95 samples were collected from 52 patients with muscle-invasive bladder cancer (20 pT1, 29 pT2, 3 pT3), 37 patients with non-muscle invasive bladder cancer and 1 patient with carcinoma in situ undergoing transurethral resection of the bladder (TURB), radical cystectomy, cytology or biopsies from metastatic sites. All samples were processed to generate organoids and tumor/organoid pairs and were characterized through H&E, immunohistochemistry, immunofluorescence for a panel of stromal and epithelial-associated markers (CK5, CK8, E-cadherin, Vimentin, SMA, Ki67) and Whole Exome Sequencing (WES). Drug testing was performed with a selected library of compounds at 8 distinct concentrations ranging between 0.01 nM and 10 µM. Therapeutic testing was carried out on organoids and matched single cells to compare efficiency of drug responses.
Out of 95 collected samples, 37 did not grow in organoid culture conditions (39%) and 40 grew short-term (i.e. maintained in culture for less than five passages, 42%). 18 samples were maintained for more than five passages and were established as long-term PDO lines (19%). Out of these long-term lines, 10 PDO lines from different bladder cancer subtypes were selected and phenotypically characterized. Selected PDO lines recapitulated the phenotype of their original patient’ tumor and were representative of relevant molecular and histological subtypes of BC. In particular, PDOs displayed either a pure luminal (CK8+), a pure basal (CK5+), a mixed basal-luminal (CK5+ and CK8+ cells), or a sarcomatoid (Vimentin+) phenotype. Five PDO lines were further characterized through WES and exhibited alterations in genes commonly altered in BC such as TP53, KMT2C, ARID1A which were shared with the corresponding patient tumor. Preliminary in vitro drug screens revealed significant differences in the response of each PDO line towards various therapeutics. The treatment on PDOs vs. single cells of the same lines showed similar response profiles, suggesting that fast-track screening for line-specific drug responses may represent a valid method.
We have successfully generated a series of PDO models, which highly resemble their original tumors and are representative of relevant histological and molecular subtypes of BC. Upon further molecular and functional characterization, our BC PDO biobank will provide a unique resource for translational research and precision oncology applications.