Last updated: 2021-02-05

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Knit directory: melanoma_publication_old_data/

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    Ignored:    data/200323_TMA_256_Hot Cold_Clinical Data_Updated Response Data_For Collaborators_latest updated_Mar_2020_for_Coxph_modeling.csv
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    Ignored:    data/density_infiltration_BlockID.csv
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    Ignored:    data/layer_1_classification_protein.csv
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File Version Author Date Message
Rmd 20a1458 toobiwankenobi 2021-02-04 adapt figure order
Rmd f9bb33a toobiwankenobi 2021-02-04 new Figure 5 and minor changes in figure order
Rmd 9442cb9 toobiwankenobi 2020-12-22 add all new files
Rmd d8c7699 Tobias Hoch 2020-10-23 adapt figure 1 and supp fig 6
Rmd 1af3353 toobiwankenobi 2020-10-16 add stuff
Rmd d8819f2 toobiwankenobi 2020-10-08 read new data (nuclei expansion) and adapt scripts
Rmd 2c11d5c toobiwankenobi 2020-08-05 add new scripts

Introduction

This script generates plots for Figure 1.

Preparations

knitr::opts_chunk$set(echo = TRUE, message= FALSE)
knitr::opts_knit$set(root.dir = rprojroot::find_rstudio_root_file())

Load libraries

library(SingleCellExperiment)
library(data.table)
library(scater)
library(ggplot2)
library(dittoSeq)
library(cba)
library(ComplexHeatmap)
library(corrplot)
library(dplyr)
library(reshape2)
library(tidyverse)
library(rms)
library(cytomapper)
library(ggrepel)
library(circlize)
library(ggbeeswarm)
library(dendextend)

Load data

sce_rna <- readRDS("data/sce_RNA.rds")
sce_prot <- readRDS("data/sce_protein.rds")

Figure 1C

Heatmap showing marker expression of celltypes

# add marker expression to cells
marker_expression <- data.frame(t(assay(sce_rna[rowData(sce_rna)$good_marker,], "asinh")))
marker_expression$cellID <- rownames(marker_expression)
dat <- data.frame(colData(sce_rna))[,c("cellID", "celltype")]
dat <- left_join(dat, marker_expression)
dat$cellID <- NULL

# aggregate the groups
dat_aggr <- dat %>%
  group_by(celltype) %>%
  summarise_all(funs(mean))
Warning: `funs()` is deprecated as of dplyr 0.8.0.
Please use a list of either functions or lambdas: 

  # Simple named list: 
  list(mean = mean, median = median)

  # Auto named with `tibble::lst()`: 
  tibble::lst(mean, median)

  # Using lambdas
  list(~ mean(., trim = .2), ~ median(., na.rm = TRUE))
This warning is displayed once every 8 hours.
Call `lifecycle::last_warnings()` to see where this warning was generated.
# number of cells per group
dat_sum <- dat %>%
  group_by(celltype) %>%
  summarise(n=n())

dat_sum <- data.frame(t(dat_sum))

# scale and center expression
dat_aggr[,-c(1)] <- scale(dat_aggr[,-c(1)])

# create matrix
m <- as.matrix(t(dat_aggr[,-c(1)]))
colnames(m) <- dat_aggr$celltype

# top annotation with number of cells
ha <- HeatmapAnnotation("Number of Cells" = anno_barplot(ifelse(as.numeric(dat_sum[2,])>100000, 101000, as.numeric(dat_sum[2,])),
                                                         height = unit(3,"cm"),
                                                         ylim = range(0,100000),
                                                         gp = gpar(fill = ifelse(as.numeric(dat_sum[2,]) > 100000, "white", "black"),
                                                                   col="white"),
                                                         axis_param = list(gp = gpar(fontsize=14))),
                        "Numbers" = anno_text(round(as.numeric(dat_sum[2,])), 
                                                   which = "column", 
                                                   rot = 0, 
                                                   just = "center", 
                                                   location = 0.5,
                                                   gp = gpar(fontsize=10,col = ifelse(as.numeric(dat_sum[2,]) > 100000, "red", "black"))),
                        annotation_name_gp = gpar(fontsize = 16))

# row_split for markers
rowData(sce_rna)$heatmap_relevance <- ""
rowData(sce_rna[rowData(sce_rna)$good_marker,])$heatmap_relevance <- "lineage"
rowData(sce_rna[grepl("CXCL|CCL|DapB", rownames(sce_rna)),])$heatmap_relevance <- "chemokine"
rowData(sce_rna[grepl("B2M|GLUT1|CD134|Lag3|CD163|cleavedPARP|pRB", rownames(sce_rna)),])$heatmap_relevance <- "other"

# plot heatmap
h <- Heatmap(m, name = "Scaled Expression",
             row_split = rowData(sce_rna[rowData(sce_rna)$good_marker,])$heatmap_relevance,
             cluster_columns = TRUE,
             show_column_dend = FALSE,
             column_names_gp = gpar(fontsize=18),
             column_names_rot = 45,
             column_names_centered = TRUE,
             show_column_names = TRUE,
             top_annotation = ha,
             row_names_gp = gpar(fontsize = 16),
             row_title_gp = gpar(fontsize = 23),
             col = colorRamp2(c(-2, 0, 2), c("blue", "white", "red")),
             heatmap_legend_param = list(at = c(-2:2), legend_width = unit(6,"cm"), 
                                         direction="horizontal", title_gp = gpar(fontsize=16),
                                         labels_gp = gpar(fontsize=12)),
             column_names_side = "top",
             height = unit(20, "cm"),
             width = unit(17,"cm"))

draw(h, heatmap_legend_side = "bottom")

Heatmap showing marker expression of celltypes

# add marker expression to cells
marker_expression <- data.frame(t(assay(sce_prot[rowData(sce_prot)$good_marker,], "asinh")))
marker_expression$cellID <- rownames(marker_expression)
dat <- data.frame(colData(sce_prot))[,c("cellID", "celltype")]
dat <- left_join(dat, marker_expression)
dat$cellID <- NULL

# aggregate the groups
dat_aggr <- dat %>%
  group_by(celltype) %>%
  summarise_all(funs(mean))

# number of cells per group
dat_sum <- dat %>%
  group_by(celltype) %>%
  summarise(n=n())

dat_sum <- data.frame(t(dat_sum))

# scale and center expression
dat_aggr[,-c(1)] <- scale(dat_aggr[,-c(1)])

# create matrix
m <- as.matrix(t(dat_aggr[,-c(1)]))
colnames(m) <- dat_aggr$celltype

# top annotation with number of cells
ha <- HeatmapAnnotation("Number of Cells" = anno_barplot(ifelse(as.numeric(dat_sum[2,])>70000, 70000, as.numeric(dat_sum[2,])),
                                                         height = unit(3,"cm"),
                                                         ylim = range(0,70000),
                                                         gp = gpar(fill = ifelse(as.numeric(dat_sum[2,]) > 70000, "white", "black"),
                                                                   col = "white"),
                                                         axis_param = list(gp = gpar(fontsize=14))),
                        "Numbers" = anno_text(round(as.numeric(dat_sum[2,])), 
                                                   which = "column", 
                                                   rot = 0, 
                                                   just = "center", 
                                                   location = 0.5,
                                                   gp = gpar(fontsize=10,col = ifelse(as.numeric(dat_sum[2,]) > 70000, "red", "black"))),
                        annotation_name_gp = gpar(fontsize = 16))

# row_split for markers
rowData(sce_prot)$heatmap_relevance <- ""
rowData(sce_prot[rowData(sce_prot)$good_marker,])$heatmap_relevance <- "lineage"
rowData(sce_prot[grepl("PDL1|CD11b|CD206|PARP|CXCR2|CD11c|pS6|GrzB|IDO1|CD45RA|H3K27me3|TCF7|CD45RO|PD1|pERK|ICOS|Ki67", rownames(sce_prot)),])$heatmap_relevance <- "other"

# plot heatmap
h <- Heatmap(m, name = "Scaled Expression",
             row_split = rowData(sce_prot[rowData(sce_prot)$good_marker,])$heatmap_relevance,
             cluster_columns = TRUE,
             show_column_dend = FALSE,
             column_names_gp = gpar(fontsize=18),
             column_names_rot = 45,
             column_names_centered = TRUE,
             show_column_names = TRUE,
             row_names_gp = gpar(fontsize = 16),
             row_title_gp = gpar(fontsize = 23),
             top_annotation = ha,
             col = colorRamp2(c(-2, 0, 2), c("blue", "white", "red")),
             heatmap_legend_param = list(at = c(-2:2), legend_width = unit(6,"cm"), 
                                         direction="horizontal", title_gp = gpar(fontsize=16),
                                         labels_gp = gpar(fontsize=12)),
             column_names_side = "top",
             height = unit(20, "cm"),
             width = unit(17,"cm"))

draw(h, heatmap_legend_side = "bottom")

Figure 1D

Cell Type Correlation between the two data sets

cur_rna <- data.frame(colData(sce_rna))
cur_prot <- data.frame(colData(sce_prot))

rna_sum <- cur_rna %>%
  group_by(Description, celltype) %>%
  summarise(n = n()) %>%
  mutate(fraction = n/sum(n)) %>%
  reshape2::dcast(Description ~ celltype, value.var = "fraction", fill = 0)

prot_sum <- cur_prot %>%
  group_by(Description, celltype) %>%
  summarise(n = n()) %>%
  mutate(fraction = n/sum(n)) %>%
  reshape2::dcast(Description ~ celltype, value.var = "fraction", fill = 0)

# Correlation Plot - shared celltypes
shared <- c("Macrophage", "Neutrophil", "Tcytotoxic", "Tumor")

corrplot(cor(rna_sum[,-1], prot_sum[,-1], method = "pearson"), 
         addCoef.col = "white",
         method = "circle",
         tl.col="black",
         tl.cex = 1.5)
# Mean Correlation between shared celltypes
mean(c(cor(rna_sum$Macrophage, prot_sum$Macrophage, method = "pearson"), 
       cor(rna_sum$Neutrophil, prot_sum$Neutrophil, method = "pearson"), 
       cor(rna_sum$Tcytotoxic, prot_sum$Tcytotoxic, method = "pearson"), 
       cor(rna_sum$Tumor, prot_sum$Tumor, method = "pearson")))
[1] 0.9389364

Figure 1E

Load masks

all_mask <- loadImages(x = "/home/ubuntu/bbvolume/Data/Analysis/melanoma_cohort_new_cp_pipeline/protein/cpout/",
                       pattern = "ilastik_s2_Probabilities_equalized_cellmask.tiff")

add the ImageNumber to masks

# we load the metadata for the images.
image_mat <- as.data.frame(read.csv(file = "data/protein/Image.csv",stringsAsFactors = FALSE))

# we extract only the FileNames of the masks as they are in the all_masks object
cur_df <- data.frame(cellmask = image_mat$FileName_cellmask,
                     ImageNumber = image_mat$ImageNumber,
                     Description = image_mat$Metadata_Description)

# we set the rownames of the extracted data to be equal to the names of all_masks
rownames(cur_df) <- gsub(pattern = ".tiff",replacement = "",image_mat$FileName_cellmask)

# we add the extracted information via mcols in the order of the all_masks object
mcols(all_mask) <- cur_df[names(all_mask),]

scale the masks

all_mask <- scaleImages(all_mask,2^16-1)

Plot two example Images

# subset masks
mask_sub <- all_mask[mcols(all_mask)$Description %in% c("H9", "L7", "H2")]
sce_prot_sub <- sce_prot[,sce_prot$Description %in% c("H9", "L7", "H2")]

# rename all cells that are not tumor and not tcytotoxic
#sce_prot_sub$celltype <- ifelse(sce_prot_sub$celltype %in% c("Tumor", "Tcytotoxic"), sce_prot_sub$celltype, "other")

col_list <- list()
col_list$`Cell Type` <- metadata(sce_prot)$colour_vectors$celltype

sce_prot_sub$`Cell Type` <- sce_prot_sub$celltype

plotCells(mask = mask_sub, 
          object = sce_prot_sub,
          cell_id = "CellNumber", img_id = "Description", 
          colour_by = "Cell Type",
          colour = col_list,
          display = "single")

Figure 1F

Barplot containing Cell Types per Image

# Create table with celltype fractions
cur_df <- data.frame(celltype = sce_prot$celltype,
                     Description = sce_prot$Description,
                     Location = sce_prot$Location)

# remove control samples 
cur_df <- cur_df %>%
  filter(Location != "CTRL") %>%
  group_by(Description, celltype) %>%
  summarise(n=n()) %>% 
  group_by(Description) %>%
  mutate(fraction = n / sum(n)) %>%
  reshape2::dcast(Description ~ celltype, value.var = "fraction", fill=0)

matrixrownames <- cur_df$Description 

# now we create a matrix from the data and cluster the data based on the cell fractions
hm_dat = as.matrix(cur_df[,-1])
rownames(hm_dat) <- as.character(matrixrownames)

# calculate distance and then cluster images based on cluster fraction
dd <- dist((hm_dat), method = "euclidean")
hc <- hclust(dd, method = "ward.D2")

# order optimal orders the "leafs" of the cluster tree optimally to achieve smooth transitions
hc$order = order.optimal(dd, hc$merge)$order
row_sorted <- hc$labels[hc$order]

# now we generate the clinical metadata and order it in the same way as the celltype data
patient_meta <- metadata(sce_prot)
patient_meta <- data.frame(patient_meta[c("Description", "MM_location_simplified", "Location")])
patient_meta <- patient_meta %>%
  filter(Location != "CTRL")

mrownames <- patient_meta$Description
patient_meta <- as.matrix(patient_meta)
rownames(patient_meta) <- mrownames
patient_meta <- data.frame(patient_meta[row_sorted,])

# generate the barplot. this is generated as the annotation for the heatmap of the Patient_ID that is generated below.
hm_dat <- hm_dat[row_sorted,]

# bring cell types in order (column order)
col_order <- c("Tumor", "Macrophage", "Neutrophil", "Tcytotoxic", "Thelper", "Tregulatory", "Bcell", "BnTcell", "pDC", "Stroma", "unknown")
hm_dat <- hm_dat[, col_order]

col_vector <- metadata(sce_prot)$colour_vector$celltype[colnames(hm_dat)]
col_vector_location <- structure(c("blue", "red", "green"), 
                                 names = c("LN", "other", "skin"))

# rename punch locations
patient_meta[patient_meta$Location == "M", ]$Location <- "margin"
patient_meta[patient_meta$Location == "C", ]$Location <- "core"

col_vector_punch <- structure(c("black", "grey"), 
                                 names = c("core", "margin"))

# create annotation with cell type proportions
ha <- rowAnnotation(`Punch Location` = patient_meta[,c("Location")],
                    `Cell Type Proportion` =
                      anno_barplot(hm_dat, 
                                   gp=gpar(fill=col_vector),
                                   bar_width = 1,
                                   height = unit(30,"cm"),
                                   width = unit(15,"cm"),
                                   show_row_names = FALSE),
                        col = list(`Punch Location` = col_vector_punch),
                    show_legend = FALSE)


dend <- as.dendrogram(hclust(dist(hm_dat, method = "euclidean"), method = "ward.D2"), ylim = c(0,10))
dend <- color_branches(dend, k = 4, groupLabels = TRUE) # `color_branches()` returns a dendrogram object
#plot(dend)

# heatmap consisting of the patient_IDs. one color per patient
h1 = Heatmap(patient_meta[,c("MM_location_simplified")], 
             col = col_vector_location, 
             width = unit(0.5, "cm"), 
             cluster_rows = dend,
             row_dend_width = unit(3, "cm"),
             height = unit(30, "cm"),
             show_heatmap_legend = FALSE, 
             heatmap_legend_param = list(title = "Met Location"),
             row_names_gp = gpar(cex=0.5),
             show_row_names = TRUE,
             right_annotation =  ha,
             column_labels = "Met Location")

# plot the data
ht = grid.grabExpr(draw(h1))
grid.newpage()
pushViewport(viewport(angle = 270))
grid.draw(ht)

Legend for Barplot

lgd1 <- Legend(labels = names(col_vector), 
             title = "Cell Type", 
             legend_gp = gpar(fill = col_vector),
             ncol = 1)

lgd2 <- Legend(labels = names(col_vector_location), 
             legend_gp = gpar(fill = unname(col_vector_location)),
             title = "Met Location",
             ncol = 1)

lgd3 <- Legend(labels = names(col_vector_punch), 
             legend_gp = gpar(fill = unname(col_vector_punch)),
             title = "Punch Location",
             ncol = 1)

pd = packLegend(lgd1, lgd2, lgd3, direction = "vertical", 
    column_gap = unit(0.5, "cm"))
draw(pd)

sessionInfo()
R version 4.0.3 (2020-10-10)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04 LTS

Matrix products: default
BLAS/LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.8.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=C             
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
 [1] grid      parallel  stats4    stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] dendextend_1.14.0           ggbeeswarm_0.6.0           
 [3] circlize_0.4.12             ggrepel_0.9.0              
 [5] cytomapper_1.3.1            EBImage_4.32.0             
 [7] rms_6.1-0                   SparseM_1.78               
 [9] Hmisc_4.4-2                 Formula_1.2-4              
[11] survival_3.2-7              lattice_0.20-41            
[13] forcats_0.5.0               stringr_1.4.0              
[15] purrr_0.3.4                 readr_1.4.0                
[17] tidyr_1.1.2                 tibble_3.0.4               
[19] tidyverse_1.3.0             reshape2_1.4.4             
[21] dplyr_1.0.2                 corrplot_0.84              
[23] ComplexHeatmap_2.4.3        cba_0.2-21                 
[25] proxy_0.4-24                dittoSeq_1.0.2             
[27] scater_1.16.2               ggplot2_3.3.3              
[29] data.table_1.13.6           SingleCellExperiment_1.12.0
[31] SummarizedExperiment_1.20.0 Biobase_2.50.0             
[33] GenomicRanges_1.42.0        GenomeInfoDb_1.26.2        
[35] IRanges_2.24.1              S4Vectors_0.28.1           
[37] BiocGenerics_0.36.0         MatrixGenerics_1.2.0       
[39] matrixStats_0.57.0          workflowr_1.6.2            

loaded via a namespace (and not attached):
  [1] readxl_1.3.1              backports_1.2.1          
  [3] systemfonts_0.3.2         plyr_1.8.6               
  [5] sp_1.4-5                  shinydashboard_0.7.1     
  [7] splines_4.0.3             BiocParallel_1.22.0      
  [9] TH.data_1.0-10            digest_0.6.27            
 [11] htmltools_0.5.0           tiff_0.1-6               
 [13] viridis_0.5.1             fansi_0.4.1              
 [15] magrittr_2.0.1            checkmate_2.0.0          
 [17] cluster_2.1.0             limma_3.44.3             
 [19] modelr_0.1.8              svgPanZoom_0.3.4         
 [21] svglite_1.2.3.2           sandwich_3.0-0           
 [23] jpeg_0.1-8.1              colorspace_2.0-0         
 [25] rvest_0.3.6               haven_2.3.1              
 [27] xfun_0.20                 crayon_1.3.4             
 [29] RCurl_1.98-1.2            jsonlite_1.7.2           
 [31] zoo_1.8-8                 glue_1.4.2               
 [33] gtable_0.3.0              zlibbioc_1.36.0          
 [35] XVector_0.30.0            MatrixModels_0.4-1       
 [37] GetoptLong_1.0.5          DelayedArray_0.16.0      
 [39] BiocSingular_1.4.0        shape_1.4.5              
 [41] abind_1.4-5               scales_1.1.1             
 [43] mvtnorm_1.1-1             pheatmap_1.0.12          
 [45] DBI_1.1.0                 edgeR_3.30.3             
 [47] Rcpp_1.0.5                xtable_1.8-4             
 [49] viridisLite_0.3.0         htmlTable_2.1.0          
 [51] clue_0.3-58               foreign_0.8-81           
 [53] rsvd_1.0.3                htmlwidgets_1.5.3        
 [55] httr_1.4.2                RColorBrewer_1.1-2       
 [57] ellipsis_0.3.1            pkgconfig_2.0.3          
 [59] nnet_7.3-14               dbplyr_2.0.0             
 [61] locfit_1.5-9.4            tidyselect_1.1.0         
 [63] rlang_0.4.10              later_1.1.0.1            
 [65] munsell_0.5.0             cellranger_1.1.0         
 [67] tools_4.0.3               cli_2.2.0                
 [69] generics_0.1.0            broom_0.7.3              
 [71] ggridges_0.5.3            fastmap_1.0.1            
 [73] fftwtools_0.9-9           evaluate_0.14            
 [75] yaml_2.2.1                knitr_1.30               
 [77] fs_1.5.0                  nlme_3.1-151             
 [79] mime_0.9                  quantreg_5.82            
 [81] whisker_0.4               xml2_1.3.2               
 [83] compiler_4.0.3            rstudioapi_0.13          
 [85] beeswarm_0.2.3            png_0.1-7                
 [87] reprex_0.3.0              stringi_1.5.3            
 [89] gdtools_0.2.3             Matrix_1.3-2             
 [91] vctrs_0.3.6               pillar_1.4.7             
 [93] lifecycle_0.2.0           GlobalOptions_0.1.2      
 [95] BiocNeighbors_1.6.0       conquer_1.0.2            
 [97] cowplot_1.1.1             bitops_1.0-6             
 [99] irlba_2.3.3               raster_3.4-5             
[101] httpuv_1.5.4              R6_2.5.0                 
[103] latticeExtra_0.6-29       promises_1.1.1           
[105] gridExtra_2.3             vipor_0.4.5              
[107] codetools_0.2-18          polspline_1.1.19         
[109] MASS_7.3-53               assertthat_0.2.1         
[111] rprojroot_2.0.2           rjson_0.2.20             
[113] withr_2.3.0               multcomp_1.4-15          
[115] GenomeInfoDbData_1.2.4    hms_0.5.3                
[117] rpart_4.1-15              rmarkdown_2.6            
[119] DelayedMatrixStats_1.10.1 git2r_0.28.0             
[121] shiny_1.5.0               lubridate_1.7.9.2        
[123] base64enc_0.1-3