This repository contains custom code to reproduce main analyses in our manuscript, "Single-cell view of tumor microenvironment gradients in Pleural Mesothelioma"

Scripts are organized by figure and bulkRNA / scRNA analysis:

FIGURE_1_S1_main.R
FIGURE_2_S2_malignant.R
FIGURE_3_S3_stromal.R
FIGURE_4_S4_myeloid.R
FIGURE_5_S5_lymphoid.R
FIGURE_5_S5_pbmc.R
FIGURE_6_S6_nk.R
bulkRNA_analyses.R # including 1E, S1H-I, 2D-E/G, S2F-J, 3H, S3I-J, 4G, 5E/K, S5D, 6A, S6A-C

To download Seurat objects inclusive of all cell annotation and additional metadata, please download it from GEO GSE190597.

Installation

To install this repository, run the following command:

git clone https://github.com/TsankovLab/PM_scRNA_atlas.git

and download the seurat objects srt_tumor.rds and srt_pbmc.rds available at GSE190597 in same folder

Installing R packages

These are the R packages required to reproduce the scRNA and bulkRNA analyses. System Requirements:

Operating System: Tested on RedHat 7.7 x86_64.
R Version: 4.2.3. R Packages:
Seurat (v4.4.0) (https://satijalab.org/seurat/)
SeuratObject (v5.0.1)
scran (v1.26.0) (https://bioconductor.org/packages/release/bioc/vignettes/scran/inst/doc/scran.html) circlize (v0.4.15) (https://jokergoo.github.io/circlize_book/book/)
ComplexHeatmap (v2.14.0) (https://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html)
clusterProfiler (v4.6.0) (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)
ggpubr (v0.6.0) (https://rdrr.io/cran/ggpubr/)
biomaRt (v2.54.0) (https://bioconductor.riken.jp/packages/3.4/bioc/vignettes/biomaRt/inst/doc/biomaRt.html)
paletteer (v1.5.0) (https://emilhvitfeldt.github.io/paletteer/)
rstatix (v0.7.2) (https://rpkgs.datanovia.com/rstatix/)
readxl (v1.4.3) (https://readxl.tidyverse.org/)
harmony (v0.1.0) (https://portals.broadinstitute.org/harmony/articles/quickstart.html)
hdWGCNA (v0.2.18) (https://smorabit.github.io/hdWGCNA/)
ggplot2 (v3.4.4) (https://ggplot2.tidyverse.org)
tidyverse (v2.0.0) (https://www.tidyverse.org/)
patchwork (v1.1.2) (https://patchwork.data-imaginist.com/)
scRepertoire (v2.0.0) (https://github.com/ncborcherding/scRepertoire)
infercnv (v1.14.2) (https://github.com/broadinstitute/inferCNV/wiki)
DirichletReg (v0.7.1) (http://cran.nexr.com/web/packages/DirichletReg/vignettes/DirichletReg-vig.pdf)

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
if (!requireNamespace("devtools", quietly = TRUE))
    install.packages("devtools")
if (!requireNamespace("remotes", quietly = TRUE)) {
  install.packages("remotes")
}

# Analysis packages
install.packages("Seurat", version = "4.4.0")  
remotes::install_github("mojaveazure/seurat-disk", ref =  = "0.0.0.9020")  
BiocManager::install("scran")  
install.packages("harmony", version = "0.1.0")  
BiocManager::install("scRepertoire")  
BiocManager::install("clusterProfiler")  
BiocManager::install("biomaRt")  
install.packages ("DirichletReg", version="0.7.1")  
install.packages("survival", version = "3.5.5")  
install.packages("survminer", version = "0.4.9")  
devtools::install_github('smorabit/hdWGCNA', ref = 'v0.3.00')  
devtools::install_github("saeyslab/nichenetr", ref = "v2.0.4")  
BiocManager::install("infercnv")  


# Data manipulation  packages
install.packages("dplyr", version = "1.1.2")  
install.packages ("tidyverse", version = "2.0.0")  
install.packages ("readxl", version = "v1.4.3")  
BiocManager::install("GenomicFeatures")  
BiocManager::install("org.Hs.eg.db")  

# Visualization packages
install.packages ("circlize", version = "0.4.15")  
BiocManager::install("ComplexHeatmap")  
BiocManager::install("ggtree")  
install.packages("ggrepel", version = "0.9.3")  
install.packages("ggpubr", version = "0.6.0")  
install.packages("rstatix", version = "0.7.2"). 

# Palettes
install.packages ("RColorBrewer")  
install.packages ("viridis")  
install.packages("paletteer", version = "1.5.0")  
install.packages('http://cran.r-project.org/src/contrib/Archive/ggplot2/ggplot2_3.4.4.tar.gz', repos=NULL, type="source")  
install.packages ("gplots", version = "3.1.3")    
install.packages("ggridges", version = "0.5.4")  
install.packages ("ggExtra", version = "0.10.1")  
install.packages ("patchwork", version = "1.1.2")  
install.packages ("scCustomize")  
install.packages("pals")  
install.packages("ggthemes")

Installing python packages

conda env create -f 'PM_scRNA_atlas/scANVI_script/environment/scvi.yml'

Run Rscript convert_to_anndata.R to convert srt_tumor.rds and srt_pbmc.rds to .h5ad before running python scripts to reproduce scANVI integration UMAP plots.

Fetal endothelial analysis

For comparison analysis of PLVAP+ EC with fetal and adult normal lungs please download data from: https://fetal-lung.cellgeni.sanger.ac.uk/scRNA.html from study: He, Peng, et al. "A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates." Cell 185.25 (2022): 4841-4860.

and https://www.synapse.org/#!Synapse:syn21041850/wiki/600865 from: Travaglini, Kyle J., et al. "A molecular cell atlas of the human lung from single-cell RNA sequencing." Nature 587.7835 (2020): 619-625.

NK cells pan-cancer atlas

For comparison analysis of NK PM cells with NKs from an immune pan-cancer study (FIGURE 6C) please download immune pan-cancer altas from here: https://zenodo.org/records/5186413#%20.YRqbJC1h2v6 from following study: Nieto, Paula, et al. "A single-cell tumor immune atlas for precision oncology." Genome research 31.10 (2021): 1913-1926.