hello，大家好，今天我们来分享一下细胞通讯的文章CellChat，其实有关这个软件已经有了很多的文章来介绍了，2020年的6月份我和Cellchat的作者聊过一次，当时感觉Cellchat这个软件很友好，其实后来做分析一直在用，文章在Inference and analysis of cell-cell communication using CellChat，2021年3月份发表于NC，影响因子12分，我们还是先来分享文章，最后看看示例代码。

Abstract

Understanding global communications among cells requires accurate representation of cell-cell signaling links and effective systems-level analyses of those links（这里其实就是需要我们先验的配受体对），We constructed a database of interactions among ligands, receptors and their cofactors that accurately represents known heteromeric molecular complexes（配受体复合物确实也需要很好的考虑），然后作者开发了Cellchat，用来推断单细胞细胞类型之间的通讯网络，CellChat predicts major signaling inputs and outputs for cells and how those cells and signals coordinate for functions using network analysis and pattern recognition approaches.通过多方面的学习和定量对比，CellChat可以对信号传导途径进行分类，并描述不同数据集中的保守途径和特定背景的途径，当然，文章把这个软件运用到了一些现有的数据中，效果很不错。（每个软件都是这么说的）。

introduction

这个地方我们提炼一下：

• While most current scRNA-seq data analysis approaches allow detailed cataloging of cell types and prediction of cellular differentiation trajectories, they have limited capability in probing underlying cellular communications that often drive heterogeneity and cell state transitions
• scRNA-seq data inherently contains gene expression information that could be used to infer such cellular communications
  现有软件的一些缺点：
• these and other similar methods usually use only one ligand /one receptor gene pairs, often neglecting that many receptors function as multi-subunit complexes.（cellphoneDB是考虑到配受体复合物的，用复合物表达最少的成分作为该配体或者受体的表达量）。
• it does so without considering other important signaling cofactors，including soluble agonists（激动剂）, antagonist（拮抗剂）, as well as stimulatory（刺激性） and inhibitory（异质性） membrane-bound co-receptors.
• can not systematically curated classification of ligand-receptor pairs into functionally related signaling pathways（当然，这就成了cellchat要改进的地方）。
• （不能）自分泌和旁分泌信号相互作用的直观可视化
• （不能）systems approaches in analyzing complex cell-cell communication。
• （不能）capability of accessing signaling crosstalk for continuous cell state trajectories given the fact that biological variability between cells can be discrete or continuous.

Cellchat的优势

• we manually curated a comprehensive signaling molecule interaction database that takes into the account the known structural composition of ligand-receptor interactions, such as multimeric ligand-receptor complexes, soluble agonists and antagonists, as well as stimulatory and inhibitory coligands and co-receptors.（我们人工整理了一个综合的信号分子相互作用数据库，其中考虑了配体-受体相互作用的已知结构组成，例如多聚体配体-受体复合物，可溶性激动剂和拮抗剂，以及刺激性和抑制性大肠菌素和共受体。），划分很精细。
• CellChat infers cell-state specific signaling communications within a given scRNA-seq data using mass action models, along with differential expression analysis and statistical tests on cell groups, which can be both discrete states or continuous states along the pseudotime cell trajectory（这一点需要大家在实践中多多检验了）。
• 可视化做的相当不错

Result

Overview of CellChat

CellChat requires gene expression data of cells as the user input and models the probability of cell-cell communication by integrating gene expression with prior knowledge of the interactions between signaling ligands, receptors and their cofactors（这一点所有软件都差不多），我们来看看分析过程。

图片.png

• 输入细胞矩阵信息
• 细胞通讯模型（这里有一个mass action，大家可以参考质量作用定律）
• 通讯分析，过程很简单，跟大多数软件一样。
  一、Cellchat配受体库的特点:
• 1、which takes into account known composition of the ligand-receptor complexes, including complexes with multimeric ligands and receptors, as well as several cofactors types: soluble agonists, antagonists, co-stimulatory and co-inhibitory membrane-bound receptors。
• 2、参考了KEGG通路
• 3、包括最近的experimental studies
• 4、contains 2,021 validated molecular interactions, including 60% of paracrine/autocrine signaling interactions, 21% of extracellular matrix (ECM)-receptor interactions and 19% of cell-cell contact interactions.（分类精细）。48% of the interactions involve heteromeric molecular complexes and 25% of the interactions are curated by us from recent literature
• 5、each interaction was manually classified into one of the 229 functionally related signaling pathways based on the literature（生物学功能通路的划分）。
  二、推断方法：
• CellChat identifies differentially over-expressed ligands and receptors for each cell group（差异基因，都是这么做的）。
• modeled by the law of mass action（质量作用定律） based on the average expression values of a ligand by one cell group and that of a receptor by another cell group as well as their cofactors。
• 挑选出来的配受体对进行置换检验。
• An intercellular communication network is a weighted directed graph composed of significant connections between interacting cell groups.（可视化）。
  三、Quantitative analysis of intercellular communications.
• it can determine major signaling sources and targets as well as mediators and influencers within a given signaling network using centrality measures from network analysis, such as out-degree, in-degree, betweenness and information metrics。
• it can predict key incoming and outgoing signals for specific cell groups as well as global communication patterns by leveraging pattern recognition approaches(这些都很简单)。
• it can groups signaling pathways by defining similarity measures and performing manifold learning from both functional and topological perspectives
• it can delineate conserved and context-specific signaling pathways by joint manifold learning of multiple networks（最后一点感觉就是凑数的）。

接下来时例子了，我们来看一下，CellChat identifies communication patterns and predicts functions for poorly studied pathways（需要注意，这些数据全是定义好的）。

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配受体的识别及可视化。

CellChat reveals continuous cell lineage-associated signaling events

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CellChat predicts key signaling events between spatially colocalized cell populations

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Joint learning of time-course scRNA-seq data to uncover dynamic communication patterns

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Joint learning of conserved and context-specific communication patterns between distinct scRNA-seq datasets

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重点来了，Method

Inference of intercellular communications

1、Identification of differentially expressed signaling genes. To infer the cell state-specific communications, we first identified differentially expressed signaling genes across all cell groups within a given scRNA-seq dataset, using the Wilcoxon rank sum test with the significance level of 0.05(这里建议大家设的再小一点)。
2、Calculation of ensemble average expression. To account for the noise effects, we calculated the ensemble average expression of signaling genes in a given cell group using a statistically robust mean method：

图片.png
where Q1, Q2 and Q3 is the first, second and third quartile of the expression levels of a signaling gene in a cell group.（这一点很重要，大家多注意）。
3、Calculation of intercellular communication probability.We modeled soluble ligand-receptor mediated signaling interactions using the law of mass action（质量作用定律，需要大家好好学习了）。
图片.png
4、Identification of statistically significant intercellular communications.
图片.png
还有很多重要的算法知识，我们就到此为止把，不然脑袋要炸了，😄

Inference and analysis of cell-cell communication using CellChat

CellChat requires gene expression data of cells as the user input and models the probability of cell-cell communication by integrating gene expression with prior knowledge of the interactions between signaling ligands, receptors and their cofactors.

Upon infering the intercellular communication network, CellChat provides functionality for further data exploration, analysis, and visualization.

Load the required libraries

library(CellChat)
library(patchwork)
options(stringsAsFactors = FALSE)

Part I: Data input & processing and initialization of CellChat object

CellChat requires two user inputs: one is the gene expression data of cells, and the other is either user assigned cell labels (i.e., label-based mode) or a low-dimensional representation of the single-cell data (i.e., label-free mode). For the latter, CellChat automatically groups cells by building a shared neighbor graph based on the cell-cell distance in the low-dimensional space or the pseudotemporal trajectory space.

Load data

For the gene expression data matrix, genes should be in rows with rownames and cells in columns with colnames. Normalized data (e.g., library-size normalization and then log-transformed with a pseudocount of 1) is required as input for CellChat analysis. If user provides count data, we provide a normalizeData function to account for library size and then do log-transformed. For the cell group information, a dataframe with rownames is required as input for CellChat.

# Here we load a scRNA-seq data matrix and its associated cell meta data
load(url("https://ndownloader.figshare.com/files/25950872")) # This is a combined data from two biological conditions: normal and diseases
data.input = data_humanSkin$data # normalized data matrix
meta = data_humanSkin$meta # a dataframe with rownames containing cell mata data
cell.use = rownames(meta)[meta$condition == "LS"] # extract the cell names from disease data

# Prepare input data for CelChat analysis
data.input = data.input[, cell.use]
meta = meta[cell.use, ]
# meta = data.frame(labels = meta$labels[cell.use], row.names = colnames(data.input)) # manually create a dataframe consisting of the cell labels
unique(meta$labels) # check the cell labels
#>  [1] Inflam. FIB  FBN1+ FIB    APOE+ FIB    COL11A1+ FIB cDC2        
#>  [6] LC           Inflam. DC   cDC1         CD40LG+ TC   Inflam. TC  
#> [11] TC           NKT         
#> 12 Levels: APOE+ FIB FBN1+ FIB COL11A1+ FIB Inflam. FIB cDC1 cDC2 ... NKT

Create a CellChat object

USERS can create a new CellChat object from a data matrix, Seurat or SingleCellExperiment object. If input is a Seurat or SingleCellExperiment object, the meta data in the object will be used by default and USER must provide group.by to define the cell groups. e.g, group.by = “ident” for the default cell identities in Seurat object.

NB: If USERS load previously calculated CellChat object (version < 0.5.0), please update the object via updateCellChat

cellchat <- createCellChat(object = data.input, meta = meta, group.by = "labels")
#> Create a CellChat object from a data matrix
#> Set cell identities for the new CellChat object
#> The cell groups used for CellChat analysis are  APOE+ FIB FBN1+ FIB COL11A1+ FIB Inflam. FIB cDC1 cDC2 LC Inflam. DC TC Inflam. TC CD40LG+ TC NKT

Add cell information into meta slot of the object

If cell mata information is not added when creating CellChat object, USERS can also add it later using addMeta, and set the default cell identities using setIdent.

cellchat <- addMeta(cellchat, meta = meta)
cellchat <- setIdent(cellchat, ident.use = "labels") # set "labels" as default cell identity
levels(cellchat@idents) # show factor levels of the cell labels
groupSize <- as.numeric(table(cellchat@idents)) # number of cells in each cell group

Set the ligand-receptor interaction database

Our database CellChatDB is a manually curated database of literature-supported ligand-receptor interactions in both human and mouse. CellChatDB in mouse contains 2,021 validated molecular interactions, including 60% of secrete autocrine/paracrine signaling interactions, 21% of extracellular matrix (ECM)-receptor interactions and 19% of cell-cell contact interactions. CellChatDB in human contains 1,939 validated molecular interactions, including 61.8% of paracrine/autocrine signaling interactions, 21.7% of extracellular matrix (ECM)-receptor interactions and 16.5% of cell-cell contact interactions.

Users can update CellChatDB by adding their own curated ligand-receptor pairs.Please check our tutorial on how to do it.

CellChatDB <- CellChatDB.human # use CellChatDB.mouse if running on mouse data
showDatabaseCategory(CellChatDB)

image.png

# Show the structure of the database
dplyr::glimpse(CellChatDB$interaction)
#> Rows: 1,939
#> Columns: 11
#> $ interaction_name   <chr> "TGFB1_TGFBR1_TGFBR2", "TGFB2_TGFBR1_TGFBR2", "TGF…
#> $ pathway_name       <chr> "TGFb", "TGFb", "TGFb", "TGFb", "TGFb", "TGFb", "T…
#> $ ligand             <chr> "TGFB1", "TGFB2", "TGFB3", "TGFB1", "TGFB1", "TGFB…
#> $ receptor           <chr> "TGFbR1_R2", "TGFbR1_R2", "TGFbR1_R2", "ACVR1B_TGF…
#> $ agonist            <chr> "TGFb agonist", "TGFb agonist", "TGFb agonist", "T…
#> $ antagonist         <chr> "TGFb antagonist", "TGFb antagonist", "TGFb antago…
#> $ co_A_receptor      <chr> "", "", "", "", "", "", "", "", "", "", "", "", ""…
#> $ co_I_receptor      <chr> "TGFb inhibition receptor", "TGFb inhibition recep…
#> $ evidence           <chr> "KEGG: hsa04350", "KEGG: hsa04350", "KEGG: hsa0435…
#> $ annotation         <chr> "Secreted Signaling", "Secreted Signaling", "Secre…
#> $ interaction_name_2 <chr> "TGFB1 - (TGFBR1+TGFBR2)", "TGFB2 - (TGFBR1+TGFBR2…

# use a subset of CellChatDB for cell-cell communication analysis
CellChatDB.use <- subsetDB(CellChatDB, search = "Secreted Signaling") # use Secreted Signaling
# use all CellChatDB for cell-cell communication analysis
# CellChatDB.use <- CellChatDB # simply use the default CellChatDB

# set the used database in the object
cellchat@DB <- CellChatDB.use

Preprocessing the expression data for cell-cell communication analysis

To infer the cell state-specific communications, we identify over-expressed ligands or receptors in one cell group and then identify over-expressed ligand-receptor interactions if either ligand or receptor is over-expressed.

We also provide a function to project gene expression data onto protein-protein interaction (PPI) network. Specifically, a diffusion process is used to smooth genes’ expression values based on their neighbors’ defined in a high-confidence experimentally validated protein-protein network. This function is useful when analyzing single-cell data with shallow sequencing depth because the projection reduces the dropout effects of signaling genes, in particular for possible zero expression of subunits of ligands/receptors. One might be concerned about the possible artifact introduced by this diffusion process, however, it will only introduce very weak communications. USERS can also skip this step and set raw.use = TRUE in the function computeCommunProb().

cellchat <- subsetData(cellchat) # subset the expression data of signaling genes for saving computation cost
future::plan("multiprocess", workers = 4) # do parallel
#> Warning: [ONE-TIME WARNING] Forked processing ('multicore') is disabled
#> in future (>= 1.13.0) when running R from RStudio, because it is
#> considered unstable. Because of this, plan("multicore") will fall
#> back to plan("sequential"), and plan("multiprocess") will fall back to
#> plan("multisession") - not plan("multicore") as in the past. For more details,
#> how to control forked processing or not, and how to silence this warning in
#> future R sessions, see ?future::supportsMulticore
cellchat <- identifyOverExpressedGenes(cellchat)
cellchat <- identifyOverExpressedInteractions(cellchat)
cellchat <- projectData(cellchat, PPI.human)

Part II: Inference of cell-cell communication network

CellChat infers the biologically significant cell-cell communication by assigning each interaction with a probability value and peforming a permutation test. CellChat models the probability of cell-cell communication by integrating gene expression with prior known knowledge of the interactions between signaling ligands, receptors and their cofactors using the law of mass action.

The number of inferred ligand-receptor pairs clearly depends on the method for calculating the average gene expression per cell group. By default, CellChat uses a statistically robust mean method called ‘trimean’, which produces fewer interactions than other methods. However, we find that CellChat performs well at predicting stronger interactions, which is very helpful for narrowing down on interactions for further experimental validations. In computeCommunProb, we provide an option for using other methods, such as 5% and 10% truncated mean, to calculating the average gene expression. Of note, ‘trimean’ approximates 25% truncated mean, implying that the average gene expression is zero if the percent of expressed cells in one group is less than 25%. To use 10% truncated mean, USER can set type = "truncatedMean" and trim = 0.1.

When analyzing unsorted single-cell transcriptomes, under the assumption that abundant cell populations tend to send collectively stronger signals than the rare cell populations, CellChat can also consider the effect of cell proportion in each cell group in the probability calculation. USER can set population.size = TRUE.

Compute the communication probability and infer cellular communication network

cellchat <- computeCommunProb(cellchat, raw.use = TRUE)
# Filter out the cell-cell communication if there are only few number of cells in certain cell groups
cellchat <- filterCommunication(cellchat, min.cells = 10)

Extract the inferred cellular communication network as a data frame

We provide a function subsetCommunication to easily access the inferred cell-cell communications of interest. For example,

• df.net <- subsetCommunication(cellchat) returns a data frame consisting of all the inferred cell-cell communications at the level of ligands/receptors. Set slot.name = "netP" to access the the inferred communications at the level of signaling pathways

• df.net <- subsetCommunication(cellchat, sources.use = c(1,2), targets.use = c(4,5)) gives the inferred cell-cell communications sending from cell groups 1 and 2 to cell groups 4 and 5.

• df.net <- subsetCommunication(cellchat, signaling = c("WNT", "TGFb")) gives the inferred cell-cell communications mediated by signaling WNT and TGFb.

Infer the cell-cell communication at a signaling pathway level

CellChat computes the communication probability on signaling pathway level by summarizing the communication probabilities of all ligands-receptors interactions associated with each signaling pathway.

NB: The inferred intercellular communication network of each ligand-receptor pair and each signaling pathway is stored in the slot ‘net’ and ‘netP’, respectively.

cellchat <- computeCommunProbPathway(cellchat)

Calculate the aggregated cell-cell communication network

We can calculate the aggregated cell-cell communication network by counting the number of links or summarizing the communication probability. USER can also calculate the aggregated network among a subset of cell groups by setting sources.use and targets.use.

cellchat <- aggregateNet(cellchat)

We can also visualize the aggregated cell-cell communication network. For example, showing the number of interactions or the total interaction strength (weights) between any two cell groups using circle plot.

groupSize <- as.numeric(table(cellchat@idents))
par(mfrow = c(1,2), xpd=TRUE)
netVisual_circle(cellchat@net$count, vertex.weight = groupSize, weight.scale = T, label.edge= F, title.name = "Number of interactions")
netVisual_circle(cellchat@net$weight, vertex.weight = groupSize, weight.scale = T, label.edge= F, title.name = "Interaction weights/strength")

image.png

Due to the complicated cell-cell communication network, we can examine the signaling sent from each cell group. Here we also control the parameter edge.weight.max so that we can compare edge weights between differet networks.

mat <- cellchat@net$weight
par(mfrow = c(3,4), xpd=TRUE)
for (i in 1:nrow(mat)) {
  mat2 <- matrix(0, nrow = nrow(mat), ncol = ncol(mat), dimnames = dimnames(mat))
  mat2[i, ] <- mat[i, ]
  netVisual_circle(mat2, vertex.weight = groupSize, weight.scale = T, edge.weight.max = max(mat), title.name = rownames(mat)[i])
}

image.png

Part III: Visualization of cell-cell communication network

Upon infering the cell-cell communication network, CellChat provides various functionality for further data exploration, analysis, and visualization.

• It provides several ways for visualizing cell-cell communication network, including hierarchical plot, circle plot, Chord diagram, and bubble plot.

• It provides an easy-to-use tool for extracting and visualizing high-order information of the inferred networks. For example, it allows ready prediction of major signaling inputs and outputs for cell populations and how these populations and signals coordinate together for functions.

• It can quantitatively characterize and compare the inferred cell-cell communication networks using an integrated approach by combining social network analysis, pattern recognition, and manifold learning approaches.

Visualize each signaling pathway using Hierarchy plot, Circle plot or Chord diagram

Hierarchy plot: USER should define vertex.receiver, which is a numeric vector giving the index of the cell groups as targets in the left part of hierarchy plot. This hierarchical plot consist of two components: the left portion shows autocrine and paracrine signaling to certain cell groups of interest (i.e, the defined vertex.receiver), and the right portion shows autocrine and paracrine signaling to the remaining cell groups in the dataset. Thus, hierarchy plot provides an informative and intuitive way to visualize autocrine and paracrine signaling communications between cell groups of interest. For example, when studying the cell-cell communication between fibroblasts and immune cells, USER can define vertex.receiver as all fibroblast cell groups.

Chord diagram: CellChat provides two functions netVisual_chord_cell and netVisual_chord_gene for visualizing cell-cell communication with different purposes and different levels. netVisual_chord_cell is used for visualizing the cell-cell communication between different cell groups (where each sector in the chord diagram is a cell group), and netVisual_chord_gene is used for visualizing the cell-cell communication mediated by mutiple ligand-receptors or signaling pathways (where each sector in the chord diagram is a ligand, receptor or signaling pathway.)

Explnations of edge color/weight, node color/size/shape: In all visualization plots, edge colors are consistent with the sources as sender, and edge weights are proportional to the interaction strength. Thicker edge line indicates a stronger signal. In the Hierarchy plot and Circle plot, circle sizes are proportional to the number of cells in each cell group. In the hierarchy plot, solid and open circles represent source and target, respectively. In the Chord diagram, the inner thinner bar colors represent the targets that receive signal from the corresponding outer bar. The inner bar size is proportional to the signal strength received by the targets. Such inner bar is helpful for interpreting the complex chord diagram. Note that there exist some inner bars without any chord for some cell groups, please just igore it because this is an issue that has not been addressed by circlize package.

Visualization of cell-cell communication at different levels: One can visualize the inferred communication network of signaling pathways using netVisual_aggregate, and visualize the inferred communication networks of individual L-R pairs associated with that signaling pathway using netVisual_individual.

Here we take input of one signaling pathway as an example. All the signaling pathways showing significant communications can be accessed by cellchat@netP$pathways.

pathways.show <- c("CXCL") 
# Hierarchy plot
# Here we define `vertex.receive` so that the left portion of the hierarchy plot shows signaling to fibroblast and the right portion shows signaling to immune cells 
vertex.receiver = seq(1,4) # a numeric vector. 
netVisual_aggregate(cellchat, signaling = pathways.show,  vertex.receiver = vertex.receiver)

image.png

# Circle plot
par(mfrow=c(1,1))
netVisual_aggregate(cellchat, signaling = pathways.show, layout = "circle")

image.png

# Chord diagram
par(mfrow=c(1,1))
netVisual_aggregate(cellchat, signaling = pathways.show, layout = "chord")
#> Note: The first link end is drawn out of sector 'Inflam. FIB'.

image.png

# Heatmap
par(mfrow=c(1,1))
netVisual_heatmap(cellchat, signaling = pathways.show, color.heatmap = "Reds")
#> Do heatmap based on a single object

image.png

For the chord diagram, CellChat has an independent function netVisual_chord_cell to flexibly visualize the signaling network by adjusting different parameters in the circlize package. For example, we can define a named char vector group to create multiple-group chord diagram, e.g., grouping cell clusters into different cell types.

# Chord diagram
group.cellType <- c(rep("FIB", 4), rep("DC", 4), rep("TC", 4)) # grouping cell clusters into fibroblast, DC and TC cells
names(group.cellType) <- levels(cellchat@idents)
netVisual_chord_cell(cellchat, signaling = pathways.show, group = group.cellType, title.name = paste0(pathways.show, " signaling network"))
#> Plot the aggregated cell-cell communication network at the signaling pathway level
#> Note: The first link end is drawn out of sector 'Inflam. FIB'.

image.png

Compute the contribution of each ligand-receptor pair to the overall signaling pathway and visualize cell-cell communication mediated by a single ligand-receptor pair

netAnalysis_contribution(cellchat, signaling = pathways.show)

image.png

We can also visualize the cell-cell communication mediated by a single ligand-receptor pair. We provide a function extractEnrichedLR to extract all the significant interactions (L-R pairs) and related signaling genes for a given signaling pathway.

pairLR.CXCL <- extractEnrichedLR(cellchat, signaling = pathways.show, geneLR.return = FALSE)
LR.show <- pairLR.CXCL[1,] # show one ligand-receptor pair
# Hierarchy plot
vertex.receiver = seq(1,4) # a numeric vector
netVisual_individual(cellchat, signaling = pathways.show,  pairLR.use = LR.show, vertex.receiver = vertex.receiver)

image.png

# Circle plot
netVisual_individual(cellchat, signaling = pathways.show, pairLR.use = LR.show, layout = "circle")

image.png

#> [[1]]
# Chord diagram
netVisual_individual(cellchat, signaling = pathways.show, pairLR.use = LR.show, layout = "chord")
#> Note: The first link end is drawn out of sector 'Inflam. FIB'.

image.png

#> [[1]]

Automatically save the plots of the all inferred network for quick exploration

In practical use, USERS can use ‘for … loop’ to automatically save the all inferred network for quick exploration using netVisual. netVisual supports an output in the formats of svg, png and pdf.

# Access all the signaling pathways showing significant communications
pathways.show.all <- cellchat@netP$pathways
# check the order of cell identity to set suitable vertex.receiver
levels(cellchat@idents)
vertex.receiver = seq(1,4)
for (i in 1:length(pathways.show.all)) {
  # Visualize communication network associated with both signaling pathway and individual L-R pairs
  netVisual(cellchat, signaling = pathways.show.all[i], vertex.receiver = vertex.receiver, layout = "hierarchy")
  # Compute and visualize the contribution of each ligand-receptor pair to the overall signaling pathway
  gg <- netAnalysis_contribution(cellchat, signaling = pathways.show.all[i])
  ggsave(filename=paste0(pathways.show.all[i], "_L-R_contribution.pdf"), plot=gg, width = 3, height = 2, units = 'in', dpi = 300)
}

Visualize cell-cell communication mediated by multiple ligand-receptors or signaling pathways

Bubble plot

We can also show all the significant interactions (L-R pairs) from some cell groups to other cell groups using netVisual_bubble.

# show all the significant interactions (L-R pairs) from some cell groups (defined by 'sources.use') to other cell groups (defined by 'targets.use')
netVisual_bubble(cellchat, sources.use = 4, targets.use = c(5:11), remove.isolate = FALSE)
#> Comparing communications on a single object

image.png

# show all the significant interactions (L-R pairs) associated with certain signaling pathways
netVisual_bubble(cellchat, sources.use = 4, targets.use = c(5:11), signaling = c("CCL","CXCL"), remove.isolate = FALSE)
#> Comparing communications on a single object

image.png

# show all the significant interactions (L-R pairs) based on user's input (defined by `pairLR.use`)
pairLR.use <- extractEnrichedLR(cellchat, signaling = c("CCL","CXCL","FGF"))
netVisual_bubble(cellchat, sources.use = c(3,4), targets.use = c(5:8), pairLR.use = pairLR.use, remove.isolate = TRUE)
#> Comparing communications on a single object

image.png

Chord diagram

Similar to Bubble plot, CellChat provides a function netVisual_chord_gene for drawing Chord diagram to

• show all the interactions (L-R pairs or signaling pathways) from some cell groups to other cell groups. Two special cases: one is showing all the interactions sending from one cell groups and the other is showing all the interactions received by one cell group.

• show the interactions inputted by USERS or certain signaling pathways defined by USERS

# show all the significant interactions (L-R pairs) from some cell groups (defined by 'sources.use') to other cell groups (defined by 'targets.use')
# show all the interactions sending from Inflam.FIB
netVisual_chord_gene(cellchat, sources.use = 4, targets.use = c(5:11), lab.cex = 0.5,legend.pos.y = 30)
#> Note: The first link end is drawn out of sector 'MIF'.

image.png

# show all the interactions received by Inflam.DC
netVisual_chord_gene(cellchat, sources.use = c(1,2,3,4), targets.use = 8, legend.pos.x = 15)

image.png

# show all the significant interactions (L-R pairs) associated with certain signaling pathways
netVisual_chord_gene(cellchat, sources.use = c(1,2,3,4), targets.use = c(5:11), signaling = c("CCL","CXCL"),legend.pos.x = 8)
#> Note: The second link end is drawn out of sector 'CXCR4 '.
#> Note: The first link end is drawn out of sector 'CXCL12 '.

image.png

# show all the significant signaling pathways from some cell groups (defined by 'sources.use') to other cell groups (defined by 'targets.use')
netVisual_chord_gene(cellchat, sources.use = c(1,2,3,4), targets.use = c(5:11), slot.name = "netP", legend.pos.x = 10)
#> Note: The second link end is drawn out of sector ' '.
#> Note: The first link end is drawn out of sector 'MIF'.
#> Note: The second link end is drawn out of sector ' '.
#> Note: The first link end is drawn out of sector 'CXCL '.

image.png

NB: Please ignore the note when generating the plot such as “Note: The first link end is drawn out of sector ‘MIF’.”. If the gene names are overlapped, you can adjust the argument small.gap by decreasing the value.

Plot the signaling gene expression distribution using violin/dot plot

We can plot the gene expression distribution of signaling genes related to L-R pairs or signaling pathway using a Seurat wrapper function plotGeneExpression.

plotGeneExpression(cellchat, signaling = "CXCL")
#> Registered S3 method overwritten by 'spatstat':
#>   method     from
#>   print.boxx cli
#> Scale for 'y' is already present. Adding another scale for 'y', which will
#> replace the existing scale.
#> Scale for 'y' is already present. Adding another scale for 'y', which will
#> replace the existing scale.
#> Scale for 'y' is already present. Adding another scale for 'y', which will
#> replace the existing scale.

image.png

By default, plotGeneExpression only shows the expression of signaling genes related to the inferred significant communications. USERS can show the expression of all signaling genes related to one signaling pathway by

plotGeneExpression(cellchat, signaling = "CXCL", enriched.only = FALSE)

Alternatively, USERS can extract the signaling genes related to the inferred L-R pairs or signaling pathway using extractEnrichedLR, and then plot gene expression using Seurat package.

Part IV: Systems analysis of cell-cell communication network

To facilitate the interpretation of the complex intercellular communication networks, CellChat quantitively measures networks through methods abstracted from graph theory, pattern recognition and manifold learning.

• It can determine major signaling sources and targets as well as mediators and influencers within a given signaling network using centrality measures from network analysis

• It can predict key incoming and outgoing signals for specific cell types as well as coordinated responses among different cell types by leveraging pattern recognition approaches.

• It can group signaling pathways by defining similarity measures and performing manifold learning from both functional and topological perspectives.

• It can delineate conserved and context-specific signaling pathways by joint manifold learning of multiple networks.

Identify signaling roles (e.g., dominant senders, receivers) of cell groups as well as the major contributing signaling

CellChat allows ready identification of dominant senders, receivers, mediators and influencers in the intercellular communication network by computing several network centrality measures for each cell group. Specifically, we used measures in weighted-directed networks, including out-degree, in-degree, flow betweenesss and information centrality, to respectively identify dominant senders, receivers, mediators and influencers for the intercellular communications. In a weighteddirected network with the weights as the computed communication probabilities, the outdegree, computed as the sum of communication probabilities of the outgoing signaling from a cell group, and the in-degree, computed as the sum of the communication probabilities of the incoming signaling to a cell group, can be used to identify the dominant cell senders and receivers of signaling networks, respectively. For the definition of flow betweenness and information centrality, please check our paper and related reference.

Compute and visualize the network centrality scores

# Compute the network centrality scores
cellchat <- netAnalysis_computeCentrality(cellchat, slot.name = "netP") # the slot 'netP' means the inferred intercellular communication network of signaling pathways
# Visualize the computed centrality scores using heatmap, allowing ready identification of major signaling roles of cell groups
netAnalysis_signalingRole_network(cellchat, signaling = pathways.show, width = 8, height = 2.5, font.size = 10)

image.png

Visualize the dominant senders (sources) and receivers (targets) in a 2D space

We also provide another intutive way to visualize the dominant senders (sources) and receivers (targets) in a 2D space using scatter plot.

# Signaling role analysis on the aggregated cell-cell communication network from all signaling pathways
gg1 <- netAnalysis_signalingRole_scatter(cellchat)
#> Signaling role analysis on the aggregated cell-cell communication network from all signaling pathways
# Signaling role analysis on the cell-cell communication networks of interest
gg2 <- netAnalysis_signalingRole_scatter(cellchat, signaling = c("CXCL", "CCL"))
#> Signaling role analysis on the cell-cell communication network from user's input
gg1 + gg2

image.png

Identify signals contributing most to outgoing or incoming signaling of certain cell groups

We can also answer the question on which signals contributing most to outgoing or incoming signaling of certain cell groups.

# Signaling role analysis on the aggregated cell-cell communication network from all signaling pathways
ht1 <- netAnalysis_signalingRole_heatmap(cellchat, pattern = "outgoing")
ht2 <- netAnalysis_signalingRole_heatmap(cellchat, pattern = "incoming")
ht1 + ht2

image.png

# Signaling role analysis on the cell-cell communication networks of interest
ht <- netAnalysis_signalingRole_heatmap(cellchat, signaling = c("CXCL", "CCL"))

Identify global communication patterns to explore how multiple cell types and signaling pathways coordinate together

In addition to exploring detailed communications for individual pathways, an important question is how multiple cell groups and signaling pathways coordinate to function. CellChat employs a pattern recognition method to identify the global communication patterns.

As the number of patterns increases, there might be redundant patterns, making it difficult to interpret the communication patterns. We chose five patterns as default. Generally, it is biologically meaningful with the number of patterns greater than 2. In addition, we also provide a function selectK to infer the number of patterns, which is based on two metrics that have been implemented in the NMF R package, including Cophenetic and Silhouette. Both metrics measure the stability for a particular number of patterns based on a hierarchical clustering of the consensus matrix. For a range of the number of patterns, a suitable number of patterns is the one at which Cophenetic and Silhouette values begin to drop suddenly.

Identify and visualize outgoing communication pattern of secreting cells

Outgoing patterns reveal how the sender cells (i.e. cells as signal source) coordinate with each other as well as how they coordinate with certain signaling pathways to drive communication.

To intuitively show the associations of latent patterns with cell groups and ligand-receptor pairs or signaling pathways, we used river (alluvial) plots. We first normalized each row of W and each column of H to be [0,1], and then set the elements in W and H to be zero if they are less than 0.5. Such thresholding allows to uncover the most enriched cell groups and signaling pathways associated with each inferred pattern, that is, each cell group or signaling pathway is associated with only one inferred pattern. These thresholded matrices W and H are used as inputs for creating alluvial plots.

To directly relate cell groups with their enriched signaling pathways, we set the elements in W and H to be zero if they are less than 1/R where R is the number of latent patterns. By using a less strict threshold, more enriched signaling pathways associated each cell group might be obtained. Using a contribution score of each cell group to each signaling pathway computed by multiplying W by H, we constructed a dot plot in which the dot size is proportion to the contribution score to show association between cell group and their enriched signaling pathways. USERS can also decrease the parameter cutoff to show more enriched signaling pathways associated each cell group.

Load required package for the communication pattern analysis

library(NMF)
#> Loading required package: pkgmaker
#> Loading required package: registry
#> Loading required package: rngtools
#> Loading required package: cluster
#> NMF - BioConductor layer [OK] | Shared memory capabilities [NO: bigmemory] | Cores 15/16
#>   To enable shared memory capabilities, try: install.extras('
#> NMF
#> ')
#> 
#> Attaching package: 'NMF'
#> The following objects are masked from 'package:igraph':
#> 
#>     algorithm, compare
library(ggalluvial)

Here we run selectK to infer the number of patterns.

selectK(cellchat, pattern = "outgoing")

image.png

Both Cophenetic and Silhouette values begin to drop suddenly when the number of outgoing patterns is 3.

nPatterns = 3
cellchat <- identifyCommunicationPatterns(cellchat, pattern = "outgoing", k = nPatterns)

image.png

# river plot
netAnalysis_river(cellchat, pattern = "outgoing")
#> Please make sure you have load `library(ggalluvial)` when running this function

image.png

# dot plot
netAnalysis_dot(cellchat, pattern = "outgoing")

image.png

Identify and visualize incoming communication pattern of target cells

Incoming patterns show how the target cells (i.e. cells as signal receivers) coordinate with each other as well as how they coordinate with certain signaling pathways to respond to incoming signals.

selectK(cellchat, pattern = "incoming")

image.png

Cophenetic values begin to drop when the number of incoming patterns is 4.

nPatterns = 4
cellchat <- identifyCommunicationPatterns(cellchat, pattern = "incoming", k = nPatterns)

image.png

# river plot
netAnalysis_river(cellchat, pattern = "incoming")
#> Please make sure you have load `library(ggalluvial)` when running this function

image.png

# dot plot
netAnalysis_dot(cellchat, pattern = "incoming")

image.png

Manifold and classification learning analysis of signaling networks

Further, CellChat is able to quantify the similarity between all significant signaling pathways and then group them based on their cellular communication network similarity. Grouping can be done either based on the functional or structural similarity.

Functional similarity: High degree of functional similarity indicates major senders and receivers are similar, and it can be interpreted as the two signaling pathways or two ligand-receptor pairs exhibit similar and/or redundant roles. The functional similarity analysis requires the same cell population composition between two datasets.

Structural similarity: A structural similarity was used to compare their signaling network structure, without considering the similarity of senders and receivers.

Identify signaling groups based on their functional similarity

cellchat <- computeNetSimilarity(cellchat, type = "functional")
cellchat <- netEmbedding(cellchat, type = "functional")
#> Manifold learning of the signaling networks for a single dataset
cellchat <- netClustering(cellchat, type = "functional")
#> Classification learning of the signaling networks for a single dataset
# Visualization in 2D-space
netVisual_embedding(cellchat, type = "functional", label.size = 3.5)

image.png

# netVisual_embeddingZoomIn(cellchat, type = "functional", nCol = 2)

Identify signaling groups based on structure similarity

cellchat <- computeNetSimilarity(cellchat, type = "structural")
cellchat <- netEmbedding(cellchat, type = "structural")
#> Manifold learning of the signaling networks for a single dataset
cellchat <- netClustering(cellchat, type = "structural")
#> Classification learning of the signaling networks for a single dataset
# Visualization in 2D-space
netVisual_embedding(cellchat, type = "structural", label.size = 3.5)

image.png

netVisual_embeddingZoomIn(cellchat, type = "structural", nCol = 2)

image.png

Part V: Save the CellChat object

saveRDS(cellchat, file = "cellchat_humanSkin_LS.rds")

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