Establishment and genomic characterization of mouse xenografts of human primary prostate tumors

Abstract

Serum prostate-specific antigen screening has led to earlier detection and surgical treatment of prostate cancer, favoring an increasing incidence-to-mortality ratio. However, about one third of tumors that are diagnosed when still confined to the prostate can relapse within 10 years from the first treatment. The challenge is therefore to identify prognostic markers of aggressive versus indolent tumors. Although several preclinical models of advanced prostate tumors are available, a model that recapitulates the genetic and growth behavior of primary tumors is still lacking. Here, we report a complete histopathological and genomic characterization of xenografts derived from primary localized low- and high-grade human prostate tumors that were implanted under the renal capsule of immunodeficient mice. We obtained a tumor take of 56% and show that these xenografts maintained the histological as well as most genomic features of the parental tumors. Serum prostate-specific antigen levels were measurable only in tumor xenograft-bearing mice, but not in those implanted with either normal prostate tissue or in tumors that likely regressed. Finally, we show that a high proliferation rate, but not the pathological stage or the Gleason grade of the original tumor, was a fundamental prerequisite for tumor take in mice. This mouse xenograft model represents a useful preclinical model of primary prostate tumors for their biological characterization, biomarker discovery, and drug testing.

Figure 1

Generation of mouse xenografts from primary localized human prostate tumors. Subrenal capsule xenografts of prostate tumor tissues (A, lower middle panel) often showed a robust vascularization that was not present in those derived from normal samples (A, upper middle panel). Hematoxylin and eosin staining of 5-micron FFPE sections of normal (A, upper right panel, ×200 magnification) and tumor (A, lower right panel, ×200 magnification) xenografts revealed that both maintain the histopathological features of the parental tissues (A, upper left and lower left panels, respectively). Tumor xenografts showed immunohistochemical markers of human PCa (PSA in B, upper panel, AMACR in B, lower panel, ×200 magnification; androgen receptor in C, lower panel, ×200 magnification, inset ×400). An example of Gleason 3 xenograft is shown. Scale bar = 10 mm. The xenografts did not show infiltration of mouse fibroblasts in the stroma, which was confirmed of human origin by both immunohistochemistry against the human androgen receptor (C, lower panel [asterisk represents human stromal cells]; C, upper panel, shows an orthotopic xenograft of human normal prostate as control [asterisk represents mouse prostate gland, double asterisk represents human prostate tissue]) and Hoechst staining (D; magnifications ×100 in the upper left panel, ×400 in the upper right and bottom panels, ×600 in the insets).

Figure 2

A: Serum total PSA was significantly increased (***P < 0.001) in tumor xenograft-bearing mice compared with those implanted with human normal tissues. The box plots show PSA values in the two groups of tumor and normal xenografts. Lower and higher whiskers indicate 10th and 90th percentile, respectively; lower and higher edges of box indicate 25th and 75th percentile, respectively; the inner line in the box indicates 50th percentile. The PSA levels increased over time as the tumor proliferated (B, graph and inset) and dropped to undetectable levels when the tumor regressed (C, graph and inset; asterisk represents calcifications).

Figure 3

Proliferation rate (percentage of Ki-67–positive nuclei) and apoptosis (percentage of Apoptag-positive nuclei) in parental prostate tumors and mouse xenografts. Tumors with higher proliferation rate (Ki-67 > 5%; A, upper middle panel, and D; **P < 0.01) grew preferentially in mice compared with those with low proliferation (Ki-67 < 5%; A, upper left panel). Representative examples of tumors with low and high apoptotic rates are reported in panel A, lower middle and lower left, respectively. Apoptotic rate, pathological stage, and GS did not affect the tumor take in mice (P > 0.05; B, C, and E). A, upper right and lower right, shows immunostaining of the xenografts derived from the tumors reported in A, upper middle and lower middle, which maintains the same proliferation rate and apoptosis of the parental tissues (×200 magnification).

Figure 4

Genome-wide analysis (aCGH) was performed after laser capture microdissection (A) and whole genome amplification (WGA; B). Genome view of a representative pair human tumor/xenograft shows that the most significant genomic aberrations (losses and gains as reported on the left and on the right of each bar, respectively) are consistent between the two profiles (C).

Figure 5

Penetrance plots of the most frequent genomic alterations occurring in the human parental tumors and in the xenografts. Chromosomes 6, 8, 13, 16, 17, and 21 are shown (A). PTEN loss is shown in a pair of human tumor and xenograft (B), where it is associated with AKT pathway activation (C). Phospho-AKT and phospho-S6 staining is heterogeneous in the parental tumor (C, upper left and right, respectively), whereas the xenograft shows a more intense and diffuse positivity for both proteins (C, middle left and right, respectively; ×400 magnification). Conversely, normal prostate epithelium and PTEN-positive tumors in three-month xenografts maintain low or absent staining for phospho-S6 (C, lower left, asterisk represents normal glands and double asterisk represents tumor, and C, lower right, respectively).

Figure 6

FISH break-apart assay showed abnormalities of the 21q22.2-3 locus in 77% of the paired human tumors and xenografts. PCa82 (PT indicates parental tumor; X, xenograft) and PCa44 are examples of TMPRSS2-ERG gene fusion by interstitial deletion and translocation, respectively. PCa41, instead, shows a rearrangement of ERG, which could have a 5′ partner different from TMPRSS2. The corresponding xenograft (PCa41 X) presents the same alteration in association with polisomy for the TMPRSS2 and ERG regions (×1000 magnification, oil immersion). Schematic representations of the rearrangements are reported on the right.