Abstract
Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance.
Highlights
Cancer is known to result from progressive accumulation of genomic and epigenomic alterations, leading to dysregulated cell growth [1, 2]
To make fluorescent signal detectable, clusters are built from adaptor-ligated single-stranded templates on magnetic beads using a procedure called “emulsion PCR” which contains millions of PCR reactions taking place in micro-scale aqueous droplets separated by mineral oil
Previous cancer investigations mainly used clinical tissues composed of mixed cancer cell populations, and the molecular interactions and interchange of proteins between individual cancer cells could not be well understood
Summary
Cancer is known to result from progressive accumulation of genomic and epigenomic alterations, leading to dysregulated cell growth [1, 2]. Due to the fact that it is able to provide a superior resolution as enhanced by NGS, single cell transcriptome (SCT) analysis has a strong potential to facilitate our understanding of cancer evolution and its transcriptional heterogeneity [7, 8], which is believed to be associated with acquired drug resistance [9]. We conducted SCT sequencing on MCF-7 breast cancer cell line to reveal their transcriptional diversity and potential clinical implications. We split the expressed genes into consistently expressed (CE) (i.e., expressed in all SCTs) and inconsistently expressed (IE) (i.e., expressed only in some but not all SCTs) genes and conducted serial analyses on these two groups together with HKGs. Results indicated a bipartite transcriptional pattern in MCF-7 single cells, with consistently expressed genes mainly coding for proteins involved in intracellular activities and inconsistently expressed genes coding for extracellular proteins, including receptors and ion channels. We suspect that diversified expression of IE genes may act as a frontline protection of cancer cell from the attack of cancer drugs, due to the fact that it is essential for cancer drugs to interact with extracellular and/or membrane-bound proteins and the absence in expression in some cells would render the cancer as a whole to survive cancer drug treatment
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