The Kaplan-Mayer survival curves for the relapse-free survival (RFS).

<p>A) The survival curves according to the CONUT score. The relapse-free survival rates were significantly worse in the high CONUT group compared to the low CONUT group (p = 0.0018). B) The survival curves according to the PNI. The relapse-free survival rates were significantly worse in the low PNI group compared to the high PNI group (p = 0.0162).</p

The receiver operating characteristic (ROC) curve for the controlling nutritional status (CONUT).

<p>We used the continuous variable Controlling Nutritional Status (CONUT) as the test variable and 5-year cancer-specific survival as the state variable. An investigation of the cut-off value for the CONUT score using the receiver operating characteristic (ROC) curve showed the most appropriate cut-off value for the CONUT score to be 3 (AUC; 0.624, 95%CI: 0.476–0.771, p = 0.076, the sensitivity was 0.5263 and the specificity was 0.7622.). We indicated the ROC curve on Fig 1. Therefore, we set 3 as the cut-off value for the CONUT score in this study and classified the patients into high CONUT (≥3) and low CONUT (≤2) groups.</p

Comparative Proteomics Analysis of Gastric Cancer Stem Cells

<div><p>Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. To date, the specific markers of CSCs remain undiscovered. The aim of this study was to identify novel biomarkers of gastric CSCs for clinical diagnosis using proteomics technology. CSC-like SP cells, OCUM-12/SP cells, OCUM-2MD3/SP cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used in this study. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. Candidate proteins detected by proteomics technology were validated by immunohistochemical analysis of 300 gastric cancers. Based on the results of LC-MS/MS, eight proteins, including RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, were up-regulated in both OCUM-12/SP cells and OCUM-2MD3/SP cells when compared to their corresponding parent cells. RT-PCR analysis indicated that the expression level of <i>RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1</i>, and <i>CK18</i> was high in OCUM-12/SP and OCUM-2MD3/SP, in compared with the control of parent OCUM-12 and OCUM-2MD3. These proteins were significantly associated with advanced invasion depth, lymph node metastasis, distant metastasis, or advanced clinical stage. RBBP6, DCTPP1, HSPA4, and ALDOA expression in particular were significantly associated with a poor prognosis in the 300 gastric cancer patients. RBBP6 was determined to be an independent prognostic factor. The motility-stimulating ability of OCUM-12/SP cells and OCUM-2MD3/SP cells was inhibited by <i>RBBP6</i> siRNA. These findings might suggest that the eight proteins, RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, utilizing comparative proteomics analysis, were perceived to be potential CSC markers of gastric cancer. Of the eight candidate proteins, RBBP6 was suggested to be a promising prognostic biomarker and a therapeutic target for gastric cancer.</p></div

Ingenuity Pathway Analysis (IPA) of Proteins Expressed in SP Cells.

<p>(A) Localization; (B) Biological processes of identified proteins. We categorized the proteins based on their functional assignments using Ingenuity Pathway Analysis. The molecular functions reported include cell death, metabolism, cellular organization, metabolism of DNA, protein degradation, processing of RNA, production of reactive oxygen species, nitric oxide, molecular transport, cell cycle, folding protein, and cellular movement. % = 100 X number of identified proteins/all 932 proteins analyzed. (C), A Venn diagram confer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110736#pone-0110736-t002" target="_blank">Table 2</a>. Forty proteins were significantly increased in OCUM-12/SP cells compared to their parent OCUM-12 cells. Thirty-five proteins were significantly increased in OCUM-2MD3/SP cells compared to their parent OCUM-2MD3cells. Eight candidate proteins, RBBP6, HSPA4, HSPA9, GLG1, DCTPP1, VPS13A, CK18 and ALDOA, overlap in both OCUM-12/SP and OCUM-2MD3/SP cells. (D), mRNA expression. RT-PCR analysis indicated that the expression level of <i>RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1</i>, and <i>CK18</i> was high in OCUM-12/SP (9.15 fold, 9.36 fold, 4.14 fold, 7.80 fold, 2.08 fold, 1.46 fold, 3.44 fold, and 1.99 fold, respectively) and OCUM-2MD3/SP (6.15 fold, 1.71 fold, 2.33 fold, 2.30 fold, 2.03 fold, 1.32 fold, 1.35 fold, and 1.31 fold, respectively), in compared with the control of parent OCUM-12 and OCUM-2MD3. (E), Correlation of Signaling Pathways between RBBP6 and Differentially-Expressed Proteins in CSC-like SP cells. RBBP6 is over-expressed (red) in CSC-like SP cells (OCUM-12/SP cells and OCUM-2MD3/SP cells). Hsp90, HSPA4, and TAGLN2 (pink) up-regulated in CSC-like SP cells were associated with the RBBP6 signaling pathway.</p

Univariate and multivariate analyses with respect to survival.

a<p>NF; Infiltration pattern of tumor. The predominant pattern of infiltrating growth into the surrounding tissue is classified as follows; INF a: The tumor shows expanding growth and a distinct border with the surrounding tissue. INF b: This category is between INF a and INF b. INF c: The tumor shows infiltrating growth and an indistinct border with the surrounding tissue.</p><p>Univariate and multivariate analyses with respect to survival.</p

Immunohistochemical Determination.

<p>(a) Expression in cancer cells. RBBP6, DCTPP1, and HSPA9 expression were observed primarily in the cytoplasm and nucleus. GLG1, VPS13A, HSPA4, ALDOA, and KRT18 were expressed in the cytoplasm. (b), Expression in epithelial cells. RBBP6, GLG1, VPS13A, DCTPP1, and HSPA9 expression were found some cells in the epithelial gland. KRT18 were expressed in most epithelial cells. HSPA4 and ALDOA expression was not found in normal cells.</p

Correlation between proteins expression and clinicopathological variables.

<p>Correlation between proteins expression and clinicopathological variables.</p

Proteins increased in both OCUM-12/SP and OCUM-2MD3/SP cells compared to their parent cells detected by QSTAR Elite LC/MS/MS.

a<p>ratio, OCUM-12/SP ratio: OCUM-12/SP cell compared with parent OCUM-12 cell. OCUM-2MD3/SP ratio: OCUM-2MD3/SP cell compared with parent OCUM-2MD3 cell.</p>b<p>↑, proteins expressed only in SP cells but not parent cells.</p>c<p>Location: C, cytoplasm; PM, plasma membrane; ES, extracellular space; N, nucleus.</p>d<p>Type: En, enzyme; K, kinase; Tp, transporter; O, other; TR, transcriptional regulator.</p><p>NA, not available.</p><p>Proteins increased in both OCUM-12/SP and OCUM-2MD3/SP cells compared to their parent cells detected by QSTAR Elite LC/MS/MS.</p

<i>siRBBP6</i> transfection into gastric cancer cells.

<p>(A), OCUM-12/SP and OCUM-2MD3/SP showed higher level of <i>RBBP6</i> mRNA expression than their parent cells, OCUM-12 and OCUM-2MD3. RBBP6 expression in OCUM-12/SP and OCUM-2MD3/SP cells was effectively downregulated by si<i>RBBP6</i> transfection. (B), Representative images of invading OCUM-12/SP cells showed the number of cancer cells invading the pore membrane filter was decreased by <i>RBBP6</i> siRNA treatment. si<i>RBBP6</i> transfection for OCUM12/SP cells significantly inhibited the invasion abilities. Data are presented as the mean and SD (error bars) of four experiments. * <i>p</i><0.05, ** <i>p</i><0.01. (C), si<i>RBBP6</i> treatment for OCUM12/SP and OCUM-2MD3/SP cells significantly inhibited the migration abilities, in comparison with that of the control of <i>negative-siRNA</i> treatment. Data are presented as the mean and SD (error bars) of four experiments. * <i>p</i><0.05, ** <i>p</i><0.01.</p