Abstract
Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31+ vascular endothelium in the core. Also, strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.
Highlights
Malignant tumors can be considered abnormal organs that arise as proliferating cancer cells recruit and subvert host vasculature, immune cells and fibroblasts to form a supportive stroma, providing the means to continue growth and to displace and invade normal tissue[1]
While 3D reconstruction from serial thin sections can yield high resolution 3D maps[8,9], current tomographic methods remain poorly matched to the demands of the clinical environment, where speed is at a premium and simple, robust methods are required
We have developed Transparent Tissue Tomography (T3) in which lightly fixed tumors are cut into thick sections that are immunostained with fluorescently labeled primary antibodies, optically cleared, and imaged by confocal microscopy[24,25,26]
Summary
Malignant tumors can be considered abnormal organs that arise as proliferating cancer cells recruit and subvert host vasculature, immune cells and fibroblasts to form a supportive stroma, providing the means to continue growth and to displace and invade normal tissue[1]. Advances in tissue optical clearing, fluorescent staining and high resolution 3D microscopic imaging of brain tissue have made significant impacts in neuroscience[10,11,12,13] Robust methods such as CLARITY14 enable mapping the distributions of multiple antigens in intact mouse brains at sub-cellular resolution, allowing reliable tracing of connections over centimeter distances. This work has revealed fundamental limitations of CLARITY and related approaches including tissue distortion, antigen loss, uneven immunostaining, limited imaging depth and low sample throughput[23] To address these challenges, we have developed Transparent Tissue Tomography (T3) in which lightly fixed tumors are cut into thick sections that are immunostained with fluorescently labeled primary antibodies, optically cleared, and imaged by confocal microscopy[24,25,26]. This work establishes T3 as a novel tool for high resolution, quantitative 3D analysis of the tumor immune microenvironment
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