Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates.

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed "organoids." Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Temporal Dynamics of Metabolic Acquisition in Grafted Engineered Human Liver Tissue.

Liver disease affects millions globally, and end-stage liver failure is only cured by organ transplant. Unfortunately, there is a growing shortage of donor organs as well as inequitable access to transplants across populations. Engineered liver tissue grafts that supplement or replace native organ function can address this challenge. While engineered liver tissues have been successfully engrafted previously, the extent to which these tissues express human liver metabolic genes and proteins remains unknown. Here, it is built engineered human liver tissues and characterized their engraftment, expansion, and metabolic phenotype at sequential stages post-implantation by RNA sequencing, histology, and host serology. Expression of metabolic genes is observed at weeks 1-2, followed by the cellular organization into hepatic cords by weeks 4-9.5. Furthermore, grafted engineered tissues exhibited progressive spatially restricted expression of critical functional proteins known to be zonated in the native human liver. This is the first report of engineered human liver tissue zonation after implantation in vivo, which can have important translational implications for this field.

Can't touch this: Stromal-mediated ductal proliferation.

Unraveling and replicating how cells communicate to regenerate organs remains one of the most compelling biological problems of our time. In this issue of Cell Stem Cell, Cordero-Espinoza et. al (2021) untangle how a subpopulation of liver mesenchymal cells residing adjacent to the bile ducts regulate biliary cell proliferation.

Vitamin D sufficiency enhances differentiation of patient-derived prostate epithelial organoids.

Vitamin D is an essential steroid hormone that regulates systemic calcium homeostasis and cell fate decisions. The prostate gland is hormonally regulated, requiring steroids for proliferation and differentiation of secretory luminal cells. Vitamin D deficiency is associated with an increased risk of lethal prostate cancer, which exhibits a dedifferentiated pathology, linking vitamin D sufficiency to epithelial differentiation. To determine vitamin D regulation of prostatic epithelial differentiation, patient-derived benign prostate epithelial organoids were grown in vitamin D-deficient or -sufficient conditions. Organoids were assessed by phenotype and single-cell RNA sequencing. Mechanistic validation demonstrated that vitamin D sufficiency promoted organoid growth and accelerated differentiation by inhibiting canonical Wnt activity and suppressing Wnt family member DKK3. Wnt and DKK3 were also reduced by vitamin D in prostate tissue explants by spatial transcriptomics. Wnt dysregulation is a known contributor to aggressive prostate cancer, thus findings further link vitamin D deficiency to lethal disease.

Erratum: Vitamin D sufficiency enhances differentiation of patient-derived prostate epithelial organoids.

[This corrects the article DOI: 10.1016/j.isci.2020.101974.].

Single-cell RNA-Seq analysis identifies a putative epithelial stem cell population in human primary prostate cells in monolayer and organoid culture conditions.

Human primary prostate epithelial (PrE) cells represent patient-derived in vitro models and are traditionally grown as a monolayer in two-dimensional culture. It has been recently demonstrated that expansion of primary cells into three-dimensional prostatic organoids better mimics prostate epithelial glands by recapitulating epithelial differentiation and cell polarity. Here, we sought to identify cell populations present in monolayer PrE cells and organoid culture, grown from the same patient, using single-cell RNA-sequencing. Single-cell RNA-sequencing is a powerful tool to analyze transcriptome profiles of thousands of individual cells simultaneously, creating an in-depth atlas of cell populations within a sample. Organoids consisted of six distinct cell clusters (populations) of intermediate differentiation compared to only three clusters in the monolayer prostate epithelial cells. Integrated analysis of the datasets allowed for direct comparison of the monolayer and organoid samples and identified 10 clusters, including a distinct putative prostate stem cell population that was high in Keratin 13 (KRT13), Lymphocyte Antigen 6D (LY6D), and Prostate Stem Cell Antigen (PSCA). Many of the genes within the clusters were validated through RT-qPCR and immunofluorescence in PrE samples from 5 additional patients. KRT13+ cells were observed in discrete areas of the parent tissue and organoids. Pathway analyses and lack of EdU incorporation corroborated a stem-like phenotype based on the gene expression and quiescent state of the KRT13+ cluster. Other clusters within the samples were similar to epithelial populations reported within patient prostate tissues. In summary, these data show that the epithelial stem population is preserved in PrE cultures, with organoids uniquely expanding intermediate cell types not present in monolayer culture.

Handling and Assessment of Human Primary Prostate Organoid Culture.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Prostate Stroma Increases the Viability and Maintains the Branching Phenotype of Human Prostate Organoids.

The fibromuscular stroma of the prostate regulates normal epithelial differentiation and contributes to carcinogenesis in vivo. We developed and characterized a human 3D prostate organoid co-culture model that incorporates prostate stroma. Primary prostate stromal cells increased organoid formation and directed organoid morphology into a branched acini structure similar to what is observed in vivo. Organoid branching occurred distal to physical contact with stromal cells, demonstrating non-random branching. Stroma-induced phenotypes were similar in all patients examined, yet they maintained inter-patient heterogeneity in the degree of response. Stromal cells expressed growth factors involved in epithelial differentiation, which was not observed in non-prostatic fibroblasts. Organoids derived from areas of prostate cancer maintained differential expression of alpha-methylacyl-CoA racemase and showed increased viability and passaging when co-cultured with stroma. The addition of stroma to epithelial cells in vitro improves the ability of organoids to recapitulate features of the tissue and enhances the viability of organoids.

The miR-183 family cluster alters zinc homeostasis in benign prostate cells, organoids and prostate cancer xenografts.

The miR-183 cluster, which is comprised of paralogous miRs-183, -96 and -182, is overexpressed in many cancers, including prostate adenocarcinoma (PCa). Prior studies showed that overexpression of individual pre-miRs-182, -96 and -183 in prostate cells decreased zinc import, which is a characteristic feature of PCa tumours. Zinc is concentrated in healthy prostate 10-fold higher than any other tissue, and an >80% decrease in zinc is observed in PCa specimens. Here, we studied the effect of overexpression of the entire 4.8 kb miR-183 family cluster, including the intergenic region which contains highly conserved genomic regions, in prostate cells. This resulted in overexpression of mature miR-183 family miRs at levels that mimic cancer-related changes. Overexpression of the miR-183 cluster reduced zinc transporter and intracellular zinc levels in benign prostate cells, PCa xenografts and fresh prostate epithelial organoids. Microarray analysis of miR-183 family cluster overexpression in prostate cells showed an enrichment for cancer-related pathways including adhesion, migration and wound healing. An active secondary transcription start site was identified within the intergenic region of the miR-183 cluster, which may regulate expression of miR-182. Taken together, this study shows that physiologically relevant expression of the miR-183 family regulates zinc levels and carcinogenic pathways in prostate cells.