A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

Abstract

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.

Figure 1

Progression of lesions in CR2-T-Ag mouse prostates recapitulates the proposed histologic progression of human prostate cancer. (A) Hematoxylin- and eosin-stained section from a nontransgenic 8-week-old mouse, demonstrating the appearance of normal prostatic epithelium. (B) Prostatic intraepithelial neoplasia (PIN) in an 8-week-old CR2-T-Ag mouse. Borders between normal-appearing epithelium and clusters of dysplastic cells contained in foci of PIN are indicated by arrows. (C) Transmission electron microscopy of a border, such as that shown in B. In contrast to normal nuclei (e.g., closed arrow), dysplastic nuclei (e.g., open arrow) have irregular size and shape, clumps of condensed chromatin, and multiple nucleoli. (D) By 12 weeks of age, invasion through the basement membrane into the surrounding stroma has begun (e.g., arrow). (E) At 16 weeks, stromal invasion is extensive, with glandular structures being replaced by tumor. (F) A 24-week-old transgenic mouse prostate consisting of sheets of neoplastic cells. High rates of mitosis (solid arrow) and apoptosis (open arrow) are evident. Spindle-shaped cells (e.g., solid arrowhead) are consistent with neuroendocrine differentiation. (G) Liver from a 24-week-old animal with evidence of metastases (e.g., arrows). Inset is a high-power view of the boxed region showing hematogenous spread of the cancer. (H) At 24 weeks, the femoral marrow space is filled with tumor cells. The tibia (located on the left) is included to show the appearance of unaffected marrow. [Bars = 50 μm (A, B, and E); 10 μm (C); 20 μm (D and F); 500 μm (G and H).]

Figure 2

Morphometric analysis of the number of foci of PIN and invasion in the prostates of 7- to 16-week-old CR2-T-Ag mice. Mean values ± SE are shown.

Figure 3

Foci of PIN are composed of SV40 T-Ag-expressing neuroendocrine cells. (A) Section from an 8-week-old CR2-T-Ag mouse prostate stained with rabbit anti-SV40 T-Ag, horseradish peroxidase-conjugated donkey anti-rabbit IgG, and diaminobenzidine, then counterstained with hematoxylin and eosin. SV40 T-Ag appears brown. The arrow points to a cluster of SV40 T-Ag-positive cells, whose morphology is distinct from their normal, SV40 T-Ag-negative epithelial neighbors (e.g., arrowheads). (B) Section from an 8-week-old CR2-T-Ag mouse showing a focus of PIN containing amplified neuroendocrine cells. The section was stained with rabbit anti-synaptophysin and Cy3-donkey anti-rabbit IgG. (C) Section from the prostate of an 8-week-old CR2-T-Ag transgenic mouse that received an injection of BrdUrd 90 min before sacrifice. The section was incubated with goat anti-BrdUrd (detected with Cy3-donkey anti-goat IgG) and rabbit anti-SV40 T-Ag (detected with FITC-donkey anti-rabbit IgG). Proliferating epithelial cells (yellow nuclei) are restricted to the cluster of SV40 T-Ag-positive cells. (Bars = 20 μm.)

Figure 4

Evidence that prostate cancer is initiated in a subset of neuroendocrine cells where nucleotides −6400 to +34 of the mouse cryptdin gene are active. (A) hGH-positive cell with prominent cytoplastic processes, in the prostate of a 12-week-old CR2-hGH transgenic mouse. The section was stained with sheep anti-hGH and Cy3-donkey anti-sheep IgG. (B–F) Multilabel confocal photomicrographs, taken in the same 1-μm-thick focal plane, of a prostate section from a 12-week-old CR2-hGH animal. The section was stained with sheep anti-hGH (detected with Cy3-donkey anti-sheep IgG), mouse anti-high molecular weight cytokeratin (detected with horseradish peroxidase-donkey anti-mouse IgG and FITC-tyramide), and rabbit antisynaptophysin (detected with indodicarbocyanine-donkey anti-rabbit IgG). (B) A single wedge-shaped hGH-positive cell is detected as red. (C) When cytokeratin is visualized (green), this cell is not evident. (D) Combined images from A and B show that the hGH-positive cell does not express cytokeratin, indicating that it does not belong to the basal cell lineage. (E) Indodicarbocyanine is assigned a green color to show synaptophysin immunoreactivity. (F) Combining the images shown in B and E reveals that the hGH-positive cell coexpresses synaptophysin, i.e., it is a member of the neuroendocrine lineage. (G) Conventional light microscopy of a 5-μm-thick section prepared from a 12-week-old bitransgenic mouse containing both CR2-hGH and CR2-T-Ag. The section was stained with sheep anti-hGH (detected with FITC-donkey anti-sheep IgG) and rabbit anti-SV40 T-Ag (detected with Cy3-donkey anti-rabbit IgG). Cells in a focus of PIN coexpress SV40 T-Ag (red-orange nuclei) and hGH (green cytoplasm). (Bars = 20 μm.)

Figure 5

Castration does not affect the growth of prostate cancers in CR2-T-Ag mice. (A) Section from a 10-week-old CR2-T-Ag prostate was stained with rabbit anti-androgen receptor, horseradish peroxidase-donkey anti-rabbit IgG, and diaminobenzidine, and then counterstained with hematoxylin and eosin. Immunoreactive androgen receptor (brown) is located in the normal glandular epithelium (e.g., open arrows) and stroma, but not in cells contained within a focus of PIN (e.g., solid arrow). (B) Hematoxylin- and eosin-stained section of prostate from a noncastrated 16-week-old CR2-T-Ag mouse. Arrows point to normal glandular epithelium. (C) Section from a CR2-T-Ag mouse castrated at 4 weeks and then sacrificed at 16 weeks of age. Comparison with B reveals that while the nonneoplastic epithelium has undergone atrophy (arrow), the histopathologic features of neoplastic foci are unaffected by castration. [Bars = 20 μm (A) and 500 μm (B and C).]