Primary Xenografts of Human Prostate Tissue as a Model to Study Angiogenesis Induced by Reactive Stroma

Characterization of the mechanism(s) of androgen-driven human angiogenesis could have significant implications for modeling new forms of anti-angiogenic therapies for CaP and for developing targeted adjuvant therapies to improve efficacy of androgen-deprivation therapy. However, models of angiogenesis by human endothelial cells localized within an intact human prostate tissue architecture are until now extremely limited. This report characterizes the burst of angiogenesis by endogenous human blood vessels in primary xenografts of fresh surgical specimens of benign prostate or prostate cancer (CaP) tissue that occurs between Days 6–14 after transplantation into SCID mice pre-implanted with testosterone pellets. The wave of human angiogenesis was preceded by androgen-mediated up-regulation of VEGF-A expression in the stromal compartment. The neo-vessel network anastomosed to the host mouse vascular system between Days 6–10 post-transplantation, the angiogenic response ceased by Day 15, and by Day 30 the vasculature had matured and stabilized, as indicated by a lack of leakage of serum components into the interstitial tissue space and by association of nascent endothelial cells with mural cells/pericytes. The angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, Vimentin, Tenascin, Calponin, Desmin and Masson's trichrome, but the reactive stroma phenotype appeared to be largely independent of androgen availability. Transplantation-induced angiogenesis by endogenous human endothelial cells present in primary xenografts of benign and malignant human prostate tissue was preceded by induction of androgen-driven expression of VEGF by the prostate stroma, and was concurrent with and the appearance of a reactive stroma phenotype. Androgen-modulated expression of VEGF-A appeared to be a causal regulator of angiogenesis, and possibly of stromal activation, in human prostate xenografts

Induction of a reactive stroma in primary xenografts of human prostate tissue.

<p>Temporal changes of protein levels of VEGF, αSMA, Calponin and Vimentin were measured by IHC-staining, and of the presence of smooth muscle cells and collagen fibers was visualized by Masson's trichrome staining, over the 14 days following xenograft transplantation. α-SMA and Calponin are early and late markers of smooth muscle, respectively. Masson's trichrome identifies smooth muscle cells (purple) and collagen fibers (green).</p

Primary xenografts of human prostate tissue undergo an explosive increase in human vessels over the initial 14 days after tissue transplantation.

<p>(<b>a–b</b>). Endothelial cells in primary xenografts of prostate tissue identified by human CD31 immuno-labeling and visualized by confocal laser scanning microscopy in initial tissue specimens (IT), and in primary xenografts of prostate tissue on Day 14 after tissue transplantation (d14). (<b>c</b>). Dual-immuno-histochemical staining with species-specific anti-human and anti-mouse CD31 antibodies in primary xenografts of prostate tissue on Day 14 after implantation. Human CD31 expression was visualized using FITC-labeled goat-anti-mouse IgG. Mouse CD31 expression was visualized using Cy3-labeled sheep-anti-rat IgG.</p

The angiogenic burst in primary xenografts of prostate tissue is preceded by androgen-modulated up-regulation of VEGF-A gene expression in the stromal compartment.

<p>(<b>a</b>). PCR analysis of expression of transcripts for pro-angiogenic factors in initial prostate tissue specimens before transplantation, and in corresponding primary xenografts after transplantation. Total RNA was extracted from initial prostate tissue (IT), and from prostate xenografts on different days after transplantation (d1–d14). GADPH was used as an internal control. (<b>b</b>). Immuno-histochemical identification of human VEGF protein in primary xenografts of prostate tissue over the 14 days after transplantation (d1–d14) in host mice pre-implanted with (+T), or not pre-implanted with (−T), sustained-release testosterone pellets. Bars = 50 µm.</p

Dependence on androgen stimulation of proliferative activity of human endothelial cells in primary xenografts of human prostate tissue.

<p>(<b>a</b>). Co-localization of huCD31 (red) and Ki-67 (brown) protein demonstrated the increased presence of vessels with proliferatively active endothelial cells over the 14 days after tissue transplantation (d2–d14). (<b>b</b>). Quantification of the complete image set is presented in (<b>a</b>). Values were expressed as a percentage of total vessels that contained at least one Ki-67-positive endothelial cells. Bars = 10 µm. (<b>c</b>). Immuno-histochemical identification of human blood vessels in initial tissue (IT) specimens before transplantation, and in corresponding primary xenografts on Day 14 after tissue transplantation. The host mice were pre-implanted with, or not implanted with, sustained-release testosterone pellets. (<b>d</b>). Quantification of MVD in prostate xenografts over the 14 days after tissue transplantation into animals pre-implanted with (open circles), or not implanted with (closed circles), sustained-release testosterone pellets.</p

Primary xenografts of human prostate tissue maintain the <i>in vivo</i> tissue architecture and expression of key prostatic markers.

<p>Immuno-histochemical identification of protein expression of androgen receptor (AR), prostate-specific antigen (PSA) and pan-cytokeratin (Cyt) visualized by peroxidase staining demonstrated the level of expression remained constant over the fourteen days post-transplantation (1–14).</p

Determination of hypoxic areas, and of expression of HIF-1α, HIF-2α and GLUT1 in primary xenografts of human prostate.

<p>Animals were administered Hypoxyprobe-1 (HyPo-P, NPI Inc.) via <i>i.p</i> injection (60 mg/100 g body weight) on select days after tissue transplantation. One hour after injection, the prostate xenografts were harvested and hypoxic areas visualized using a monoclonal antibody specific for Hypoxyprobe-1. Immuno-histochemical identification of changes in human HIF-1α, HIF-2α and GLUT1 protein levels in primary xenografts of human prostate tissue over the 4 days after tissue transplantation (1–4). Hypoxic areas, and human HIF-1α, HIF-2α, and GLUT1 protein, were visualized using DAB and hydrogen peroxide.</p