This repository contains the pipelines developed for quantifying and determining spatial relationships bteween subtypes of immune cells and fibroblasts in tissues stained using multiplex immunofluorescence technology

1. Project outline

1.1. Dataset 1:

The dataset comprises three-channel images - DAPI (DNA), FITC (CD8+ T-cell), Texas Red (SMA+ fibroblasts). Images were received in three sets; high-contrast images, low-contrast images, and other images (composite/merged images).

1.1.1. Objectives:

1. Count the number of CD8+ cells
2. Measure the percentage of tissue area that is SMA+
3. Determine spatial relationship between CD8+ cells and SMA+ areas, i.e., measure the average distances of CD8+ cells from nearest SMA+ areas, and the number of CD8+ cells within a given distance (20, 50, 100, and 200 um) of the SMA+ areas

1.1.2. Challenges

1. Variable intensity of SMA-expression in the fibroblasts
2. Accurate segmentation of fibroblasts (fibrillar, elongated, non-circular cells)
3. Accurate segmentation of clumped nuclei

1.2. Dataset 2:

This dataset comprises three-channel images - DAPI (DNA), FITC (CD3+ T-cells), Cy5 (FOXP3+ T-cells).

1.2.1. Objectives:

1. Count the number of CD3+ cells
2. Count the number of CD3-FOXP3 double-positive cells and percentage of CD3+ cells that are CD3-FOXP3 double-positive

1.2.2. Additional objectives

• Ability to use the pipeline regardless of fluorophore for each stain

2. Files in this repository:

1. Pipelines

(i) Pipelines for Dataset 1

1. CellProfiler pipeline without plugins: CD8-SMA-tumor_CellProfiler.cpproj; CD8-SMA-tumor_CellProfiler.cppipe
2. CellProfiler pipeline with RunStarDist plugin: CD8-SMA-tumor_RunStarDist.cpproj; CD8-SMA-tumor_RunStarDist.cppipe

(ii) Pipelines for Dataset 2

1. CellProfiler pipeline without plugins: CD3-FoxP3-tumor_CellProfiler.cpproj; CD3-FoxP3-tumor_CellProfiler.cppipe
2. CellProfiler pipeline with RunStarDist plugin: CD3-FoxP3-tumor_RunStarDist.cpproj; CD3-FoxP3-tumor_RunStarDist.cppipe

2. Description of all the steps with example images are provided for both pipelines