Figure 1.
Primary xenografts of human prostate tissue maintain the in vivo tissue architecture and expression of key prostatic markers.
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).
Figure 2.
Primary xenografts of human prostate tissue undergo an explosive increase in human vessels over the initial 14 days after tissue transplantation.
(a–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). (c). 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.
Figure 3.
Time course of angiogenic activity in primary xenografts of human prostate.
(a–g) Immuno-histochemical identification of blood vessels in initial tissue specimens before transplantation (IT), and in corresponding primary xenografts of prostate tissue during the fourteen days after transplantation (d1–d14). (h). Quantification of MVD in primary xenografts of prostate tissue, and RCC tissue, over the 14 days after tissue transplantation. MVD was quantitated by immuno-staining with anti-human CD31. (i). Quantification of MVD in prostate and renal tissue xenografts represented by fold-increase in human-CD31 positive cells in primary xenografts of prostate tissue, and RCC, on Day 14 (diagonal lines bars) and Day 30 (horizontal lines bars) after tissue transplantation, compared to the IT (solid bars). Bars = 50 µm.
Figure 4.
Dependence on androgen stimulation of proliferative activity of human endothelial cells in primary xenografts of human prostate tissue.
(a). 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). Quantification of the complete image set is presented in (a). Values were expressed as a percentage of total vessels that contained at least one Ki-67-positive endothelial cells. Bars = 10 µm. (c). 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. (d). 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.
Figure 5.
Vascular integrity and maturation of newly formed vessels in primary xenografts of human prostate tissue.
(a–f). Immediately before xenograft harvest, the neo-vasculature in human prostate xenografts was labeled in vivo with biotin-conjugated lectin injected i.v. into the host mice. In vivo labeling studies demonstrated anastomosis of the prostate vasculature to the host vasculature by Day 7 after transplantation, and maturation of the human neo-vasculature by Day 30 after transplantation. (g–i). Confocal laser scanning microscopic visualization of dual-immuno-labeling of alpha-smooth muscle actin (αSMA, green) and huCD31 (red). Endothelial cells were associated with αSMA-positive peri-endothelial cells (indicated by arrowheads) on Day 30 post-transplantation.
Figure 6.
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.
(a). 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). 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.
Figure 7.
Determination of hypoxic areas, and of expression of HIF-1α, HIF-2α and GLUT1 in primary xenografts of human prostate.
Animals were administered Hypoxyprobe-1 (HyPo-P, NPI Inc.) via i.p 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.
Figure 8.
Induction of a reactive stroma in primary xenografts of human prostate tissue.
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).
Figure 9.
Schematic representation of timeframe of the transplantation-induced biological processes in primary xenografts of human benign and malignant prostate tissue.
(a) Temporal changes of VEGF-A expression (brown line), angiogenesis (blue line), microvessel density (red line) and expression of a reactive stroma phenotype after xenograft transplantation. (b) The data from graph (a) suggests two hypothetical models of the cause-effect relationship of VEGF-A expression with angiogenesis and reactive stroma generation in primary xenografts of human prostate tissue.