Single-cell RNA sequencing in cancer research

Abstract

Single-cell RNA sequencing (scRNA-seq), a technology that analyzes transcriptomes of complex tissues at single-cell levels, can identify differential gene expression and epigenetic factors caused by mutations in unicellular genomes, as well as new cell-specific markers and cell types. scRNA-seq plays an important role in various aspects of tumor research. It reveals the heterogeneity of tumor cells and monitors the progress of tumor development, thereby preventing further cellular deterioration. Furthermore, the transcriptome analysis of immune cells in tumor tissue can be used to classify immune cells, their immune escape mechanisms and drug resistance mechanisms, and to develop effective clinical targeted therapies combined with immunotherapy. Moreover, this method enables the study of intercellular communication and the interaction of tumor cells and non-malignant cells to reveal their role in carcinogenesis. scRNA-seq provides new technical means for further development of tumor research and is expected to make significant breakthroughs in this field. This review focuses on the principles of scRNA-seq, with an emphasis on the application of scRNA-seq in tumor heterogeneity, pathogenesis, and treatment.

Keywords: Immune escape; Invasion and metastasis; Single‐cell RNA sequencing; Tumor heterogeneity; Tumor microenvironment.

Fig. 1

Schematic overview of five scRNA-seq methods Summary of the Tang method, Smart-seq, and the UMI-based sequencing methods STRT-seq, CEL-seq, Drop-seq. Comparative differences of the processes of these methods are outlined: scRNA-seq, reverse transcription, cDNA amplification, purifying and filtration, and library construction. Tang method is the earliest scRNA-seq technology. Single cells are separated by micromanipulation. The overall sequencing sensitivity and accuracy are relative low. In Smart-seq, RNA is reverse transcribed by Moloney mouse leukemia virus(MMLV). The sequencing range can reach the full-length cDNA. It has higher sensitivity and accuracy. STRT-seq and STRT/C1-seq introduce UMI on the basis of Smart-seq and labele with biotin at the 5 ‘end, which can be recovered by magnetic beads. This sequencing method improves the sensitivity and accuracy, but has a strong 5’ end bias. CEL-seq obtains 3 ‘terminal fragment by IVT. The sequencing sensitivity is high, but there is a strong 3’ end bias and the accuracy is low. Drop-seq uses microfluidic technology to package a single cell into an independent droplet, which greatly increases the capture capacity and library capacity of single cell. It has great advantages in detecting a large number of single cell sequencing samples, but the sequencing sensitivity is low

Fig. 2

Application of scRNA-seq in tumorigenesis In the process of normal tissues developing from precancerous lesions and carcinoma in situ to metastatic carcinoma, scRNA-seq plays an important role in tumor prevention, diagnosis and treatment

Fig. 3

scRNA-seq for immunocyte typing Advantages of scRNA-seq technology to classify the tumor infiltrating T cells, and to provide a detailed landscape of the overall tumor infiltrating immune cells

Fig. 4

Application of scRNA-seq to evaluate tumor immune escape. a scRNA-seq is used to detect and screen for neoantigens. b Detection of transcriptional activity at immune checkpoints and evaluation of ICI drug resistance mechanisms by scRNA-seq. c scRNA-seq detection of immune escape caused by MHC mutations. d Identification of TCR sequences within different T cells by scRNA-seq