DOI: 10.5061/dryad.w6m905r0b
title: Data From: Matrix tropism influences endometriotic cell attachment patterns

description: Due to the extended period for clinical diagnosis, the etiology of
 endometriotic lesion initiation is not well understood or characterized.
 Endometriotic lesions are most often found on pelvic tissues and organs,
 especially the ovaries. To investigate the role of tissue tropism on
 ovarian endometrioma initiation, we adapted a well-characterized
 polyacrylamide microarray system to investigate the role of
 tissue-specific extracellular matrix and adhesion motifs on endometriotic
 cell attachment, morphology, and size. We report the influence of cell
 origin (endometriotic vs. non-endometriotic), substrate stiffness
 mimicking aging and fibrosis, and the role of multicellular
 (epithelial-stromal) cohorts on cell attachment patterns. We identify
 multiple ovarian-specific attachment motifs that significantly increase
 endometriotic (vs. non-endometriotic) cell cohort attachment that could be
 implicated in early disease etiology. 
description: (As per described methods in the manuscript)  <br>Details
 on cell culture, Polyacrylamide (PA) microarray fabrication, and PA
 microarray experimentation were provided in the methods section of the
 associated paper. Slides were immunostained for actin and cell nuclei,
 cells prior to seeding were treated with Cell Tracker. Cellular
 microarrays were imaged at 10x and 20x magnification on the Zeiss
 AxioScan.Z1 Slide Scanner. Images of entire arrays were converted to 8-bit
 TIFF files per output channel using Fiji (ImageJ) and cropped into single
 island images utilizing dextran-rhodamine marker islands to identify the
 location of arrayed conditions within each image. CellProfiler (version
 4.2.6) was used to identify and measure nuclei and fluorescent regions
 using the IdentifyPrimaryObjects, IdentifySecondaryObjects, and
 MeasureObjectSizeAndShape modules. Single-cell fluorescent intensity was
 quantified using the MeasureObjectIntensity module. The output from
 CellProfiler was loaded into RStudio for analysis. Cell location for
 patterning analysis was determined using coordinates output by
 CellProfiler. Cell location was assigned based on distance from the
 centroid. For co-culture analysis, the fluorescent intensity of the
 CellTracker dyes were rescaled to stretch each image to full intensity
 range using RescaleIntensity module and then binned for the expression of
 the pink and green channels by mean intensity using ClassifyObjects.
 Additionally, the comparison of mean intensity between the pink and green
 channels were classified as low-low, low-high, high-low, and high-high for
 mean intensity using the ClassifyObjects module. These classifications
 were used to create a decision tree that reliably assigned 95.41% of the
 total cells as either Stromal or Epithelial, with the remaining cells
 being categorized as Unassigned due to not fitting the criteria of the
 decision tree. For analysis, the number of cells attached to each island
 were counted and individual cell measurements were averaged together. Each
 representative island for a condition on a slide were averaged together to
 provide a per slide measurement.  Each island serves as a technical
 replicate of a condition on a slide. The multiple slides per experimental
 run serve as technical replicates of the culture conditions. 
description: # Data From: Matrix tropism influences endometriotic cell attachment
 patterns
 [https://doi.org/10.5061/dryad.w6m905r0b](https://doi.org/10.5061/dryad.w6m905r0b) ## Description of the data and file structure (As per described methods in the manuscript)    Endometriotic or non-endometriotic cells were cultured as described.  PA Microarrays were fabricated as previously described.   ### Files and variables For Cell Profiler Output files, there are many columns included in the CSV, this is the raw output from our analysis pipeline in Cell Profiler analyzing the cropped images of the slides included in this data.  A brief explanation of these files are included below: * ImageNumber & ObjectNumber - Metadata associating measurements with specific image and object  * Columns Starting with "Metadata_" - Metadata for future processing including Cell Type, Experiment, Scene, Row and Column. * Columns Starting with "AreaShape_" - These are measurements from the MeasureObjectSizeShape module in Cell Profiler. Measurements are in pixels, see the Cell Profiler manual for detailed explanation of measurements ([https://cellprofiler-manual.s3.amazonaws.com/CellProfiler-4.2.8/modules/measurement.html](https://cellprofiler-manual.s3.amazonaws.com/CellProfiler-4.2.8/modules/measurement.html))  * Columns Starting with "Intensity_" - These are measurements of intensity from the MeasureObjectIntensity module in Cell Profiler. These measurements are based on pixel intensities within an object. See the Cell Profiler manual for detailed explanation of measurements ([https://cellprofiler-manual.s3.amazonaws.com/CellProfiler-4.2.8/modules/measurement.html](https://cellprofiler-manual.s3.amazonaws.com/CellProfiler-4.2.8/modules/measurement.html))  * Columns Starting with "Location*" -* These are pixel coordinates (X,Y) of object centroids for each primary object (nuclei) and the center of mass for each secondary object (fluorescent stain). Additionally, there are Location_Center columns for the X and Y pixel coordinates for the center of the image. These are outputs from the IdentifyPrimaryObjects and IdentifySecondaryObjects modules in Cell Profiler. For ds_e_pheno CSVs, the unlabelled column is a row number from exporting a dataframe in R to a CSV. Metadata_ECMs is the protein condition that the cells were cultured on, Group is the assigned organ group those protein combinations are representative of (Liver, Ovary, or Ovary & Liver (or Common)). Metadata_Stiffness is the stiffness of the polyacrylamide gel (units of kPa) either 1 kPa or 6 kPa. Metadata_Experiment is the experiment (biological replicate) for each condition. Metadata_CellType is the cell type of each measurement: 12z or EndoE (endometriotic epithelial cells), hEEC or NonEndoE (non-endometriotic epithelial cells), hESC or NonEndoS (non-endometriotic stromal cells), and iEC-ESC or EndoS (endometriotic stromal cells). Mean_Blue is the average fluorescent intensity of the nuclei stain (DAPI) and SD_Blue is the standard deviation of the intensity of the nuclear stain (this also applies to the other fluorescent channels i.e. Mean_Green is the average fluorescent intensity of the green channel's stain). Mean_Object_Counts and SD_Object_Counts are the average number of cells per island and the standard deviation of this measurement. Abb_ECMs is the abbreviated form of the Metadata_ECMs for ease of use in graphing. #### File Figure_2.zip **Description:** Contains files for cell counts (nuclei counts), data summarized to slides, and raw image (.tiff) files pertaining to Figure 2. This zip folder contains 2 subfolders. ***Subfolder “CadherinCAM”*** – Includes files related to Cell Adhesion Molecule (CAM) and Cadherin troubleshooting. This subfolder contains 27 items. * File “Cell_Profiler_Output_blue_objects” – This includes the blue objects (nuclei) counts output from Cell Profiler  * File “Summarized_Results_ds_e_pheno” – This includes data summarized to slides   * Files “.tiff files” – include all 25 images taken of the slides to be processed in Cell Profiler and MatLab (Images TIFF files) ***Subfolder “Full ECM Studies”*** – Includes files related to down-selection studies. There are 179 total items. * File “e1_blue_objects” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 1 for the Full ECM Studies.  * File “e2_blue_objects” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 2 for the Full ECM Studies.  * File “full_ds_e_pheno_combined” - This includes data summarized to slides combining experiments 1 & 2 from Cell Profiler from experiment 1 for the Full ECM Studies.  * Files “.tiff files” – include all 175 images taken of the slides separated by array replicate (scene) to be processed in Cell Profiler and MatLab (Images TIFF files)  #### File Figure_6_Co-culture.zip **Description:** Contains subfolders for cell counts (nuclei counts), data summarized to slides, and raw image (.tiff) files pertaining to co-culture studies in Figure 6. This zip folder contains 1 subfolder and 7 CSV files. * File “blue_objects” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 1-3 for the co-culture studies.  * File “green_objects” - This includes the green objects (stromal cells) counts output from Cell Profiler from experiment 1-3 for the co-culture studies.  * File “pink_objects” - This includes the pink objects (epithelial cells) counts output from Cell Profiler from experiment 1-3 for the co-culture studies.  * File “red_objects” - This includes the red objects (actin) counts output from Cell Profiler from experiment 1-3 for the co-culture studies.  * File “decision.csv” - This includes the decisions made to differentiate the two cell types from each other including the label “epithelial”, “stromal”, or “not identified” from experiment 1-3 for the co-culture studies. This is a similar layout to the blue_objects output from Cell Profiler but with an additional module readout starting with "Classify_" which is an output of the ClassifyObjects module which grouped each object into one of four classifications based on fluorescent intensity of the green and pink channels or binned the fluorescent intensity of each object into 4 levels in each channel. After output from Cell Profiler, the following columns were created through analysis in R Studio. The PinkClass, GreenClass, and GroupClass columns summarize these columns into abbreviations. PinkInt and GreenInt are the average intensity of the respective channel for the secondary object while PinkIntNuc and GreenIntNuc is the average intensity of the respective channel for the primary object (nuclei). IntClass is the assignment based on the comparison of PinkClass and GreenClass. NucIntClass is the assignment based on the comparison of GreenIntNuc and PinkIntNuc. Final_Cell_Assignment is the result of a decision tree based on IntClass and NucIntClass. IntClassPlusNuc is the concatenation of the results of IntClass and NucIntClass for checking the Final_Cell_Assignment column result. Epithelial, Stromal, and Unassigned are the final categorization of each object with a 1 representing that is the assignment of each cell with 0 in the other two columns.  * File “ds_e_pheno” - This includes data summarized to slides from Cell Profiler from all experiments for the co-culture studies.  * File “ds_e_MCred” - This includes summarized to slides for the “red objects” from Cell Profiler from all experiments for the co-culture studies.  ***Subfolder “Images”*** - Contains subfolders divided by experiment for co-culture studies.  * Subfolder “e1” - Contains 241 raw image (.tiff) files pertaining to experiment 1 of the co-culture studies.  * Subfolder “e2” - Contains 243 raw image (.tiff) files pertaining to experiment 2 of the co-culture studies.  * Subfolder “e3” - Contains 240 raw image (.tiff) files pertaining to experiment 3 of the co-culture studies.   #### File Figures_3-5_Monoculture.zip **Description:** Contains subfolders for cell counts (nuclei counts), data summarized to slides, and raw image (.tiff) files pertaining to monoculture studies in Figures 3-5.  ***Subfolder “Attachment & Morphology”*** - Contains files for cell counts (nuclei counts) and data summarized to slides pertaining to Monoculture Studies. There are 9 total files in this subfolder. * File “blue_objects_e1” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 1 for the Monoculture Studies.  * File “cell_e1” - This includes the red objects (actin) counts output from Cell Profiler from experiment 1 for the Monoculture Studies.  * File “blue_objects_e2” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 2 for the Monoculture Studies.  * File “cell_e2” - This includes the red objects (actin) counts output from Cell Profiler from experiment 2 for the Monoculture Studies.  * File “blue_objects_e3” - This includes the blue objects (nuclei) counts output from Cell Profiler from experiment 3 for the Monoculture Studies.  * File “cell_e3” - This includes the red objects (actin) counts output from Cell Profiler from experiment 3 for the Monoculture Studies.  * File “e1_ds_e_pheno” - This includes data summarized to slides from Cell Profiler from experiment 1 for the Monoculture Studies.  * File “e2_ds_e_pheno” - This includes data summarized to slides from Cell Profiler from experiment 2 for the Monoculture Studies.  * File “e3_ds_e_pheno” - This includes data summarized to slides from Cell Profiler from experiment 3 for the Monoculture Studies.  ***Subfolder “Images”*** - Contains 3 subfolders that contain raw image (.tiff) files pertaining to Monoculture Studies.  * Subfolder “e1” - Contains 201 raw image (.tiff) files pertaining to experiment 1 of the monoculture studies.  * Subfolder “e2” - Contains 228 raw image (.tiff) files pertaining to experiment 2 of the monoculture studies.  * Subfolder “e3” - Contains 210 raw image (.tiff) files pertaining to experiment 3 of the monoculture studies.   ## Code/software The analysis was performed on R Studio using openly available libraries. Packages include: tidyr, ggplot, ggprism, ggpubr, dplyr, viridis, lme4, sjPlot. Additionally, Cell Profiler was used to perform the image analysis and to generate the data to be analyzed in R. R Projects for Analysis By Figure:  1. No R Projects (Illustrative Figure) 2. Cadherins/CAM and Full Scale Arrays 1. HT12-10-22 - Cadherins/CAM Arrays 2.  HTOLe1 - Full Array e1 analysis 3. HTOLe2 R Analysis - Full Array e2 analysis 4. HTOL Combined R Analysis - Combining biological replicates of full arrays 3. Figures 3-5 Monoculture Experiments 1. HTOL Reduced e1 R Analysis  2. HTOL Reduced e2 R Analysis 3. HTOL Reduced e3 R Analysis 4. HTOL Reduced Combined Analysis - Combining biological replicates of reduced monoculture arrays 4. Figure 6 Coculture Experiments 1. CoCultureAnalysis - Combined biological replicates of coculture experiments including cell assignment work and comparison to monoculture 2. Actin Analysis - Phenotype Analysis utilizing actin stain ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A ## Usage Notes R Studio is a free and open software platform that can be used to look at the software files included in this repository. The images included in this repository are all in TiFF format which can be opened in any standard image viewer, for detailed information users can open the files in ImageJ (FIJI) which is a free and open image analysis software. Cell Profiler is also a free and open software that can be used for further analysis on the images. All data files are CSVs so that the data can be accessed using any software that reads CSVs.  ## Human subjects data The cell lines used in this work were originally obtained from human subjects by other researchers and were de-identified at that time. There are no human subjects in this work as we used previously de-identified cell lines.