{'fraction_non_alphanumeric': 0.03131597466572836, 'fraction_numerical': 0.017593244194229415, 'mean_word_length': 5.73696682464455, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Abstract B29: Multiplex three-dimensional optical mapping of tumor immune microenvironment

Recent advances in optical tissue clearing and microscopic imaging have facilitated three-dimensional (3D) visualization of large tissues and whole organs at a high resolution. However, to expand its applications to oncology, current challenges such as slow and complicated tissue clearing and uneven immunostaining must be addressed. Here we describe transparent tissue tomography (T3), a tool for rapid, three-dimensional, multiplex immunofluorescence tumor imaging. Cutting tumors into 400 μm 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 programmed death-ligand 1 (PD-L1) distribution in transgenic mouse mammary tumors, with high expression limited to Her2+ 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 at the tumor margin was determined by T3 analysis of the whole tumors. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 to immunolocalization of cytotoxic lymphocytes in whole core needle biopsies obtained from mouse mammary tumors and human head & neck tumors. We spatially and quantitatively map granzymeB-producing CD3+CD8+ cytotoxic T cells and CD49b+CD31+ natural killer cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by chromogenic immunohistochemistry (IHC). We anticipate that T3 can be applied broadly to facilitate preclinical studies of tumor immunology and immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy. Citation Format: Steve Seung-Young Lee, Vytautas Bindokas, Stephen Kron. Multiplex three-dimensional optical mapping of tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B29.