Abstract 1675: Parallel genome and transcriptome-wide profiling of PD-L1 expressing tumor and infiltrating immune single cells in NSCLC
Authors: Casonato Stefano , Bassi Giulio , Bolognesi Chiara , Mangano Chiara , Monaco Valentina del , Buson Genny , Forcato Claudio , Ferrarini Alberto , Bischoff Farideh , Medoro Gianni , Menaresi Nicolò , Fontana Francesca 
Published: July 2017

Abstract
Introduction Targeting immune checkpoint has demonstrated contrasting clinical response in non-small cell lung cancer (NSCLC) due to the lack of robust biomarkers for patients stratification. This is partially due to a poor understanding of the role of the different cellular players (tumor, stromal, infiltrating immune cells) within the tumor microenvironment. Here we present an innovative workflow using the DEPArray™ sorting system, to isolate different classes of epithelial and hematopoietic viable single cells from fresh tissue biopsies for integrated genome and transcriptome sequencing analysis.
Methods Freshly resected NSCLC tissue was dissociated mechanically and enzymatically down to single cell suspension, and labelled with immunofluorescent antibodies targeting PD-L1 (28-8), EpCAM, CD8, CD45 along with Hoechst 33342 for nuclei detection. Distinct pure single cells, along with pools of precise number of cells were digitally isolated using DEPArray™ platform based on marker expression. For each cell recovery, mRNA was physically separated from genomic DNA and then amplified using the Ampli1™ WTA Kit to generate a library of high quality, full-length double stranded cDNA. Amplified cDNA is used for NGS library preparation and sequencing on Illumina® MiSeq System. In parallel, genomic DNA is collected with magnetic beads and eluted directly into the reaction mixture for whole genome amplification by Ampli1™ WGA kit. The WGA products were used to profile genome-wide copy-number aberrations (CNA) using Ampli1™ LowPass kit.
Results As a first step, we identified two main cell populations, the CD45+ infiltrating hematopoietic cells and the EpCAM+ epithelial cells. The hematopoietic fraction was further investigated to evaluate the presence of different subpopulations. Interestingly the PD-L1+ population, corresponding to immune suppressive hematopoietic cells, represented 47% of the total CD45+ fraction while CD8+ cells corresponding to cytotoxic T cells represented 9% of the total CD45. In the EpCAM+ population the PD-L1+ fraction represented 29% of the cells. Single viable cells along with matching pools of precise number of cells from each population were digitally isolated with DEPArray™ system for downstream analysis. Results of RNA-Seq and genomic DNA copy-number aberrations profiling will be presented at the conference.
Conclusion We show here a new approach to isolate pure, single PD-L1+/- tumor and tumor infiltrating hematopoietic cells from NSCLC tissue for integrated genome and transcriptome sequencing. The direct comparison of gene expression data to its corresponding genomic data in the same tumor cell, along with the transcriptomic profiling of immune infiltrate and stroma, may add a powerful tool to disentangle the tumor microenvironment biology. This will potentially offer a novel tool to develop new biomarkers and identify therapeutic leads.
Citation Format: Stefano Casonato, Giulio Bassi, Chiara Bolognesi, Chiara Mangano, Valentina del Monaco, Genny Buson, Claudio Forcato, Alberto Ferrarini, Farideh Bischoff, Gianni Medoro, Nicolò Menaresi, Francesca Fontana. Parallel genome and transcriptome-wide profiling of PD-L1 expressing tumor and infiltrating immune single cells in NSCLC [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 1675. doi:10.1158/1538-7445.AM2017-1675