A novel histological examination with dynamic three-dimensional reconstruction from multiple immunohistochemically stained sections of a PD-L1-positive colon cancer.

Abstract

Programmed cell death-ligand 1 (PD-L1) expression is observed in patients with microsatellite instability-high (MSI-H) colon cancer, which is susceptible to immune checkpoint blockade. The aim of this study was to investigate the interrelationship between PD-L1-positive cells and cytotoxic T cells, lymphatic vessels and vascular endothelium by using histological examination with the three-dimensional (3D) reconstruction of a PD-L1-positive colon cancer. Serial sections of MSI-H colon cancer tissue were stained with haematoxylin and eosin (H&E) and Masson trichrome stains; immunohistochemical analysis of PD-L1, CD8, D2-40 and CD31 was performed. Several 3D models of MSI-H colon cancer were reconstructed with a 3D data visualisation system. Moreover, 18 serial sections were stained with PD-L1, cytokeratin AE1/AE3, CD45, CD31, CD68 and H&E in the same case to confirm that PD-L1 was expressed on tumour cells, CD31-positive cells and macrophages in the invasive frontal region. Notably, there was a peak in the expression of PD-L1 and CD31 in the invasive frontal region. D2-40-positive cells were abundant in the overall tumour stroma, and CD8-positive cells infiltrated the tumour parenchyma. PD-L1 was expressed on tumour cells in the parenchyma and other cells in the stroma. Additional staining of 18 consecutive sections revealed that the other cells were CD68-positive and CD45-positive macrophages and CD31-positive proliferating vascular endothelial cells. We confirmed that PD-L1 was highly expressed in the invasive frontal region in 3D models of MSI-H colon cancer tissue. This method can be useful for accurately evaluating the localisation of immune checkpoint molecules.