Abstract 4707: LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment
Authors: Zheng Jingtian , Phillips Evan , Wu Yi-Chien , Lee Steve Seung-Young , Bindokas Vytautas 
Published: April 2023

Abstract
Multiplexing in immunofluorescence imaging is important for the spatial profiling of cells and molecules in tumor tissue samples. Cyclic immunofluorescence (IF) methods using oxidants (e.g. hydrogen peroxide) and enzymes (e.g. DNase) localize a great number of cellular makers and proteins in a tissue section while repeating a process of IF staining, imaging, and fluorescence deactivation. However, the repeated use of chemicals and enzymes might cause artifacts in tissue and cell morphologies. Furthermore, these methods are restricted to thin tissue sections (~5 μm thick) which are inappropriate to provide comprehensive structural information on tissue samples. Although reconstruction of two-dimensional (2D) images from serial tissue sections can provide a certain volumetric tissue image, it takes a huge amount of time and effort. Here we introduce a three-dimensional (3D) multiplex IF imaging method using LED photobleaching. We built high-power LED illuminators with 100W warm (emission wavelength: 480-700 nm), green (430-520 nm), and red (600- 680 nm) LED chips, which can efficiently bleach a broad or selected wavelength of fluorescence signals in tissue samples. We integrated this LED photobleaching with the Transparent Tissue Tomography (T3) protocol and created a 3D cyclic IF method involving tissue macrosectioning (400 μm), three-color IF staining, D-fructose-based tissue clearing, 3D confocal fluorescence microscopy, LED photobleaching, tissue washing, and three-color IF staining for other biomarkers, and repeating the process. By applying this method to mouse mammary tumor tissues, we could perform 8-plex fluorescence microscopy for visualizing cell nuclei (DAPI), vascular (CD31, SMA) and structural (ER-TR7) cells, immune cells (CD3, CD8, CD45), and cancer cells (CK8) in the tumor macrosections in 3D at tissue and cellular resolution. To validate the method as an evaluation tool for immunotherapy, we treated the mouse mammary tumor with a STING agonist (DMAXX) intratumorally and collected the tumor tissue 1 day after the treatment, and processed it for the 3D cyclic IF protocol. The quantitative multiplex image data showed immune-driven-cancer eradication and high tumor infiltration of a large number of CD3+CD8+CD45+ cytotoxic T cells. We also examined that Red and Green LED illumination can selectively bleach fluorophores in tissues, which would be useful for patterning fluorescence in tissue as well as studying fluorescent drug-cell interaction in a tissue. In summary, this chemical and enzyme-free 3D cyclic IF imaging method will be a powerful tissue assay tool to provide comprehensive spatial information of tissue (tumor) samples including cell types, cellular and molecular location, and their 3D organization in a tissue sample.
Citation Format: Jingtian Zheng, Evan Phillips, Yi-Chien Wu, Steve Seung-Young Lee, Vytautas Bindokas. LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4707.