The Evolution of Single-Cell Analysis and Utility in Drug Development

Abstract

This review provides a brief history of the advances of cellular analysis tools focusing on instrumentation, detection probes, and data analysis tools. The interplay of technological advancement and a deeper understanding of cellular biology are emphasized. The relevance of this topic to drug development is that the evaluation of cellular biomarkers has become a critical component of the development strategy for novel immune therapies, cell therapies, gene therapies, antiviral therapies, and vaccines. Moreover, recent technological advances in single-cell analysis are providing more robust cellular measurements and thus accelerating the advancement of novel therapies.Graphical abstract.

Keywords: drug development; flow cytometry; single cell.

Figure 1

Conventional flow cytometry. In conventional flow cytometry, cells are labeled with fluorophore-conjugated probes, typically monoclonal antibodies. Within the flow cell, samples then enter the sample stream focused such that a single cell is interrogated by one or more lasers resulting in light dispersion (forward side scatter (FSC)) and excitation of the fluorophores. Emitted fluorescence is then directed to the photodetectors (usually photomultiplier tubes (PMTs)) via a series of long pass (LP), band pass filters and dichroic mirrors. Spectral overlap between fluorophores on the same cell is subtracted by applying spectral overlap compensation (figure adapted from reference (12))

Figure 2

Using high-dimensional cytometry to reveal immune cell populations. A An example of peripheral whole blood labeled with nine fluorophore-conjugated probes. The number of probes influences the type of cells and subsets which can be identified. Hierarchical gating and bivariate plots are used to reveal, leucocytes, lymphocytes, T lymphocytes, CD8⁺ T lymphocytes, and finally the memory subset of CD8⁺ T lymphocytes. B Uniform Manifold Approximation and Projection (UMAP) multidimensional analysis of CD8⁺ T cells from 29 colors flow cytometry assay acquired on BD FACSSymphony. Labeling the samples with a greater number of probes reveals additional cellular subsets and greater information about the biology (figure adapted from reference (18))

Figure 3

Evolution of instruments and probes. A schematic representation of the timelines of the emergence of new technologies and probes used in cytometry (figure adapted from reference (20)). ICP Impulscytophotometer, FACS fluorescence-activated cell sorter, PE phycoerythrin, APC allophycocyanin

Figure 4

Mass cytometry. In mass cytometry, cells are labeled with antibodies tagged with heavy metals. Cells are introduced into the inductively coupled plasma (ICP) by nebulization and are atomized and ionized. After removal of abundant ions, the heavy element composition is determined and signals corresponding to markers are then correlated and data analysis can be performed using any cytometry data analysis software (figure adapted from reference (20))

Figure 5

Spectral Cytometry. Sample preparation is similar to that of conventional flow cytometry; detection antibodies conjugated with fluorophores are used for labeling. Within the flow cell, sample then enters the sample stream focused such that a single cell is interrogated by one or more lasers resulting in light dispersion and activation of the fluorophores. Emitted fluorescence is then collected by a detector array. Spectral overlap between multiple fluorophores on the same cell then separated using spectral unmixing, enabling the simultaneous detection of fluorophores with overlapping emission spectra (figure adapted from reference (12); emission spectra from www.cytekbio.com)

Figure 6

Multi-omics. A schematic representation of the simultaneous measurement of gene and protein expression using a combination of oligo-conjugated antibodies (Abseq) and capture beads. A Samples are labeled with antibodies conjugated with oligonucleotides. Structure of oligo-conjugated antibody (PCR polymerase chain reaction). B Workflow. Labeled samples are mixed with barcoded beads and lysed. mRNA and antibody oligos bound to beads are then converted to cDNA prior to proceeding to generation of mRNA derived library and AbSeq libraries and next-generation sequencing (NGS).