Abstract 833: Linking phenotype and genotype: Multimodal analysis of surface proteins, intracellular proteins, and SNV in single cells
Authors: Singh Prithvi , Parikh Saurabh , Lai Tim , Dhingra Dalia , Ooi Aik 
Published: June 2022

Cancers evolve via processes of clonal expansion, selection, and somatic variation, which contribute to tumor heterogeneity. While bulk analysis has improved our understanding of cancer, the heterogeneity of a tumor is masked with the average readout provided by a bulk measurement. This problem could be overcome by employing single-cell technologies. One of the most common single-cell technologies is single-cell RNA sequencing (scRNA-seq). Transcriptomic data collected by scRNA-seq are used to imply activity of gene products based on RNA levels. Signaling pathways related to cancer initiation and progression have been mapped solely based on scRNA-seq results. This approach, however, is not an accurate measurement of proteins that are the functional molecules responsible for key events in cancer. Recent breakthroughs of multimodal approaches such as CITE-seq and REAP-seq partially addressed this problem by allowing simultaneous analysis of transcriptomes and surface protein expression levels. These methods still lack a couple components that are required to fully dissect the biology of cancer. First, the methods are limited to the measurements of cell surface proteins, whereas proteins involved in cancer-related signal transduction and transcriptional pathways are mostly localized in the intracellular compartments. Second, the methods do not allow direct analysis of DNA sequences, hence unable to provide an accurate readout of genotypic information such as single-nucleotide variants and copy number variations. Here we describe a technology that could overcome these hurdles. To achieve this, cells are first treated with oligonucleotide-barcoded antibodies targeting surface proteins. Cells are then fixed and permeabilized, followed by incubation with barcoded antibodies for intracellular proteins. The resulting cells are processed on the Mission Bio TapestriⓇ platform, a device that enables encapsulation of single cells in droplets. Targeted DNA sequencing libraries are generated from the single cells using a multiplex panel of primers targeting regions of interest. Protein sequencing libraries are separately generated from the oligonucleotides off the antibodies. The inclusion of intracellular protein detection enables measurement of pivotal proteins in cancer mechanisms, including apoptosis (BCL2 family proteins), transcription factors (GATA3), tumor suppressors (TP53), as well as phosphorylated proteins involved in cell growth signaling pathways (phosphorylated ERK and STAT proteins). This is the first ever method that provides a solution to effectively link surface and intracellular protein measurement with targeted DNA analysis. With this approach, single-cell readout of genotypic and phenotypic information can be collected together, allowing concurrent complex analyses of cancer clonal evolution and driver protein expression.

Citation Format: Prithvi Singh, Saurabh Parikh, Tim Lai, Dalia Dhingra, Aik Ooi. Linking phenotype and genotype: Multimodal analysis of surface proteins, intracellular proteins, and SNV in single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 833.