Assessment of sample fixation methods in single-cell RNA-sequencing and single-cell analyses of nasopharyngeal carcinoma

Abstract

Single-cell RNA-seq (scRNA-seq) won its popularity for offering high-resolution information on the samples and is being used extensively in recent research. Regarding this technology, we first evaluated the effect of methanol fixation in sample preservation and demonstrated the feasibility and data alternation carried by the scRNA-seq data that is generated from fixed samples. Our result showed that while fixed data can faithfully preserve biological information, the effect of fixation can be observed consistently. Additionally, this effect is distributed unevenly on the transcriptome and the longer transcript with higher GC-content is more seriously affected. Except for the evaluation of applying methanol fixation on sample preservation for scRNA-seq, we also applied this technology to the nasopharyngeal carcinoma (NPC) and attempted to explore the heterogeneity of this cancer, starting with the cell line models. Our scRNA-seq data delineated the status of the NPC43 cell line with or without 12-o-tetradecanoylphorbol-13-acetate (TPA) treatment and revealed a subgroup of cells that can retain the untreated characteristics as compared with the rest. That specific cell group expressed SOX2 and NTRK2 which was originally abundant in the untreated cells, yet much decreased once treated with TPA. By exploring the significance of this gene combination in the clinical samples, we discovered that the expression of SOX2 and NTRK2 is associated with tumorigenesis and the higher expression of those genes indicates a worse survival outcome. We later demonstrated that the cells enriched with SOX2 and NTRK2 showed stronger sphere-forming ability, and migration ability and they are capable of repopulating the original culture once isolated from the parental cells. Additionally, NTRK2 expression is influenced by SOX2 since by turning on or turning down the expression of SOX2, NTRK2 expression is regulated in the same pattern. Conclusively, our work evaluated the effect of the most widely used fixation method for scRNA-seq and utilized this technique to explore the heterogeneity of NPC.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Angela Ruohao WU (Supervisor)