Extended Data Fig. 4: Multispectral imaging workflow and spatial distribution comparisons.

(a) Schematic showing workflow for multispectral immunohistochemistry imaging panel and downstream analysis using Biorender.com (b) Composite and single channel images for each marker used in the panel to demonstrate marker specificity and quality of unmixing. Images represent a single region of interest (ROI) with fairly consistent results across each channel observed for all ROIs (>1200) in the mIHC dataset (c) Median cell frequencies for immune cell subsets by mIHC in ER+ tumors (n = 115 patients) compared to TNBC (n = 21 patients) in stroma (left panel) and tumor bed (right panel). Each circle represents the median value across all ROIs for each patient and bar represents group median. Multiple Mann Whitney non-parametric two-sided T test was used for statistical analysis with Holm-Sidak correction for multiple comparison.(d) Median immune cell frequency distributions in ER+ IDC and ILC and TNBC after excluding HER2+ER+ IDC and ILC sample across stromal (left panel) and tumor (right panel) compartments. Each circle represents the median value across all ROIs for each patient and bar represents group median. Multiple Mann Whitney non-parametric two-sided T test was used for statistical analysis with Holm-Sidak correction for multiple comparison. (e) Pie charts demonstrating median composition of tumor cells and immune cells across all ROIs in ER+IDC (n = 50 patients) (top panel), ILC (n = 65 patients) (middle panel) and TNBC (n = 21 patients) (bottom panel).