Identification of gastric cancer stem cells with CD44 and Lgr5 double labelling and their initial roles on gastric cancer malignancy and chemotherapy resistance

Accumulating evidences have indicated that cancer stem cells (CSCs) can initiate tumor progression and cause recurrence after therapy. However, specific markers of gastric CSCs (GCSCs) from different origins have not been comprehensively revealed. Here, we further detected whether cell populations labelled with CD44 and Lgr5, well-recognized stem markers for gastric cancer (GC), can better emphasize cancer initiation, therapeutic resistance and recurrence. Flow cytometry was utilized to sort the CD44 + Lgr5 + and CD44 + Lgr5- cells from GC cell line HGC-27 and primary GC cells. The influences of CD44 and Lgr5 GCSCs on the malignant behaviors and their potential mechanisms was investigated, respectively. In our study, we reported the identification and validation of CD44 + Lgr5 + cells that presented stronger stemness characteristics, as evidenced by increase of sphere forming ability, elevation of stem cell transcriptional activity. Additionally, CD44 + Lgr5 + double positive cells have lower apoptosis, greater chemotherapy resistance, and higher EMT capacity and LC3 density compared with CD44 + Lgr5- cells. Tumor xenograft experiments also verified the faster carcinogenesis of CD44 + Lgr5 + GCSCs. Furthermore, a series of key proteins in the Wnt, Hedgehog, Notch, and TGF-beta pathways were elevated in the CD44 + Lgr5 + double positive subpopulation, except for Notch 1 and Smad 1. In conclusion, the binding of CD44 and Lgr5 can serve as a precise GCSCs marker that initiate malignant progression and chemotherapy resistance in GC by activating Wnt, Hedgehog, Notch, TGF-beta pathways. Those evidences raise the needs to target both markers simultaneously as a potential approach for the GC treatment.Graphical abstract1. CD44+Lgr5+ GCSCs exhibited the activation of Hedgehog pathway, and Wnt/beta-catenin pathway.2. In terms of the Notch pathway, all Notch proteins and NICD were elevated in CD44+Lgr5+ GCSCs except Notch 1 protein.3. TGF/beta components including TGF-beta 1 and Smad 2, 3, and 4 were involved in stemness phenotypes of CD44+Lgr5+ GCSCs.Graphical abstract1. CD44+Lgr5+ GCSCs exhibited the activation of Hedgehog pathway, and Wnt/beta-catenin pathway.2. In terms of the Notch pathway, all Notch proteins and NICD were elevated in CD44+Lgr5+ GCSCs except Notch 1 protein.3. TGF/beta components including TGF-beta 1 and Smad 2, 3, and 4 were involved in stemness phenotypes of CD44+Lgr5+ GCSCs.Graphical abstract1. CD44+Lgr5+ GCSCs exhibited the activation of Hedgehog pathway, and Wnt/beta-catenin pathway.2. In terms of the Notch pathway, all Notch proteins and NICD were elevated in CD44+Lgr5+ GCSCs except Notch 1 protein.3. TGF/beta components including TGF-beta 1 and Smad 2, 3, and 4 were involved in stemness phenotypes of CD44+Lgr5+ GCSCs.