Screening
phenotypic-based screening assays is the extensive use of human primary cells, often derived from the patient with the disease under investigation. Such cells, which can provide robust understanding of disease pathologies, are optimal for drug discov- ery, as they express the targets, disease-causing mutations and signal pathways involved in the dis- ease pathology. The widespread availability of such cells has contributed to a renaissance in phe- notypic screening, facilitated by the rapid develop- ment of robust, high throughput, high content cel- lular imaging systems. This is particularly relevant in assays directed at difficult drug targets as well as in diseases for which the molecular mechanisms are unknown. Furthermore, rapid technological advances in signal detection and microfluidic han- dling systems now enable phenotypic assays to be conducted using human cells in two mutually exclusive formats. The first is single-cell analysis, in which several analytical studies can be directed at a single-cell (genomic, proteomic, etc). The sec- ond is in multicellular populations, where aggre- gate responses are measured. In this format, the cells are cultured in three-dimensional assemblies (spheroid, organoids, tissues, etc) in a manner that more accurately resembles, or translates to, human tissues in vivo1.
The question: phenotypic or target-based screening? Single-cell or multicell analysis? Consequently, one can now pose the question of which assay approach, single-cells or multicellular complexes grown in three-dimensional structures, is optimal when conducting a phenotypic (ie unbi- ased) screening campaign. It is increasingly evident that both target-based and phenotypic screening share the same fundamental problem: the assay format’s biology does not fully recapitulate the physiological or pathophysiological microenviron- ment of the cells in vivo, and thus the preclinical data may not adequately model the human clinical outcomes. Conversely, multicellular analysis, by definition, which deals with population responses, may obscure target sensitivity to compound phar- macology as well as the contribution of rare cells to the signal from the heterogeneous population. This question of whether phenotypic screening is best conducted by single or multicellular analysis is highly relevant when the disease target is incom- pletely defined, difficult to screen at, or simply ‘undruggable’ from medicinal, chemistry or biolog- ic perspectives. The goal of this brief review is, therefore, to address the use of single-cell as opposed to three-
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dimensional cell culture in the context of pheno- typic screening. It is suggested that the use of both approaches can facilitate disease understanding to identify novel, validated drug targets. They can also identify novel biomarkers that drive patient stratification and help predict therapeutic respon- sivity. While the use of both approaches is optimal, it is surprising that few, if any, studies have been undertaken that compare data from single-cell to multicellular screens using phenotypic readouts.
One answer: single-cell analysis The basic concept of single-cell screening is to enable a better understanding of assay responses from an individual cell, as opposed to measure- ment of population averages and the resulting het- erogeneity of measurements from genomics, tran- scriptomic, epigenomic, proteomic and pharmaco- logic analysis. Screening for compound action at the single-cell
level also reveals previously
obscured individual cell-to-cell variations, fre- quently masked when whole population responses are taken. The increasing use of single-cell analysis in
screening demonstrates that many cell populations exhibit a level of heterogeneity, even among those cells of the same phenotype, such as cells growing within a single tumour. The ability to define this heterogeneity is important in understanding the complexities of polygenic diseases and their opti- mal treatment. Subtle, but significant, differences among patient cells are illustrated by the realisa- tion that responses of individual cells to oncologic drugs cause the emergence of drug-resistant responses, even though only a small percentage (~0.3%) of cells possess the ability for tumour recurrence2. More generally, a small cell population within
the larger heterogeneous population often domi- nate the assay response being measured3. Specifically, next generation sequencing (NGS) technologies are classically used to genomically characterise bulk cell populations. However, NGS is increasingly being used to focus on characteris- ing single-cells. Transcriptomic and epigenomic analysis of single-cells can reveal novel biological pathways masked by the heterogeneous nature of large cell populations. Single-cell RNA sequenc- ing (scRNA-seq), for example, reveals complex and rare cell populations, uncovers regulatory interactions between key genes, and tracks the trajectories of distinct cell lineages during devel- opment4. Historically, screening for novel compounds using single-cell responses has been limited both
Drug Discovery World Fall 2019
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