Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer

Abstract

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

Keywords: bladder cancer; clonal evolution; drug response; patient-derived organoids; patient-derived xenografts.

Figure 1. Establishment of patient-derived bladder tumor organoids and xenografts

(A) Overview of experimental plan. (B) Bright-field images of organoids together with H&E staining of parental tumors, patient-derived organoids, orthotopic xenografts generated from organoids, and organoids derived from the xenografts. Scale bars indicate 50 μm. (C) Ultrasound imaging (left) of orthotopic xenografts (dashed lines) and whole-mount images of corresponding bladders (right). Scale bars indicate 2 mm. See also Figure S1.

Figure 2. Molecular alterations in bladder organoid lines

(A) Summary of mutations and DNA copy number alterations identified in organoid lines by deep targeted sequencing, together with their percentage representation in this dataset. Representative genes detected in the TCGA study and known to be mutated in bladder cancer are shown. Passage numbers of the organoid lines were: SCBO-1, P11; SCBO-2, P11; SCBO-3, P14; SCBO-3.2, P14; SCBO-4, P13; SCBO-5, P15; SCBO-6, P9; SCBO-7, P11; SCBO-7.2, P1; SCBO-8, P19; SCBO-9, P17; SCBO-10, P17; SCBO-11, P20; SCBO-11.2, P8; SCBO-11.3, P2; SCBO-12, P15; SCBO-13, P7; SCBO-14, P19; SCBO-15, P12; SCBO-16, P7; SMBO-1, P8; SMBO-2, P10. (B) Concordance of mutations detected in the parental tumor and corresponding organoid lines. Passage numbers are the same as in panel B. (C) Detection of a FGFR3-TACC3 fusion transcript in the SCBO-10 organoid line. Results from RT-PCR in SCBO-7, SCBO-8 and SCBO-10 organoids are shown (top), together with the junction sequences on the mRNA and the reading frame at the breakpoint (bottom).

Figure 3. Tumor evolution during organoid culture and xenograft establishment

(A) Summary of mutations detected by deep targeted sequencing of parental tumors (a), patient-derived organoids at early (b) and late passages (c), orthotopic xenografts generated from the organoids (d), and organoids derived from xenografts (e). See Figure S2 for details of variant allele fractions and passage numbers analyzed. (B) Variant allele fractions during clonal evolution of SCBO-3.2 (top) and SCBO-5 (bottom) as determined by targeted sequencing analysis. (C) Mutational signature decomposition analysis based on whole-exome sequencing. From the 29 signatures tested, the top signatures representing the majority of mutations with p < 0.05 are shown. (D) Phylogenetic trees based on whole-exome sequencing. Orthotopic xenografts were converted from SCBO-3 at P8, SCBO-3.2 at P4, SCBO-5 at P5, and SCBO-6 at P7 respectively. See also Figures S2 and S3 and Table S1.

Figure 4. Phenotypic stability and plasticity in organoid culture

(A, B) H&E and immunostaining for the indicated markers in SCBO-10 (A) and SCBO-7 (B) parental tumors, organoids at the indicated passages, orthotopic xenografts generated from organoids, and organoids derived from xenografts. Scale bars indicate 50 μm. (C, D) Molecular subtypes of parental tumors and corresponding organoids at the indicated passages were analyzed using the BASE47 (C) and the MDACC classifiers (D). Heatmaps show normalized gene expression of organoid lines and parental tumors organized by the luminal and basal classifier genes. Unsupervised clustering analyses were performed by the z-score of normalized gene reads from RNA-seq data. (E) Summary of tumor subtypes as determined by the BASE47 and MDACC classifiers, as well as by immunofluorescence detection of markers. See also Figures S4, S5, S6, and Table S2.

Figure 5. Drug response of organoid lines

(A) Heatmap of logIC₅₀ values calculated from drug response analyses of patient-derived organoids by applying nonlinear regression (curve fit). Putative drug targets of the tested compounds are listed at left. Specific values for IC₅₀, Hill slope, and AUC are listed in Table S3. Passage numbers of the organoid lines were: SCBO-1, P8; SCBO-2, P9; SCBO-3, P8; SCBO-3.2, P9; SCBO-4, P10; SCBO-5, P8; SCBO-6, P9; SCBO-11, P5; SCBO-11.2, P6; SCBO-8, P12; SCBO-10, P8. (B) Dose response curves for SCBO-5 and SCBO-10 organoids treated with the MEK inhibitor trametinib, and SCBO-5 and SCBO-6 organoids with the mTOR inhibitor AZD8055. Each data point corresponds to three biological replicates; error bars correspond to one standard deviation. (C) Western blotting performed with the indicated antibodies of lysates from organoids treated for 8 hours with 10 nM or 500 nM of trametinib (left), or with 10 nM or 500 nM of AZD8055 (right). (D) Dose response curves for SCBO-3 and SCBO-3.2 organoids as well as SCBO-11 and SCBO-11.2 organoids treated with the indicated compounds. (E, F) Dose response curves for SCBO-6 organoids treated with the indicated drugs individually or in combination. See also Figures S7 and S8 and Table S3.

Figure 6. In vivo validation of drug response in orthotopic xenografts

(A) Dose response curves for selected organoid lines treated with the indicated drugs in culture. (B) Overview of drug treatment of xenografts. Passage numbers of the organoid lines were: SCBO-6, P10; SCBO-3, P11; and SCBO-5, P9. (C, E, G) Ultrasound images of orthotopic tumors (dashed lines), whole-mount images of bladder, H&E-stained sections of xenografts, and immunofluorescence detection of the indicated markers in xenografts. Scale bars indicate 2 mm for ultrasound images, and 50 μm for H&E and immunofluorescence images. (D, F, H) Tumor volumes as determined by ultrasound imaging at the indicated time points, together with quantitation of Ki67 immunostaining (D, F) or of cleaved caspase-3 immunostaining (H).