Abstract 4749: Simultaneous multi-omic measurement of nucleic-acids and proteins at 800-plex using single-molecule optical barcodes: Application to cancer immunotherapy
Authors: Beechem Joseph M. , Geiss Gary , Filanoski Brian , Birditt Brian 
Published: August 2015

Abstract
Both ENCODE and TCGA projects highlighted the value of quantifying multiple biomarker classes (DNA, RNA, protein) from cancer tumor samples. In the case of cancer immunotherapy, the importance of measuring non-DNA markers (e.g., mRNA and proteins) becomes crucial, since cell-signaling, tumor microenvironment, and protein-protein interactions dominate over pure SNP-based (DNA) driver mutations in determining therapeutic response. Combining multiple data types together into a single correlated analysis, however, is adversely effected by the drastically different methodologies utilized for measurement. For example, the fluorescence signal intensity obtained from a camera imaging a protein array (e.g., RPPA) is very difficult to correlate directly with an RNA-Sequencing count of a clonally amplified, cDNA-converted, mRNA molecule. New developments in multiple biomarker-class optical barcode counting significantly reduce this problem. Recent work from the Weissleder-lab [1] has shown how optical barcode technology can be utilized for multiplexed digital counting of proteins, and be combined with simultaneous digital counting of nucleic-acids on a single platform.
In this study we describe single-molecule digital counting of mRNA and proteins in a single simultaneous reaction using ∼ 50,000 cells as input (∼ 1000 cells if only protein-targets). Since multiplexed digital protein counting with barcodes is relatively new, a detailed comparison study versus flow cytometry was performed. Short photo-cleavable 60-mer single-stranded DNA-tags that index, and hybridize to, NanoString Optical barcodes were covalently labeled on the following antibody targets: EGFR, pEGFR, HistoneH3, CD45, FoxP3, pAKT, PCNA, GAPDH, Her3, PD-L1, pS6, Her2, ERK, pERK, and a rabbit monoclonal control. Single-target flow cytometry was performed on three cell-lines (A431, A565, H520) using the unlabeled primary antibodies and detected using FITC-dye labeled goat anti-rabbit. The flow-data were then compared to simultaneous 15-plex optical barcode detection. In all cases, high correlation coefficients (&gt; 0.9) were obtained when comparing any single-target (flow vs. 15-plex optical barcode) across all cell-lines (for above-background markers). Additionally, post-translational modifications were both imaged (F-IHC) and digitally counted, revealing a high correlation in response. These multiplexed protein measurements can now be simultaneously combined with the PanCancer Immune Profiling Panel, consisting of 770 mRNAs representing 24 infiltrating immune cell-types, as well as a number of antigen processing pathways, yielding an unprecedented multi-omics measurement of tumor immune response.
[1] Ullal et al. Science Translational Medicine 6:219 (Jan 15 2014)
Citation Format: Joseph M. Beechem, Gary Geiss, Brian Filanoski, Brian Birditt. Simultaneous multi-omic measurement of nucleic-acids and proteins at 800-plex using single-molecule optical barcodes: Application to cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4749. doi:10.1158/1538-7445.AM2015-4749