Spatial TME analysis

Spatial-PHLEX is the final submodule of TRACERx-PHLEX. It is a nextflow-based pipeline for the analysis of spatially resolved single cell data. It is designed for the highly granular phenotypic outputs of TYPEx, but owing to its simple input format can be used with any single cell coordinate data where single cells are assigned a phenotype or class label with minimal effort.

Cell type niche analysis via density-based spatial clustering
Cellular barrier scoring

Workflow options

Spatial-PHLEX provides three primary workflow options for spatial cell data:

Workflow 1: Spatial clustering
Cellular barrier scoring
Workflow 3: Barrier scoring to spatial clusters

Workflow 1: Spatial clustering

Spatial clustering with Spatial-PHLEX. — Overview of the Spatial-PHLEX spatial clustering workflow.

Workflow 2: Cellular barrier scoring

Definition of a barrier

Workflow 3: Barrier scoring to spatial clusters

Example usage

## Spatial-PHLEX
nextflow run ./main.nf \
     --metadata <path/to/metadata>\
     --metadata_delimiter '\t'\
     --objects '/path/to/cellobjects.csv'\
     --objects_delimiter '\t'\
     --phenotyping_column 'majorType' \
     --graph_type 'nearest_neighbour' \
     --outdir '../../results' \
     --release 'PHLEX_test' \
     --workflow_name 'clustered_barrier' \

Input Files

Required Inputs

cell_objects.csv
- A plaintext, delimited file containing single cell-level coordinate data for a set of images, plus their phenotypic identities.
metadata.csv
- A plaintext, delimited file containing metadata information about the images in cell_objects.csv. To run the pipeline this file must contain, for each image, an image identifier (‘imagename’), and the width and height in pixels for every image as columns with the header ‘image_width’ and ‘image_height’.

The Spatial-PHLEX input dataframe. — The Spatial PHLEX input dataframe has a simple format allowing cell coordinate results from other imaging modalities to be processed with Spatial-PHLEX.

Outputs

Cell type specific spatial clusters
Barrier scores

Output from Spatial-PHLEX has the following directory structure.

results
├── graph
│   ├── aggregated_barrier_scoring
│   └── raw_barrier_scoring
└── spatial_clustering

Note

The name of the raw_barrier_scoring directory will vary depending on which Spatial-PHLEX workflow is specified.

Parameters

Spatial PHLEX parameters are defined in the nextflow.config file in the Spatial PHLEX base directory.

Spatial PHLEX input parameter definitions.
Spatial-PHLEX param	Definition	Input options
barrier_cell_type	The type of cell forming the barrier in the barrier scoring calculation.	Myofibroblasts
barrier_phenotyping_level	Column name in the objects table used to derive cell types for barrier scoring.	e.g. cellType
barrier_source_cell_type	The source cell type to compute barrier scores for.	CD8 T cells
barrier_target_cell_type	The target cell type to compute barrier scores for.	Epithelial cells_tumour
dev	Development mode; sample a subset of input images.	true, false
graph_type	The method of graph construction from cell positional data.	‘nearest_neighbour’,’neighbourhood’,’spatial_neighbours’
metadata	Path to the metadata file containing image-level metadata about images to be analysed.	e.g. ‘/path/to/metadata.txt’
metadata_delimiter	Delimiter of the metadata file.	e.g. ‘t’
n_neighbours	Number of nearest neighbours for nearest_neighbour graph construction.	10
neighborhood_input	globbable path to csv files containing neighbouRhood output produce by CellProfiler module.	e.g. ‘/path/to/results/segmentation///neighbourhood.csv’
neighbourhood_module_no	Module number of the neighbouRhood proces sin the CellProfiler pipeline	e.g. 865
number_of_inputs	Number of images to process the data for in development mode.	2
objects	Path to the cell objects dataframe.	e.g. ‘/path/to/objects.csv’
objects_delimiter	Character delimiting the objects dataframe.	e.g. ‘t’
outdir	Root output directory where results will be created.	../../results
overwrite	Overwrite results published to the results directory, if they already exist.	true
phenotyping_level	The column name in the objects dataframe defining the phenotypes of the cells.	e.g. ‘cellType’; ‘Ki-67+ve’
publish_dir_mode	Way Nextflow generates output in the publish directory.	default: ‘copy’
release	Release directory. Identifier for the data analysis run.	e.g. ‘2022-08-23’
workflow_name	Spatial PHLEX workflow to run on the data.	Options: ‘default’,’spatial_clustering’, ‘graph_barrier’