Abstract 5915: Three-dimensional, 3-D, multiplex imaging of biomarkers in tumor tissue
Authors: White Sharla L. , McCurdy Sarah , Goodman Laurie J. 
Published: July 2017

Abstract
Background: Obtaining high-resolution information from solid tumors, while maintaining the spatial perspective needed to understand the heterogeneous tumor microenvironment, represents a key challenge for preclinical and clinical cancer applications.
Methods: We utilized a novel method CLARITY that has been shown to transform intact rodent tissues into an optically transparent and permeable hybridized-hydrogel form that can undergo immunostaining followed by 3-D imaging of nucleic acids and proteins markers, without the need to separately analyze hundreds of conventional FFPE thin sections. In this study, human breast and lung cancer excision biopsy samples were obtained commercially. Murine orthotopic MCF-7 xenograft and patient-derived-xenograft (PDX) breast tumors were explanted and prepared as fresh samples. The intact tissues were incubated in a mixture of 4% paraformaldehyde/4% acrylamide/0.05% bis-acrylamide for 48 hours, and polymerized in the presence of a thermal initiator, VA-044, for 3 hours at 37°C to form an intact hydrogel/tissue matrix. The tissues were then lipid-cleared in a solution of 0.2M borate buffer containing 8% SDS, pH 8.5 at 45-55°C from 5-35 days, depending on sample size. The cleared tissues were then stained with two multiplex antibody panels that were selected to highlight the microenvironment of the tissue including, immune cells (CD-3 or CD-8), vasculature (Lectin-Texas Red or CD-31), and tumor (cytokeratin [8+18] or PD-L1, directly conjugated to Alexa Fluor® 647). For non-directly conjugated antibodies, the tissues were then subjected to a secondary goat anti-mouse or goat anti-rabbit antibody conjugated to either Alexa Fluor® 568 or 700 followed by a nuclear counterstain, Sytox Blue. Samples were then placed in an appropriate refractive index solution for imaging on a laser scanning confocal microscope.
Results: The tissues remained intact throughout the procedure and the cellular morphology of the tissue was well preserved. The individual components of the microenvironment could be identified, demonstrating that the tissues could be successfully interrogated with a multiplex of indirect and directly conjugated commercially available antibodies. These preliminary studies, provide evidence for the use of this novel technology for detecting the heterogeneity of biomarker expression within the tumor microenvironment. Most notably, the analysis of a lymph node from a patient with metastatic breast cancer revealed variation across the tissue which may have implications for the detection of micro-metastasis in patients with early stage disease when using conventional thin section histological methods.
Conclusion: These results implicate CLARITY as a powerful next generation tissue processing technology for profiling the intact tumor microenvironment, eliminating the need to recapitulate this spatial and quantitative information with standard thin section techniques.
Citation Format: Sharla L. White, Sarah McCurdy, Laurie J. Goodman. Three-dimensional, 3-D, multiplex imaging of biomarkers in tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5915. doi:10.1158/1538-7445.AM2017-5915