Fig 1.
Histology of human prostate xenografts collected at 7, 30, and 90 days, demonstrating phenotypic tissue changes after a single estradiol dose in the “one-hit” dosing paradigm.
(A) At 7 days post-implantation, control xenografts display a cellular mesenchyme and small primordial glands, which is representative of normal human fetal tissue. (B) Estrogen-treated xenograft at 7 days post-implantation demonstrates minor ductal branching with some vacuolization. (C) At 30 days post-implantation, control xenografts present with both well-developed glands and stroma. (D) Estrogen-treated xenografts at 30 days display significant basal cell hyperplasia (*) encompassed by an un-developed mesenchyme. (E-F) At 90 days, both control- and estrogen-treated xenografts have well-developed tortuous glands with no visible differences between control and treated. (A-F) Gestational age of human fetal prostate prior to implantation is 20 weeks. (Hematoxylin and Eosin staining; scale bar = 50 μm).
Fig 2.
Histology and prostate specific antigen (PSA) staining of 200-day human prostate xenografts exposed to varying initial (corn-oil or estradiol) and later-life (control placebo capsules or estrogen pellets and testosterone capsules) exposures.
(A) Control human prostate xenografts (control/control) contained phenotypically normal prostatic ducts surrounded by mature stromal cells, (E) with proper PSA staining in the luminal cells of ducts. (B) Human prostate xenografts given an initial treatment of estrogen, with no subsequent treatment (estrogen/control) demonstrated a mature phenotype containing both nicely developed glands and a normal cellular stroma, (F) with normal PSA staining similar to C/C-treated xenografts. (C) In comparison, xenografts given only a secondary treatment of estrogen and testosterone (control/estrogen), appeared to have areas of immature or undeveloped tissue (G) with a greater amount of PSA staining in the lumen compared to C/C-xenografts. (D) An initial and secondary exposure to estrogen (estrogen/estrogen) exhibited an extensive amount of glandular hyperplasia (*), (H) as well as a significant amount of PSA staining. Figure headings represent treatment conditions as initial/secondary exposure. (A-H) Gestational age of human fetal prostate before implantation is 20 weeks. (Hematoxylin and Eosin staining and counter-stain; scale bar = 50 μm).
Fig 3.
Immunohistochemical staining of the epithelial basal cell marker p63 in 7, 30, and 200-day control and estrogen-treated human prostate xenografts.
Basal cell hyperplasia is evident in both (A) control and (B) estrogen-treated 7-day xenografts. At 30 days post-implantation basal cell hyperplasia is reduced in (C) control xenografts but is still prominent in the (D) estrogen-treated xenografts. At 200-days post-implantation, (E) control/control xenografts appear phenotypically mature with p63 lining the basal cell layer of adult prostatic ducts. Similarly, (F) the prostatic basal cells appear mature in 200-day estrogen/estrogen-treated xenografts while the luminal cells appear hyperplastic. (A-F) Gestational age of human fetal prostate before implantation is 20 weeks. (Hematoxylin counterstain; scale bar = 50 μm).
Fig 4.
Immunohistochemical staining of the adult stromal muscle marker caldesmon in 7, 30, and 200-day control and estrogen-treated human prostate xenografts.
Caldesmon staining is minimally present in both 7-day (A) control and (B) estrogen-treated xenografts, as these both present with a phenotypically fetal stromal environment. At 30-days, (C) control xenografts stain strongly for caldesmon and display a more mature stromal environment and the presence of smooth muscle bundles. In comparison, (D) the 30-day estrogen-treated xenografts stain for caldesmon with an immature muscle structure compared to control. At 200-days, (E&F) both control and estrogen-treated xenografts display a significant amount of caldesmon staining indicative of normal adult stromal tissue. Gestational age of human fetal prostate before implantation is 20 weeks. (Hematoxylin counterstain; scale bar = 50 μm).
Fig 5.
Ki-67 proliferative index in both epithelial and stromal compartments of human prostate xenografts at 7, 30, 90, and 200 days post-implantation.
While an increasing percent of epithelial cells is proliferating over time, estrogen treatment does not affect prostate xenograft growth in the 7, 30 or 90-day xenografts in either the (A) epithelial or (B) stromal compartments. At 200 days post-implantation (C) there is a significant increase in epithelial proliferation in xenografts given an initial and secondary treatment of estrogen compared to control, (D) while there was no significant change in the stromal compartment. (C-D) The x-axis on the 200-day bar graphs are depicted as initial/secondary treatment in which C = control, and E = estrogen treatment. Ki-67 positive nuclei were measured as a percent of the total number of nuclei in each respective compartment. Line and bar graphs indicate the mean % Ki-67 positive cells ± SEM, ** indicates t-test significance relative to control of p<0.01. Legend: —●— Control, -■- Estrogen. Age of human fetal prostate prior to implantation was 15–21.5 weeks gestation. Sample size (n) for each group is as follows: 7D control (3), 7D estrogen (2), 30D control (3), 30D estrogen (4), 90D control (4), 90D estrogen (5), 200D control/control (4), 200D estrogen/control (4), 200D control/estrogen (2), 200D and estrogen/estrogen (4).
Table 1.
Summary of PCR array results from human prostate xenografts at 200-days post-implantation.
Fig 6.
Overview schematic adapted from the KEGG prostate cancer pathway describing the progression of prostate cancer and the events that occur within the cell type coinciding with each step in the xenografts disease transformation.
PCR array results from 200-day human xenografts given both an initial and secondary treatment of estrogen demonstrate changes that coincide with the PI3K-Akt signaling pathway, depicting the stage of hyperplasia. This is a transitional stage from normal prostate epithelium to the formation of prostatic intraepithelial neoplasia (PIN) lesions. There is a reduction in the gene expression of the critical tumor suppressor, PTEN (−1.29) and NKX3.1 (−2.04, non-significant), and an increase in PKB/Akt that inhibits, via phosphorylation (+P), expression of both CASP9 (−6.54) and CDKN1A (−1.45; also referred to as p21). This results in the inhibition of apoptosis, concurrent with an increase in cell cycle progression. Genes circled in red are down-regulated, while those circled in green are up-regulated. Analysis was performed (n = 4, age of human fetal prostate prior to implantation was 16–21.5 weeks gestation) using a LIMMA statistical test, with the circled genes found to be significant (p<0.05).
Fig 7.
DNA methylation of compartmental epithelial and stromal LCM-microdissected human prostate xenografts.
The number of differentially hypomethylated and hypermethylated CpG sites (89 total CpG sites) is shown for the epithelial (white bars) and stromal (black bars) compartments. The number above each bar graph represents the number of genes that correspond to the number of sites found in each group. Stromal hypomethylated (57) CpG sites predominated over epithelial (2) CpG sites. There was no significant epithelial CpG hypermethylation sites (0) found compared to the stromal region (30). Gestational age of human fetal prostate prior to implantation was 16–21.5 weeks old. Sample size (n) for each group is as follows: control/control (3), and estrogen/estrogen (4), with n representing different individual human fetal prostate samples prior to implantation. Significant difference (q<0.05) was seen in 89 CpG sites between control (C/C) and estradiol benzoate-treated (E/E) samples.
Table 2.
Stromal-compartment methylation associated gene-changes.
Table 3.
Functional gene clusters of 200-day LCM-separated epithelial and stromal human prostate xenograft tissue DNA methylation.