Generation and characterization of FGF-8b transgenic mice. (A) The transgene construct. (B) Transgene expression was localized to VP of TG mice by in situ hybridization with FGF-8b-specific riboprobes (a, b) and by FGF-8b immunostaining (c, d). Expression of FGF-8b was absent or low in VP samples of WT mice. (C) Representative pictures of prostate blocks from a 13.5-month-old WT and a 14-month-old FGF-8b TG mouse showing the abnormal, cystic appearance of TG mouse prostate. Bl indicates bladder; LP, lateral prostate; SV, seminal vesicle.

Aggregates of inflammatory cells in TG prostates are associated with epithelial changes. (A) Detection of inflammatory cell types by IHC staining. B-cell-specific CD45R IHC in a TG VP presenting pronounced inflammation in the prostatic stroma (a). T-cell-specific CD3 IHC in the same region as in subpanel a (b). Macrophage-specific CD68 IHC staining in WT mouse VP (c) and in TG mouse VP (d). Magnification, x200. Scale bars, 100 µm. (B) Degree of inflammation in TG mouse groups (9–20 months old) defined by the histologic status of prostatic epithelium (groups as in Figure 2C). Evaluation and scoring (scores 0–3); the degree of inflammation was performed as described in the Materials and Methods section. The bars show the means ± SEs. Statistical significance was assessed with the Mann-Whitney U tests, and Bonferroni corrections were applied. **P < .01, *P < .05.

Up-regulation of CTGF and SPP1 expression in TG mouse prostates. (A) qRT-PCR analysis of relative expression levels of CTGF and SPP1 mRNA in FGF-8b-TG and WT mouse VPs in three age groups. The lowest expression value among WT mouse samples was used as a reference and set to 1. Statistical significance was assessed with the t test (CTGF) or the Mann-Whitney U tests (SPP1). **P < .01, *P < .05. (B) Immunohistochemical staining of SPP1 in WT mouse VP (a), in TG mouse VP with stromal proliferation (b), in TG mouse VP with mPIN (c), and in TG mouse prostate with carcinosarcoma (d). Magnification, x200. Inserts, x400. Scale bars, 100 µm.

A proposed model for FGF-8 action in prostate. (A) Increased levels of FGF-8b are secreted by the epithelial cells. It affects both the epitheliumand the stroma (autocrine and paracrine effects, pink arrows). (B) The affected stroma presents features of reactive stroma, such as increased amount of fibroblasts, inflammation, and angiogenesis. Altered stroma secretes connective tissue growth factor (CTGF), SPP1, and other (unidentified) factors that can affect the epithelium. The disturbed cross talk (indicated by green arrows) between stromal and epithelial compartment together with increased amounts of FGF-8b induce proliferation and dedifferentiation of the epithelium, leading to the development of preneoplastic mPIN lesions. (C) Prolonged FGF-8b exposure and disturbed epithelial-to-stromal cross talk induces the epithelium and the stroma to develop atypical and malignant changes. AR-positive nuclei are presented by dark gray; AR-negative nuclei, light gray color.

FGF-8b transgenic mice develop advancing histologic changes in prostate epithelium and stroma. (A) Representative pictures of histologic changes in hematoxylin and eosin-stained sections (a, WT; b-i, TG). Normal VP histology, WT, 16-month-old (a); stromal hypercellularity and increased vasculature in VP, TG, 4-month-old (b); hypercellularity of VP epithelium, TG, 6-month-old (c); mucinous metaplasia in VP, TG, 13-month-old (d); advanced mPIN lesion in VP, TG, 16-month-old (e); adenocarcinoma in VP, TG, 13-month-old (f); atypical stromal hypercellularity showing phyllodes growth pattern and inflammation, TG, 14.5-month-old (g); sarcoma in VP, TG, 13-month-old (h); and carcinosarcoma in VP, TG, 18.5-month-old (i). Images were taken at x100 to x200 magnification, and inserts were taken at x400 magnification. Scale bars, 100 µm. (B) Frequencies (%) of histologic changes in the oldest age group (12-21 months old) of TG and WT mice. Carcinosarcomas were included in the sarcoma group. (C) Immunohistochemical staining of FGF-8b in neoplastic lesions of FGF-8b TG mouse prostates. VP glands with normal-appearing histology located next to a big carcinosarcoma lesion in a TG prostate (a), mPIN lesions in the VP of a TG mouse (b), adenocarcinoma in the VP of a TG mouse (c), and carcinosarcoma in the same prostate as the normal-appearing glands in subpanel a (d). Images were taken at x200 magnification. Scale bars, 100 µm. (D) Association between epithelial and stromal changes in TG prostates. Mean scores of stromal phenotypes in TG mouse groups (9–20 months old) defined by the histologic status of prostatic epithelium. Numeric values (1–4) for stromal phenotype were given as indicated in the Materials and Methods section. The bars show the means ± SEs. Statistical significance was assessed with the Mann-Whitney U tests, and Bonferroni corrections were applied. ***P < .001, *P < .05.

Altered staining for cytokeratins, laminin, and smooth muscle actin in FGF-8b TG mouse prostates. (A) Immunohistochemical staining for cytokeratins (CKs), laminin, smooth muscle α-actin (SMA), and Masson trichrome staining. VP samples from WT mice (left panel), TG mice with mPIN and stromal hypercellularity (middle panel), and a TG mouse with carcinosarcoma (right panel). Magnification, x200. Scale bar, 100 µm. (B) Masson trichrome staining of VP sections of prepubertal (28-day-old) WT and TG mice. A thicker layer of blue green-stained stroma (arrow) was detected around the acini of TG mice administrated 14 days with testosterone-propionate (Te) than around those of the WT mice with the corresponding treatment (lower panel). Control mice (upper panel) were treated in the similar manner without testosterone. Magnification, x200. Inserts, x400. Scale bars, 100 µm.

FGF-8b TG mouse prostates present altered AR staining. (A) IHC staining of AR in VP of a WTmouse (a), in TG prostate with stromal hypercellularity and inflammation (b), in an mPIN focus of a TG mouse (c), in adenocarcinoma (d), in sarcoma (e), and in carcinosarcoma (f). Magnification: all, x100; d, x200. Inserts, x200. Scale bars, 100 µm. (B) Staining grades in AR IHC in VP epithelium (upper graph) and stroma (lower graph) of WT and TG mice with normal and altered histology. Grades were evaluated as proportion of AR-positive cells in the VP areas with respective changes as indicated in the Materials and Methods section.