Supplementary Material for: Transcriptome Profiling of Panc-1 Spheroid Cells with Pancreatic Cancer Stem Cells Properties Cultured by a Novel 3D Semi-Solid System

<b><i>Background/Aims:</i></b> Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs <i>in vitro</i> as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation. <b><i>Methods:</i></b> We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity <i>in vitro</i> and <i>in vivo</i> through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells. <b><i>Results:</i></b> We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both <i>in vitro</i> and <i>in vivo</i>. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS. <b><i>Conclusions:</i></b> Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.