The hepatic cellular environment is altered with PLD inhibitors.

<p>Liver sections were incubated with hematoxylin and eosin (<b>A-D</b>) or antibodies specific for Arg1 (<b>E-H</b>), F4/80 (<b>J-M</b>) or LY6G (<b>N-Q</b>) for immunofluorescence microscopy. Xenotransplanted MDA-MB-231 cells in the presence of vehicle, FIPI or VU0155072-2, respectively, and no xenotransplantation in the presence of vehicle. (<b>A-D</b>) Scale bar = 50 μm. (<b>E-Q</b>) Scale bar = 200 μm. (<b>I</b>) ImageJ quantification of the negative effect of PLD inhibitors on the total area of Arg1 positive foci in liver tissues in terms of μm² x 10³. Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Decreased Ly6G⁺ neutrophils in the presence of PLD inhibitors using IF microscopy of lung sections.

<p>Lung sections from MDA-MB-231 xenotransplanted mice were incubated with a TRITC-Ly6G antibody specific for TANs (<b>A-D</b>) or biological duplicates from independent experiments stained with Ly6G-R-PE (<b>A’-D’</b>) TANs (<b>A-D</b>) or biological duplicates from independent experiments stained with Ly6G-R-PE or with an isotype control-R-PE IgG antibody (<b>E-H</b>). (<b>A,A’,E</b>) Tissues from non-xenographed controls; (<b>B,B’,F</b>) from Xenotransplanted controls with Alzet pumps delivering vehicle; (<b>C,C’,G</b>) Xenotransplanted with Alzet pumps delivering vehicle FIPI or (<b>D,D’,H</b>) VU0155072-2. Scale bar = 200 μm. ImageJ quantification on the total area of positive foci for TANs using Ly6G staining (<b>I</b>) in lung tissues in terms of μm² x 10³. Also, total mean fluorescence of each field of view is presented in panel (<b>J</b>). In both those quantifications the background isotype controls have been subtracted. The use of TRITC- or R-PE labeling yielded virtually similar results. Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Inhibitory effects of FIPI or VU0155072-2 “NOPT” in mouse mammary tumors following xenotransplantation of human breast cancer cells.

<p>(<b>A-C</b>) SCID mice were xenotransplanted with human MDA-MB-231 breast cancer cells following implantation of Alzet pumps containing 2 different PLD-small molecule inhibitors of activity: 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) or N- [2- (4- oxo- 1- phenyl- 1, 3, 8- triazaspiro[4, 5]dec- 8- yl)ethyl]- 2- naphthalenecarboxamide (VU0155072-2) [also referred to as “NOPT” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166553#pone.0166553.ref031" target="_blank">31</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166553#pone.0166553.ref040" target="_blank">40</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166553#pone.0166553.ref042" target="_blank">42</a>]]. Approximately 45 days post xenotransplantation, primary and axillary breast tumors, lungs and livers were harvested from mice that received MDA-MB-231 cancer cells and fixed in formalin, which were then embedded in paraffin, sectioned and mounted onto glass microscope slides. (<b>D,E</b>) Negative effect of small molecule inhibitors on PLD2 in mouse primary mammary and metastatic axillary tumors. (<b>F,G</b>) Images were quantified in terms of total mean fluorescence per field of view for PLD2 using ImageJ software. (<b>H</b>) PLD enzyme activity and (<b>I</b>) PLD2 gene expression. (<b>J</b>) PLD2 protein expression and (<b>K</b>) densitometry of same Western blot. Results are mean relative PLD gene expression (-fold) ± SEM, or mean PLD activity ± SEM, in terms of percent of control (100% = 1,254 ± 117 dpm/mg protein). Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Presence of tumor-associated macrophage (TAM) F4/80 and tumor-associated neutrophil (TAN) Ly6G in xenotransplanted mice are suppressed by PLD inhibitors.

<p>(<b>A,C</b>) Immunofluorescence staining of mouse primary breast tumor sections using either a rat anti-mouse F4/80 AlexaFluor 488 IgG antibody specific for TAMs (<b>A</b>) or a rat anti-mouse Ly6G-R-PE IgG antibody specific for TANs (<b>C</b>). Each set of three panels represents results from Xenotransplanted MDA-MB-231 cells in the presence of vehicle, FIPI or VU0155072-2, respectively. Scale bar = 200 μm. (<b>B,D</b>) ImageJ quantification of the negative effect of PLD inhibitors on the total mean fluorescence of each field of view for either F4/80 (<b>B</b>) or Ly6G (<b>D</b>) in primary metastatic breast tumors. Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Differential effects of PLD small-molecule inhibitors on mouse leukocyte chemotaxis.

<p>(<b>A</b>) Gene expression by quantitative PCR of MCF-7 cells stably overexpressing (O/E) PLD2 (O/E-PLD2-MCF7) or MDA-MB-231 stably silenced with shRNA for PLD2 (shPLD2-MB-MB-231). Non-cancerous control cells, HMEC (human mammary epithelial cells). (<b>B-E</b>) Functional assay of non-tumor associated leukocytes. Peripheral blood PMNs (<b>B,C</b>) or peritoneal macrophages (<b>D,E</b>) from mice treated with either vehicle, FIPI or VU0155072-2 were used for chemotaxis assays (<b>B,D</b>) and PLD activity assays (<b>C,E</b>) in response to either 30 nM IL-8 (for PMNs) or 30 nM MCP-1 (for macrophages). For chemotaxis, the numbers of cells that migrated to the lower were calculated by counting four fields in duplicate. Triplicate results are mean ± SEM and are expressed in terms of either number of cell migrated per field or number of invading cells per insert. For PLD assay, the amount of TLC-isolated [³H]-PBut that co-migrated with PBut standards was measured by scintillation spectrometry. Results are mean ± SEM and are expressed in terms of percent of control. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Induction of the M2 macrophage protein Arg1 in xenotransplanted mouse mammary tumors.

<p>(<b>A-B</b>) Mouse primary breast tumors. (<b>C-D</b>) metastatic axillary tumors. (<b>E-G</b>) Lungs showing xenotransplant vs. no-xenotransplant controls. Sections showing Arg1 expression. Nuclei were stained with DAPI for reference. Scale bars = 200 μm. (<b>B,D,F</b>) Quantification of the negative effect of PLD inhibitors on Arg1 staining. Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples. <b>(H-I)</b> Presence of Arg1 in necrotic areas in xenotransplanted primary tumors. A boundary of non-necrotic versus necrotic cells within the primary tumors is delineated by the white, dashed line in the FIPI-treated sample <b>(I).</b></p

Lack of NOS2 induction in xenotransplanted mouse mammary tumors.

<p>(<b>A-D</b>) Primary breast tumor sections were incubated with rabbit anti-NOS2 IgG antibody followed by goat anti-rabbit IgG TRITC antibody and DAPI for reference. (<b>A</b>) Xenotransplanted MDA-MB-231 cells, vehicle. (<b>B</b> Xenotransplanted MDA-MB-231 cells, FIPI. (<b>C</b>) Xenotransplanted MDA-MB-231 cells, VU0155072-2. Scale bar = 100 μm. Sample sizes were n = 5 for each set of animals used and 6–8 fields were viewed for each section. (<b>D</b>) Quantification of NOS2 from IF images shown in (<b>A-C</b>). Statistical analyses for the increases (*, p<0.05, ANOVA) in xenotransplants and the decreases (#, p<0.05, ANOVA) in PLD inhibitors-treated samples.</p

Neutrophils adhered to the apical surface of polarized-MDCK cells augment AdV entry without decreasing the TER.

<p>A) MDCK-CAR^Ex8 cells were either mock- or DOX-induced. A neutrophil adhesion assay was performed with increasing numbers of neutrophils, as indicated. Immediately post-neutrophil adhesion, MDCK-CAR^Ex8 epithelia were infected with AdV5-β-gal for 1 h from the apical surface. 24 h later, viral entry was determined by qPCR analysis. Fold change in viral genomes, relative to AdV5-βGal entry in the absence of DOX and neutrophils, is shown. AdV entry from the apical surface was quantitated by qPCR analysis of polarized B) MDCK-CAR^Ex8 C) MDCK-mCherry and D) MDCK-CAR^Ex7 cells that were uninduced (circles), uninduced with adhered neutrophils (squares), or induced with DOX for 24 h prior to neutrophil adhesion (triangles). E) AdV5-β-gal entry from the apical surface of MDCK-CAR^Ex8 epithelia in the presence or absence of neutrophils and AdV5 FK or AdV3 FK. F) TER of mock- or Dox-induced MDCK-CAR^Ex8 epithelia was measured in the presence or absence of neutrophils. Error bars represent standard error of the mean (SEM) from three independent experiments. No significant difference was detected by one-way ANOVA. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.001 by one-way ANOVA and Bonferroni post hoc test.</p

Schematic of IL-8-mediated enhancement of AdV entry into polarized epithelia.

<p>1) Pathogenic microbes that invade the airway 2) cause both the resident macrophages and the epithelial cells to secrete IL-8. 3) IL-8 exposure causes intracellular signaling within the epithelial cells that augments <i>de novo</i> protein synthesis and apical localization of CAR^Ex8. 4) IL-8 simultaneously recruits neutrophils that transmigrate through the epithelium from the basal surface to the apical surface and 5) bind to CAR^Ex8 at the apical surface of the epithelium. 6) AdV entering the airway hijacks the host innate immune response and apical CAR^Ex8 to gain entry into the host cell.</p

Apical CAR^Ex8 protein expression increases apical adhesion of infiltrating neutrophils.

<p>Neutrophil transmigration assays were performed in the basal-to-apical direction in MDCK stable cells exposed to the neutrophil chemoattractive peptide fMLP on the apical surface. A) % neutrophil adhesion and B) % neutrophil transmigration were quantitated by measuring the fluorescence intensity of fluorescently-labeled neutrophils imaged by fluorescence microscopy. Error bars represent the SEM from three independent experiments; *p < 0.05 or **p < 0.01 by one-way ANOVA.</p