Combination therapy.

<p>Mouse pancreatic tumor cells (panc2) were implanted subcutaneously and treated with two doses of gemcitabine, alone or in combination with halofuginone. A– Tumor volume during 30 days. B– Tumor weight at the end of the experiment. C– Effects of halofuginone and gemcitabine on the number of host cells in the tumor. D– Effects of halofuginone and gemcitabine on apoptosis of all cells within the tumor, or of the cells that expressed αSMA. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Origin of the tumor collagen.

<p>A & B– Biopsies of subcutaneous panc2 tumors 21 days post-transplantation were hybridized with collagen type I probe (black) and were immunostained with GFP antibodies (green). Nuclei were stained with DAPI (blue). Arrows point to: A– cell expressing GFP and mRNA for collagen α1(I); B– cell expressing mRNA for collagen α1 but not GFP; C– Subcutaneous tumors were established by implanting 10⁶ human MiaPaca2 into male C57/GFP mice. Biopsies were immunostaining with human (green) and mouse (red) collagen antibodies. Collagen from both human and mouse origin is observed within the tumor.</p

Effect of halofuginone on host cells migrating into the tumor.

<p>Biopsies of subcutaneous panc2 tumors 21 days post-transplantation, with and without halofuginone treatment three times weekly at 15 µg/mouse, were: A– stained with GFP antibodies or double-stained with GFP plus SM22α, GFP plus Cygb/STAP, or macrophages plus mast cell antibodies; Halofuginone inhibited the number of total stroma cell within the tumor and reduced the number of macrophages and myofibroblasts expressing SM22α, Cygb/STAP but not the number of mast cells. B– immunostained with P4Hβ or SM22α antibodies or double-stained with both antibodies; C– immunostained with P4Hβ or αSMA antibodies or double-stained with both antibodies. Halofuginone inhibited the number of stroma cells expressing both the collagen cross-linking enzyme and smooth muscle genes. At least 20 photographs of tissue sections from different mice were taken for each analysis. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Collagen and P4Hβ-expressing cells in the tumor.

<p>A– Biopsies from panc2 subcutaneous tumors were stained with Sirius red for collagen (red) and immune-stained with P4Hβantibodies (red, indicated by arrows). Halofuginone treatment three times weekly at 15 µg/mouse resulted in major reductions in collagen content and in the total number of P4Hβ-expressing cells. B– Biopsies were double-immunostained with GFP-antibodies (green) and P4Hβ antibodies (red), and nuclei were stained with DAPI (blue). A stroma cell not expressing P4Hβ (white arrow) is adjacent to a tumor cell expressing P4Hβ (yellow arrow). C– Stroma cell expressing P4Hβ (white arrow). D– Halofuginone treatment reduced the total number of stroma cells expressing P4Hβ. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Mouse pancreatic tumor cells (panc2) were implanted subcutaneously and treated with either halofuginone three times weekly at 15 µg/mouse or gemcitabine twice weekly at 20 mg/kg, starting 7 days after tumor cell implantation.

<p>At the end of the experiment, i.e., 30 days after cell implantation: A– tumors were taken for immunohistochemistry with pSmad3 antibodies; B– at least 20 photographs from five different mice from each group were taken for each analysis. The results are expressed as arbitrary pSmad3/Smad units ± SE. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Pancreatic fibrosis and tumor development.

<p>C57/GFP mice were treated with halofuginone or cerulein, alone or in combination, and untreated mice served as controls. After 8 weeks, pancreatic fibrosis was validated by Sirius red (SR) staining and quantified by image analysis. At that time, the cerulein and halofuginone treatments were terminated, and mCherry-labeled panc2 cells were injected into the spleen; after a further 2 weeks, mouse pancreas was photograph with the Maestro imaging system. Pancreas sections were stained with mCherry, annexin II, PCNA and anti-GFP antibodies, and positive signals were quantified. In the fibrotic tissue more stroma (GFP-positive) and tumors cells (annexin II–positive) were observed. Halofuginone treatment that lessens tissue fibrosis reduced both the tumor and stroma cell number. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Inhibition of tumor development by halofuginone.

<p>Mouse (mCherry-labeled Panc2) and human (colo357) pancreatic tumor cells were transplanted subcutaneously into C57/GFP and nude mice, respectively. Halofuginone (15 µg/mouse) was injected ip three times weekly, starting 3 days post-transplantation. During tumor progression tumor size was monitored by caliper. After 21 days (Panc2) and 36 days (colo 357) the tumors were removed, weighted and photographed. Results are means ± SE and columns with different letters within each experiment differ significantly (<i>P</i><0.05).</p

Effect of halofuginone on vaginal infection.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl or 10 µg/100 µl of halofuginone solution or diluent of halofuginone and, in selected experiments, were treated intravaginally with 10 pg of mouse rIL-17. (A) Evaluation of IL-17 concentration by ELISA in supernatants of vaginal fluids obtained at different days after vaginal <i>Candida</i> infection and halofuginone treatment. Results are expressed as mean±SD (n = 9 mice, 3 mice for each of three separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice). At day 4, 14 and 25 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC) and treated with halofuginone or diluent. Images are representative of 5 out of 10 mice in two different experiments. (C) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes of mice (n = 10) treated with halofuginone or diluent. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice).</p

IL-17 and IL-23 concentration in murine vaginal washes of mice infected with <i>Candida albicans</i>.

<p>Evaluation of IL-17 (A–C) and IL-23 (D) concentration by ELISA test on supernatants of vaginal fluids obtained at different times after vaginal infection with different doses of <i>Candida albicans</i> gLUC59 (A–B) or CA1399 (D). Results are expressed as mean±SD (n = 12 mice, 4 mice for each of three separate experiments). * <i>p</i><0.05, (infected mice vs non infected mice).</p

Effect of halofuginone treatment on vaginal β defensin production.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl of halofuginone solution or diluent of halofuginone and were treated intravaginally with 10 pg of mouse rIL-17. At day 4, 8 and 14 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane. (A) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. After 4, 8, 14, 21, 28 days from challenge, the vaginal lumen was washed with 150 µl of saline and vaginal cells were recovered for β-defensin analysis. (B) Mean of fluorescence MIF of β-defensin 1, β-defensin 2 and β-defensin 3 cells evaluated by FACS analysis. The vaginal cells recovered by vaginal washes were stained with rabbit anti-mouse BD1, goat anti-mouse BD2 or goat anti-mouse BD3 and goat anti-rabbit TRIC conjugate or rabbit anti-goat PE conjugate respectively. (C) Percentage of epithelial cells producing d β-defensin 2. Data are the mean±SD (n = 8 mice, 4 mice for each of two separate experiments). Cells of vaginal washes were stained with FITC anti mouse pan-cytokeratin and goat anti-mouse BD2 and rabbit anti-goat PE conjugate. Data are representative of one out of two independent experiments (total 8 mice). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (infected halofuginone+IL-17 treated mice vs infected halofuginone treated mice).</p