Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics

Abstract

The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from Trop2(hi) basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo.

Fig. 1.

Trop2 has progenitor characteristics in the murine prostate. (A) Frozen prostate tissue sections from 8- to 12-week-old mice were stained with antibodies against Trop2 in combination with CD49f, cytokeratin 5, or cytokeratin 8. Sections were counterstained with DAPI (blue) nuclear stain. (B) FACS analysis for Trop2 on total dissociated prostate cells. (C) Trop2^hi and Trop2^lo prostate cells were isolated by FACS from 8- to 12-week-old mice. Cells were plated on top of a thin layer of Matrigel. Graph shows the colonies formed after 10 days versus the number of cells plated (1,000, 10,000). (D) Trop2^hi and Trop2^lo prostate cells were isolated by FACS from 8- to 12-week-old mice. Graph shows the spheres formed in Matrigel after 10 days versus the number of cells plated (1,000, 10,000).

Fig. 2.

Trop2 separates the Lin⁻Sca-1⁺CD49f^hi fraction into 2 basal subpopulations. (A) FACS plots show gates drawn for sorting of Trop2^hi LSC (Lin⁻Sca-1⁺CD49f^hi) and Trop2^lo LSC subpopulations from 8- to 12-week-old mice. (B) RNA was isolated from total cells, non-LSC (Lin⁻ not Sca-1⁺CD49f^hi), Trop2^hi LSC, and Trop2^lo LSC fractions in duplicate experiments. RNA was synthesized into cDNA and subjected to qRT-PCR. Graph shows fold-enrichment over the total prostate cells for each gene. GAPDH was used as the reference gene.

Fig. 3.

Trop2 enriches for sphere-forming cells from the mouse and human prostate. (A) Total prostate, LSC, Trop2^hi LSC, and Trop2^lo LSC cells were isolated by FACS from 8- to 12-week-old mice. Graph shows the percentage of sphere-forming cells, based on the spheres formed in Matrigel from each population per 2,500 cells plated (5,000 cells plated from total prostate) after 8 days of growth. Spheres from the Trop2^hi LSC subpopulation were dissociated and replated for 3 successive generations. Data from several experiments were pooled. (B) Frozen human prostate tissue sections were stained with antibodies against Trop2 and CD49f. Sections were counterstained with DAPI (blue) nuclear stain. (C) FACS plots show gates drawn for sorting of human prostate cells into 4 subpopulations based on expression of Trop2 and CD49f. (D) Total prostate, CD49f⁻Trop2⁻, CD49f^hiTrop2⁻, CD49f^loTrop2^hi, and CD49f^hiTrop2^hi subpopulations were isolated by FACS from 3 patient samples. Graph shows the percentage of sphere-forming cells, based on the spheres formed in Matrigel from each population per 2,500 cells plated after 7 days of growth. Spheres from the CD49f^hiTrop2^hi subpopulation were dissociated and passaged for 3 successive generations.

Fig. 4.

Trop2^hi LSC cells give rise to basal, luminal, and neuroendocrine cells in vivo. (A) A total of 3 × 10³ Trop2^hi LSC or Trop2^lo LSC cells from β-actin DsRed mice, 10⁵ total prostate cells from β-actin GFP mice, and 2 × 10⁵ UGSM cells were combined in Matrigel and injected s.c. into SCID mice. Grafts were harvested after 8 weeks, and overview images were taken by using transillumination (TI) and DsRed or GFP fluorescence. (B) Tissue sections from regenerated tissue were stained for antibodies against cytokeratin 5 (K5), cytokeratin 8 (K8), androgen receptor (AR), p63, and synaptophysin. Sections were counterstained with DAPI (blue) nuclear stain. Sections from 8- to 12-week-old mice were stained for comparison of neuroendocrine cell morphology and marker expression pattern (synaptophysin). (C) In tissue sections from regenerated tissue, DsRed and GFP fluorescence (13 and 16) were visualized before antigen retrieval caused by a loss of signal after treatment. The remaining images demonstrate synaptophysin staining and DAPI nuclear counterstain after antigen retrieval.