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.

Keywords: cancer stem cells; differentiation; prostate cancer; prostatospheres; self-renewal; sphere-formation assay.

Figure 1

Schematic illustrating strategy of drug treatments in sphere-formation assay. After isolating single cell suspension from PCa tissues or PCa cell lines, drugs can be added to every generation in the sphere-formation assay (A), or drugs can be added to the first generation of spheres only (G1D0) and then spheres can be serially propagated to investigate whether the effect is permanent or reversal (B), or spheres can be formed and serially passaged so that you have extensively grew and enriched for stem cells, and then drugs are added at G5D0 to potentially target those cells (C). The sphere-formation efficiency has to be calculated for each generation to assess self-renewal ability of sphere-forming cells. At each generation, spheres could be processed for immune-histochemical analysis to check for differentiation markers, or proteins and total RNA could also be extracted to assess for differentiation.

Figure 2

The sphere-forming assay with murine and human prostate cells. Representative bright-field images of mouse wt, Pten^−/−TP53^−/−, PLum-AD, and PLum-AI prostate spheres (upper panel) and human DU145, PC3, 22RV1, and RWPE1 (lower panel) prostate spheres are shown. Zeiss Axiovert microscope was used for the acquisition of bright field images. Scale bars = 100 μm.

Figure 3

Immunophenotype of prostatospheres. Immunofluorescent images of confocal cross sections from mouse wt prostate spheres stained for prostate lineage epithelial markers CK8, CK5, and CK14 (left panel) and stem cell markers: p63, SOX2, CD49f, and SCA-1 (right panel). Prostatospheres displayed a heterogenous population of cells displaying intermediate cytokeratin profiles, where major population of cells co-expressed CK8 (luminal prostate cell marker) and either CK5 or CK14 (basal prostate cell marker). Furthermore, positive expression of the neuroendocrine marker β3 tubulin was detected in a minor population of cells within the prostatospheres. On the other hand, co-expression of p63 [basal prostate cell marker and believed to be a marker of the stem cells of developing prostate epithelium (46)] and SOX2 [essential embryonic stem cell gene involved in prostate tumorigenesis (47)] was also detected, besides expression of the stem cell marker CD49f and SCA-1, which have been shown to identify putative prostate stem-like cells (48, 49). The nuclei were stained with anti-fade reagent Fluorogel II with DAPI. Scale bars = 100 μm. Representative confocal microscopy images were acquired using the 63x oil objective and images were processed using the Zeiss ZEN 2012 image-analysis software. Microscopic analysis was performed using Zeiss LSM 710 laser scanning confocal microscope (Zeiss). DAPI, 40,6-diamidino-2-phenylindole.