Abstract 5399: Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing

Abstract

Abstract Single-cell RNA sequencing (RNA-seq) is a powerful tool for investigating rare tumor subpopulations and resolving intra-tumor heterogeneity, but is low throughput, expensive, and requires fresh tissue samples. To address these limitations, we developed a 5’ high-throughput single-nucleus RNA sequencing (SNRS) approach that uses nanogrid technology to perform single-cell imaging and sequencing of 500-2500 nuclei in parallel. The automated image scanning procedure allowed us to exclude doublets and select live cells with DAPI/PI staining. This approach allows the transcriptomic profiling of frozen tissue samples, in which the cytoplasmic membrane is ruptured in cells, but leaves the nuclear membrane intact. We validated SNRS in a breast cancer cell line (SK-BR-3) and compared the transcriptomes of 500 nuclei to 500 whole cells, which revealed a high concordance in the number of genes expressed as well as their expression levels. We also performed bulk RNA-seq of isolated nuclear and cellular fractions from 5 breast cancer cell lines, which showed a high concordance in genes and expression levels. Differentially expressed genes in the nucleus mainly included lincRNAs, pseudogenes and mitochondria genes, but did not affect most cancer genes and pathway analysis. We further applied SNRS to sequence 500 nuclei from a triple-negative breast cancer patient and identified diverse phenotypes in tumor cells, including variation in cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition. These studies demonstrated the technical feasibility of using a nanogrid platform to perform high-throughput single-cell RNA sequencing and showed that nuclei from cell lines and tumors can be used to study signaling pathways and gene networks that play an important role in tumor progression. Citation Format: Ruli Gao, Charissa Kim, Emi Sei, Jie Yang, Leo Chan, Maithreyan Srinivasan, Hong Zhang, Funda Meric-Bernstam, Nicholas E. Navin. Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing [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 5399. doi:10.1158/1538-7445.AM2017-5399