---
title: "Tools for IMC data analysis"
date: "`r BiocStyle::doc_date()`"
package: "`r BiocStyle::pkg_ver('imcRtools')`"
author:
- name: Nils Eling
affiliation:
- Department for Quantitative Biomedicine, University of Zurich
- Institute for Molecular Health Sciences, ETH Zurich
email: nils.eling@dqbm.uzh.ch
- name: Lasse Meyer
affiliation:
- Department for Quantitative Biomedicine, University of Zurich
- Institute for Molecular Health Sciences, ETH Zurich
email: lasse.meyer@@uzh.ch
- name: Daniel Schulz
affiliation:
- Department for Quantitative Biomedicine, University of Zurich
- Institute for Molecular Health Sciences, ETH Zurich
email: daniel.schulz@@uzh.ch
output:
BiocStyle::html_document:
toc_float: yes
bibliography: library.bib
abstract: |
This R package supports the handling and analysis of imaging mass cytometry
and other highly multiplexed imaging data. The main functionality includes
reading in single-cell data after image segmentation and measurement, data
formatting to perform channel spillover correction and a number of spatial
analysis approaches. First, cell-cell interactions are detected via spatial
graph construction; these graphs can be visualized with cells representing
nodes and interactions representing edges. Furthermore, per cell, its direct
neighbours are summarized to allow spatial clustering. Per image/grouping
level, interactions between types of cells are counted, averaged and
compared against random permutations. In that way, types of cells that
interact more (attraction) or less (avoidance) frequently than expected by
chance are detected.
vignette: |
%\VignetteIndexEntry{"Tools for IMC data analysis"}
%\VignetteEncoding{UTF-8}
%\VignetteEngine{knitr::rmarkdown}
editor_options:
markdown:
wrap: 72
---
```{r, echo=FALSE, results="hide"}
knitr::opts_chunk$set(error=FALSE, warning=FALSE, message=FALSE, crop = NULL)
library(BiocStyle)
```
```{r library, echo=FALSE}
library(imcRtools)
```
# Introduction
This vignette gives an introduction to handling and analyzing imaging
mass cytometry (IMC) and other highly-multiplexed imaging data in R. The
`imcRtools` package relies on expression and morphological features
extracted from multi-channel images using corresponding segmentation
masks. A description of data types and segmentation approaches can be
found below ([data types](#dataTypes), [segmentation](#segmentation)).
However, due to shared data structures, the functionalities of the
`imcRtools` package are applicable to most highly multiplexed imaging
modalities.
## Overview
The `imcRtools` package exports functions and example data to perform
the following analyses:
1. [Read in pre-processed data](#read-in-data)
2. [Perform spillover correction for IMC data](#spillover)
3. [Build](#build-graph) and [visualize](#viz-cells) spatial graphs
4. [Aggregate across neighbouring cells](#aggregate-neighbors) for
spatial clustering
5. [Detect spatial patches](#patch-detection) of similar cell-types
6. [Test the attraction or avoidance between
celltypes](#test-neighborhood)
To highlight the usability of these function, the `imcRtools` package
also exports a number of [test data files](#test-data).
## Highly multiplexed imaging
Highly multiplexed imaging techniques [@Bodenmiller2016] such as imaging
mass cytometry (IMC) [@Giesen2014], multiplexed ion beam imaging (MIBI)
[@Angelo2014] and cyclic immunofluorescence techniques [@Lin2018,
@Gut2018] acquire read-outs of the expression of tens of protein in a
spatially resolved manner. After technology-dependent data
pre-processing, the raw data files are comparable: multi-channel images
for the dimensions `x`, `y`, and `c`, where `x` and `y` define the
number of pixels (`x * y`) per image and `c` the number of proteins
(also refered to as "markers") measured per image. More info on the
[data types](#dataTypes) and [further pre-processing](#segmentation) can
be found below.
Increased multiplexity compared to epitope-based techniques is achieved
using single-cell resolved spatial transcriptomics techniques including
MERFISH [@Chen2015] and seqFISH [@Lubeck2014]. However, data produced by
these techniques required different pre-processing steps. Nevertheless,
analysis performed on the single-cell level is equally applicable.
### Imaging mass cytometry
IMC [@Giesen2014] is a highly multiplexed imaging approach to measure
spatial protein abundance. In IMC, tissue sections on slides are stained
with a mix of around 40 metal-conjugated antibodies prior to laser
ablation with $1\mu{}m$ resolution. The ablated material is transferred
to a mass cytometer for time-of-flight detection of the metal ions
[@Giesen2014]. At an ablation frequency of 200Hz, a 1mm x 1mm region can
be acquired within 1.5 hours.
### Data types {#dataTypes}
In the case of IMC, the raw data output are .mcd files containing all
acquired regions per slide. During image pre-prcoessing these files are
converted into individual multi-channel .tiff and
[OME-TIFF](https://docs.openmicroscopy.org/ome-model/5.6.3/ome-tiff/)
files. These file formats are supported by most open-source and
commercial image analysis software, such as
[Fiji](https://imagej.net/software/fiji/),
[QuPath](https://qupath.github.io/) or [napari](https://napari.org/).
In R, these .tiff files can be read into individual
[Image](https://bioconductor.org/packages/release/bioc/vignettes/EBImage/inst/doc/EBImage-introduction.html#3_Image_data_representation)
objects or combined into a
[CytoImageList](https://www.bioconductor.org/packages/release/bioc/vignettes/cytomapper/inst/doc/cytomapper.html#5_The_CytoImageList_object)
object supported by the
[cytomapper](https://www.bioconductor.org/packages/release/bioc/html/cytomapper.html)
package.
### Segmentation and feature extraction {#segmentation}
The pixel resolution of most highly multiplexed imaging technologies
including IMC support the detection of cellular structures. Therefore, a
common processing step using multi-channel images is object
segmentation. In this vignette, objects are defined as cells; however,
also larger scale structures could be segmented.
There are multiple different image segmentation approaches available.
However, `imcRtools` only supports direct reader functions for two
segmentation strategies developed for highly multiplexed imaging
technologies:
1. The
[ImcSegmentationPipeline](https://github.com/BodenmillerGroup/ImcSegmentationPipeline)
has been developed to give the user flexibility in how to perform
channel selection and image segmentation. The underlying principle
is to train a pixel classifier (using
[ilastik](https://www.ilastik.org/)) on a number of selected
channels to perform probabilistic classification of each pixel into:
background, cytoplasm and nucleus. Based on these classification
probabilities a [CellProfiler](https://cellprofiler.org/) pipeline
performs cell segmentation and feature extraction.
2. A containerized version of this pipeline is implemented in
[steinbock](https://github.com/BodenmillerGroup/steinbock).
`steinbock` further supports segmentation via the use of `Mesmer`
from the `DeepCell` library [@Greenwald2021].
While the output of both approaches is structured differently, the
exported features are comparable:
1. per cell: channel intensity, morphology and location
2. cell-cell interactions exported as graph
By combining these with availabel channel information, the data can be
read into a `r BiocStyle::Biocpkg("SpatialExperiment")` or
`r BiocStyle::Biocpkg("SingleCellExperiment")` object (see
[below](#read-in-data)).
# Example data {#test-data}
The `imcRtools` package contains a number of example data generated by
the Hyperion imaging system for different purposes. The following
section gives an overview of these files.
## For spillover correction
To highlight the use of the `imcRtools` package for spillover
correction, we provide four .txt files containing pixel intensities of
four spotted metals.
Please refer to the [spillover correction](#spillover) section for full
details.
These files are accessible via:
```{r access-spillover-files}
path <- system.file("extdata/spillover", package = "imcRtools")
list.files(path, recursive = TRUE)
```
## Raw data in form of .txt files
IMC generates .mcd files storing the raw data or all acquired regions of
interest (ROI). In addition, the raw pixel values are also stored in
individual .txt files per ROI.
To highlight reading in raw data in form of .txt files, the `imcRtools`
contains 3 sample acquisitions:
```{r access-txt-files}
txt_files <- list.files(system.file("extdata/mockData/raw",
package = "imcRtools"))
txt_files
```
## ImcSegmentationPipeline output data
IMC data preprocessing and segmentation can be performed using the
[ImcSegmentationPipeline](https://github.com/BodenmillerGroup/ImcSegmentationPipeline).
It generates a number of `.csv` files containing object/cell-specific
and image-specific metadata.
The `imcRtools` package exports the `read_cpout` function as convenient
reader function for outputs generated by the `ImcSegmentationPipeline`.
For demonstration purposes, `imcRtools` contains the output of running
the pipeline on a small example dataset:
```{r imcsegmentationpipeline-data}
path <- system.file("extdata/mockData/cpout", package = "imcRtools")
list.files(path, recursive = TRUE)
```
## steinbock output data
The [steinbock](https://github.com/BodenmillerGroup/steinbock) pipeline
can be used to process, segment and extract features from IMC data. For
more information, please refer to its
[documentation](https://bodenmillergroup.github.io/steinbock/).
To highlight the functionality of `imcRtools` to read in single-cell
data generated by `steinbock`, we provide a small toy dataset available
at:
```{r steinbock-data}
path <- system.file("extdata/mockData/steinbock", package = "imcRtools")
list.files(path, recursive = TRUE)
```
The example data related to the `ImcSegmentationPipeline` and
`steinbock` can also be accessed
[online](https://github.com/BodenmillerGroup/TestData/tree/main/datasets/210308_ImcTestData).
# Read in IMC data {#read-in-data}
The `imcRtools` package supports reading in data generated by the
[ImcSegmentationPipeline](https://github.com/BodenmillerGroup/ImcSegmentationPipeline)
or [steinbock](https://github.com/BodenmillerGroup/steinbock) pipeline.
To read in the outpout data into a
`r BiocStyle::Biocpkg("SpatialExperiment")` or
`r BiocStyle::Biocpkg("SingleCellExperiment")`, the `imcRtools` package
exports the `read_cpout` function.
By default, the single-cell data is read into a
`r BiocStyle::Biocpkg("SpatialExperiment")` object. Here, the extracted
channel- and cell-specific intensities are stored in the `counts(spe)`
slot. All morphological features are stored in `colData(spe)` and the
spatial locations of the cells are stored in `spatialCoords(spe)`. The
interaction graph is stored in `colPair(spe, "neighbourhood")`.
Alternatively, the data can be read into a
`r BiocStyle::Biocpkg("SingleCellExperiment")` object. The only
difference is the lack of `spatialCoords(sce)`. Here, the spatial
coordinates are stored in `colData(spe)$Pos_X` and `colData(spe)$Pos_Y`.
## Read in CellProfiler output
The
[ImcSegmentationPipeline](https://github.com/BodenmillerGroup/ImcSegmentationPipeline)
produces a number of output files. By default, all single-cell features
are measured and exported. To browse and select the features of
interest, the `imcRtools` package provides the `show_cpout_features`
function:
```{r show_cpout_features}
path <- system.file("extdata/mockData/cpout", package = "imcRtools")
show_cpout_features(path)
```
By default, `read_cpout` will read in the mean intensity per channel and
cell from "hot pixel" filtered image stacks specified via
`intensities = "Intensity_MeanIntensity_FullStackFiltered"`. Please
refer to `?read_cpout` for the full documentation.
```{r read_cpout}
cur_path <- system.file("extdata/mockData/cpout", package = "imcRtools")
# Read as SpatialExperiment
(spe <- read_cpout(cur_path, graph_file = "Object_relationships.csv"))
# Read as SingleCellExperiment
(sce <- read_cpout(cur_path, graph_file = "Object_relationships.csv",
return_as = "sce"))
```
## Read in steinbock output
Single-cell data and all associated metadata (e.g. spatial location,
morphology and interaction graphs) as produced by the
[steinbock](https://github.com/BodenmillerGroup/steinbock) pipeline can
be read in using the `read_steinbock` function:
```{r read_steinbock}
cur_path <- system.file("extdata/mockData/steinbock", package = "imcRtools")
# Read as SpatialExperiment
(spe <- read_steinbock(cur_path))
# Read as SingleCellExperiment
(sce <- read_steinbock(cur_path, return_as = "sce"))
```
For more information, please refer to `?read_steinbock`.
## Read raw .txt files into Image objects
For reading in and visualization of multi-channel images and
segmentation masks, please refer to the
`r BiocStyle::Biocpkg("cytomapper")` package. The `imcRtools` package
however supports reading in raw .txt files generated by the Hyperion
imaging system into a `CytoImageList` object; a data container exported
by `cytomapper`.
The user needs to provide a path from which all .txt files will be read
in:
```{r read-txt-1}
path <- system.file("extdata/mockData/raw", package = "imcRtools")
cur_CytoImageList <- readImagefromTXT(path)
cur_CytoImageList
```
By specifying the `pattern` argument, individual or a subset of files
can be read in. For more information, please refer to
`?readImagefromTXT`.
# Spillover correction {#spillover}
When acquiring IMC images, pixel intensities can be influenced by
spillover from neighboring channels. To correct for this, Chevrier *et
al.* have developed a staining protocol to acquire individually spotted
metal isotopes [@Chevrier2017]. Based on these measurements, spillover
into neighboring channels can be quantified to correct pixel
intensities.
The `imcRtools` package provides helper functions that facilitate the
correction of spillover for IMC data. For a full tutorial, please refer
to the [IMC data analysis
book](https://bodenmillergroup.github.io/IMCDataAnalysis/spillover-correction.html).
## Read in the single-spot acquisitions
In the first step, the pixel intensities of individually spotted metals
need to be read into a `SingleCellExperiment` container for downstream
use with the `r BiocStyle::Biocpkg("CATALYST")` package. For this, the
`readSCEfromTXT` function can be used:
```{r read-single-metals}
path <- system.file("extdata/spillover", package = "imcRtools")
sce <- readSCEfromTXT(path)
sce
```
Here, the example metal spot files are read in. The spot information are
stored in the `colData(sce)` slot and channel information are stored in
`rowData(sce)`. Each column represents a single pixel.
## Quality control on single-spot acquisitions
In the next step, it is crucial to identify potentially mislabeled spots
or spots with low pixel intensities. The `imcRtools` package exports the
`plotSpotHeatmap` function, which visualizes the aggregated (default
`median`) pixel intensities per spot and per metal:
```{r plotSpotHeatmap, fig.width=5, fig.height=5}
plotSpotHeatmap(sce)
```
Here, high median pixel intensities can be observed in each spot and
their corresponding channels (visualized on the `log10` scale by
default). To quickly identify spot/channel combinations with low signal,
the `threshold` parameter can be set:
```{r plotSpotHeatmap-2, fig.width=5, fig.height=5}
plotSpotHeatmap(sce, log = FALSE, threshold = 200)
```
## Consecutive pixel binning
If pixel intensities are low, spillover estimation might not be robust.
Therefore, the `binAcrossPixels` function can be used to sum consecutive
pixels and enhance the acquired signal. This step is optional for
spillover estimation.
```{r pixel-binning, fig.width=5, fig.height=5 }
sce2 <- binAcrossPixels(sce, bin_size = 5)
plotSpotHeatmap(sce2, log = FALSE, threshold = 200)
```
## Pixel filtering
Prior to spillover estimation, the `r BiocStyle::Biocpkg("CATALYST")`
package provides the `assignPrelim`, `estCutoffs` and `applyCutoffs`
functions to estimate the spotted mass for each pixel based on their
channel intensities. For more information on the spillover estimation
and correction, please refer to the [CATALYST
vignette](https://bioconductor.org/packages/release/bioc/vignettes/CATALYST/inst/doc/preprocessing.html#compensation).
This estimation can be used to identify pixels that cannot be easily
assigned to their spotted mass, potentially indicating pixels with weak
signal. To remove these pixels, the `filterPixels` function can be used.
This function further removes pixels assigned to masses, which only
contain very few pixels.
```{r assign-pixels}
library(CATALYST)
bc_key <- as.numeric(unique(sce$sample_mass))
assay(sce, "exprs") <- asinh(counts(sce)/5)
sce <- assignPrelim(sce, bc_key = bc_key)
sce <- estCutoffs(sce)
sce <- applyCutoffs(sce)
# Filter out mislabeled pixels
sce <- filterPixels(sce)
table(sce$bc_id, sce$sample_mass)
```
## Estimating the spillover matrix
Finally, the pre-processed `SiingleCellExperiment` object can be used to
generate the spillover matrix using the `CATALYST::computeSpillmat`
function:
```{r estimate-spillover}
sce <- computeSpillmat(sce)
metadata(sce)$spillover_matrix
```
This spillover matrix can be directly applied to compensate the
summarized pixel intensities per cell and per channel as described
[here](https://bioconductor.org/packages/release/bioc/vignettes/CATALYST/inst/doc/preprocessing.html#compcytof-compensation-of-mass-cytometry-data).
# Spatial analysis
The following section will highlight functions for spatial analyses of
the data.
## Constructing graphs {#build-graph}
When following the `ImcSegmentationPipeline` or `steinbock` and reading
in the data using the corresponding functions, the generated graphs are
automatically stored in the `colPair(spe, "neighborhood")` slot.
Alternatively, the `buildSpatialGraph` function in the `imcRtools`
package constructs interaction graphs using either (i) cell-centroid
expansion, (ii) k-nearest neighbor search or (iii) delaunay
triangulation.
```{r buildSpatialGraph, message=FALSE}
library(cytomapper)
data("pancreasSCE")
pancreasSCE <- buildSpatialGraph(pancreasSCE, img_id = "ImageNb",
type = "expansion",
threshold = 20)
pancreasSCE <- buildSpatialGraph(pancreasSCE, img_id = "ImageNb",
type = "knn",
k = 5)
pancreasSCE <- buildSpatialGraph(pancreasSCE, img_id = "ImageNb",
type = "delaunay")
colPairNames(pancreasSCE)
```
When setting `type = "knn"`, by default a directional graph will be
build. Setting `directed = FALSE` will create bi-directional edges for
each pair of cells that are connected by at least one edge in the
directed setting.
## Graph/cell visualization {#viz-cells}
The cells' locations and constructed graphs can be visualized using the
`plotSpatial` function. Here, cells are referred to as "nodes" and
cell-cell interactions are referred to as "edges". All visual attributes
of the nodes and edges can be set. Either by specifying a variable in
`colData(spe)`, a marker name or a single entry using the `*_fix`
parameters. By default the `plotSpatial` function will visualize equal physical
units on the x- and y-axis with an aspect ratio of 1. The example data are
located in different regions of an image and we therefore set `scales = "free"`
for simpler visualization.
```{r plotSpatial}
library(ggplot2)
library(ggraph)
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "CellType",
node_shape_by = "ImageNb",
node_size_by = "Area",
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = FALSE,
scales = "free")
# Colored by expression and with arrows
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "PIN",
assay_type = "exprs",
node_size_fix = 3,
edge_width_fix = 0.2,
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = TRUE,
arrow = grid::arrow(length = grid::unit(0.1, "inch")),
end_cap = ggraph::circle(0.05, "cm"),
scales = "free")
# Subsetting the SingleCellExperiment
plotSpatial(pancreasSCE[,pancreasSCE$Pattern],
img_id = "ImageNb",
node_color_by = "CellType",
node_size_fix = 1,
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = TRUE)
```
The returned object can be further modified using the `ggplot2` logic.
This includes changing the node color, shape and size using
`scale_color_*`, `scale_shape_*` and `scale_size_*`. Edge attributes can
be altered using the `scale_edge_*` function exported by `ggraph`,
## Neighborhood aggregation {#aggregate-neighbors}
The `aggregateNeighbors` function can be used to aggregate features of
all neighboring cells for each individual cell. This function operates
in two settings.
1. `metadata`: when aggregating by cell-specific metadata, the function
computes the relative frequencies of all entries to
`colData(sce)[[count_by]]` within the direct neighborhood of each
cell.\
2. `expression`: the expression counts of neighboring cells are
aggregated using the specified `statistic` (defaults to `mean`).
Each cell's neighborhood is defined as endpoints of edges stored in
`colPair(sce, colPairName)`.
```{r aggregateNeigbors}
pancreasSCE <- aggregateNeighbors(pancreasSCE,
colPairName = "knn_interaction_graph",
aggregate_by = "metadata",
count_by = "CellType")
head(pancreasSCE$aggregatedNeighbors)
pancreasSCE <- aggregateNeighbors(pancreasSCE,
colPairName = "knn_interaction_graph",
aggregate_by = "expression",
assay_type = "exprs")
head(pancreasSCE$mean_aggregatedExpression)
```
The returned entries can now be used for clustering to group cells based
on their environment (either by aggregated categorical features or
expression).
```{r aggregateNeigbors-clustering}
set.seed(22)
cur_cluster <- kmeans(pancreasSCE$aggregatedNeighbors, centers = 3)
pancreasSCE$clustered_neighbors <- factor(cur_cluster$cluster)
# Visualize CellType and clustered_neighbors
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "CellType",
node_size_fix = 4,
edge_width_fix = 2,
edge_color_by = "clustered_neighbors",
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free") +
scale_color_brewer(palette = "Set2") +
scale_edge_color_brewer(palette = "Set1")
# Visualize clustered_neighbors
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "clustered_neighbors",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free")+
scale_color_brewer(palette = "Set1")
```
## Spatial context analysis {#spatial-context}
The single cell assignments derived from clustering cells based on their
environment can be interpreted as **cellular neighborhoods (CNs)**, which can
represent sites of unique local processes [@Schurch2020].
Downstream of CNs, imcRtools exports three functions to detect and
analyze the **spatial context (SC)** of each cell.
1. `detectSpatialContext`: for the function to detect SCs
2. `filterSpatialContext`: for the function to filter SCs
3. `plotSpatialContext`: for the function to plot SC graphs
The term SC was coined by Bhate and colleagues [@Bhate2022] and
describes tissue regions in which distinct CNs may be interacting,
which can lead to specialized local biological events.
The `detectSpatialContext` function relies on CN fractions for each cell in
a spatial interaction graph (originally a k-nearest neighbor (KNN) graph).
We can retrieve the CN fractions using the above-described `buildSpatialGraph`
and `aggregateNeighbors` functions. The window size (k for KNN) for
`buildSpatialGraph` should reflect a length scale on which biological signals
can be exchanged and depends, among others, on cell density and tissue area.
In view of their divergent functionality, we recommend to use a larger
window size for SC (interaction between local processes) than for CN
(local processes) detection.
Subsequently, the CN fractions are sorted from high-to-low and the SC of each
cell is assigned the minimal combination of SCs that additively surpass a
user-defined threshold. The default threshold of 0.9 aims to represent the
dominant CNs, hence the most prevalent signals, in a given window.
For more details, please refer to [@Bhate2022].
```{r detectSpatialContext}
# Generate k-nearest neighbor graph
pancreasSCE <- buildSpatialGraph(pancreasSCE, img_id = "ImageNb",
type = "knn",
name = "knn_spatialcontext_graph",
k = 15)
colPairNames(pancreasSCE)
# Aggregate based on clustered_neighbors
pancreasSCE <- aggregateNeighbors(pancreasSCE,
colPairName = "knn_spatialcontext_graph",
aggregate_by = "metadata",
count_by = "clustered_neighbors",
name = "aggregatedNeighborhood")
# Detect spatial contexts
pancreasSCE <- detectSpatialContext(pancreasSCE,
entry = "aggregatedNeighborhood",
threshold = 0.9,
name = "spatial_context")
# Define SC color scheme
col_SC <- setNames(c("#A6CEE3", "#1F78B4", "#B2DF8A", "#33A02C", "#FB9A99", "#E31A1C", "#FDBF6F"),
sort(unique(pancreasSCE$spatial_context)))
# Visualize spatial contexts on images
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_context",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "knn_spatialcontext_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free") +
scale_color_manual(values = col_SC)
```
After SC assignment for each individual cell, the `filterSpatialContext`
function allows to filter detected SCs based on user-defined thresholds
for number of group entries (usually image or patient ID) and/or total
number of cells per SC.
In addition to a new column entry to the `colData(object)`, the function
also returns a `data.frame` entry to `metadata(object)` containing filtered group
and cell counts per SC.
```{r filterSpatialContext}
# Filter spatial contexts
# By number of group entries
pancreasSCE <- filterSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb",
group_threshold = 2,
name = "spatial_context_filtered_group")
metadata(pancreasSCE)$filterSpatialContext
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_context_filtered_group",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "knn_spatialcontext_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free") +
scale_color_manual(values = col_SC)
# By total number of cells
pancreasSCE <- filterSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb",
cells_threshold = 15,
name = "spatial_context_filtered_cells")
metadata(pancreasSCE)$filterSpatialContext
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_context_filtered_cells",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "knn_spatialcontext_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free") +
scale_color_manual(values = col_SC)
```
Lastly, the `plotSpatialContext` function plots directed *SC graphs*,
akin to *CN combination maps* in [@Bhate2022], based on symbolic
edge-lists and vertex metadata, which operates on cohort-level.
```{r plotSpatialContext}
## Plot spatial context graph
# Default
plotSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb")
# Colored by name and size by n_cells
plotSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb",
node_color_by = "name",
node_size_by = "n_cells",
node_label_color_by = "name")
# Colored by n_cells and size by n_group
plotSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb",
node_color_by = "n_cells",
node_size_by = "n_group",
node_label_color_by = "n_cells")+
scale_color_viridis()
```
The returned object can be further modified using the `ggplot2` logic.
This includes changing the node color and size using `scale_color_*` and
`scale_size_*`. Edge attributes can be altered using the `scale_edge_*`
function exported by `ggraph`.
Furthermore, setting `return_data = TRUE` returns the symbolic edge-list
and vertex metadata used for graph construction in a `list` of two
`data.frames`.
```{r plotSpatialContext - return data}
# Return data
plotSpatialContext(pancreasSCE,
entry = "spatial_context",
group_by = "ImageNb",
return_data = TRUE)
```
## Community detection
In addition to cellular neighborhood and spatial context analysis, `imcRtools` exports
the `detectCommunity` function to detect the spatial community of each cell as
proposed by [@Jackson2020]. Here, each cell is clustered based on its interactions
as defined by a spatial object graph.
In more detail, the spatial community detection procedure is as follows:
1. Create an igraph object from the edge list stored in
\code{colPair(object, colPairName)}.
2. Perform community detection using the specified \code{cluster_fun} algorithm
(defaults to "louvain").
3. Store the community IDs in a vector and replace all communities
with a size smaller than \code{size_threshold} by NA.
```{r detectCommunity}
## Detect spatial community
set.seed(22)
pancreasSCE <- detectCommunity(pancreasSCE,
colPairName = "expansion_interaction_graph")
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_community",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "expansion_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free")
## Detect spatial community - specify size_threshold
set.seed(22)
pancreasSCE <- detectCommunity(pancreasSCE,
colPairName = "expansion_interaction_graph",
size_threshold = 20)
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_community",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "expansion_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free")
```
It is also possible to use different community detection algorithms from the `igraph`package.
```{r detectCommunity-walktrap}
## Detect spatial community - walktrap community detection
set.seed(22)
pancreasSCE <- detectCommunity(pancreasSCE,
colPairName = "expansion_interaction_graph",
cluster_fun = "walktrap")
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_community",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "expansion_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free")
```
Moreover, the user can specify \code{group_by} to perform spatial community
detection separately for all unique entries to \code{colData(object)[,group_by]}
e.g. for tumor and non-tumor cells.
```{r detectCommunity-bygroup}
## Detect spatial community - specify group_by
pancreasSCE <- detectCommunity(pancreasSCE,
colPairName = "expansion_interaction_graph",
group_by = "CellType",
size_threshold = 10,
BPPARAM = BiocParallel::SerialParam(RNGseed = 22))
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "spatial_community",
node_size_fix = 4,
edge_width_fix = 1,
draw_edges = TRUE,
colPairName = "expansion_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free")
```
## Border cells
To exclude cells that are close to the image border, the `imcRtools`
package exports the `findBorderCells` function.
```{r findBorderCells}
pancreasSCE <- findBorderCells(pancreasSCE,
img_id = "ImageNb",
border_dist = 10)
plotSpatial(pancreasSCE[,!pancreasSCE$border_cells],
img_id = "ImageNb",
node_color_by = "CellType",
node_size_fix = 4,
edge_width_fix = 2,
edge_color_by = "clustered_neighbors",
draw_edges = TRUE,
colPairName = "knn_interaction_graph",
directed = FALSE,
nodes_first = FALSE,
scales = "free") +
scale_color_brewer(palette = "Set2") +
scale_edge_color_brewer(palette = "Set1")
```
Excluding border cells can be useful when incorrectly connected cells
are observed at image borders.
## Patch detection {#patch-detection}
An alternative and supervised way of detecting regions with similar
types of cells is available via the `patchDetection` function. Here, the
user defines which cells should be used for patch detection via the
`patch_cells` parameter. A patch is defined as a set of cells as defined
by `patch_cells` which are weakly connected in the graph in
`colPair(object, colPairname)`.
Below, the `patchDetection` function is demonstrated using the
previously computed `expansion` graph and defining cells of `celltype_B`
as the cells of interest. Here, the function additionally draws a
concave hull around the detected patch, expands the hull by 20$\mu{}m$
and defines all cells within this expanded hulls as patch cells.
```{r patchDetection}
pancreasSCE <- patchDetection(pancreasSCE,
patch_cells = pancreasSCE$CellType == "celltype_B",
colPairName = "expansion_interaction_graph",
expand_by = 20,
img_id = "ImageNb")
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "patch_id",
scales = "free")
```
Patches that only consist of 1 or 2 cells cannot be expanded.
For each patch larger than 2 cells, the spatial area can be computed using the
`patchSize` function:
```{r patchSize}
patchSize(pancreasSCE)
```
## Minimal distances to cells of interest
Calculate the minimal distance for each cell to a given cell type or class of
cells of interest, the function `minDistToCells` is available. Cells of interest
are defined via the `x_cells` parameter as `logical` and distances to for all
cells to those cells will be reported in a new column in the `colData` of the
`SingleCellExperiment`.
If the cells of interest form patches (many cells of the same type next to each
other) or similarly if a patch detection has previously been performed the
positive distances reflect the distances from cells outside of patches to the
closest patch border and the negative distances reflect the distances from cells
inside the patches to the patch border. If `return_neg` is set to `FALSE`
negative distances are set to 0.
```{r minDistToCells}
pancreasSCE <- minDistToCells(pancreasSCE,
x_cells = pancreasSCE$CellType == "celltype_B",
coords = c("Pos_X","Pos_Y"),
img_id = "ImageNb")
plotSpatial(pancreasSCE,
img_id = "ImageNb",
node_color_by = "distToCells",
scales = "free") +
scale_color_viridis()
```
## Neighborhood permutation testing {#test-neighborhood}
The following section describes how to observe and test the average
number of interactions between cell labels (e.g. cell-types) within
grouping levels (e.g. images). For full descriptions of the testing
approaches, please refer to [Shapiro et al., Nature
Methods](https://www.nature.com/articles/nmeth.4391) [@Shapiro2017] and
[Schulz et al., Cell
Systems](https://www.sciencedirect.com/science/article/pii/S2405471217305434)
[@Schulz2018]
The `imcRtools` package exports the `countInteractions` and
`testInteractions` functions, which summarize all cell-cell interactions
per grouping level (e.g. image). As a result, a table is returned where
each row represents one of all possible cell-type/cell-type interactions
among all grouping levels. Missing entries or `NA`s indicate missing
cell-type labels for this grouping level. The next section gives details
on how interactions are summarized.
### Summarizing interactions
The `countInteractions` function counts the number of edges
(interactions) between each set of unique cell labels per grouping
level. Simplified, it counts for each cell of type A the number of
neighbors of type B. This count is averaged within each unique grouping
level (e.g. image) in three different ways:
1. `method = "classic"`: The count is divided by the total number of
cells of type A. The final count can be interpreted as "How many
neighbors of type B does a cell of type A have on average?"
2. `method = "histocat"`: The count is divided by the number of cells
of type A that have at least one neighbor of type B. The final count
can be interpreted as "How many many neighbors of type B has a cell
of type A on average, given it has at least one neighbor of type B?"
3. `method = "patch"`: For each cell, the count is binarized to 0 (less
than `patch_size` neighbors of type B) or 1 (more or equal to
`patch_size` neighbors of type B). The binarized counts are averaged
across all cells of type A. The final count can be interpreted as
"What fraction of cells of type A have at least a given number of
neighbors of type B?"
The `countInteractions` returns a `DataFrame` containing the summarized
counts (`ct`) for all combinations of `from_label`, `to_label` and
`group_by`.
```{r countInteractions}
out <- countInteractions(pancreasSCE,
group_by = "ImageNb",
label = "CellType",
method = "classic",
colPairName = "knn_interaction_graph")
out
```
### Testing for significance
In the next instance, one can test if the obtained count is larger or
smaller compared to what is expected from a random distribution of cell
labels. For this, the `testInteractions` function permutes the cell
labels `iter` times and counts interactions as described above. This
approach generates a distribution of the interaction count under a
random distribution of cell labels. The observed interaction count is
compared against this Null distribution to derive empirical p-values:
`p_gt`: fraction of perturbations equal or greater than the observed
count
`p_lt`: fraction of perturbations equal or less than the observed count
Based on these empirical p-values, the `interaction` score (attraction
or avoidance), overall `p` value and significance by comparison to
`p_treshold` (`sig` and `sigval`) are derived. All results are returned
in form of a `DataFrame`.
We set the seed within the `SerialParam` (or `MulticoreParam`) function for
reproducibility.
```{r testInteractions}
out <- testInteractions(pancreasSCE,
group_by = "ImageNb",
label = "CellType",
method = "classic",
colPairName = "knn_interaction_graph",
BPPARAM = BiocParallel::SerialParam(RNGseed = 123))
out
```
# Contributions
Large chunks of the code of the `imcRtools` package is based on the work
of [Vito Zanotelli](https://github.com/votti). Vito has co-developed the
spillover correction approach and translated the interaction testing
approach developed by [Denis Shapiro](https://github.com/DenisSch) and
[Jana Fischer](https://github.com/JanaFischer) into R (formerly known as
the [neighbouRhood](https://github.com/BodenmillerGroup/neighbouRhood) R
package). Jana has furthermore added the "patch" method for interaction
counting and testing. [Tobias Hoch](https://github.com/toobiwankenobi)
has written the first version of reading in the
`ImcSegmentationPipeline` output and the `patchDetection` function.
[Daniel Schulz](https://github.com/SchulzDan) has build the
`aggregateNeighbors` and `minDistToCells` functions and contributed to developing the spatial
clustering approach. [Lasse Meyer](https://github.com/lassedochreden) has
implemented the functions for spatial context analysis.
# Session info {.unnumbered}
```{r sessionInfo, echo=FALSE}
sessionInfo()
```
# References