Stem Cell Antigen-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

Abstract

Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (stem cell antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1(+) luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1(+) luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1(+) luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1(+) luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer.

Keywords: Organoid; Progenitor cells; Prostate; Sca-1.

Figure. 1. Sca-1 defines a distinct population of prostate luminal cells

A–B: FACS plots of prostate cell lineages in intact (A) and castrated (B) adult mice. Bar graphs show means ± s.d. of percentages of individual cell lineages from 3 independent experiments. C: Co-immunostaining of Sca-1, cytokeratin 14 (K14), and cytokeratin 8 (K8) on cytospins of individual FACS-sorted prostate lineages. Bars=10µm. D: qRT-PCR analysis of lineage marker expressions in individual FACS-sorted prostate cell lineages. Results show means ± s.d. from 3 independent experiments. E: Co-immunostaining of Sca-1 and androgen receptor (AR) in proximal and distal prostatic ducts. F: qRT-PCR analysis of expression of prostate secretory proteins in FACS-sorted Sca-1⁺ and Sca-1⁻ luminal cells. Results show means ± s.d. from 3 independent experiments. *:p<0.05, **:p<0.01, ***:p<0.001.

Figure. 2. Sca-1⁺ luminal cells reside in proximal prostatic ducts

A: Schematic illustration of distal, middle, and proximal anterior prostatic ducts. B: FACS plots of prostate cell lineages in proximal, middle, and distal anterior prostatic ducts. C: Bar graphs show means ± s.d. of percentages of individual cell lineages from 3 independent FACS experiments. D–F: Co-immunostaining of Sca-1 and K5 (D), Sca-1 and K8 (E), Sca-1 and Nkx3.1 (F) in anterior prostate lobes. Yellow bars=100µm. White bars=25µm.

Figure. 3. Sca-1 fractionates CD24^lowCD49f^high luminal cells

A: FACS plot shows that CD24 separates stromal cell contamination from Sca-1-expressing luminal cells. Bar graph shows the identity of FACS-sorted cells based on immunostaining of cytospins. B: FACS plot shows that Sca-1 fractionates CD24^lowCD49f^high luminal cells. C: Bar graph representing the purity of individual FACS-sorted prostate lineages quantified based on immunostaining of cytospins.

Figure. 4. Different lineages of prostate epithelial cells generate distinct organoids in vitro

A: IHC analysis of lineage marker expression in the 4 types of organoids. White arrow points to P63-expressing basal cells inside an organoid. Bars=50µm. B: Pie chart quantifies composition of organoids derived from wild type prostate epithelial cells. C: Transilluminating images of prostate organoids. Enlarged inset shows a type III budding organoid. Yellow bar=100µm; white bar=50µm. D: Individual prostate epithelial lineages form different types of organoids at distinct efficiencies. Dot plot shows means ± s.d. of organoid-forming units of different lineages from 3 independent experiments. Pie charts quantify the types of organoids in different cultures. *:p<0.001. E: Dot plot shows means ± s.d. of organoid size in different cultures. *:p<0.001.

Figure. 5. Distinct types of organoids retain their histological and morphological features upon passaging

A: Prostate organoid cells can be serially passaged as dissociated cells. Dot plot shows means ± s.d. of organoid-forming units during serial passaging. Pie charts show composition of organoid types. B: Type I and type III organoids can be passaged and only generate organoids of parental types. Dot plot shows means ± s.d. of OFUs from 3 independent experiments.

Figure. 6. The Sca-1⁺ luminal cells possess in vivo bipotent differentiation capacity in the prostate regeneration assay

A: Bar graph shows means ± s.e.m. of gland-forming capacity of FACS-sorted basal cells, Sca-1⁻ luminal cells, and Sca-1⁺ luminal cells. UGSM cells serve as negative control. B: H&E staining and IHC analysis of K5 and K8 in regenerated glands. C: Dot plot shows means ± s.e.m. of diameters of regenerated glands. Bars=50µm. ***:p<0.001.