Abstract P5-03-02: Three-dimensional (3D) imaging of biomarkers in human core needle biopsies of normal and cancerous breast tissue
Authors: Chen Y , Shen Q , Goodman LJ , Gökmen-Polar Y , Badve SS 
Published: February 2018

Abstract
Background: The tumor microenvironment is spatially and compositionally very heterogenous, which introduces great challenges to characterize the underlying factors using standard 2D diagnostic methodologies. Capturing high resolution 3D quantitative biomarker data, while simultaneously preserving morphology of the tumor microenvironment, could lead to a better understanding of key spatial relationships and may lead to better prognostic and predictive clinical outcomes. In this study, we utilized a novel technique, CLARITY, to transform core needle biopsies from patients with breast cancer, into optically transparent tissues, followed by multiplex immunostaining and 3D imaging of molecular markers. This data was compared to the conventional methods of immunohistochemistry and immunofluorescence staining on FFPE thin sections.
Methods: Formalin-fixed (less than 24 hours) human breast cancer core needle biopsy tissue pairs (tumor and adjacent normal) were obtained from patients undergoing excisional surgery. Tissues were subsequently embedded in 4% paraformaldehyde containing a 4%/0.05% ratio of acrylamide/BIS for 48 hours, and polymerized to form an intact hydrogel/tissue matrix. The samples were sectioned to a thickness of 200µm, 400 µm, and 1000µm and lipid-cleared in a solution of 0.2M borate buffer containing 8% SDS, pH 8.5 at 45°C. The tissues were then immunostained for various cellular markers (Pan-cytokeratin, Her2, CD3, CD31) and a nuclear marker (Ki67) and counterstained with DAPI. Samples were refractive index matched prior to 3D imaging on a Leica SP8 laser scanning confocal microscope or a LaVision BioTec Ultramicroscope II, light sheet microscope.
Results: During the process, the samples remained intact and the cellular morphology was well preserved suggesting that a pre-fixation step following by hydrogel embedding/Immunostaining was feasible. The average passive lipid-clearing time for breast cancer core needle biopsy tissue was 5-20 days depending on the size of the tumor. The majority of the samples reached visual optical transparency, with the exception of some regions that contained heavy fibrotic tissue. Preliminary results demonstrated that specific staining of various cellular and nuclear markers was successful as evidenced by 3D imaging depth up to 1000 µm. As compared to the images obtained from 2D thin sections, the CLARITY procedure followed by 3D imaging yielded significant imaging depth, with the potential to greatly enhance the understanding of the heterogeneity of the tumor microenvironment.
Conclusion: This is the first study demonstrating that other than fresh or frozen tissues, pre-fixed clinical tissue from patients with breast cancer, can be successfully processed by the CLARITY methods and 3D imaged, indicating the potential power of the technique for core needle biopsy tissue processing and in the identification of biomarkers based on tumor cell heterogeneity.
Citation Format: Chen Y, Shen Q, Goodman LJ, Gökmen-Polar Y, Badve SS. Three-dimensional (3D) imaging of biomarkers in human core needle biopsies of normal and cancerous breast tissue [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-02.