Mouse and human urothelial cancer organoids: A tool for bladder cancer research

Abstract

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.

Keywords: bladder cancer; organoid; urothelium.

Fig. 1.

Establishment of murine urothelial organoid cultures. (A) Schematic of organoid culture procedure of murine bladder. (B) Representative bright-field images showing the morphology of murine bladder organoids. (Scale bars, 100 µm.) (C) IHC analysis of murine bladder (Top) and murine urothelial organoids (Bottom) for H&E, Ck5, Ck20, and UPKIII as indicated. (Scale bars, 100 µm.) (D) IF staining of murine bladder organoids with Ck5, Phalloidin (F-actin), and DAPI (DNA) as indicated. (Scale bars, 100 µm.) (E) IF staining of murine bladder organoids with CD44, Phalloidin (F-actin), and DAPI (DNA) as indicated. (Scale bars, 100 µm.)

Fig. 2.

Establishment of murine ureter and suprabasal bladder organoids. (A) Schematic of organoid culture procedure of murine ureter and suprabasal bladder organoids. (B) IHC analysis of murine ureter (Top) and murine suprabasal bladder organoids (Bottom) for H&E, Ki67, Ck5, Ck20, and UPKIII as indicated. (Scale bars, 100 µm.) (C) Schematic of CRISPR experiment in murine basal bladder organoids. (D) Images of transfected (p53 and Stag2 gRNAs) and control bladder organoids in absence and presence of 5 µM Nutlin in the culture media. (E) Sanger sequencing of the mouse Trp53 (Top) and Stag2 (Bottom) genomic loci. In both cases, a Sanger sequencing trace of a wild-type (wt) organoid is compared with a knockout (ko) organoid. (F) Western blot analysis of resulting organoid wt and p53/Stag2 ko clones for indicated antibodies. Asterisk (*) indicates a nonspecific background band generated by the Stag2 antibody.

Fig. 3.

Establishment of human urothelial cancer organoids. (A and B) Schematic overview and representative images of organoids derived from (A) human bladder tissue obtained from radical cystectomies and (B) biopsy material obtained from TUR procedures. (Scale bars: tissue, 1 cm; organoids, 500 µm.) (C) Overview of the composition of the human bladder cancer biobank indicating the proportion of samples obtained from each procedure (TUR and cystectomy) and the gender distribution of the bladder samples. (D) Overview of bladder cancer organoids lines; each bar represents a single organoid line.

Fig. 4.

Morphology and histology of human bladder cancer organoids. (A) Representative images of human bladder organoid lines. (Scale bar, 500 µm.) (B) IHC of normal human bladder tissue (HBL8N Tissue) and six representative human bladder cancer organoid lines. Tissue and organoids were stained for H&E, Ck5, Ki67, Ck20, and TP63 as indicated. (Scale bar, 50 µm.)

Fig. 5.

Cellular composition of human bladder cancer organoids. (A) IF staining of two representative human bladder cancer organoid lines with Ck5, Ck20, Phalloidin (F-actin), and DAPI (DNA) as indicated. (Scale bars, 50 µm.) (B) IF staining of two representative human bladder cancer organoid lines with Ck5, CD44, Phalloidin (F-actin), and DAPI (DNA) as indicated. (Scale bars, 50 µm.)

Fig. 6.

Functional analysis of the human bladder cancer organoid biobank. (A) Images of human bladder cancer organoid cultures in the presence and absence of Nutlin in the culture media. (B) Images of human bladder cancer organoid cultures in presence and absence of FGF in the culture media. (C) Sanger sequencing of the FGFR3 genomic locus in human bladder cancer organoids. (D) Karyotype analysis of human bladder cancer organoids as indicated. (E) RT-qPCR for basal (KRT5 and KRT6) and luminal markers (KRT20, UPK1A and UPK3A) in human bladder cancer organoid. (Scale bars, 500 μm.)

Fig. 7.

Drug response to chemotherapeutic agents. Organoids were subjected to the chemotherapy drugs (A) epirubicin, (B) mitomycin C, (C) gemcitabine, (D) vincristine, (E) doxorubicin, and (F) cisplatin. Cell viability was measured and plotted as percentage of untreated organoids.