Generation of prostate cancer assembloids modeling the patient-specific tumor microenvironment

Abstract

Prostate cancer (PC) is the most frequently diagnosed malignancy among men and contributes significantly to cancer-related mortality. While recent advances in in vitro PC modeling systems have been made, there remains a lack of robust preclinical models that faithfully recapitulate the genetic and phenotypic characteristics across various PC subtypes-from localized PC (LPC) to castration-resistant PC (CRPC)-along with associated stromal cells. Here, we established human PC assembloids from LPC and CRPC tissues by reconstituting tumor organoids with corresponding cancer-associated fibroblasts (CAFs), thereby incorporating aspects of the tumor microenvironment (TME). Established PC organoids exhibited high concordance in genomic landscape with parental tumors, and the tumor assembloids showed a higher degree of phenotypic similarity to parental tumors compared to tumor organoids without CAFs. PC assembloids displayed increased proliferation and reduced sensitivity to anti-cancer treatments, indicating that PC assembloids are potent tools for understanding PC biology, investigating the interaction between tumor and CAFs, and identifying personalized therapeutic targets.

Fig 1. Establishment of patient-specific PC organoids from LPC and CRPC specimens.

(A) Schematic diagram illustrating the establishment of patient-derived PC organoids. Created in BioRender. Kim, Y. (2025) https://BioRender.com/d84u314. (B) Establishment of nine PC organoids from patients with LPC and CRPC. The histopathology of PC organoids and parental tissues was analyzed using haematoxylin and eosin (H&E) staining and immunostaining for luminal (KRT8) and basal (KRT5) markers. Scale bars = 100 μm. (C) Bright-field and H&E-stained images showing the morphological phenotype of PC organoids. (D) Quantification of the proportion of each organoid morphology at day 20 of culture. (E) Quantification of the average size of organoids at 5, 10, and 20 days. (F) Bar chart illustrating the stratification of all tumors and all derived organoid lines based on Gleason score (left), T stage (middle), and PSA level (ng/mL) (right). Gleason scores, T stage, and PSA levels were determined from the parental tumor tissues.

Fig 2. PC organoids represent genomic profiles of the parental tumor tissues.

(A) Correlation heatmap of pairwise genotypic concordance among samples, calculated as the Pearson's correlation coefficient (r) based on allele frequency data within the coding regions. (B) Distribution of somatic mutations and oncoplots depicting somatic variants in tumor organoids and parental tissues. (C) Proportion of nucleotide substitution types among total SNVs in organoids and corresponding parental tissues. (D) Frequency of somatic variants in tumor organoids and corresponding parental tissues. Sample annotations are indicated on the right side of panel A. SNV = single-nucleotide variant.

Fig 3. Generation of patient-specific PC assembloids by three-dimensionally reconstituting tumor organoids with matched CAFs.

(A) Schematic diagram illustrating the generation of PC assembloids reconstituted with tumor organoids and CAFs. Created in BioRender. Kim, Y. (2025) https://BioRender.com/k74v593. (B) Representative bright-field images of PC organoids and assembloids derived from LPC-5 and CRPC. Scale bar = 1 mm. (C) Histopathology of PC organoids, assembloids, and parental tumor tissues derived from patients with LPC and CRPC: LPC-5 and CRPC analyzed by H&E staining and immunostaining to mark luminal (KRT8) cells, basal (KRT5) cells, and CAFs (vimentin). Scale bar = 100 μm.

Fig 4. CAFs promote tumor proliferation and attenuate drug sensitivity in PC assembloids.

(A) Representative bright-field images of PC organoids and assembloids derived from LPC-5 and CRPC. Dotted lines demarcate the border between organoids and CAFs. Scale bars = 500 μm. (B) Graphs illustrating the average size of tumors at 0, 10, and 20 days. (C) Immunofluorescence images of PC organoids and assembloids stained with KRT8, Ki67, and DAPI. Scale bars = 200 μm. (D) Graph showing the proportion of Ki67-positive tumor cells in KRT8-positive PC cells of PC organoids or assembloids (n = 6). (E) Graphs depicting the proportion of cleaved caspase-3-positive tumor cells in PC organoids and assembloids treated with enzalutamide (1 and 10 μM), bicalutamide (1 and 10 μM), docetaxel (1 μM), and vehicle control for 72 h (n = 6). Scale bars = 100 μm. Data are mean +/− SEM. n.s., not significant; * p-value < 0.05, ** p-value < 0.01, and **** p-value < 0.0001 as determined by unpaired t-test. DMSO = dimethyl sulfoxide; Bic = bicalutamide; Enz = enzalutamide; Doc = docetaxel.