Alignment, Segmentation and Neighborhood Analysis in Cyclic Immunohistochemistry Data Using CASSATT

Abstract

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. In addition, cycIHC benefits from the speed and simplicity of brightfield microscopy for data collection, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets (greater than 50 GB) currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the logodds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact - or do not interact - at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT was first tested using a newly generated cycIHC dataset consisting of six GBM tissue sections processed through eight cycles of AEC based IHC staining. Further validation was completed on a previously published lung cancer tissue microarray dataset consisting of 107 cores processed through eighteen cycles of staining and imaging. CASSATT is a fully open-source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique.

An immunosuppressed microenvironment distinguishes lateral ventricle–contacting glioblastomas

“…Multiplex IHC. Cyclic IHC was performed as reported (48). Formalin-fixed, paraffin-embedded tissue was sequentially stained with a panel of validated antibodies (CD3, CD4, CD8, FOXP3, PD-1, CD68, IBA1, PD-L1) with colorimetric detection using 3-amino-9-ethylcarbazole via the Translational Pathology Shared Resource at Vanderbilt University Medical Center.…”

“…Between rounds of staining, images were digitally acquired using a Leica SCN400 slide scanner. The Cyclic Analysis of Single-Cell Subsets and Tissue Territories image analysis pipeline registered sequential images and identified immune populations within the tissue (48).…”