CCL2 and/or CCL12 blockade impact on tumor growth.

<p>(A) Pleural tumors from mice with LLC-induced MPE treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a> were stained with Hoechst 33258, anti-Caspase-3, and PCNA antibodies. Shown are summary of data for PCNA staining (left) and representative images from pleural tumors of an IgG2a and a combination-treated mouse (right). Scale bar = 100 µm, Å = 400. Arrows indicate rare caspase-3 positive cells. (B,C) Volume of flank tumors induced by subcutaneous injection of LLC and MC38 cells after treatment with IgG2a control or anti-CCL2/CCL12 combination therapy. Arrows indicate the day of antibody therapy start. (D) Volume of flank tumors induced by subcutaneous injection of LLC cells stably expressing random or anti-CCL2-specific shRNAs (sh166 and sh436). (E) Tumor volumes from flank tumor experiments at four weeks. (F) Photograph of mouse thoracic-abdominal border (dashed line) four hours after intraperitoneal Evans’ blue delivery. <i>Columns and squares</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; ns and ***, P>0.05 and P<0.001 compared with saline and/or IgG2a or random shRNA controls.</p

CCL2 and/or CCL12 neutralization inhibits MPE-precipitating vascular hyperpermeability.

<p>(A,B) Pleural fluid Evans’ blue levels from mice with LLC- and MC38-induced MPE treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a>. Mice received intravenous Evans’ blue before sacrifice, followed by quantification of the albumin tracer in MPE. *, ** and ***: P<0.05, P<0.01, and P<0.001 compared with saline and/or IgG2a. (C) Summary of data (<i>n</i> = 5) and photographs of representative skin test sites from C57BL/6 mice that received intradermal injections of IgG2a or cell-free MPE fluid admixed with IgG2a or anti-CCL2/12 antibodies, followed immediately by intravenous delivery of Evans’ blue. Mice were euthanized after one hour, followed by skin inversion and imaging. Shown is test spot area relative to the control test spot on the same mouse. ##: P<0.01 compared with IgG2a; * and **: P<0.05 and P<0.01 compared with MPE. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size.</p

CCL2 and/or CCL12 neutralization impacts new vessel assembly in pleural tumors.

<p>(A) Microvessel density of pleural tumors from mice with LLC- and MC38-induced MPE treated with IgG2a or anti-CCL2/12 combination as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071207#pone-0071207-g001" target="_blank">Figure 1A</a>. Shown is summary of data and representative images of factor-VIII-associated protein (F8A) immunoreactivity. Scale bar = 100 µm; Å = 400. Arrows indicate new vessels. (B) Representative chorioallantoic membranes and summary of data obtained from six membranes/group that were incubated with IgG2a or cell-free MPE fluid admixed with IgG2a or anti-CCL2/12 antibodies. Scale bar = 5 mm. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; ns: P>0.05; #: P<0.05 compared with IgG2a; *, **, and ***, P<0.05, P<0.01, and P<0.001 compared with MPE.</p

Impact of anti-CCL2 and/or anti-CCL12 monoclonal antibody treatment on syngeneic models of malignant pleural effusion (MPE).

<p>(A) Photographs of intrapleural injection technique (x indicates the injection site). (B) Transdiaphragmatic photograph of mouse pleural space before (left) and four hours after (right) intrapleural Evans’ blue delivery. The dashed lines indicate the pleural confines. (C) Graphical outline of <i>in vivo</i> experiments. C57BL/6 mice received intrapleural Lewis lung carcinoma (LLC) or MC38 colon adenocarcinoma cells (grey arrow) followed by intraperitoneal treatment with normal saline, IgG2a, anti-CCL2, anti-CCL12, or anti-CCL2 plus anti-CCL12 every three days (white arrows). Mice were terminated after 12 days (black arrow). Primary end-points were MPE incidence and volume. Secondary end-points were pleural tumor number and survival. (D) MPE incidence (left), volume (middle), and pleural tumor number (right) of mice with LLC-induced MPE after regular-dosed antibody treatment. (E) MPE incidence (left), volume (middle), and pleural tumor number (right) of mice with LLC-induced MPE after high-dose antibody treatment. (F) MPE incidence (left), volume (middle), and pleural tumor number (right) of mice with MC38-induced MPE treated with high-dose IgG2a control or anti-CCL2/12 combination regimen. (G) Transdiaphragmatic photographs of representative MC38-induced MPEs from an IgG2a and an anti-CCL2/12 combination-treated mouse. Dashed lines outline MPEs and <i>t</i> designates pleural tumors. (H,I) Fractional survival of mice with LLC- and MC38-induced MPE. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; <i>P</i>, probability by χ² test; ns, *, **, ***: P>0.05, P<0.05, P<0.01, and P<0.001 compared with saline and/or IgG2a control.</p

CCL2 blockade limits MPE induced by A549 human lung adenocarcinoma.

<p>(A) Development of a novel model of MPE induced by A549 cells. Shown are transdiaphragmatic photographs of SCID mouse indicating the normal anatomy (top left) and SCID mouse with A549-induced MPE (top right; dashed lines); retrieved MPE and blood samples (middle left); hematoxylin & eosin-stained visceral (middle right; scale bars = 400 µm; Å = 100) and parietal (bottom left; scale bars = 800 µm; Å = 50) pleural tumor tissue sections indicating the pleural cavity (pc), tumors (t), lung (l) and the chest wall (cw); and May-Gruenwald-Giemsa-stained pleural fluid cell cytocentrifugal specimen (scale bar = 100 µm; Å = 400) showing a mixture of cancer (cc), mononuclear (m), polymorphonuclear (pmn), and lymphoid (l) cells (bottom right). (B) Cellular and biochemical composition of A549-induced MPE in SCID mice (values given represent mean ± SD; n = 10). (C) Experimental set-up of host-directed anti-CCL2 trial using the xenogeneic A549/SCID MPE model. (D) Kaplan-Meier survival curve of SCID mice after intrapleural delivery of A549 cells followed by IgG2a or anti-CCL2 antibody treatment. (E,F) Representative transdiaphragmatic photographs and experimental end-points of A549/SCID MPE trial. Dashed lines outline MPEs; <i>t</i>, tumor; <i>l</i>, lung. (G) Summary of data and representative images of F8A-stained pleural tumors from the A549/SCID MPE model. Scale bar = 100 µm; Å = 400. Arrows indicate new vessels. <i>Columns</i>, mean; <i>bars</i>, SD; <i>n</i>, sample size; <i>P</i>, probability; ns, *, and **: P>0.05, P<0.05, and P<0.01 compared with IgG2a.</p

Cellular Adhesion Promotes Prostate Cancer Cells Escape from Dormancy

<div><p>Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of <i>in vitro</i> models. Here, we characterized <i>in vitro</i> PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant <i>in vitro</i> model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.</p></div

CDK6 overexpression induced proliferation of LuCaP PDX cells <i>in vitro</i>.

<p>LuCaP 86.2, 92 and 93 cells were lentivirally transduced to overexpress CDK6 and cultured <i>in vitro</i> to assess proliferation. Positive Ki67 indicated that CDK6 overexpression facilitated proliferation in these cells. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm.</p

Constitutive activation of MLCK promotes proliferation of LuCaP PDX cells via upregulation of CDK6.

<p>A) LuCaP cells were infected with lentivirus containing A-tMK (constitutively activate MLCK) showed positive Ki67 staining, whereas cells transduced with an empty vector did not. B) In LuCaP 86.2, 92, and 93, ectopic expression of A-tMK induced an upregulation of CDK6 and a concurrent downregulation of E2F4 when compared to that of the empty vector-transduced cells. Inhibition of MLCK with the MLCK inhibitor ML-7 suppressed proliferation by C) abolishing Ki67 expression, D) decreasing cell viability assessed by WST-1 assay and E) downregulating CDK6 expression. E2F4 expression was not altered by the ML-7. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm. **p< 0.01 as compared to the DMSO control. CDK6: cyclin-dependent kinase 6; E2F4: E2F transcription factor 4.</p

Genes associated with cellular movement were downregulated in proliferating LuCaP cells.

<p>A) Heat map of hierarchically clustered differential gene expression in NG and G LuCaP PDX cells. Green, downregulated; red, upregulated. B) Ingenuity pathway analysis showing cellular movement was the top molecular and cellular function altered between NG and G cells. C) List of eight genes that were involved in the decreased activation of cellular movement in G when compared to NG cells. D) EDN1 was predicted to be the top regulator that affected the cell movement via downregulation of FN1, CDC42, and FOSL1. E) Quantitative real-time PCR showed a downregulation of FN1, CDC42 and TGFb2 in growing LuCaP lines. Data were normalized to the levels of housekeeping gene RPS15. NG: not growing; G: growing.</p