Additional file 1 of mir-145-5p is a suppressor of colorectal cancer at early stage, while promotes colorectal cancer metastasis at late stage through regulating AKT signaling evoked EMT-mediated anoikis

Supplementary Material

Additional file 2 of mir-145-5p is a suppressor of colorectal cancer at early stage, while promotes colorectal cancer metastasis at late stage through regulating AKT signaling evoked EMT-mediated anoikis

Supplementary Material

Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target for Hepatocellular Carcinoma

<div><p>Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.</p> </div

tHSC-CM promoted proliferation, migration and invasion of PLC/PRF/5 cells.

<p>(A and B) tHSC-CM significantly promoted HCC cell growth with time- and concentration-dependent manner. (C) Representative images of wound migration assays (left panel). Results are expressed as the percentage of the wounded area (right panel). (D) Representative images of invasion assays performed by transwell chamber (left panel). Results are expressed as the number of cells per field (right panel). *p < 0.05; **p < 0.01. </p

tHSC-CM induces EMT-like phenotype in HCC cells.

<p>(A) The morphology changes in PLC/PRF/5 cells (magnification, ×200). (B) Western blot analysis of E-cadherin and vimentin expressions in PLC/PRF/5 cells (left panel). Results are expressed as the fold value of protein levels compared with GAPDH (right panel). *p < 0.05. (C and D) Representative images of E-cadherin and vimentin expressions in PLC/PRF/5 cells by immunoflurescence staining (magnification, ×400). </p

tHSCs promote HCC growth, and icaritin effectively inhibits tHSC-induced HCC-promoting effects in vivo.

<p>(A) The tumor volumes were monitored every 4 days up to 23 days after cell implantation. (B) The tumor weights at the end of experiments (*p < 0.05). (C) Representative immunohistochemical staining of α-SMA, E-cadherin, Ki-67, and CD34 expressions in different xenografts. (D) Representative images of TUNEL assay in different xenografts (magnification, ×200). PLC: PLC/PRF/5.</p

tHSCs are associated with E-cadherin expression, HCC cell invasion in human HCC specimens, and poor survival outcome.

<p>(A) Representative immunostaining of HSCs in normal liver tissues and HCC tissues with α-SMA and desmin antibodies. (B) Representative samples of the tHSC density determined by α-SMA immunostaining (magnification, ×200). Typical grades 0, 1, 2, and 3 are shown in a, b, c, and d, respectively. (C) Representative pictures of α-SMA and E-cadherin expressions in human HCC tissues detected by immunostaining (magnification, ×400). tHSC-rich is accompanied by deccreased E-cadherin expression in case 1 (upper panel), and tHSC-poor is accompanied by increase E-cadherin expression in case 2 (lower panel). (D) Immunofluorescence staining separately with Hep Par 1 and E-cadherin antibody on serial sections of one HCC sample (magnification, ×400). The white arrows indicated the tumor cells with both Hep Par 1-positive and E-cadherin-negative staining that have infiltrated from their nests into the surrounding stroma. (E) The impact of tHSCs on the survival of HCC patients. (a) tHSC-associated overall survival rate. (b) tHSC-associated recurrence-free survival rate.</p

Additional file 1 of Preoperative serum CA19-9 should be routinely measured in the colorectal patients with preoperative normal serum CEA: a multicenter retrospective cohort study

Additional file 1: FigureS1. Association between preoperative CA19-9 status and overall survival. (a) overall population. (b) patients with normal preoperative CEA. (c) patientswith elevated preoperative CEA. Solid yellow lines are unadjustedhazard ratios, with dashed yellow lines showing 95% confidence intervalsderived from restricted cubic spline regressions. Reference lines for noassociation are indicated by the solid bold lines at a hazard ratio (HR) of 1.0. Dashed blue curves show the fraction of the population with different levels of preoperative CA19-9. Arrows indicate the concentration of preoperative CA19-9 with HR of 1.0. CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; CI, confidence interval; E, number of events; HR, hazard ratio; N, number of patients. FigureS2. Kaplan‐Meier curves for overall survival according to the preoperative CA19-9 group. (a) overall population. (b) patients with normal preoperative CEA. (c) patientswith elevated preoperative CEA. CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen. FigureS3. Kaplan‐Meier curves according to the joint group of preoperative CEA and CA19-9 in colorectal cancer patients. (a) recurrence-free survival. (b) overall survival. CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; OS, overall survival; RFS, recurrence-free survival. FigureS4. Forest plot for recurrence-free survival of preoperative CA 19-9 groups stratified by clinicopathological features based on the Cox models. P values for interaction were calculated using Cox regression model. HR and 95%CIs were given and visually represented by the squares and error bars. CA 19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; CI, confidence interval; HR, hazard ratio. FigureS5. Forest plot for performance overallsurvival of preoperative CA19-9 groups stratified by clinicopathological features based on the Cox models. P values for interaction were calculated using Cox regression model. HR and 95%CIs were given and visually represented by the squares and error bars. CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; CI, confidenceinterval; HR, hazard ratio. FigureS6. Kaplan‐Meier curves according to the joint group of preoperative CEA and CA19-9 in patients with stage II colorectal cancer. (a) recurrence-free survival.(b) overall survival. CA 19-9, carbohydrate antigen 19-9;CEA, carcinoembryonic antigen; OS, overall survival; RFS, recurrence-freesurvival

Additional file 2 of Preoperative serum CA19-9 should be routinely measured in the colorectal patients with preoperative normal serum CEA: a multicenter retrospective cohort study

Additional file 2: Table S1.Baseline characteristics by participant site. Table S2.Multivariate analyses of recurrence-free survival in total population (Cox model). Table S3.Multivariate analyses of overall survival in total population (Cox model). Table S4.Interaction between preoperative CEA and CA19-9 with risk of outcomes. Table S5.Multivariate analyses of recurrence-free survival in colorectal cancer subgroup with CEA < 5 ng/ml (Cox model). Table S6.Multivariate analyses of recurrence-free survival in colorectal cancer subgroup with CEA ≥ 5 ng/ml (Cox model). Table S7. Multivariate analyses of overall survival in colorectal cancer subgroup with CEA < 5 ng/ml (Cox model). Table S8.Multivariate analyses of overall survival in colorectal cancer subgroup with CEA ≥ 5 ng/ml (Cox model). Table S9.A frailty model analysis of preoperative CA19-9 (cutoff: 37 U/ml) on colorectal cancer outcomes in total population. TableS10.Cox proportional hazard regression analysis of preoperative CA19-9 (cutoff:74 U/ml) on colorectal cancer outcomes in total population. Table S11.Relationship between preoperative CA19-9 and benefit from adjuvant chemotherapyin patients with stage II colorectal cancer