Illustrating Heterogeneity in AML By Leveraging Multimodal Analysis of DNA, Surface Proteins, and Intracellular Proteins in Single Cells

Introduction: Recent advancements in precision medicine, although highly promising, present a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multiomics analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights into cellular and genetic composition.

Methods: Using a two-step droplet microfluidic technology, the Mission Bio TapestriⓇ platform enables high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a novel workflow was developed to detect protein expression in addition to DNA genotypes in the same single cells. In this approach, cells are labeled with oligonucleotide-conjugated antibodies prior to single-cell encapsulation and DNA target amplification on the Tapestri system. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. The number of protein targets can range from a few targets to over 40, which is beyond the limit for a single flow cytometry run.

Results: This workflow has been successfully demonstrated on cell lines as well as clinical samples. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data were able to clearly distinguish CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs from CD34hiCD11blo and CD34loCD11bhi subpopulations that did contain the same pathogenic SNVs and CNVs.

Conclusion: We further developed this workflow to include simultaneous detection of intracellular proteins and surface proteins along with targeted sequencing in single cells. The inclusion of intracellular protein detection enables measurement of pivotal proteins in cancer mechanisms. We believe that this novel multiomic technology will facilitate new discoveries inthe complex relationship between genotype and phenotype, enabling a better understanding of disease biology, and subsequently improve the design of diagnostics and therapies.

Singh:Mission Bio: Current Employment, Current holder of stock options in a privately-held company. Dhingra:Mission Bio: Current Employment, Current holder of stock options in a privately-held company. Parikh:Mission Bio: Current Employment, Current holder of stock options in a privately-held company. Sciambi:Mission Bio, Inc: Current Employment, Current equity holder in private company, Other: Co-founder & Director of Engineering & Mission Bio, and has a patent pending for a portion of the described work in the submitted abstract.. Ooi:Mission Bio: Current Employment, Current holder of stock options in a privately-held company.