Association of BDNF, TrkB, and co-expression of BDNF/TrkB with clinicopathological variables.

<p>TrkB mRNA expression indicates mRNA transcript levels of both TrkB.FL and TrkB.T1.</p

BDNF increases cell viability in TrkB-expressing CRC cells.

<p>BDNF/TrkB-expressing DLD1 (A), SW480 (B), and LoVo (C) cells were used to examine the effect of BDNF, K252a, and their combination on tumor cell viability. Each DLD1, LoVo, and SW480 cell was treated with BDNF (100 ng/ml), K252a (100 nM), or K252a followed by BDNF for 48 h. Exogenous BDNF increased tumor cell viability in TrkB-expressing CRC cells, and K252a inhibited tumor cell viability. The data were obtained from similar results of at least three independent experiments. Results are presented as mean ±SE. *; P<0.05.</p

Kaplan-Meier plots of overall survival according to BDNF, TrkB, and co-expression of BDNF/TrkB.

<p>(A): Comparison between the overall survival of patient groups with high BDNF mRNA expression (n = 102) and those with low BDNF mRNA expression (n = 121). (B): Comparison between the overall survival of patient groups with high TrkB mRNA expression (n = 171) and those with low TrkB mRNA expression (n = 52). (C): Comparison between the overall survival of patients groups with high co-expression of BDNF and TrkB (n = 94) and those without it (n = 129). TrkB mRNA expression indicates mRNA transcript levels of both TrkB.FL and TrkB.T1.</p

<i>In Vivo</i> Characterization of Neutrophil Extracellular Traps in Various Organs of a Murine Sepsis Model

<div><p>Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model <i>in vivo</i> and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.</p></div

Circulating cell-free NETs.

<p>Circulating cell-free NETs (red) were observed in the blood flow of either postcapillary venules of the cesum or hepatic sinusoids of the liver (i, ii) at 24 h after LPS (20 mg/kg) intraperitoneal administration (n = 10). NETs were anchored to the leukocyte adhering to the vascular endothelium (iii; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s010" target="_blank">Movie S5</a>). Thereafter, NETs were leaving away from leukocyte, changing shape from solid to cotton-like, and flowing down the vessel. These cotton-like NETs were arrested far away from the leukocyte (iv). They were not observed in control mice.</p

<i>In vivo</i> NETs in postcapillary venules of the cecum.

<p>The postcapillary venules of the cecum were observed at 24 h after LPS (20 mg/kg) intraperitoneal administration (n = 10). NETs (red) were detected by SYTOX Orange, Alexa Fluor 594-labeled anti-histone antibody, or Alexa Fluor 594-labeled anti-NE antibody, respectively. NETs were characterized as reticular structures anchored to leukocytes (A-i; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s006" target="_blank">Movie S1</a>), reticulolinear structures (A-ii), spot-like structures anchored to leukocytes (A-iii, iv), membranous structures on the surface of leukocytes (A-v) and linear structures anchored to leukocytes (A-vi; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s007" target="_blank">Movie S2</a>). The number of leukocyte-endothelial interactions and NETs per FOV was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#s2" target="_blank">materials and methods</a>, respectively. The number of leukocyte-endothelial interactions per FOV (B) were significantly greater in LPS treated mice than control mice (14.6±1.6 vs 0.6±0.2). The number of NETs per FOV (C) was also significantly greater in LPS treated mice than control mice (4.4±0.4 vs 0.2±0.1). Data was presented as mean+standard error. **P<0.01 versus control.</p

K252a suppresses the peritoneal metastasis of BDNF/TrkB-co-expressing CRC cells <i>in vivo</i>.

<p>(A): The experimental schedule of the <i>in vivo</i> peritoneal metastasis assay. DLD1 cells (5×10⁷ cells/500 µl PBS) were injected intraperitoneally (day 0). Seven days later (day 7), K252a (500 µg/kg) or PBS (control) was injected three times a week. Four weeks later (day 28), mice were sacrificed, and then the size and number of peritoneal metastatic nodules were evaluated (for each group; n  =  5). (B): Representative images of control mice and K252a-treated mice. (C): Representative images of peritoneal metastases in control mice and K252a-treated mice. (D): The bar graph indicates that the amount of peritoneal metastasis was significantly lower in K252a-treated mice than in control mice. Each value represents the mean ±SE. *; P<0.05.</p

BDNF and TrkB protein expression in primary and metastatic colorectal cancer.

<p>Representative images of immuno-reactive BDNF and TrkB protein expression in primary CRC and peritoneal metastasis are shown (original magnification: 100×). Anti TrkB antibody (R&D Systems, Foster City, CA, USA) detected both TrkB.FL and TrkB.T1 proteins, which was confirmed by Western blotting analysis. (A): The immunoreactive BDNF protein is located in the cytoplasm of the tumor cells of the primary CRC. (B): The immunoreactive BDNF protein is located in the cytoplasm of the tumor cells in the corresponding peritoneal metastasis. (C): The immunoreactive TrkB protein is located in the nucleus of the tumor cells of the primary CRC. (D): The immunoreactive TrkB protein is located in the nucleus of the tumor cells in the corresponding peritoneal metastasis. These expression patterns for the BDNF and TrkB proteins were confirmed in CRC patients (n = 5) whose primary and peritoneal metastatic nodules were available for immunohistochemistry.</p

BDNF promotes migration in TrkB-expressing CRC cells.

<p>(A): Representative images from migration assays for DLD1 cells are shown (original magnification; 100×). Tumor cells were treated with medium (control), BDNF (100 ng/ml), K252a (100 nM), or K252a followed by BDNF. (B): The bar graph of these results indicates that exogenous BDNF enhanced tumor cell motility in TrkB-expressing CRC cells, and that K252a inhibited the migratory ability of these tumor cells. The data were obtained from similar results of at least three independent experiments. Results are presented as mean ±SE. *; P<0.01.</p

BDNF promotes invasion in TrkB-expressing CRC cells.

<p>(A): Representative images of an invasion assay with SW480 cells are shown (original magnification; 100×). Tumor cells were treated with medium (control), BDNF (100 ng/ml), K252a (100 nM), or K252a followed by BDNF. (B): The bar graph of these results indicates that exogenous BDNF enhanced tumor cell invasion in TrkB-expressing CRC cells, and that K252a inhibited the invasive ability of these tumor cells. The data were obtained from similar results of at least three independent experiments. Results are presented as mean ±SE. *; P<0.05.</p