Abstract 4347: Integrated single-cell DNA and RNA analysis of intratumoral heterogeneity and immune lineages in colorectal and gastric tumor biopsies
Authors: Billy Lau, Noemi Andor, Anuja Sathe, Christina Wood-Bouwens, George Poultsides, Hanlee Ji
Journal: Cancer Research (2018)

Abstract Intratumoral heterogeneity and immune cellular infiltration are important indicators for both prognosis and prediction of treatment efficacy. Intratumoral heterogeneity, arising from the backdrop of ongoing mutation accrual and genome instability, drives Darwinian evolutionary selection for therapy-resistant cells. Infiltrating immune cell lineages are repressed in their function, thus enabling tumors to evade the immune system. Currently, conventional bulk sequencing does not resolve features from individual cancer cells and other cellular components present in a tumor. Thus, one loses critical granular information describing the overall complexity of the tumor and its local microenvironment. To achieve a complete analysis of intratumoral heterogeneity and immune response to a tumor, we developed and applied a strategy for parallel single cell DNA-Seq and single cell RNA-Seq on excised tumors - our approach used tumor biopsies obtained directly after surgery with immediate processing and dissociation into single cell suspensions for genomic analysis. For thousands of cells, single-cell DNA-Seq analysis was used to generate genome wide copy number results and total quantification of chromosomal aneuploidy. With these results, we determined the intratumoral phylogenetic distributions at single cell resolution. Single-cell copy number variant detection performance in cancer cell lines was cross-validated with traditional whole genome sequencing and karyotyping from established cell lines. Remarkably, we distinguished between replicating and non-replicating cells - this was defined by discrete and localized fluctuations in read coverage across the genome. Our findings were confirmed by flow cytometry analysis. Single-cell RNA-Seq provided an overview of the different subpopulations with distinct transcriptional phenotypes. We resolved multiple subsets of tumor epithelial cells reflecting transcriptional heterogeneity as well as stromal components including endothelial, muscle, fibroblast and immune cells. Among immune cells, we further resolved phenotypes including T, B, NK, dendritic cells and monocytes. We also detected a small number of cells with expression of markers of immune exhaustion. Overall, our study successfully demonstrated the feasibility of conducting integrated single-cell DNA-Seq and RNA-Seq on patient tumors. As supported by our results, single cell genomics offers an unprecedented perspective on the genomic and cellular complexities of primary tumor biopsies. Citation Format: Billy Lau, Noemi Andor, Anuja Sathe, Christina Wood-Bouwens, George Poultsides, Hanlee Ji. Integrated single-cell DNA and RNA analysis of intratumoral heterogeneity and immune lineages in colorectal and gastric tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4347.