Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Background: Gliomas and neuroblastomas pose a great health burden worldwide with a poor and moderate prognosis, respectively. Many studies have tried to find effective treatments for these primary malignant brain tumors. Of interest, the AMP-activated protein kinase (AMPK) pathway was found to be associated with tumorigenesis and tumor survival, leading to many studies on AMPK drugs, especially Metformin, and their potential role as anti-cancer treatments. Cancer stem cells (CSCs) are a small population of slowly-dividing, treatment-resistant, undifferentiated cancer cells that are being discovered in a multitude of cancers. They are thought to be responsible for replenishing the tumor with highly proliferative cells and increasing the risk of recurrence.Methods: Metformin and 9-β-d-Arabinofuranosyl Adenine (Ara-a) were used to study the role of the AMPK pathway in vitro on U251 (glioblastoma) and SH-SY5Y (neuroblastoma) cell lines.Results: We found that both drugs are able to decrease the survival of U251 and SH-SY5Y cell lines in a 2D as well as a 3D culture model. Metformin and Ara-a significantly decreased the invasive ability of these cancer cell lines. Treatment with these drugs decreased the sphere-forming units (SFU) of U251 cells, with Ara-a being more efficient, signifying the extinction of the CSC population. However, if treatment is withdrawn before all SFUs are extinguished, the CSCs regain some of their sphere-forming capabilities in the case of Metformin but not Ara-a treatment.Conclusion: Metformin and Ara-a have proved to be effective in the treatment of glioblastomas and neuroblastomas, in vitro, by targeting their cancer stem/progenitor cell population, which prevents recurrence.

Cell lines representing the progression of prostate cancer (PC) from an androgen-dependent to an androgen-independent state are scarce. In this study, we used previously characterized prostate luminal epithelial cell line (Plum), under androgen influence, to establish cellular models of PC progression. Cells derived from orthotopic tumors have been isolated to develop an androgen-dependent (PLum-AD) versus an androgen-independent (PLum-AI) model. Upon immunofluorescent, qRT-PCR and Western blot analyses, PLum-AD cells mostly expressed prostate epithelial markers while PLum-AI cells expressed mesenchymal cell markers. Interestingly, both cell lines maintained a population of stem/progenitor cells. Furthermore, our data suggest that both cell lines are tumorigenic; PLum-AD resulted in an adenocarcinoma whereas PLum-AI resulted in a sarcomatoid carcinoma when transplanted subcutaneously in NOD-SCID mice. Finally, gene expression profiles showed enrichment in functions involved in cell migration, apoptosis, as well as neoplasm invasiveness and metastasis in PLum-AI cells. In conclusion, these data suggest that the newly isolated cell lines represent a new in vitro model of androgen-dependent and –independent PC.

Organoids represent one of the most important advancements in the field of stem cells during the past decade. They are three-dimensional in vitro culturing models that originate from self-organizing stem cells and can mimic the in vivo structural and functional specificities of body organs. Organoids have been established from multiple adult tissues as well as pluripotent stem cells and have recently become a powerful tool for studying development and diseases in vitro, drug screening, and host–microbe interaction. The use of stem cells—that have self-renewal capacity to proliferate and differentiate into specialized cell types—for organoids culturing represents a major advancement in biomedical research. Indeed, this new technology has a great potential to be used in a multitude of fields, including cancer research, hereditary and infectious diseases. Nevertheless, organoid culturing is still rife with many challenges, not limited to being costly and time consuming, having variable rates of efficiency in generation and maintenance, genetic stability, and clinical applications. In this review, we aim to provide a synopsis of pluripotent stem cell-derived organoids and their use for disease modeling and other clinical applications.

Cancer stem cells (CSCs), including those of advanced prostate cancer, are a suggested reason for tumor resistance toward conventional tumor therapy. Therefore, new therapeutic agents are urgently needed for targeting CSCs. Despite the minimal understanding of their modes of action, natural products and herbal therapies have been commonly used in the prevention and treatment of many cancers. Berberis libanotica Ehrenb (BLE) is a plant rich in alkaloids which may possess anti-cancer activity and a high potential for eliminating CSCs. We tested the effect of BLE on prostate cancer cells and our data indicated that this extract induced significant reduction in cell viability and inhibited the proliferation of human prostate cancer cell lines (DU145, PC3 and 22Rv1) in a dose- and time-dependent manner. BLE extract induced a perturbation of the cell cycle, leading to a G0-G1 arrest. Furthermore, we noted 50% cell death, characterized by the production of high levels of reactive oxidative species (ROS). Inhibition of cellular migration and invasion was also achieved upon treatment with BLE extract, suggesting a role in inhibiting metastasis. Interestingly, BLE extract had a major effect on CSCs. Cells were grown in a 3D sphere-formation assay to enrich for a population of cancer stem/progenitor cells. Our results showed a significant reduction in sphere formation ability. Three rounds of treatment with BLE extract were sufficient to eradicate the self-renewal ability of highly resistant CSCs. In conclusion, our results suggest a high therapeutic potential of BLE extract in targeting prostate cancer and its CSCs.