Abstract 3032: Quantitative three-dimensional imaging cytometry of tumor immune microenvironment
Authors: Lee Steve Seung-Young , Kron Stephen J. , Bindokas Vytautas P. 
Published: August 2018

Abstract
The tumor microenvironment is a three-dimensional (3D) system of diverse cellular and non-cellular components whose heterogeneous structure is typically defined by haphazard growth of cancer cells and a disordered microvasculature. Tumor-immune cell interactions occur within this context, providing a challenge for analysis of the infiltrate and anti-tumor immune responses by two-dimensional (2D) methods such as immunohistochemistry (IHC). Toward overcoming the limitations of 2D methods, we introduce Transparent Tissue Tomography (T3) as a tool for quantitative 3D imaging cytometry of the tumor immune microenvironment. For T3 imaging cytometry, tumors were sliced into 400 μm macrosections to facilitate immunofluorescence staining, optical clearing, and confocal microscopic imaging. Macrosections were stained overnight with panels of four to six directly labeled fluorescent primary antibodies then cleared by D-fructose. The full volume of each macrosection was scanned in each channel using a confocal microscope and tumor images were tomographically reconstructed from the macrosection images. The tumor images were segmented to discriminate cell types, map biomarkers, and perform spatial analysis. As an application of T3 imaging cytometry, we examined the distribution of programmed death-ligand 1 (PD-L1) expression in spontaneous Her2+ mammary tumors formed in BALB-NeuT mice. T3 analysis of whole tumors determined PD-L1 expression by tumor cells at the periphery and CD31+ vascular endothelium in the core. For the first time, 3D tomographic projection disclosed vascular PD-L1 expression localized between the endothelium and inner layer of smooth muscle cells. In turn, T3 revealed a strong spatial correlation between CD45+ immune cell distribution and PD-L1 expression. Toward translation, T3 was adapted to analyze whole core needle biopsies. Using T3 to map CD3+CD8+ cytotoxic T cells (CTLs) in BALB-NeuT tumors revealed marked inhomogeneity across single 18 gauge needle cores, confirmed by serial section IHC assays after T3 imaging. Modeling on-treatment biopsy analysis, T3 confirmed a compound effect of radiation and anti-PD-L1 therapy on infiltration of effector cytotoxic lymphocytes such as granzymeB+ NK cells and T cells. Applying T3 imaging cytometry to analyze needle cores from EGFR+ human head and neck cancer tissue, we mapped the distribution of CD3+CD8+ CTLs with respect to the CD31+ microvasculature and tumor cells. By assessing multiple tumor parameters simultaneously at cellular resolution, T3 provides a unique window into the heterogeneity of the tumor immune microenvironment. In particular, spatial, multiparameter T3 analysis may serve as a tool to improve diagnostic, prognostic and predictive testing of patient biopsies as part of evaluation for immune checkpoint blockade therapy.
Citation Format: Steve Seung-Young Lee, Stephen J. Kron, Vytautas P. Bindokas. Quantitative three-dimensional imaging cytometry of tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3032.