phenoptr contains several functions which analyze and report on the spatial relationship between cells in a single field.
find_nearest_distance function finds per-cell nearest neighbor distances. For each cell in a sample, it finds the nearest neighbor cell in each of the provided phenotypes and reports the cell ID and distance to the nearest neighbor cell. Results are returned in a
tibble with a
Distance to and
Cell ID column per phenotype.
For example, the
phenoptr sample data
sample_cell_seg_data contains 5 unique phenotypes:
# A tibble: 5 x 2 Phenotype n <chr> <int> 1 CD68+ 417 2 CD8+ 228 3 CK+ 2257 4 FoxP3+ 228 5 other 2942
other cells are not of interest, so first filter them out.
csd <- csd %>% filter(Phenotype!='other')
find_nearest_distance on this file returns a
tibble with two columns for each phenotype and one row for each cell. The
Distance to <phenotype> columns give the distance to the nearest cell of the phenotype; the
Cell ID <phenotype> columns identify the nearest cell.
Rows: 3,130 Columns: 8 $ `Distance to CD68+` <dbl> 29.529646, 38.082148, 36.674242, 73.119765, 51... $ `Cell ID CD68+` <int> 108, 41, 262, 99, 217, 69, 236, 217, 262, 110,... $ `Distance to CD8+` <dbl> 18.03469, 64.37585, 67.57403, 91.44397, 37.947... $ `Cell ID CD8+` <int> 101, 5068, 423, 189, 128, 188, 280, 128, 423, ... $ `Distance to CK+` <dbl> 36.830694, 109.317199, 3.605551, 4.031129, 5.5... $ `Cell ID CK+` <int> 192, 5127, 45, 58, 4943, 209, 636, 87, 30, 45,... $ `Distance to FoxP3+` <dbl> 16.347783, 40.140379, 30.870698, 65.408333, 77... $ `Cell ID FoxP3+` <int> 117, 214, 229, 138, 229, 102, 66, 229, 229, 95...
To create a combined data frame, use
csd_with_distance <- bind_cols(csd, distances)
Note: The origin for cell positions in a cell seg data file may be the slide origin or the field origin, depending on the inForm version and the selected option. The field origin is the top-left corner of the field, so calling
find_nearest_distance on a merged data file with field positions will compute incorrect values. One way to add distance columns to such a merged data file is to use
dplyr::group_by to process each field separately. For example, if
merged contains merged cell seg data with field positions, add distance columns with this code:
Once the nearest neighbors have been computed per cell, standard aggregation, analysis and plotting commands can be used to examine the results. For example, find the mean nearest neighbor distances by phenotype:
csd_with_distance %>% group_by(Phenotype) %>% select(Phenotype, starts_with('Distance to')) %>% summarize_all(~round(mean(.), 1))
# A tibble: 4 x 5 Phenotype `Distance to CD6~ `Distance to CD~ `Distance to CK~ `Distance to Fo~ <chr> <dbl> <dbl> <dbl> <dbl> 1 CD68+ 14.2 30.6 23.1 24.8 2 CD8+ 23.1 17.5 19.3 28.4 3 CK+ 44.3 48.2 7.9 50.5 4 FoxP3+ 19.1 29.1 23.3 22.3
Show the distribution of distances in a density plot:
Cell ID <phenotype> column allows visualizing nearest neighbors by joining the combined cell seg table with itself. For example, to show the nearest CK+ cell for each CD8+ cell, join the
Cell ID CK+ field to the original
Cell ID field. This example filters the data before joining.
# Filter to just CD8+ and CK+ cells cd8_cells = csd_with_distance %>% filter(select_rows(csd_with_distance, 'CD8+')) ck_cells = csd_with_distance %>% filter(select_rows(csd_with_distance, 'CK+')) # For each CD8+ cell, join with the data for the nearest CK+ cell cd8_to_ck = cd8_cells %>% left_join(ck_cells, by=c('Cell ID CK+'='Cell ID'), suffix=c('', '.CK'))
Show the CD8+ and CK+ cells with the nearest neighbors connected:
# Read a background image and make a base plot background_path = system.file("extdata/sample/Set4_1-6plex_[16142,55840]_composite_image.jpg", package='phenoptr') background = jpeg::readJPEG(background_path) %>% as.raster() xlim = c(0, 934) ylim = c(0, 700) base_plot = ggplot(mapping=aes(`Cell X Position`, `Cell Y Position`)) %>% phenoptr:::add_scales_and_background(background, xlim, ylim, scale_color='white') + labs(x='Cell X Position', y='Cell Y Position') + scale_color_manual('Phenotype', values=c('CD8+'='red', 'CK+'='cyan2')) # Add lines and points base_plot + geom_segment(data=cd8_to_ck, aes(xend=`Cell X Position.CK`, yend=`Cell Y Position.CK`), color='white') + geom_point(data=ck_cells, aes(color='CK+'), size=1) + geom_point(data=cd8_cells, aes(color='CD8+'), size=1) + labs(title='Nearest CK+ to each CD8+')
To show the nearest CD8+ for each CK+, join in the other direction; for each CK+ cell, find the nearest CD8+ cell:
ck_to_cd8 = ck_cells %>% left_join(cd8_cells, by=c('Cell ID CD8+'='Cell ID'), suffix=c('', '.CD8')) base_plot + geom_segment(data=ck_to_cd8, aes(xend=`Cell X Position.CD8`, yend=`Cell Y Position.CD8`), color='white') + geom_point(data=ck_cells, aes(color='CK+'), size=1) + geom_point(data=cd8_cells, aes(color='CD8+'), size=1) + labs(title='Nearest CD8+ to each CK+')
Mutual nearest neighbors are cells which have each other as nearest neighbors; i.e. cells where the nearest neighbor of the nearest neighbor is the starting cell:
mutual = ck_to_cd8 %>% filter(`Cell ID`==`Cell ID CK+.CD8`)
This data set contains 126 mutual nearest neighbor pairs between CD8+ and CK+ cells.
base_plot + geom_segment(data=mutual, aes(xend=`Cell X Position.CD8`, yend=`Cell Y Position.CD8`), size=1, color='white') + geom_point(data=ck_cells, aes(color='CK+'), size=1) + geom_point(data=cd8_cells, aes(color='CD8+'), size=1) + labs(title='Mutual nearest neighbors - CD8+ and CK+')
count_within function looks at the number of cells within a radius of another cell and returns summary measures. For example, use
count_within to find the number of
CD68+ cells having a
CK+ cell within 25 microns:
count_within(csd, from='CD68+', to='CK+', radius=25)
# A tibble: 1 x 5 radius from_count to_count from_with within_mean <dbl> <int> <int> <int> <dbl> 1 25 417 2257 274 3.28
In this result,
to_count are the total numbers of eligible cells. They agree with the counts in the first table in this tutorial.
from_with is the number of
CD68+ cells having at least one
CK+ cell within 25 micron.
within_mean is the average number of
CK+ cells found within 25 micron of each
Note there are some subtleties to
count_within. Most importantly, it is not symmetric. In this example, the number of
CK+ cells with a
CD68+ within 25 microns is not the same as the number of
CD68+ cells with a
CK+ cell within 25 microns.
count_within(csd, from='CK+', to='CD68+', radius=25)
# A tibble: 1 x 5 radius from_count to_count from_with within_mean <dbl> <int> <int> <int> <dbl> 1 25 2257 417 664 0.606
help(count_within) for details.
You may want to run
count_within on an entire directory of cell seg data files, or to count multiple combinations of phenotypes. Both of these are possible using
count_within_batch. This function takes the path to a directory that contains multiple cell seg data files. The
category parameters are lists and may contain multiple entries.
For example, the following commands will count
FoxP3+ cells with a
CK+ cell within 10 or 25 microns. Separate counts are returned for each
to phenotype and for
Stroma tissue categories. Counts will be calculated for all
cell_seg_data.txt files in
spatial_distribution_report function is a bit different from the other functions mentioned in this tutorial. Rather than calculate and return distance metrics, it creates a report which shows visually the nearest neighbor relations between two phenotypes in a single field. Because the result is a stand-alone HTML file, it can’t easily be demonstrated in a tutorial. For an example, copy and paste this code into your own copy of R. It will create a sample report in your user directory.
This example requires the
phenoptrExamples package, which includes extended sample data.
library(phenoptrExamples) cell_seg_path = system.file("extdata", "samples", "Set4_1-6plex_[16142,55840]_cell_seg_data.txt", package = "phenoptrExamples") pairs <- list(c('CD68+', 'CD8+')) colors <- c('CD68+'='magenta', 'CD8+'='yellow') out_path <- path.expand('~/spatial_distribution_report.html') spatial_distribution_report(cell_seg_path, pairs, colors, output_path=out_path)
To create reports for all cell seg data files in a directory, first define
colors as above. Use
list_cell_seg_files to find all the files. Then call
spatial_distribution_report for each file. This will create reports in the same directory as the data files.