Abstract 3073: Bladder cancer patient-derived organoids to decipher cellular plasticity and cancer progression
Authors: Garioni Michele , Roma Luca , Mével Renaud , Vlajnic Tatjana , Pueschel Heike , Iber Dagmar , Seifert Hans-Helge , Rentsch Cyrill A. , Bubendorf Lukas , Le Magnen Clémentine 
Published: June 2022

Abstract
Introduction: Muscular Invasive Bladder Cancer (MIBC) is a highly heterogeneous disease. A consensus classification, based on gene expression profiling, has identified six MIBC subgroups, ranging from basal/squamous (Ba/Sq) to luminal papillary (LumP). This classification is a static representation of the disease and does not account for cellular plasticity, which may induce phenotype switch and cancer progression. Here, we aimed at identifying factors involved in cellular plasticity using bladder cancer patient-derived organoids (PDO) models.
Methods: Bladder cancer RNA sequencing data from bulk and single cell experiments were downloaded from public repositories. Samples from both datasets were classified using the consensus molecular classification. Pathway activation scores and cell-cell interaction scores were calculated for each subtype. Differential gene expression analysis was performed with the R packages edgeR and Seurat. MIBC samples were collected from bladder cancer patients undergoing surgical treatment. The samples were mechanically and enzymatically digested to generate a cell suspension which was seeded in Matrigel and immersed in growth-factor supplemented medium. Phenotypical characterization of long-term PDO lines was performed through immunofluorescence (IF). The PDOs were classified as Luminal, Basal or Mixed, based on the number of cells expressing the luminal marker CK8 or the basal marker CK5. Three selected organoids lines were cultured in alginate, immersed in a serum-free chemically-defined medium.
Results: Long-term PDO lines were successfully generated from 6 out of 13 MIBC samples. Organoids from the same line showed a consistent expression of basal and luminal markers. One line was classified as Basal, 2 lines as Mixed and 3 lines as Luminal. Exploring bulk RNAseq and scRNAseq datasets, we identified several genes whose expression was significantly altered in Ba/Sq and LumP MIBC subtypes. In particular, SHH and related BMP genes were significantly less expressed in Ba/Sq samples than in LumP. In addition, we observed an overexpression of BMP3 in LumP samples and of TGFβ2 in Ba/Sq samples. As preliminary test, we formulated a chemically-defined medium containing inhibitors of the proteins encoded by the identified genes (TGFβ inhibitor and Noggin); using this medium, we were able to maintain organoids from a Luminal, Basal and Mixed subtype in culture for up to two weeks. Interestingly, after two weeks of culture, IF analysis highlighted the presence of cells expressing luminal markers in the basal organoid line, suggesting culture-induced change of phenotype.
Conclusions: Bladder cancer PDOs represent promising models to study MIBC plasticity. The integration of RNA sequencing data and the successful culture of PDOs in chemically-defined culture conditions may allow to identify candidate factors impacting the phenotype of bladder cancer cells.
Citation Format: Michele Garioni, Luca Roma, Renaud Mével, Tatjana Vlajnic, Heike Pueschel, Dagmar Iber, Hans-Helge Seifert, Cyrill A. Rentsch, Lukas Bubendorf, Clémentine Le Magnen. Bladder cancer patient-derived organoids to decipher cellular plasticity and cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3073.