GSE200941
Dosage effect of TGF-beta on EMT induction of MCF10A cells
Epithelial-mesenchymal transition (EMT) is a key cellular process involved in development and disease progression. Single-cell transcriptomes can reveal intermediate EMT states observed in tumors, but previous studies focused on time-dependent responses in epithelial cells after stimulation with EMT signals as an in vitro model for understanding EMT progression, so it was unclear how transcriptomes can change in response to different levels of EMT inducer. Here, we performed single-cell RNA-sequencing with human mammary epithelial cells treated with various concentrations of TGF-ß. We found that the dose-dependent EMT harbors multiple intermediate states at the single-cell level after two weeks of treatment, suggesting a stable EMT continuum. After correcting batch effects from experiments, we performed comparative analyses of the dose- and time-dependent EMT. We found that the dose-dependent EMT shows a stronger anti-correlation between epithelial and mesenchymal transcriptional programs and a better resolution of stages of EMT compared to the time-dependent progress. These properties enable more power to detect genes whose expressions are associated with EMT. Nonetheless, our analyses showed cell clusters unique to the time-dependent EMT, which correspond to en route cell populations that do not appear at steady states. Furthermore, combining dose- and time-dependent cell clusters gave rise to more accurate prognosis for cancer patients compared to individual EMT spectrum. Overall design: Normal human mammary epithelial cell MCF10A was stimulated with TGFbeta  at concentrations of 0, 12.5, 25, 50, 100, 200, 400, 800 pM for 14 days and all samples were pooled before loading into 10x Genomics. Single-cell 3'RNA-seq library was prepared according to the manufacturer's instructions. RNA-Seq library was prepared using 10x Chromium Single Cell 3' –end Reagent Kits User Guide (v2 Chemistry).  Libraries were sequenced using paired-end sequencing (26bp Read 1 and 98bp Read 2) with a single sample index (8bp) on the Illumina HiSeq 2500.