Abstract 3771: 3D correlated multi-scale cellular and molecular architecture of solid tumors

Abstract A solid tumor presents heterogeneous 3D structures at tissue, cell, and molecule levels. To develop advanced cancer diagnostics and therapies, it is important to understand in-depth cellular and molecular architecture of a tumor at multiple scales. Here we introduce novel optical microscopy methodologies for in-depth 3D spatial analysis of tumor tissues: 1) LED photobleaching-mediated 3D multiplex immunofluorescence (IF) microscopy, 2) Correlated 3D multi-resolution optical microscopy, and 3) 3D tissue region-based cell type-selective multi-omics. We built a high-power LED light irradiator with a broad emission spectrum (490-700 nm) that bleaches a broad wavelength of fluorescence signals simultaneously throughout an optically cleared, 400 µm-thick tumor tissue. Cyclic workflow involving IF staining, optical tissue clearing, 3D confocal microscopy, and LED photobleaching enables visualization of multiple cell types in the same whole 400 µm-thick tumor tissue after computational 3D image registration. The constructed multiplex image offers comprehensive 3D cellular landscape of a tumor. We also developed a method for tracking regions of interest (ROIs) in the middle of a large tumor tissue using a UV-activated visible dye in light sheet microscopy. Light sheet microscopy allows imaging an optically cleared large-sized tumor tissue at a low spatial, tissue resolution. To mark the ROI positions on a tumor, the dye is infused into agarose gel surrounding the tumor tissue and be activated and present purple-colored lines where are exposed to UV light sheets in light sheet microscopy. The tumor tissue was physically sectioned at the marked ROI positions using a vibratome after reversing tissue clearing process. Sequential IF staining and high-resolution confocal microscopy permit for visualizing detailed cellular compositions of the ROIs in the tumor. The tissue and cellular-resolution 3D images from light sheet and confocal microscopy are correlatively integrated to display ‘Google Earth’-like multi-scale view of a tumor tissue. To further analyze the ROIs of a tumor at molecular level, we have developed optical microscope-mediated fluorescence lithography methods that allow fluorescently labeling of a selected cell type in ROIs of an optically cleared 3D tumor tissue. After wash free from clearing solution and dissociation of the tumor into cells, fluorescence-marked, intact cells from the ROIs are collected through flow cell sorting. Proteins and mRNAs are extracted from the collected cells and sent to LC-MS/MS analysis and RNA sequencing. The spatial multi-omics method provides in-depth molecular information of selected cells in the ROIs of a tumor. We believe all these 3D microscopy methods will provide powerful spatial analysis tools for better understanding cellular and molecular architecture of solid tumors. Citation Format: Steve Seung-Young Lee, Yi-Chien Wu, Jingtian Zheng, Elie Abi Khalil, Samuel Wang, Alexander Lippert, Joshua Plank. 3D correlated multi-scale cellular and molecular architecture of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3771.