MP06-06: Establishment and evaluation of a simplified approach to patient derived bladder cancer organoids using urine

Paul Pollehne, Ivonne Aidee Montes Mojarro, Johannes Schneider, Bastian Amend, Falko Fend, Arnulf Stenzl, Wilhelm K. Aicher, Niklas Harland*, Tuebingen, Germany

Introduction: Patient derived organoids (PDO) are a promising tool for research and individualized therapy. The cells that are used to culture organoids are commonly retrieved from invasive surgery, requiring general anesthesia. In this study we evaluate a simplified approach through PDOs out of rinsing urine from patients with urothelial carcinoma.

Methods: Urine cells were isolated and after using an erythrocyte lysis buffer were resuspended with Matrigel® (BME) and covered with an PDO medium containing the wnt-agonist r-spondin, fibroblast-growth- factors 2, 7 and 10, the ROCK-inhibitor Y-27632 dihydrochloride and epidermial growth factor. The growth of the PDOs was observed by measuring the diameters by light microscopy. When the PDOs ceased to grow, the cells were passaged. After 5 passages the PDOs were confirmed as established organoid cell lines. For characterization the cells were transferred into chamber slides and stained using primary antibodies against urothelial and epithelial markers (GATA-3, AE1/AE3, CK5, CK7), tumor markers (p53, TP63, CD276, CK20, FGFR-3, Vimentin, S-100-P, Ki67) and stem cell markers (CD24, CD44).

Results: In 35 out of 35 samples (100%), cells were isolated without any contamination in cell cultures. Spheroid growth was observed in 27 samples in primary culture (77%), in 18 samples in 1st passage (51%) and in 10 samples in 2nd passage (29%).  One of the samples (3%) reached the 5th passage (UCO#33). In 23 associated solid tumor samples were retrieved during transurethral resection of a bladder tumor. Out of these, PDOs were established in 19 samples in primary culture (83%), 9 samples in 1st passage (39%) and 3 samples in 2nd passage (13%)).  None of the samples (0%) reached the 5th passage.  Compared tocell cultures which ceased growth before the 5th passage UCO#33 increased in diameter in later passages and showed a significant higher expression of CD276 and as the only culture a coexpression of CD24 and CD44.  The expression of CK7 and Ki67 increased from 3rd to 5th passage while the expression of TP63, CK20, Vimentin and CD24 decreased.

Conclusions: In this project we were able to grow PDOs out of rinsing urine from patients with bladder cancer, whose growth are comparable to the ones out of solid tumor samples. We identified immune checkpoint CD276 and stem cell markers CD24/CD44 as potential markers for the success of organoid growth out of urine. PDOs out of rinsing urine are promising tools for research and as well for clinical use.

Source of Funding: The work was funded by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 2543/I).