The Functional Role and Molecular Characterization of the Therapeutic Target CLDN6 in Germ Cell Tumors

ABSTRACT Background The tight junction protein CLDN6 has been identified as a cancer‐associated cell surface marker that is rarely expressed in healthy tissues. In testicular germ cell tumors (GCT), CLDN6 is particularly detectable in seminomas, embryonal carcinomas, and choriocarcinomas. However, little is known about the biological function of CLDN6 in GCT. Objectives This study aimed at characterizing the functional and molecular role of CLDN6 in GCT. Materials and Methods Using CRISPR/Cas9 gene editing, CLDN6 ‐deficient GCT cell lines (TCam‐2, 2102EP, NCCIT, and JAR) were generated and validated on gene (PCR), mRNA (qRT‐PCR), and protein level (flow cytometry). The functional impact of CLDN6 ‐deficiency was assessed via proliferation, migration, adhesion, and aggregation assays in both 2D and 3D cell culture models and visualized by electron microscopy. Further, transcriptome‐ and proteome‐wide analyses were performed using RNA‐sequencing and mass spectrometry, respectively. Results Compared to their respective parental controls, CLDN6 ‐deficient GCT cells showed diminished proliferative, migratory, and adhesive potential, as well as reduced capacity to form cellular aggregates, which could be attributed to disrupted cell‐cell contacts. The molecular analyses revealed only very few transcriptome‐ or proteome‐wide changes upon CLDN6 loss, suggesting a minor role in influencing gene expression. Discussion and conclusion This study highlights the role of CLDN6 in cell adhesion, migration, and proliferation of GCT cells, suggesting that, in addition to its suitability as a therapeutic target, CLDN6 may also play a putative role during tumor progression.