In this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged.

Background: Bladder cancer (BC) can be characterized clinically as either non-muscle-invasive (NMIBC) or muscle-invasive (MIBC). While NMIBC generally has a favorable prognosis, MIBC is characterized by high morbidity and mortality. Understanding the molecular determinants of tumor invasion is critical, yet research is hampered by the limitations of current experimental models. Standard assays such as the Boyden chamber lack physiological complexity, while porcine bladder models suffer from tissue contamination and genetic variability. There is an urgent need for reliable models that mimic the intact tissue architecture. Methods: We established a unique ex vivo tissue invasion model (EXTIM) to evaluate the invasive capacity of BC cells within a largely intact tissue context, using freshly prepared bladders from mice. The invasiveness of human BC cells (RT4, T24, UMUC3) and the immortal urothelial cell strain (Y235T) was comparably evaluated using EXTIM, the Boyden chamber, and porcine models. Gene knockdown or ectopic expression of GJB3 or ORP3 indicated the suitability of EXTIM to investigate the impact of specific factors on tumor cell invasion. To identify novel genetic regulators of cell invasion, we combined EXTIM with a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen. Additionally, we utilized the EXTIM to perform a pharmacological screen of a small molecule library comprising 90 substances to identify compounds capable of suppressing BC cell dissemination. Results: Importantly, by combining EXTIM with genomewide CRISPR-Cas9 screening, we identified several candidate genes involved in BC progression. Notably, discoidin domain receptor tyrosine kinase 1 (DDR1) was identified as a functional inhibitor of tumor cell invasion. Furthermore, the small-molecule screen revealed that PD-156707, a selective antagonist of the endothelin receptor A (ETA), significantly suppresses cancer cell invasion within the EXTIM environment. Conclusions: EXTIM serves as a robust and physiologically relevant tool for assessing tumor cell invasion and migration under ex vivo conditions. EXTIM can be used to identify factors involved in the progression of invasive BC by high-throughput genetic screenings in an ex vivo organ culture system, by culturing cells after transmigration through the bladder tissue. Moreover, the impact of specific genetic factors in the process of tumor cell dissemination can be assessed by placing bladders from genetically modified mice into the EXTIM.