Single-Cell Transcriptomic Analysis of Tumor Heterogeneity

Abstract

Intratumoral heterogeneity is among the greatest challenges in precision cancer therapy. However, developments in high-throughput single-cell RNA sequencing (scRNA-seq) may now provide the statistical power to dissect the diverse cellular populations of tumors. In the future these technologies might inform the selection of targeted combination therapies and enrollment criteria for clinical trials.

Figure 1 Key Figure. Single-cell transcriptomics technologies and applications in precision medicine

Panel 1: technologies: A) In conventional scRNA-Seq, individual cells are flow sorted into multi-well plates. RNA-Seq libraries of individual cells are prepared, barcoded in separate wells (per cell reaction volume: ~ 10 μL) and pooled together for next generation sequencing. B) In high-throughput scRNA-Seq with microfabricated microwell arrays, individual cells are co-encapsulated with individual uniquely barcoded mRNA capture beads in physically-isolated microwells (per cell reaction volume: ~ 100 pL). Reverse transcription of bead-captured mRNA molecules results in the incorporation of a bead-specific barcode to each cDNA molecule. The barcoded cDNA molecules from all cells are then pooled together and converted into a single RNA-Seq library. C) High-throughput scRNA-Seq with droplet-based microfluidics is similar to B) except that the co-encapsulation occurs in droplets (per cell reaction volume: ~ 1 nL). D) In conventional RNA-FISH, multiple singly-labeled fluorescent probes are hybridized to transcripts encoding a specific target gene in situ. The fluorescent probes for each transcript share the same color. The number of transcript species that can be measured scales linearly with the number of imaging cycles/channels. E) In highly multiplexed RNA-FISH, probes against multiple genes are distinguished by unique combinations of secondary probes and read out by sequential hybridization. The number of transcript species that can be measured scales exponentially with the number of imaging cycles/channels. Panel 2: applications Simplified illustrations of the potential application of single-cell transcriptomics in precision medicine: A) identification of distinct malignant subpopulations that could be effectively targeted by different drugs in combination and B) identification of distinct malignant subpopulations or cell types co-expressing targets in multiple redundant pathways that could be targeted by different drugs in combination.