Abstract P4-06-09: Pick-Seq®: Multi-parameter imaging combined with RNA sequencing of tissue section micro-regions for spatial analysis in breast carcinoma
Authors: Ericson Nolan , Podyminogin Rebecca , Chow Jennifer , Lin Jia-Ren , Chen Yu-An , Maliga Zoltan , Sorger Peter , Teplitz Kyla , Kaldjian Eric , George Tad 
Published: March 2020

Abstract
Multi-parameter tissue imaging allows for a contextual understanding of tumor cells in relation to the tumor microenvironment. Pick-Seq is a novel workflow uniquely enabled by the RareCyte CyteFinder® Instrument that combines interrogation of multiple protein markers with investigation of gene expression from selected micro-regions on tissue slides. In this study, formalin-fixed, paraffin-embedded tonsil sections and OCT-embedded, frozen breast cancer sections were stained with epithelial and lymphocyte markers by multi-parameter immunofluorescence (IF) and scanned with the CyteFinder Instrument to identify regions of interest. Based on IF staining patterns, 40 μm diameter micro-regions were retrieved with the integrated CytePicker® Retrieval Module. Micro-region RNA was isolated for whole transcriptome amplification, library preparation, and sequencing. Resulting data were analyzed for differential gene expression, cell composition, and T cell receptor (TCR) sequences. Transcriptomic analysis of tonsil micro-regions confirmed increased expression of B cell markers in follicles and T cell markers in the T cell zone. CIBERSORT revealed distinct cellular compositions between T cell zones and the B cell follicles. Principle component analysis of gene expression found that micro-regions retrieved from the two follicles clustered independently from each other, and from the T cell zone micro-regions. Fragments per kilobase million (FPKM) analysis revealed differing expression of genes, particularly CD19 and CD21, between the adjacent follicles. Subsequent staining confirmed differential protein expression, indicating that only one follicle contained a germinal center. In breast carcinoma, regions were identified for CytePicker retrieval that included (1) tumor cells, (2) tumor cells with interspersed tumor infiltrating lymphocytes (TIL), or (3) adjacent lymphoid aggregates. CIBERSORT analysis demonstrated the presence of T cell-associated transcriptomic profiles in lymphoid aggregates and in TIL-containing micro-regions that were proportional to the number of T cells retrieved. Aligned RNA-seq reads were further analyzed to identify TCR α and β chain sequences from retrieved TILs. These data establish the potential of combining multi-parameter IF microscopy with deep RNA sequencing as a powerful tool for investigation and biomarker discovery in the immuno-oncology of breast cancer.
Citation Format: Nolan Ericson, Rebecca Podyminogin, Jennifer Chow, Jia-Ren Lin, Yu-An Chen, Zoltan Maliga, Peter Sorger, Kyla Teplitz, Eric Kaldjian, Tad George. Pick-Seq®: Multi-parameter imaging combined with RNA sequencing of tissue section micro-regions for spatial analysis in breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-09.