Handling and Assessment of Human Primary Prostate Organoid Culture

McCray, Tara; Richards, Zachary; Marsili, Joseph; Prins, Gail S.; Nonn, Larisa

doi:10.3791/59051-v

Cited by 9 publications

(8 citation statements)

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“…For the mono-culture organoid assay, 700 cells/well were mixed in 300 μL of 5 μg/ml of Matrigel (Corning; Cat # CB40230) in the media and seeded on a 150 μL of 5 μg/ml Matrigel base layer. The assay was performed as per the manufacturer's protocol, Corning LifeSciences (30) and previously published protocols (19,(31)(32)(33). The cultures were maintained in 500 μL of media that was changed every 2-3 days.…”

Section: Organoid Assaymentioning

confidence: 99%

TIAM1 signaling drives prostatic budding and branching phenotypes and is a potential therapeutic target for BPH

Khedmatgozar,

Dutta,

Dominguez

et al. 2024

Preprint

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Benign prostatic hyperplasia (BPH) is the most prevalent urologic disease in men aged over 50 years. However, the molecular mechanisms that drive BPH pathophysiology remain elusive. In this study, we integrated bioinformatic and experimental analyses of human BPH to identify TIAM1-RAC1 signaling pathway as a promising candidate for a molecular-based approach for BPH therapy. First, elevated TIAM1 expression in a BPH transcriptomic signature that was generated from the analysis of RNA-seq data from three independent BPH patient cohorts was validated at the protein level in a fourth patient cohort. Additional bioinformatic analyses of the BPH transcriptomic signature pointed to TIAM1-RAC1 pathway as the potential lead therapeutic pathway; and NSC23766 - a small molecule inhibitor of TIAM1 signaling - as a developmental lead compound for BPH therapy. Next, a proof-of-concept pharmacological approach of TIAM1-RAC1 inhibition in human prostatic cells using NSC23766 resulted in attenuated organoid budding and branching - a developmental program associated with prostatic nodule formation and BPH pathogenesis. Finally, shRNA-based genetic knock-down of TIAM1 in human prostatic cells led to a reduction in budding and branching phenotypes thereby phenocopying the effects of NSC23766. Together, our observations implicate elevated TIAM1 as a driver of budding and branching in BPH, and our studies pave the way for TIAM1-RAC1 based targeted approach for the treatment of the disease.

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Section: Organoid Assaymentioning

confidence: 99%

TIAM1 signaling drives prostatic budding and branching phenotypes and is a potential therapeutic target for BPH

Khedmatgozar,

Dutta,

Dominguez

et al. 2024

Preprint

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“…To check the expression pattern of multiple well-known signature genes in each group (control, 5 g, 8 g, and lung met samples), we first extracted 21/22 genes for mesenchymal/epithelial, 22/31 genes for basal/luminal, 17 genes related to AR, 11 genes for NEPC 31,33,67 . Besides, we used four different gene signatures to assess the stemness feature of the tumor tissues [68][69][70][71] . Next, we calculated the average of z scores for genes in each signature and visualized them with library 'ggpubr'(v.0.4.0) in R environment (v4.1.0).…”

Section: Rnaseq Analysismentioning

confidence: 99%

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

Thomsen,

Cai,

Zhang

et al. 2024

Preprint

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Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men, and treatment options for metastatic disease remain limited. In this study, we employed Adeno-associated virus (AAV) delivery and CRISPR-Cas9 technology to simultaneously mutate multiple genes in the mouse prostate, in order to investigate positive genetic interactions during PCa progression. By targeting five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL), we induced advanced prostate tumors in mice without metastasis, and mice reached humane endpoint at eight weeks. When three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16) were further depleted, tumor progression remained identical, but metastases were observed in the lung of all mice. Whole genome sequencing of metastatic tumors revealed few genetic alterations, confirming that mutations in the targeted genes are sufficient to transform prostatic cells into metastatic cancer. Mechanistic analysis uncovered multiple altered pathways, including impaired p-Src/p-Lyn-cMyc signaling, underscoring the importance of cMyc signaling in PCa metastasis. RNA sequencing revealed a significant dysregulation of overall transcriptome and increased expression of numerous genes in a genomic region at chr5qE1, which is highly conserved to human chr4q13.3 and regulated by KMT2C. Intriguingly, the depletion of Odam and Cabs1 in this gene cluster dismissed metastasis formation, indicating that they have critical functions for secondary tumor formation, which has not been reported before. Further genetic analysis demonstrated that Kmt2c deficiency is sufficient to facilitate lung metastasis, identifying Kmt2c as an essential gatekeeper for metastatic formation. Interestingly, the gene expression signature from mutations of the three epigenetic regulators were predictive of progression-free and overall survival and able to distinguish primary and metastatic human prostate cancer. Overall, this study highlights the positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in the progression and onset of metastatic disease, providing predictions and insights into possible treatments for metastatic PCa.

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“…Primary advantage of 3D culture is its broader specialisation of cells dictated by three-dimensional architecture compared to conventional 2D cultures. In fact, prostate organoids derived from primary prostate cells contain additional intermediate differentiation cell types compared to the same cells grown in a Petri dish [ 64 ]. scRNA-seq allowed to compare newly established organoid models with matched liver [ 65 ], intestine [ 66 ], endometrium [ 67 ], epididymis [ 68 ], biliary epithelium [ 69 ], salivary gland [ 70 ], and heart [ 34 ] tissues (Fig.…”

Section: Single-cell Transcriptomics Of Organoidsmentioning

confidence: 99%

Gene expression in organoids: an expanding horizon

2023

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Recent development of human three-dimensional organoid cultures has opened new doors and opportunities ranging from modelling human development in vitro to personalised cancer therapies. These new in vitro systems are opening new horizons to the classic understanding of human development and disease. However, the complexity and heterogeneity of these models requires cutting-edge techniques to capture and trace global changes in gene expression to enable identification of key players and uncover the underlying molecular mechanisms. Rapid development of sequencing approaches made possible global transcriptome analyses and epigenetic profiling. Despite challenges in organoid culture and handling, these techniques are now being adapted to embrace organoids derived from a wide range of human tissues. Here, we review current state-of-the-art multi-omics technologies, such as single-cell transcriptomics and chromatin accessibility assays, employed to study organoids as a model for development and a platform for precision medicine.

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Handling and Assessment of Human Primary Prostate Organoid Culture

Cited by 9 publications

References 0 publications

TIAM1 signaling drives prostatic budding and branching phenotypes and is a potential therapeutic target for BPH

TIAM1 signaling drives prostatic budding and branching phenotypes and is a potential therapeutic target for BPH

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression

Gene expression in organoids: an expanding horizon

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