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

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

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

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

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

Effect of NADPH oxidase in local and systemic accumulation of MDSCs in tumor-bearing mice.

<p>A) Representative quantification of MDSCs. Splenocytes from WT and p47<i>^phox−/−</i> mice at day 42 and 90 after MOSEC administration were analyzed for MDSC accumulation. Gating on myeloid (CD11b⁺) cells, the proportion of monocytic MDSCs (R1; Ly6C⁺Ly6G⁻) and granulocytic MDSCs (R2; Ly6G⁺Ly6C^Low) significantly increased at day 90 versus day 42. All gates were set based on isotypes. This approach was used to quantify MDSCs in PECs, lymph nodes, and spleens. B) Proportion of MDSCs in myeloid PECs on day 42 and 90. The proportion with granulocytic and monocytic MDSC markers was greater in advanced (day 90) versus early (day 42) stage tumor burden in both genotypes. C) In draining lymph nodes, there was a trend toward increased monocytic MDSC accumulation in p47<i>^phox−/−</i> versus WT mice at day 42 but not at day 90. There was no effect of NADPH oxidase on granulocytic MDSC accumulation at either time point. D) In spleens, there was an increased accumulation of MDSCs, particularly granulocytic MDSCs, in mice with advanced versus early disease, but no effect of mouse genotype. Data (± SEM) are from at least 3 mice per genotype per time point, and are representative of 3 separate experiments. Comparison between genotypes: p = NS.</p

Peritoneal and splenic granulocytic MDSCs from tumor-bearing mice suppress T cell proliferation independently of NADPH oxidase.

<p>Ly6G-enriched PECs and splenocytes from MOSEC-bearing WT and p47<i>^phox−/−</i> mice (day 90) were co-cultured with splenocytes from non-tumor-bearing WT mice (E∶T ratio: 1∶1). A) Ly6G-enriched PECs completely suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. PECs from 3 mice per genotype were evaluated. B) In Ly6G-enriched splenocytes, the majority of cells had a granulocytic morphology (arrows), and 86% of CD11b⁺ cells expressed granulocytic MDSC markers (Ly6G⁺Ly6C^low). C) Ly6G-enriched splenocytes from WT and p47<i>^phox−/−</i> mice modestly suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. N = 3 mice per genotype were used in this experiment, and results are representative of 3 experiments. Comparison between genotypes: p = NS.</p

Analysis of peritoneal exudate cells from MOSEC-bearing mice after enrichment for myeloid cells and granulocytic cells.

<p>Myeloid cells and granulocytic cells from PECs of MOSEC-bearing WT and p47<i>^phox−/−</i> mice were column-purified using anti-CD11b and anti-Ly6G, respectively. Analysis of post-enriched fractions showed concordance between cytology and flow cytometry. Representative analysis of PECs from p47^phox−/− mice collected on 90 after MOSEC administration is shown. A) The CD11b-negative fraction contained a preponderance of tumor cells (based on cytology), while myeloid cells were rare. B) The CD11b-enriched fraction contained a mixed myeloid cell population, with a preponderance of macrophages based on cytology and surface markers (CD11b⁺F4/80⁺). C) The Ly6G-enriched fraction contained a preponderance of granulocytic cells (arrows), with the majority of myeloid cells expressing granulocytic MDSC markers (CD11b⁺Ly6G⁺Ly6C^low).</p

The p47<i>^phox</i> component is required for NADPH oxidase activity in granulocytic MDSCs.

<p>PECs from WT and p47<i>^phox−/−</i> mice harvested at day 90 after MOSEC challenge were stimulated with PMA, and intracellular ROI production in CD11b⁺Ly6G⁺ cells was assessed by H₂DCFDA fluorescence. A) Gating on all non-aggregated cells (Gate 1), CD11b⁺Ly6G⁺ cells (Gate 2) were defined using respective isotype controls. B and C) Stimulated ROI production was detectable in WT (B), but not in NADPH oxidase-deficient (C), granulocytic MDSCs. White plot = PMA stimulation; shaded plot = no-stimulation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069631#s3" target="_blank">Results</a> are representative of two experiments.</p