Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

Joint Olink/customer paper reporting out from the “single-cell panel” project. The need for single-cell studies is growing as the heterogeniety within cell populations is better understood, which can have great pathophysiological consequences – e.g. during tumor development. Genomic and transcriptomic technologies have been developed to address single cell studies, but methods to examine proteins at the single-cell level have suffered from throughput and multiplexing issues. A prototype PEA-based panel designed for use in single cell assays was used to study the effects of the experimental therapeutic agent BMP4 on early passaged tumor cells taken from a gliobastoma patient. A panel of 75 “cancer pathways” assays with sensitivity down to at least the 10 cell level was constructed and results compared to RNA analysis with TaqMan Gene Expresion assays (22 markers overlapped for both types of analysis). The protein assays were reliable and reproducible. RNA-protein correlations were much poorer than has previously been reported from large pools of cells, indicating that RNA levels are less accurate indicators of cell states than proteins (factors such as short mRNA half-lives and burst-like transcription patterns may contribute to this observation). Examining control and BMP4-treated cells, RNA & protein analysis could separate non-treated/treated cells well, with protein markers performing better than RNA, but with a combination of the two being optimal. BMP4 did not significantly reduce cell proliferation, but activation of known BMP4 response genes was observed, as well as reduction in some neural stem cell markers. Futhermore, the combined analysis could clearly identify distinct sub-groups within both the control and treated cell populations. For treated cells, two sub-groups showed differential expression of a set of proliferation markers and cell-cycle regulators, suggesting that one sub-group may partially escape the effects of the drug and maintain proliferation levels, despite intitial activation of the BMP4 pathway. In conclusion: 1) the study demonstrates the highest degreee of multiplex protein analysis that has been reported in single cells 2)PEA shown to be a valuable complement to single-cell RNA analysis and that of the two, protein analysis may be a more reliable differentiatior at the single cell level 3) the combined RNA/protein analysis shows a significant heterogeneity within low-passage glioblastoms cells for both control and BMP4 populations 4) results support a role for single-cell studies using RNA & protein biomarkers to identify phenotypic variations and analyze cellular responses, to aid prognostics and design personalized treatments.