Characterization of Tumor-immune Microenvironment by High-throughput Image Analysis of CD8 Immunohistochemistry Combined With Modified Masson's Trichrome

Abstract

With the advent of checkpoint inhibitors, there is increasing need to study the dynamics of CD8+ T-cells in the tumor microenviroment. In this article, we describe a semi-automated method to quantify and interrogate spatial relationships between T-cells and collagenous stroma in human and mouse tissue samples. The assay combines CD8 immunohistochemistry with modified Masson's trichrome. Slides are scanned and digital images are analyzed using an adjustable MATLAB algorithm, allowing for high-throughput quantification of cytotoxic T-cells and collagen. This method provides a flexible tool for unbiased quantification of T-cells and their interactions with tumor cells and tumor microenvironment in tissue samples.

Keywords: CD8; collagen; dual staining; immunohistochemistry; trichrome.

Figure 1.

CD8-trichrome staining of mouse xenograft models and human urinary bladder cancer. CD8-trichrome-stained slides showing interactions between CD8+ and collagen fibers in mouse xenografts (A–C). Pancreatic adenocarcinoma showing intratumoral collagen (arrowheads) with T-cells distributed between tumor cells and throughout the collagenous stroma (A). Colorectal carcinoma showing prevalence of immature collagen within the tumor center (arrowheads) and a thin peritumoral capsule of mature collagen (arrows). CD8+ cells are present primarily within tumor cell nests (B). Breast carcinoma showing ample mature collagen both peripherally and scattered throughout the tumor with CD8+ cells located at tumor periphery (C). Representative examples of immune phenotype categories in human urinary bladder cancer. Excluded category shows extensive colocalization of CD8+ cells with collagen fibers (arrowheads) and rare CD8+ cells in contact with tumor cells (arrows) (D). Inflamed type 2 tumor shows the majority of CD8+ cells within stromal bands (arrowheads) but with “spillover” into tumor cell nests (arrows) (E). Inflamed type 1 tumor shows a diffuse infiltrate of CD8-positive cells that involves tumor cell nests (arrows) (F). Scale bar, 100 µm.