Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics.

To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

Probing single-cell metabolism reveals prognostic value of highly metabolically active circulating stromal cells in prostate cancer.

Despite their important role in metastatic disease, no general method to detect circulating stromal cells (CStCs) exists. Here, we present the Metabolic Assay-Chip (MA-Chip) as a label-free, droplet-based microfluidic approach allowing single-cell extracellular pH measurement for the detection and isolation of highly metabolically active cells (hm-cells) from the tumor microenvironment. Single-cell mRNA-sequencing analysis of the hm-cells from metastatic prostate cancer patients revealed that approximately 10% were canonical EpCAM+ hm-CTCs, 3% were EpCAM- hm-CTCs with up-regulation of prostate-related genes, and 87% were hm-CStCs with profiles characteristic for cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells. Kaplan-Meier analysis shows that metastatic prostate cancer patients with more than five hm-cells have a significantly poorer survival probability than those with zero to five hm-cells. Thus, prevalence of hm-cells is a prognosticator of poor outcome in prostate cancer, and a potentially predictive and therapy response biomarker for agents cotargeting stromal components and preventing epithelial-to-mesenchymal transition.

Single-Cell Analysis Using Droplet Microfluidics.

Droplet microfluidics has revolutionized the study of single cells. The ability to compartmentalize cells within picoliter droplets in microfluidic devices has opened up a wide range of strategies to extract information at the genomic, transcriptomic, proteomic, or metabolomic level from large numbers of individual cells. Studying the different molecular landscapes at single-cell resolution has provided the authors with a detailed picture of intracellular heterogeneity and the resulting changes in cellular phenotypes. In addition, these technologies have aided in the discovery of rare cells in tumors or in the immune system, and left the authors with a deeper understanding of the fundamental biological processes that determine cell fate. This review aims to provide a detailed overview of the various droplet microfluidic strategies reported in the literature, taking into account the sometimes subtle differences in workflow or reagents that enable or improve certain protocols. Specifically, approaches to targeted- and whole-genome analysis, as well as whole-transcriptome profiling techniques, are reviewed. In addition, an up-to-date overview of new methods to characterize and quantify single-cell protein levels, and of developments to screen secreted molecules such as antibodies, cytokines, or metabolites at the single-cell level, is provided.

Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.