The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis

Abstract

Sca-1 (stem cell antigen-1) enriches for murine prostate cells capable of regenerating tubular structures containing basal and luminal cell lineages in a dissociated cell prostate regeneration system. Sca-1(+) fractions are enriched for cells at the G(0) stage of the cell cycle, and Sca-1(+) cells cluster in the proximal region of prostatic tubules where replication-quiescent cells have been localized. Castration-induced enrichment for androgen-independent cells results in a concomitant enrichment for Sca-1(+) cells. Genetic perturbations of PTEN/AKT signaling in prostate-regenerating cells leads to the initiation of tumorigenesis, and cancer progression is associated with a dramatic increase in Sca-1(+) cells. Sca-1-enriched prostate-regenerating cells possess multiple stem/progenitor cell properties and can serve as targets for cancer initiation.

Fig. 1.

Sca-1 enriches for cells with prostate-regenerating activity. (A) Comparison of regenerative activity of Sca-1⁺ and Sca-1^–/– prostate cell fractions. Single cell suspensions prepared from 8- to 10-week-old murine prostate tissue were separated into Sca-1⁺ and Sca-1^–/– fractions by using magnetic bead sorting. Sca-1⁺ or Sca-1^–/– cells (1 × 10⁵) were combined with 1 × 10⁵ UGSM cells. Regenerated grafts were harvested 10 weeks later, and regenerative activity was compared by transillumination microscopy (Upper) and H&E staining of tissue sections (Lower). Original magnification of H&E staining: ×10. (Scale bars, 100 μm.) (B) Strategy for investigation of differentiation potential of Sca-1-enriched PRCs. Dissociated prostate epithelial cells were prepared from 8-week-old C57BL/6 mice and β-actin GFP transgenic mice, as shown schematically. Cells from β-actin GFP mice were sorted into Sca-1⁺ and Sca-1^–/– fractions by using magnetic beads. Sca-1⁺GFP⁺ or Sca-1^–GFP⁺ prostatic epithelial cells (5 × 10⁴) were mixed with 5 × 10⁴ cells from C57BL/6 mice and 1 × 10⁵UGSM cells and incubated under the kidney capsule of SCID mice. Transillumination (TI) and fluorescent (GFP) images were taken of grafts harvested after a 10-week incubation. GFP signal from each graft was quantified by using a charge-coupled device camera. Frozen tissue sections were counterstained with PI and analyzed by fluorescence microscopy. Original magnification: ×40 (Scale bars, 100 μm.) (C) Single Sca-1-enriched PRCs can regenerate prostatic tubular structures. High-magnification fluorescent images of tissue sections from chimeric tissue grafts show that each prostatic tubule consists exclusively of single donor cell type. Confocal microscopy analysis of sections reveals fluorescence at sites within GFP⁺ tubules where GFP expression is low. Original magnification: ×100. (Scale bars, 100 μm.) (D) Sca-1-enriched PRCs can give rise to both basal and luminal cells. Paraffin sections were prepared from each graft and stained with antibodies against p63 (arrow) and the AR as described in Materials and Methods. Original magnification: ×200. (Scale bars, 50 μm.)

Fig. 2.

Sca-1⁺ prostate cell fractions contain increased numbers of replication-quiescent, androgen-independent cells that cluster in the proximal region of murine prostatic tubules. (A) FACS analysis of cell cycle status of Sca-1⁺ and Sca-1^–/– cell fractions. Dissociated prostate cells from C57BL/6 mice were separated into Sca-1⁺ and Sca-1^–/– fractions by using magnetic beads. Each fraction was stained and analyzed by FACS for simultaneous quantification of DNA and RNA content as described in Materials and Methods. (B) FACS analysis of Sca-1 expression in alternative regions of the murine prostate. The proximal and distal regions of prostatic ducts from 8-week-old C57BL/6 mice were microdissected as shown. Dissociated cells from each region were stained with antibody against the Sca-1 antigen and compared by FACS. (C) Immunofluorescent analysis of Sca-1 expression. Longitudinal prostate tissue sections were stained with antibody against Sca-1 as described in Materials and Methods. Fluorescence microscopy images show the expression of Sca-1 in the proximal and distal regions of the murine prostate. Sections were counterstained with PI (red). Original magnification: 1, 3, 5, and 7, ×100; 2, 4, 6, and 8, ×200. (Scale bars: 1, 3, 5, and 7, 100 μm; 2, 4, 6, and 8, 50 μm.) (D) Sca-1-positive cells are enriched in prostate tissue from castrated mice. Cells were dissociated from the prostate tissue of 10-week-old castrated and intact C57BL/6 mice. FACS analysis of Sca-1 expression was performed by using a FITC-conjugated anti-Sca-1 antibody.

Fig. 3.

Expression of constitutively active AKT1 in PRCs is sufficient to initiate tumorigenesis in the regeneration system. (A) Transillumination (TI) and fluorescent (GFP) microscopy analysis of grafts regenerated from prostate cells infected by control (Upper) or AKT1 (Lower) lentivirus. A blue arrow indicates sites of extensive angiogenesis. Frozen sections from each graft were counterstained with PI (GFP/PI) and analyzed by fluorescence microscopy. Original magnification: ×100. (Scale bars, 100 μm.) (B) (1) Histological and immunohistochemical analysis of tissue regenerated from cells infected by AKT1 lentivirus. (2) H&E staining of mPIN lesion (green arrow) and adenocarcinoma. (3) Phosphorylated AKT1 is only present in mPIN lesions (green arrow). (Inset) Clear plasma membrane staining characteristic of phospho-Akt1. (4) Immunohistochemical analysis of AR (red) and p63 (brown) within mPIN lesions. Original magnification: 1 and 2, ×100; 3 and 4, ×200; Inset, ×400. (Scale bars, 100 μm.)

Fig. 4.

Enrichment of PRCs by using the Sca-1 marker confirms the oncogenic potential of PRCs. (A) Regeneration of AKT1 lentivirus-infected Sca-1⁺ and Sca-1^–/– prostate epithelial cells. Cells from 8-week-old C57BL/6 mouse prostate tissue were separated into Sca-1⁺ and Sca-1^–/– fractions by using magnetic bead sorting and infected with AKT1 lentivirus. Infected Sca-1⁺ or Sca-1^–/– cells (5 × 10⁴) were mixed with 1 × 10⁵ UGSM cells and implanted under the kidney capsule of SCID mice. Regenerated grafts were harvested after a 6-week incubation and analyzed by transillumination and fluorescence microscopy. (B) Bar graph comparing the fluorescence signal emitted from each graft measured by a charge-coupled device camera. (C) Histological analyses of regenerated cancer tissue grafts. H&E staining shows mPIN lesions in sections from Sca-1⁺ grafts (Upper) and normal tubular structures in sections from Sca-1^–/– grafts (Lower). (D) Immunohistochemical staining for AR and p63 on representative sections from Sca-1⁺ grafts. (Scale bars, 50 μm.) (E) Prostate cancer tissues contain increased percentages of Sca-1⁺ cells. Dissociated cells from 8-week-old murine prostate tissue were infected with AKT1 lentivirus and incubated under the kidney capsule of SCID mice for 10 weeks. FACS analysis was performed on cells stained with a phycoerythrin-conjugated antibody against Sca-1.