Epcam, CD44, and CD49f Distinguish Sphere-Forming Human Prostate Basal Cells from a Subpopulation with Predominant Tubule Initiation Capability

Abstract

BackgroundHuman prostate basal cells expressing alpha-6 integrin (CD49fHi) and/or CD44 form prostaspheres in vitro. This functional trait is often correlated with stem/progenitor (S/P) activity, including the ability to self-renew and induce differentiated tubules in vivo. Antigenic profiles that distinguish tubule-initiating prostate stem cells (SCs) from progenitor cells (PCs) and mature luminal cells (LCs) with less regenerative potential are unknown.Methodology/Principle FindingsProstasphere assays and RT-PCR analysis was performed following FACS separation of total benign prostate cells based upon combinations of Epcam, CD44, and/or CD49f expression. Epithelial cell fractions were isolated, including Epcam+CD44+ and Epcam+CD44+CD49fHi basal cells that formed abundant spheres. When non-sphere-forming Epcam+CD44− cells were fractionated based upon CD49f expression, a distinct subpopulation (Epcam+CD44−CD49fHi) was identified that possessed a basal profile similar to Epcam+CD44+CD49fHi sphere-forming cells (p63+ARLoPSA−). Evaluation of tubule induction capability of fractionated cells was performed, in vivo, via a fully humanized prostate tissue regeneration assay. Non-sphere-forming Epcam+CD44− cells induced significantly more prostate tubular structures than Epcam+CD44+ sphere-forming cells. Further fractionation based upon CD49f co-expression identified Epcam+CD44−CD49fHi (non-sphere-forming) basal cells with significantly increased tubule induction activity compared to Epcam+CD44−CD49fLo (true) luminal cells.Conclusions/SignificanceOur data delineates antigenic profiles that functionally distinguish human prostate epithelial subpopulations, including putative SCs that display superior tubule initiation capability and induce differentiated ductal/acini structures, sphere-forming PCs with relatively decreased tubule initiation activity, and terminally differentiated LCs that lack both sphere–forming and tubule-initiation activity. The results clearly demonstrate that sphere-forming ability is not predictive of tubule-initiation activity. The subpopulations identified are of interest because they may play distinct roles as cells of origin in the development of prostatic diseases, including cancer.

Prostate Cancer Stem Cell-Targeted Efficacy of a New-Generation Taxoid, SBT-1214 and Novel Polyenolic Zinc-Binding Curcuminoid, CMC2.24

“…However, accumulated data suggest that tumor-initiating cells can represent a heterogeneous population. It was shown recently that Epcam + /CD44 + and Epcam + /CD44 + /CD49f high basal cells can form abundant PrC spheres; in contrast, Epcam + /CD44 − cells cannot form spheres, but possess the Epcam + /CD44 − /CD49f high subpopulation with a basal profile similar to Epcam + /CD44 + /CD49f high sphere-forming cells, which are p63 + /AR low /PSA − [23]. After >10 passages, the majority of the PPT2 cells expressed cell surface markers commonly used for the isolation of tumor-initiating cells from different types of human cancer, including CD133, CD44, CD44v6, EpCAM, CD166, CD49f, and presently, they retain these features after more than 26 passages.…”

“…At the molecular level, there are currently no definitive markers to prove the malignant or nonmalignant nature of prostate cells, and to distinguish between normal and cancer stem cells. Growing evidence also suggests that CSCs might represent a heterogeneous subpopulation of the tumor-initiating cells [17-23]. Nevertheless, a combination of multiple cell surface markers for initial cell sorting followed by thorough functional characterization of the isolated cell phenotypes can lead to the identification of the most functionally significant (i.e.…”

Isolation and analysis of discreet human prostate cellular populations

“…To better understand the castration-resistant cell types capable of regenerating prostate epithelium, a number of cell surface markers have recently been discovered that enrich for prostate stem cell activity including α2β1 integrin, CD49f, CD44, TROP2, CD133, CD117, CD338, and Sca-1 (mice only) (Garraway et al, 2010, Goldstein et al, 2010, Goldstein et al, 2008, Guo et al, 2012, Lawson et al, 2007, Lawson et al, 2010, Leong et al, 2008, Lukacs et al, 2010, Mulholland et al, 2009, Richardson et al, 2004, Shi et al, 2014, Xin et al, 2005, Xin et al, 2007). Fractions with these markers are enriched for cells that display increased clonogenicity, serial passaging as spheroids, and tissue regenerative capacity as xenografted recombinants with inductive urogenital sinus mesenchyme.…”

Enrichment of Human Stem-Like Prostate Cells with s-SHIP Promoter Activity Uncovers a Role in Stemness for the Long Noncoding RNA H19

“…To circumvent this difficulty, several studies used nonmalignant-immortalized or malignant cell lines that contain sub-populations of cells with stem cell properties [3]. Enrichment in stem-like cell subpopulations could be achieved by multiple approaches: by modifying cell culture conditions such as cultivating cells in low-calcium serum-free defined medium [4] or under hypoxia [5]; by using a combination of cell surface markers, such as CD133 [6], CD44 [7], CD166 [8,9], or TROP2 [10]; by developing functional assays on cytoprotective activity such as the side population assay based on the efflux of Hoechst 33342 fluorescent dye by the ATP-binding cassette transporter and the ALDEFLUOR assay based on activity of aldehyde dehydrogenases detoxifying enzyme activity [11][12][13]; or finally by using reporter vectors containing fluorescent protein driven by stemness gene promoters [14,15].…”