Abstract 76: Correlated multi-scale 3D optical microscopy of tumor tissues

Abstract Three-dimensional (3D) optical microscopy in combination with advanced tissue clearing methods permits the interrogation of intact large-sized tumor tissues. There are many optical microscopy methods (e. g. light-sheet and confocal microscopy) offering 3D tumor images at varying levels of spatial resolution (e. g. organ, tissue, cell, and molecule level). However, it is a challenge to correlatively integrate various resolution 3D images obtained by different optical microscopy of tumor tissue. In this project, we developed a correlated multi-scale 3D optical microscopy method that enables the acquisition of macroscopic to microscopic spatial information from a single tumor tissue in 3D. Firstly, we used light sheet microscopy to image a whole large-sized, cleared tumor tissue and analyzed the results to identify regions of interests (ROIs). After defining ROIs in the 3D whole tumor image, we added a UV-activatable visible dye, spiropyran, in the agarose gel surrounding the cleared tumor and exposed the sample to a UV light sheet horizontally at the height (z-axis) of the tumor where the ROIs present. The activated dye showed ‘purple’ colored lines in the agarose gel and we physically marked the gel along the visible colored lines. We further sectioned the tumor at the ROI positions using a vibratome after reversing tissue clearing process. After immunofluorescence staining and aqueous-based tissue clearing, confocal microscopy allowed the localization of various cell types in the ROIs of tumor macrosections. To validate the method for multi-scale evaluation of cancer immunotherapy, we tested it with control and DMXAA-treated mouse mammary tumor tissues. We successfully acquired the 3D images of whole tumors by capturing autofluorescence signals with a 633 nm excitation laser and its matched emission filter. Based on the autofluorescence tumor images, we defined two ROIs for each control and treated tumor and performed optical marking and physical sectioning of the tumors as described above. Next, we stained the macrosections (400 µm thick) with DAPI, CK8-DL488, CD45-DL550, and ER-TR7-DL680 to visualize cancer, immune, and stromal cells in the ROIs at cellular resolution. We quantitatively analyzed the correlated multi-scale 3D image data for assessment of the effects of DMXAA treatment on the tumor microenvironment including eradiation of cancer cells, immune infiltration, and remodeling of tumor stroma. In summary, this new microscopy method will provide comprehensive spatial information of tumor tissues and benefit a broad cancer research field such as the discovery of new tumor biomarkers and the development of effective cancer therapies. Citation Format: Jingtian Zheng, Steve Seung-Young Lee. Correlated multi-scale 3D optical microscopy of tumor tissues 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 (6Suppl): Abstract nr 76.