Robust 3D Models are Important Research Tools

Two-dimensional (2D) cell culture is the backbone of many research programs for a number of reasons – they are historically well-established, simple and a relatively inexpensive way to culture a variety of cell types. However, because cells in these systems are grown on flat, planar surfaces, they fail to recapitulate the cytoarchitecture of in vivo tissues or tumors, which limits their clinical predictivity in drug discovery and toxicology programs. This has driven scientists to invest in more complex and physiologically relevant 3D tissue models like spheroids and organoids, which can more accurately model in vivo mechanisms.

Prostate epithelial cells from both normal and cancer tissues, grown in 3D culture to form spheroids and organoids, represent promising in vitro models to study tissue homeostasis and disease progression (i.e., prostate cancer). This week on the blog, we will review some of the featured publications from years past that have used 3D prostate models across a wide range of research areas from studying prostate cancer to determining toxicity effects of environmental pollutants.

The first blog post featured a publication by Zhang and Colleagues. The authors developed a Matrigel-based in vitro prostate organoid model from Lifeline’s normal Human Prostate Epithelial Cells (hPrECs) cultured in ProstaLife™ Medium in their study to identify the role of CEACAM20 and CEACAM1 in normal prostate luminal and ductal differentiation. Phenotypic analysis of the hPrEC-formed organoids on-Matrigel demonstrated they could develop spherical ancini and tubule-like structures that closely resemble the acinar-budding-branching morphogenesis observed in normal prostate development. Immunofluorescence staining and knockdown studies determined that CEACAM20 and CEACAM1 are necessary for the differentiation observed in the organoids and it is their loss of function that drives prostate cancer initiation. This in vitro model system is able to recapitulate normal prostate morphogenesis and is therefore well-suited to further studies aimed at examining prostate epithelial differentiation and cancer initiation mechanisms.

The second blog post highlighted research by Hu and Colleagues to develop a novel prostasphere-based BrdU (a DNA-intercalating molecule) label retention assay to identify and isolate prostate stem cells.  Based on the immortal strand hypothesis, BrdU should bind the DNA of the slower cycling prostate stem cells while it is diluted out and eventually lost in daughter cells that become terminally differentiated. First, the authors established 3D spheroids (prostaspheres) derived from Lifeline’s hPrECs grown in ProstaLife medium to enriched in stem and progenitor cells, which were then incubated with BrdU to label the rare stem cell population. Their stem cell phenotype was confirmed using immunostaining for NT10B and KRT13 and demonstrated in in vitro and in vivo regenerative assays. Finally, the authors utilized this assay to label stem-like cells from human prostate cancer samples, colorectal (HCT116) and breast (MCF-7) cancer cell lines to perform genetic and molecular analysis, further demonstrating the utility of this BrdU assay to isolate prostate stem cells to study prostate homeostasis and mechanisms involved in prostate tumorigenesis.

Prins and Colleagues sought to determine the effects of chronic low-dose Bisphenol A (BPA)—a widespread chemical—on the prostate epithelium. The full details of the publication were covered in another Lifeline blog here. The authors evaluated the prostate histopathology in a set of Sprague-Dawley rats dosed with BPA to determine its impact on epithelial stem and progenitor cells behavior. To confirm their findings in the animal model with human cells, the authors utilized the human prostasphere-based BrdU label retention assay described by Hu and Colleagues above to enrich for and assess stem and progenitor cell dynamics resulting from BPA exposure. The 3D prostaspheres generated from Lifeline’s hPrECs exposed to 2.5 mM BPA for 7 days had increased numbers of longer-term BrdU-labeled cells compared with 25 nM BPA and the vehicle control. The 10-fold higher BPA dose (25 nM) was shown to disrupt normal prostate stem and progenitor cell dynamics, which the authors speculate could play a role in increased prostate cancer risk.

The final study, which was featured on our blog in September 2020, also examines the impact of an environmental toxin, inorganic arsenic (iA) on prostate homeostasis and tumorigenesis. The authors isolated prostate stem and progenitor cells from 3D prostaspheres derived from Lifeline’s normal hPrEC and exposed the cells to iA to study its impact on prostate homeostasis. At environmentally relevant doses of iA (1μM) homeostasis was affected, manifesting in increased cellular proliferation, suppression of epithelial differentiation, and activation of oncogenic pathways including the KEAP1/NRF2 pathway. iA appears to cause dysfunction in lysosomal acidification, preventing autophagy in prostate progenitor cells, and this disruption in homeostasis is preserved and genetically transmitted to later generations of daughter epithelial cells, which predisposes them to a carcinogenic state and could make an individual more susceptible to developing prostate cancer later in life.

These studies were made possible with products from Lifeline Cell Technology and underscore the importance of accurate 3D cellular models upon which scientific conclusions can be made. For more information on normal human prostate epithelial cells or male reproductive cells, please click on the links below:

• Normal Human Prostate Epithelial Cells
• Prostate Smooth Muscle Cells
• Human Prostate Fibroblasts
• Normal Human Vas Deferens Fibroblasts
• Human Seminal Vesicle Epithelial Cells

Have you used Lifeline Cell Technology products to create in vitro 3D models to improve the accuracy of your research? Connect with us and your study could be featured here on our blog!