Abstract 489: Multi-parameter tissue section imaging and retrieval of image-defined micro-regions for RNA sequencing using the RareCyte® platform
Authors: Podyminogin Rebecca , Ericson Nolan , Lin Jia-Ren , Chen Yu-An , Sorger Peter , Kaldjian Eric , George Tad , U'Ren Lance 
Published: August 2019

Abstract
Background: Tumor tissue imaging allows for a contextual understanding of tumor cells in relation to the immune microenvironment. The ability to interrogate tissues for multiple proteins that define microscopic regions of interest (ROI) and investigate gene expression of cells in those regions is needed for advancing immuno-oncology therapeutic and biomarker discovery. The RareCyte CyteFinder® instrument provides integrated multi-parameter imaging and micro-region retrieval capabilities for sequencing and transcript-level analysis.
Methods: Formalin-fixed, paraffin-embedded (FFPE) sections of tonsil and breast carcinoma sections were stained with a panel of antibodies to CD3, CD8, CD20, cytokeratin, and Ki-67. Frozen sections were stained with antibodies to CD3, CD4, CD8, CD19, and Ki-67. All sections were also stained with SYTOX Orange (nuclear dye). Whole-slide six-color scanning and ROI identification was performed with the CyteFinder instrument. For frozen sections, 40µm diameter micro-regions were retrieved directly from the antibody-stained section using the CytePicker® module. For FFPE sections, ROI were identified on the antibody-stained sample and micro-regions were retrieved from the same location on an adjacent section stained with DRAQ5 only. RNA from retrieved micro-regions was amplified and sequenced and differential gene expression analysis was performed.
Results: Tonsil staining distinctly identified crypt lining (cytokeratin), T cell (CD3, CD4, CD8) and B cell (CD19, CD20) compartments; Ki-67 was prominently expressed in germinal centers. Breast carcinoma staining distinctly identified tumor cells (cytokeratin) and CD8-positive T cells that were prominent at the periphery of the tumor with smaller numbers interspersed. Ki-67 staining was variably present in tumor cell nuclei. Comparative RNA analysis of four frozen tonsil T cell zone micro-regions against four B cell zone micro-regions resulted identified genes with expected differential gene expression pattern, including T cell zone upregulation of CD8a, CCL19, and CCL21 and downregulation of CD38, CR2, and CXCL13. Analysis of FFPE tonsil micro-regions comparing four germinal centers with areas outside the follicle identified genes differentially regulated within the germinal center, including B cell markers CD19, CD22 and CD79a. Out of 20 selected genes preferentially expressed in the germinal center, 16 (80%) were confirmed by interrogation of the Human Protein Atlas to be specific or enriched in germinal centers by immunohistochemical staining.
Conclusions: This study demonstrates high quality six-parameter imaging of tonsil and breast carcinoma tissues. Confirmation of tonsil immune cell type by gene expression analysis supports use of the RareCyte platform for retrieval of defined tissue micro-regions for RNA sequencing in frozen and FFPE samples.
Citation Format: Rebecca Podyminogin, Nolan Ericson, Jia-Ren Lin, Yu-An Chen, Peter Sorger, Eric Kaldjian, Tad George, Lance U'Ren. Multi-parameter tissue section imaging and retrieval of image-defined micro-regions for RNA sequencing using the RareCyte® platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 489.