In Vivo and FACS-based CRISPR/Cas9 Screening Uncovers VENTX As a Crucial Element in Maintaining Seminoma Stemness.

Seminoma, which is the most frequent testicular germ cell tumor, has stem cell-like features related to treatment resistance and recurrence, yet the molecular mechanisms that preserve its stemness are not well understood. This study aimed to discover the tumorigenesis and stemness regulation of seminoma. In vivo CRISPR/Cas9 knockout library screening was performed using Tcam-2 seminoma cells implanted into NSG mice to identify tumorigenesis drivers. Parallel FACS-based screening enriched CD117/CD133 double-negative cells to assess the regulators of seminoma stemness. Functional validation included clonogenicity assays, sphere formation tests, and tumor initiation experiments in vivo. RNA sequencing analyzed downstream pathways. In vivo and FACS-based CRISPR/Cas9 screening uncovers VENTX as a crucial element in maintaining seminoma stemness. Functional validation showed that VENTX knockout decreased clonogenicity, sphere formation, and CD117+/CD133+ populations in Tcam-2 cells, and also hindered tumor initiation in vivo. RNA sequencing identified a connection between VENTX and pluripotency pathways, demonstrating a downregulation of ID1/2/3 (essential differentiation inhibitors) and an imbalance in Wnt/β-catenin and TGF-β signaling. VENTX expression showed a significant association with DNA methylation-based stemness scores in testicular germ cell tumors. In conclusion, VENTX is a key regulator of seminoma stemness, presenting a promising target for therapy to address recurrence and resistance in seminoma.