Extended Data Fig. 5: Generation of bladder tumour assembloids recapitulating the histopathology, genomic alterations and tumour subtypes of human urothelial carcinomas.

a, Eight bladder tumour organoid lines were established from patient-derived invasive urothelial carcinoma samples. Resulting tumour organoids were analysed by H&E staining and immunostaining to mark basal (KRT5) and luminal (KRT18) cells. Scale bars, 100 μm. b–i, The relative expressions of luminal (UPK1A, UPK2, ERBB2, FOXA1, and GATA3) and basal (KRT5, KRT14, CDH3, and KRT6A) markers in eight patient-derived bladder tumour organoids. j, Summary of molecular subtypes of eight bladder tumour organoids based on gene expression analysis. k, Experimental scheme for reconstituting patient-derived bladder tumour assembloids. l, m, Histopathology of tumour organoids, tumour assembloids, xenografts, and parental tumours derived from P-3 and P-6 were analysed by H&E staining and immunostaining. Scale bars, 100 μm. n, Bladder tumour assembloids derived from P-1 were analysed by immunostaining to mark tumour cells, CAFs, and endothelial cells (arrowheads, interconnected regions). Scale bars, 100 μm. o, Tumour growth was quantified by measuring tumour areas using the Image J program. p, Bladder tumour assembloids reconstituted without or with endothelial cells were analysed by immunostaining. Scale bars, 100 μm. q, Comparative analysis for mutations detected by WES of parental tumours, tumour organoids, and tumour assembloids. r, Parental tumours, tumour organoids at late passages (late organoid), and tumour assembloids at late passages (late assembloid) were analysed by immunostaining. Scale bars, 50 μm. s, Relative expression of luminal (UPK1A, UPK2, ERBB2, FOXA1, and GATA3) and basal (KRT5, KRT14, CDH3, and KRT6A) markers in parental tumours, late organoids, and late assembloids. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).

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