "Cell culture" section of the methods, HDMEC from Promocell are listed, however, the manuscript and figures show data from HPMEC. Both EC subtypes are available from Promocell, however, HDMEC are from dermal origin.
    15. Vascular endothelial-cadherin should be abbreviated "VE-CAD" or "CDH5" and not "VEC", as this is not a standard or gene notation, and will likely be confused with the more common abbreviations for venous or vascular EC. It seems as though "CDH5" is used most commonly throughout manuscript, so this should be used throughout.
    16. The authors refer to "activated NOTCH" when describing antibodies in the methods, however, it would be clearer to the reader to simply refer to the antibody target (NICD), and mention that this reflects canonical NOTCH downstream activation.
    17. The sentence in the "Immunostaining" methods "CDH5 is a lineage marker..." should be moved to results/discussion as these details are out of place in methods.
    18. How were the 3 areas captured per wells designated? Were these locations the automated, and the same for all wells?
    19. "Appendix - Figure" notation should be revised to "Appendix Figure" for consistency and to avoid confusion.
    20. How were artifacts and mis-segmented cell objects excluded?
    21. In "Statistical analysis" "Tuckey's" should be "Tukey's". "HSD" should be defined "honestly significant difference" or simply removed, as Tukey's is most common name.
    22. In "Statistical analysis", "significative" should be "significant" or "statistically significant".
    23. Scale bars should be added to micrographs.
    24. Could the authors comment on the necessity of µclear plates, which substantially increases the cost per plate/experiment.
    25. Were other seeding densities and times investigated?
    26. More description on potentially novel observations between these three primary EC subtypes would be informative for the readership to appreciate
    27. The references do not appear in chronological order. Further, consistency of reference formatting should be reviewed, and appropriate journal name abbreviations should be used.

    Significance

    • This manuscript presents a conceptual and technical advance, introducing a high throughput imaging platform to assess endothelial phenotypes
    • Within the field of angiogenesis, several tools exist, either proprietary, or leveraging ImageJ software to assist in assessment of cells. The ECPT provides a more complex analysis platform to integrate analysis of multiple endpoints
    • This work would be of interest to vascular biology laboratories to adopt a more comprehensive view of heterogeneous endothelial phenotypes in vitro
    • As a vascular biology researcher, I have had extensive experience with in vitro culture of various endothelial cell subtypes from human and mouse. My field of expertise gives me the perspective of the nuances of the direct handling and phenotyping of ECs, and have worked specifically worked with HUVEC, HAoEC and HPMEC, and assessed the impact of key factors relevant in angiogenesis such as VEGF, Notch and other mediators.
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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Chesnais et al reports development of workflow for analysis of cultured endothelial cells , which they call Endothelial Cell Profiling tool (ECPT). Using ECPT they analyse several parameters in three different endothelial cell types (HuAEC, HUVEC and HPMEC), such as cell morphology, activation of cytoskeleton, VE-cadherin junctions, cell proliferation and Notch activation, under steady conditions and upon treatment with VEGF. The analysis allows to observe some predicted changes, such as increase in cell cycle and junctional activation in cells treated with VEGF-A, and such changes are highly heterogeneous. Overall, this is a potentially useful albeit not revolutionary tool for batch analysis of cultured endothelial cell phenotypes.

    I have the following comments:

    1. To make their case the authors should provide a comparison with other currently used approaches for EC phenotypic analysis in vitro - what is the advantage of using ECPT? The authors repeatedly use the term "single-cell level of analysis ", but this is in fact the case of any IF based analysis of cultured cells.
    2. I strongly recommend to stain HPMECs for PROX1, these cells are frequently 100% lymphatic endothelial cells. In this case the authors compare different lineages and not blood endothelial cells from different locations.
    3. Please provide evidence for specificity of NICD antibody.
    4. Figure 1: HPMEC picture appears out of focus
    5. Figure 3 A - it is not entirely clear what is the difference between activated and stressed phenotype, they look quite similar.
    6. Figure 5 - what is the difference in NICD localization between "high" and "On" conditions?
    7. Since the authors make a correlation between Notch activity and junctional stabilization, it would be important to confirm this by other means, such as analysis of Notch target genes.

    Technical and minor

    1. Methods mentions HDMECs (human dermal microvascular endothelial cells) but the authors discuss HPMEC throughout the text
    2. Please add scale bars on all microscopy pictures.
    3. Please provide the information on what isoform of VEGF-A was used for stimulation and the rationale for selecting the concentration.

    Significance

    The authors provide a workflow for the phenotypic analysis of cultured cells. Such tool is potentially useful, although the examples the authors show do not reveal striking examples of why such analysis is better in comparison to existing approaches. My guess is that the analysis may be faster and less tedious, once the training sets are generated, but this is not specified. My speciality is endothelial cells biology.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors highlight the importance of endothelial heterogeneity using endothelial cells from different tissues. They examined aortic and pulmonary endothelium as well as HUVECs. They cultured the cells in identical conditions and also stimulated them with a physiological concentration of vascular endothelial growth factor as well high concentrations as would be found in cancers. They developed a profiling tool that allowed analysis of individual endothelial cells within a monolayer and quantification of inter-endothelial junctions, Notch activation, proliferation and other features.

    Major comments

    1. It would be useful to apply this technology one step beyond two-dimensional culture, to use vessels opened up longitudinally so that one can see the monolayer of endothelial cells and assess whether it is relevant in primary material in situ. I think this would be a major utility of the whole approach.
    2. There are some very nice images here but disappointed not see a field that could show staining and markers for several of the target proteins and thus show the heterogeneity and randomness or organisation of the endothelial cells. For example are any clusters of a subtype of endothelial cells around proliferating cells.
    3. The Notch signalling is an important aspect of this work, particularly evidence of lateral inhibition would have been of value. For example, one might expect cells adjacent to each other to have alternating high and low NICD. NICD staining alone does score the extent of the signalling because of many factors that can influence the transport of the cleaved NICD. Really a marker of Notch signalling downstream e.g. HES or HEY family ,DLL4 fis needed to give more information about this critical aspect.
    4. I really do not think that in Figure 5 it is justified to have a red line drawn through the cloud of points. The correlation coefficient is so low that this is meaningless. The failure to distinguish a P value from biological relevant is worrying. Much better comparison would have been between NICD staining and a downstream gene regulated by notch.
    5. It is important to know that the antibodies used for staining have be validated by the investigators. They would need to show a single band on Western blots or be able to block staining on immunohistochemistry. We all know the manufacturers can be unreliable and use high concentrations of proteins for Western blots. These should be added as a supplementary figure.

    Significance

    This represents a valuable and thorough methodology likely to be highly useful to many groups and show new insights into endothelial biology.

    Wide audience, cancer, cardiology, vascular disease-covid.

    My expertise >100 papers on angiogenis in cancer, basic mechanism, therapy models, bioinformatics IHC, patients, clinical trial. H score 190 Google Scholar

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