Handling and Assessment of Human Primary Prostate Organoid Culture

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

doi:10.3791/59051

Cited by 12 publications

(10 citation statements)

References 21 publications

(31 reference statements)

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“…After gently washing with 1X PBS 3 times, organoids were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 30 min at room temperature (RT), and washed and stored in PBS until immunostaining. Immunofluorescent staining used modifications of previous methods [ 25 , 26 , 27 , 28 ]. Briefly, to reduce auto-fluorescence, 250 µL of 50 mM NH 4 Cl in 1X PBS were added into each well of the slides at RT for 30 min while gently shaking.…”

Section: Methodsmentioning

confidence: 99%

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Liu

Anderson

Deng³

et al. 2020

Genes

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We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.

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Section: Methodsmentioning

confidence: 99%

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Liu

Anderson

Deng³

et al. 2020

Genes

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“…Organoids were grown as previously described by our group (McCray et al, 2019b;Richards et al, 2019). Briefly, epithelial cells were thawed and grown to passage 2 on collagen-coated dishes to ~70% confluency.…”

Section: Organoid Culturementioning

confidence: 99%

“…Briefly, epithelial cells were thawed and grown to passage 2 on collagen-coated dishes to ~70% confluency. Epithelial cells were plated singly and sparsely (100-1,000 cells per well, depending on patient-specific growth ability) in 100 μL of 10%-50% growth factor-reduced Images of organoid cultures were acquired and analyzed as previously described by our group (McCray et al, 2019b;Richards et al, 2019). Briefly, images were captured at 4x magnification using the Evos FL Auto 2 Imaging System (Thermo Fisher Scientific, MA), and up to 50 z-planes were captured per quadrant of a 96-well plate.…”

Section: Organoid Culturementioning

confidence: 99%

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Vitamin D Sufficiency Enhances Differentiation of Patient-Derived Prostate Epithelial Organoids

McCray

Pacheco

Baumann

et al. 2020

Preprint

Self Cite

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Vitamin D is an essential steroid hormone that regulates systemic calcium homeostasis and cell fate decisions, including differentiation in many cell types. The prostate gland is hormonally regulated, requiring steroids for proliferation and terminal differentiation of secretory luminal cells. Vitamin D deficiency is associated with higher risk of lethal prostate cancer with an aggressive dedifferentiated pathology, linking vitamin D sufficiency to epithelial differentiation homeostasis. To determine regulation of prostate epithelial differentiation by vitamin D status, patient-derived benign prostate epithelial organoids were maintained in vitamin D deficient (vehicle) or sufficient (10 nM 1,25-dihydroxyvitamin D) conditions and assessed by phenotype and single cell RNA sequencing. Mechanistic validation demonstrated that vitamin D sufficiency promoted organoid growth and accelerated differentiation of lineage-committed cells by inhibiting canonical Wnt activity and Wnt family member DKK3. Wnt dysregulation is a known contributor to aggressive prostate cancer, thus these findings further link vitamin D deficiency to lethal disease.

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“…Tumor microenvironments are heterogenous and consist of different cell types such as endothelial cells, stromal cells, and immune cells. Studies have shown that stromal cells in the tumor microenvironment (TME) help promote PCa progression and spread [ 19 , 20 ]. One of the main components of the TME are fibroblasts, specifically cancer-associated fibroblasts (CAFs).…”

Section: Introductionmentioning

confidence: 99%

Overcoming TRAIL-resistance by sensitizing prostate cancer 3D spheroids with taxanes

et al. 2021

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Three-dimensional spheroid cultures have been shown to better physiologically mimic the cell-cell and cell-matrix interactions that occur in solid tumors more than traditional 2D cell cultures. One challenge in spheroid production is forming and maintaining spheroids of uniform size. Here, we developed uniform, high-throughput, multicellular spheroids that self-assemble using microwell plates. DU145 and PC3 cells were cultured as 2D monolayers and 3D spheroids to compare sensitization of TRAIL-resistance cancer cells to TRAIL mediated apoptosis via chemotherapy based on dimensionality. Monocultured monolayers and spheroids were treated with soluble TRAIL alone (24 hr), DTX or CBZ alone (24 hr), or a combination of taxane and TRAIL (24 + 24 hr) to determine the effectiveness of taxanes as TRAIL sensitizers. Upon treatment with soluble TRAIL or taxanes solely, monolayer cells and spheroids exhibited no significant reduction in cell viability compared to the control, indicating that both cell lines are resistant to TRAIL and taxane alone in 2D and 3D. Pretreatment with CBZ or DTX followed by TRAIL synergistically amplified apoptosis in 2D and 3D DU145 cell cultures. PC3 spheroids were more resistant to the combination therapy, displaying a more additive effect in the DTX + TRAIL group compared to 2D. There was a downregulation of DR4/5 expression in spheroid form compared to monolayers in each cell line. Additionally, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were cocultured with both PCa cell lines as spheroids to determine if CAFs confer additional resistance to chemotherapy. We determined that co-cultured spheroids show similar drug resistance to monocultured spheroids when treated with taxane plus TRAIL treatment. Collectively, these findings suggest how the third dimension and cocultures of different cell types effect the sensitization of androgen-independent prostate cancer cells to TRAIL, suggesting therapeutic targets that could overcome TRAIL-resistance in metastatic castration-resistant prostate cancer (mCRPC).

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Handling and Assessment of Human Primary Prostate Organoid Culture

Cited by 12 publications

References 21 publications

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis

Vitamin D Sufficiency Enhances Differentiation of Patient-Derived Prostate Epithelial Organoids

Overcoming TRAIL-resistance by sensitizing prostate cancer 3D spheroids with taxanes

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