Abstract LB-316: Innovative multimodal single-cell workflow to interrogate cellular responses to cancer therapy

Abstract

Abstract Elucidation of biological processes that drive cellular migration, proliferation, stemness and differentiation have powered our view of tumor initiation and promotion. These avenues of research are reliant on engaging key biomarkers to measure and quantify cell systems. Widely practiced methodologies include metabolomic assays, immunoassays, imaging platforms and genomic tools. Understanding the genomic landscape of cancer has proven valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. For sampling convenience, genomic measurements are typically applied to bulk cell samples. Bulk samples often obscure important rare cell types that define disease progression. Single-cell genomic analysis can highlight rare cells. Such efforts commonly generate one “modal” of information, most often next generation sequencing datasets containing RNA transcriptomic expression profiles. While transcriptomic information has great utility for plotting global patterns of response, correlating RNA dynamics to genomic variation and protein levels has proven difficult, especially at the single-cell level. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using high-throughput droplet microfluidics paired with next generation sequencing readouts. Our procedure determines targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts and DNA amplicons. We employ oligo-conjugated antibody panels to probe cell surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with cocktails of oligonucleotide-antibodies. Cells are loaded onto the Mission Bio Tapestri microfluidic cartridge for generation of a droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA/mRNA. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced yielding a multimodal readout. We applied this technique to several dynamic immunology and oncology in vitro models. We explored the relationship between genotype-to-phenotype in breast carcinoma cell lines cultured under different drug regimen conditions, PBMC cell population responses to mitogenic perturbations and imatinib impact on retinoblastoma and BCR-ABL driven leukemic cells. We note CNV influences (such as ERRB) on gene and protein expression as well as RNA-protein concordances. Our pilot studies demonstrate the utility of multimodal resolution in further illuminating cellular states and biological responses in cancer cells. Citation Format: David Ruff, Dalia Dhingra, Pedro Mendez, Aik Ooi, Adam Sciambi, Shu Wang. Innovative multimodal single-cell workflow to interrogate cellular responses to cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-316.