Zhang et al 2017 SUSD2 ENCa Data files

FACS histograms, Western blot, qPCR and cell death prism files, photographs of cell senescence

Downregulation of endometrial mesenchymal marker SUSD2 causes cell senescence and cell death in endometrial carcinoma cells

<div><p>The cause of death among the majority of endometrial cancer patients involves migration of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into neighbouring organs. It is, thereby, important to identify factors that mediate metastasis. Cell adhesion and migration are modified by the mesenchymal stem cell (MSC) marker Sushi domain containing 2 (SUSD2), a type I transmembrane protein that participates in the orchestration of cell adhesion and migration through interaction with its partner Galactosidase-binding soluble-1 (LGALS1). MSCs have emerged as attractive targets in cancer therapy. Human endometrial adenocarcinoma (Ishikawa) cells were treated with TGFβ (10 ng/ml) for 72h. <i>SUSD2</i>, <i>LGALS1</i> and <i>MKI67</i> transcript levels were quantified using qRT-PCR. The proportion of SUSD2 positive (SUSD2+) cells and SMAD2/3 abundance were quantified by FACS and Western blotting, respectively. Senescent cells were identified with β-galactosidase staining; cell cycle and cell death were quantified using Propidium Iodide staining. Treatment of endometrial cancer cells (Ishikawa cells) with TGFβ (10 ng/ml) significantly decreased <i>SUSD2</i> transcript levels and the proportion of SUSD2 positive cells. Silencing of <i>SUSD2</i> using siRNA resulted in senescence and cell death of Ishikawa cells <i>via</i> activation of SMAD2/3. These findings suggest that SUSD2 counteracts senescence and cell death and is thus a potential chemotherapeutic target in human endometrial cancer.</p></div

Influence of SUSD2 knockdown on cell senescence.

<p>(A) Representative pictures of Ishikawa cells stained for β-Galactosidase after SUSD2 knockdown. Scale bar 100μm. (B) Arithmetic means ± SEM (n = 3–8) of relative abundance of β-Galactosidase. *P< 0.05 indicates statistically significant difference from control cells using Student’s t-test.</p

Titration and Time-course of the TGFβ effect on <i>SUSD2</i>+ Ishikawa cells.

<p>(A) Representative original FACS plots showing the effect of increasing TGFβ concentrations (Control, 1, 5, 10, 20, 50 ng/ml) for 72h on the percentage of SUSD2+ expressing cells. (B) Representative original FACS plots showing increasing abundance of SUSD2+ cells at 24h, 48h and 72h time points with TGFβ (10 ng/ml) or untreated (Control).</p

Effect of SUSD2 on cell death.

<p>(A) Representative FACS plots showing cell cycle progression of Ishikawa cells characterized by Propidium Iodide (PI) staining after 72h TGFβ (10 ng/ml) or Non-targeting (NT) or with SUSD2 siRNA treatment as indicated. Sub G0/G1 represents the apoptotic fraction. (B) Arithmetic means ± SEM (n = 5–6) of percentage cells in different cell cycle phases. *P < 0.05, **P < 0.01 indicates statistically significant difference from control cells using Student’s t-test.</p

Diagnostic performance of CTLA-4, carcinoembryonic antigen and CYFRA 21-1 for malignant pleural effusion

<p><b>Objectives</b>: The diagnosis of malignant pleural effusion (MPE) remains a clinical challenge. As a negative regulator of T-cell activation, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has been associated with many malignant diseases. However, there is limited data about the relationship between CTLA-4 and MPE. The present study aims to investigate whether CTLA-4 levels may correlate with presence of MPE and to assess its potential diagnostic accuracy relative to that of the established markers carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21–1).</p> <p><b>Methods</b>: Pleural effusion samples were collected from 36 patients with MPE and 48 patients with benign pleural effusion (BPE). Pleural levels of CTLA-4 were measured by ELISA; levels of CEA and CYFRA 21-1, by electrochemiluminescence immunoassay. Receiver operating characteristic curves were calculated to evaluate the ability of CTLA-4, CEA and CYFRA 21-1 to differentiate MPE from BPE.</p> <p><b>Results</b>: Pleural levels of CTLA-4 were significantly higher in MPE than in BPE patients (471.73 ± 378.86 vs. 289.22 ± 173.67 pg/ml, p = 0.004). At a cut-off value of 351.25 pg/ml, the sensitivity and specificity of CTLA-4 in diagnosing MPE were 58.30% and 83.30%, respectively, and the area under the curve was 0.72. Pleural levels of CEA and CYFRA 21-1 were also higher in MPE. Using the combination of CTLA-4, CEA and CYFRA 21-1 increased diagnostic sensitivity to 88.89% and the area under the curve to 0.92.</p> <p><b>Conclusion</b>: The results of this preliminary study suggest that increased levels of CTLA-4 correlate with MPE, and that CTLA-4 may have some diagnostic usefulness when used in combination with conventional tumor markers such as CEA and CYFRA 21-1. These results justify larger, more rigorous studies to validate our findings.</p

Effect of SUSD2 knockdown on SMAD2/3 expression.

<p>(A) Representative original FACS plots showing SUSD2 knockdown. (B) Arithmetic means ± SEM (n = 5) of SUSD2+ Ishikawa cells. Data are depicted as fold induction relative to non-targeting siRNA (Control) samples. ****P<0.0001 indicates statistically significant difference from control cells using Student’s t-test. (C) Arithmetic means ± SEM (n = 4) of <i>SUSD2</i> transcript levels normalized to <i>L19</i> transcript levels in Ishikawa cells. Data are depicted as fold induction relative to transcript levels of control samples. *P< 0.05 indicates statistically significant difference from control cells using Student’s t-test. (D) Representative original Western blot of SMAD2/3 and GAPDH in Ishikawa cells. (E) Arithmetic means ± SEM (n = 7) of SMAD2/3 ratio normalized to GAPDH in Ishikawa cells. *P< 0.05 indicates statistically significant difference from control cells using Student’s t-test.</p

Additional file 1: Fig. S1. of Is neuron-specific enolase useful for diagnosing malignant pleural effusions? evidence from a validation study and meta-analysis

Serum and pleural levels of neuron specific enolase in patients standardized by pleural protein levels. After standardized by pleural protein levels, both serum and pleural levels of neuron specific enolase were higher than that in patient with benign pleural effusion. NSE: Neuron specific enolase; BPE: Benign pleural effusion; MPE: Malignant pleural effusion; LAC-MPE: Lung adenocarcinoma-malignant pleural effusion; LSCC-MPE: Lung squamous cell carcinoma- malignant pleural effusion; SCLC-MPE: Small cell lung cancer- malignant pleural effusion (TIFF 2523 kb

mTOR was activated during the process of pulmonary fibrosis in vivo and vitro.

<p>A) mTOR activation in fibroblast foci of lung tissue in IPF patients. a,b, H&E staining with normal control and IPF lung tissues; Immunohistochemical staining performed with α-SMA (c,d) and p-S6 (e,f) antibodies showed an increase in α-SMA and p-S6 in IPF lung tissues (d, f) compared with the control (c, e). Scale bar = 100 μm. B) mTOR activation in the lung tissues of C57BL/6J mice after bleomycin intra-tracheal injection. a,b, H&E staining with lungs of saline and bleomycin-treated mice; Immunohistochemical staining was performed with α-SMA (c,d) and p-S6 (e,f) antibodies in saline- and bleomycin-treated mouse lung tissues. NS, normal saline. Scale bar = 100 μm. C) mTOR signaling pathway was activated in primary lung fibroblasts isolated from normal controls treated with TGF-β1(5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated primary lung fibroblasts (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against GAPDH and (c) with p-S6 normalized against S6. **, P<0.01; *, P<0.05. n = 3. D) mTOR signaling pathway was activated in MRC5 cells (a human fetal lung fibroblast cell line) treated with TGF-β1 (5 ng/ml) for 48 h. Western blot analysis of α-SMA and p-S6 in control and TGF-β1-treated MRC5 cells (a). Densitometric quantification of the Western blot in (a) is shown in (b) with α-SMA normalized against β-actin and (c) with p-S6 normalized against S6. **, p<0.01; *, p<0.05. n = 3.</p

Rapamycin-induced autophagyin the bleomycin-mediated lung injury and fibrosis model.

<p>A) Rapamycin decreased the death caused by bleomycin. Chloroquine, an autophagy inhibitor, reversed the benefit of rapamycin in the bleomycin-mediated lung injury model (Bleo+Rapa+CQ vs Bleo+Rapa, p = 0.0158). B) Western blot analysis of p62 and p-S6 were performed in the bleomycin-mediated lung injury and fibrosis model. p62, a protein inversely correlated with autophagy activity, was decreased in lungs of mice treated with rapamycin alone. p62 expression was higher with combined rapamycin and chloroquine treatment than with rapamycin alone. S6 and β-actin were used as controls. C) Western blot ananlysis of LC3 I and LC3 II were performed in the bleomycin-mediated lung injury and fibrosis mice model. D) Relative density of LC3 II/LC3 I of bands in Fig 5C. Autophagy was significantly decreased in bleomycin-mediated lung injury and fibrosis model (*bleomycin vs normal saline, p < 0.05). E) Electron microscope images of lung tissues show autophagosomes in the bleomycin-mediated lung injury model. Arrows indicate autophagosomes. Rapamycin treatment alone induced an increased number of autophagosomes. Left panel, original magnification: 6,000X and right panel, original magnification: 11,500X. F) Statistical results for the autophagosomes in Fig 5E. The statistical results indicate the percent area of autophagosomes in a cell.</p