Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells

Abstract

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.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem‐like cells

“…Therefore, recent efforts are being directed towards targeting CSCs . We evaluated the ability of ST1926 to target this sub‐population of stem/progenitor cells, in DU145 and PC3 cells, using a 3D sphere formation assay . Interestingly, a recent study elucidated the role of retinoic acid, one of the products of ALDH isozymes, and other vitamin A derivatives such as ATRA, in suppressing ALDH activity among CSCs .…”

“…53 We evaluated the ability of ST1926 to target this sub-population of stem/progenitor cells, in DU145 and PC3 cells, using a 3D sphere formation assay. 4,[28][29][30] Interestingly, a recent study elucidated the role of retinoic acid, one of the products of ALDH isozymes, and other vitamin A derivatives such as ATRA, in suppressing ALDH activity among CSCs. 54 We showed that treatment with ST1926 at a concentration as low as 0.01 µmol/L inhibited drastically the SFU as well as the sphere size over five generations.…”

“…This assay was performed according to the protocol previously described by our laboratory . Two thousand cells/well of 24‐well plates were suspended in cold growth factor‐reduced Matrigel/serum‐free medium (1:1) in a total volume of 50 μL.…”

“…This assay was performed according to the protocol previously described by our laboratory. [26][27][28][29][30] Two thousand cells/well of 24-well plates were suspended in cold growth factor-reduced Matrigel/serumfree medium (1:1) in a total volume of 50 μL. PC3 and DU145 cells were seeded uniformly in a circular manner around the bottom rim of the well and allowed to solidify in the incubator at 37°C for 1 hour.…”

Specific Inhibition of GSK-3β by Tideglusib: Potential Therapeutic Target for Neuroblastoma Cancer Stem Cells

“…The sphere formation assay was used as previously reported by our lab [19, 20]. In brief, single SH-SY5Y and SK-N-SH cell suspensions (10×10 3 cells/well) were seeded in Matrigel™/serum free DMEM Ham’s F-12 (1:1).…”