Expression of CD151 in Gastric Cancer Tissues and Its Clinical Significance

Objective:To explore the expression of CD151 in gastric cancer tissues and its clinical significance.Methods:Immunohistochemistry was employed to detect the expression of CD151 in gastric cancer tissues and adjacent normal tissues.The relationship between CD151 expression and the clinicopathological characteristics of gastric cancer was analyzed.Results:The expression of CD151 in gastric cancer tissues was significantly higher than in adjacent normal tissues(P<0.05).It was associated with the degree of differentiation,depth of invasion,lymph node metastasis,and TNM staging of gastric cancer.The survival time of patients with high CD151 expression was significantly shorter than that of those with low expression(P<0.05).Conclusion:High expression of CD151 in gastric cancer tissues is correlated with the malignant biological behavior of gastric cancer and can serve as an indicator for evaluating the prognosis of gastric cancer.

Increased CD4/CD8 Lymphocyte ratio predicts favourable neoadjuvant treatment response in gastric cancer:A prospective pilot study

BACKGROUND Despite optimal neoadjuvant chemotherapy only 40%of gastric cancer tumours achieve complete or partial treatment response.In the absence of treatment response,neoadjuvant chemotherapy in gastric cancer contributes to adverse events without additional survival benefit compared to adjuvant treatment or surgery alone.Additional strategies and methods are required to optimize the allocation of existing treatment regimens such as FLOT chemotherapy(5-Fluorouracil,Leucovorin,Oxaliplatin and Docetaxel).Predictive biomarkers detected using immunohistochemistry(IHC)methods may provide useful data regarding treatment response.AIM To investigate the utility of CD4,CD8,Galectin-3 and E-cadherin in predicting neoadjuvant FLOT chemotherapy tumour response in gastric adenocarcinoma.METHODS Forty-three adult patients with gastric adenocarcinoma,of which 18 underwent neoadjuvant chemotherapy,were included in a prospective clinical cohort.Endoscopic biopsies were obtained from gastric cancer and normal adjacent gastric mucosa.Differences in expression of Galectin-3,Ecadherin,CD4^(+)and CD8^(+)molecules between tumours with and without treatment response to neoadjuvant chemotherapy were assessed with IHC.Treatment response was graded by clinical pathologists using the Tumour Regression Score according to the College of American Pathologists criteria.Treatment response was defined as complete or near complete tumour response,whereas partial or poor/no response was defined as incomplete.Digital IHC images were annotated and quantitatively assessed using QuPath 0.3.1.Biomarker expression between responsive and incomplete response tumours was assessed using a two-sided Wilcoxon test.Biomarker expression was also compared between normal and cancer tissue and between 15 paired tumour samples before and after chemotherapy.We performed a preliminary multivariate analysis and power analysis to guide future study.Statistical analyses were completed using R 4.1.2.RESULTS The ratio between CD4^(+)and CD8^(+)lymphocytes was significantly greater in treatment responsive tumours(Wilcoxon,P=0.03).In univariate models,CD4^(+)/CD8^(+)ratio was the only biomarker that significantly predicted favourable treatment response(Accuracy 86%,P<0.001).Using a glmnet multivariate model,high CD4^(+)/CD8^(+)ratio and low Galectin-3 expression were the most influential variables in predicting a favourable treatment response.Analyses of paired samples found that FLOT chemotherapy also results in increased expression of CD4^(+)and CD8^(+)tumour infiltrating lymphocytes(Paired Wilcoxon,P=0.002 and P=0.008,respectively).Our power analysis suggests future study requires at least 35 patients in each treatment response group for CD8 and Galectin-3 molecules,whereas 80 patients in each treatment response group are required to assess CD4 and E-cadherin biomarkers.CONCLUSION We demonstrate that an elevated CD4^(+)/CD8^(+)Ratio is a promising IHC-based biomarker to predict favourable treatment response to FLOT neoadjuvant chemotherapy in locally advanced gastric cancer.

Ardisia gigantifolia ethanolic extract inhibits cell proliferation and targets cancer stem cells in gastric cancer

Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay.Non-adherent culture(3D)model was used to evaluate the effect of the extract on tumorsphere size and number.Moreover,the expression of CD44,ALDH,and p21 was determined by immunofluorescence analysis.Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH.Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes.Results:Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC_(50)of 55.7μg/m L in MKN45 cells and 123.6μg/m L in MKN74 cells.The extract also arrested cell cycle in the G_(0)/G_(1)phase as well as significantly reduced the size and number of tumorspheres.The markedly increased expression of p21 was observed at both m RNA and protein levels in the extract-treated adherent cells and tumorspheres.In addition,Ardisia gigantifolia extract significantly reduced the number of CD44-and/or ALDH-expressing gastric CSC.Conclusions:The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.

CD93 serves as a potential biomarker of gastric cancer and correlates with the tumor microenvironment

BACKGROUND The tumor microenvironment(TME)plays an important role in the growth and expansion of gastric cancer(GC).Studies have identified that CD93 is involved in abnormal tumor angiogenesis,which may be related to the regulation of the TME.AIM To determine the role of CD93 in GC.METHODS Transcriptomic data of GC was investigated in a cohort from The Cancer Genome Atlas.Additionally,RNA-seq data sets from Gene Expression Omnibus(GSE118916,GSE52138,GSE79973,GSE19826,and GSE84433)were applied to validate the results.We performed the immune infiltration analyses using ESTIMATE,CIBERSORT,and ssGSEA.Furthermore,weighted gene co-expression network analysis(WGCNA)was conducted to identify the immunerelated genes.RESULTS Compared to normal tissues,CD93 significantly enriched in tumor tissues(t=4.669,95%CI:0.342-0.863,P<0.001).Higher expression of CD93 was significantly associated with shorter overall survival(hazard ratio=1.62,95%CI:1.09-2.4,P=0.017),less proportion of CD8 T and activated natural killer cells in the TME(P<0.05),and lower tumor mutation burden(t=4.131,95%CI:0.721-0.256,P<0.001).Genes co-expressed with CD93 were mainly enriched in angiogenesis.Moreover,11 genes were identified with a strong relationship between CD93 and the immune microenvironment using WGCNA.CONCLUSION CD93 is a novel prognostic and diagnostic biomarker for GC,that is closely related to the immune infiltration in the TME.Although this retrospective study was a comprehensive analysis,the prospective cohort studies are preferred to further confirm these conclusions.

Application of CD34 expression combined with three-phase dynamic contrast-enhanced computed tomography scanning in preoperative staging of gastric cancer

BACKGROUND Accurate preoperative staging of gastric cancer(GC),a common malignant tumor worldwide,is critical for appropriate treatment plans and prognosis.Dynamic three-phase enhanced computed tomography(CT)scanning for preoperative staging of GC has limitations in evaluating tumor angiogenesis.CD34,a marker on vascular endothelial cell surfaces,is promising in evaluating tumor angiogenesis.We explored the value of their combination for preoperative staging of GC to improve the efficacy and prognosis of patients with GC.Medical records of 106 patients with GC treated at the First People's Hospital of Lianyungang between February 2021 and January 2023 were retrospectively studied.All patients underwent three-phase dynamic contrast-enhanced CT scanning before surgery,and CD34 was detected in gastroscopic biopsy specimens.Using surgical and pathological results as the gold standard,the diagnostic results of three-phase dynamic contrast-enhanced CT scanning at different T and N stages were analyzed,and the expression of CD34-marked microvessel density(MVD)at different T and N stages was determined.The specificity and sensitivity of three-phase dynamic contrast-enhanced CT and CD34 in T and N staging were calculated;those of the combined diagnosis of the two were evaluated in parallel.Independent factors affecting lymph node metastasis were analyzed using multiple logistic regression.RESULTS The accuracy of three-phase dynamic contrast-enhanced CT scanning in diagnosing stages T1,T2,T3 and T4 were 68.00%,75.00%,79.41%,and 73.68%,respectively,and for diagnosing stages N0,N1,N2,and N3 were 75.68%,74.07%,85.00%,and 77.27%,respectively.CD34-marked MVD expression increased with increasing T and N stages.Specificity and sensitivity of three-phase dynamic contrast-enhanced CT in T staging were 86.79%and 88.68%;for N staging,89.06%and 92.86%;for CD34 in T staging,64.15%and 88.68%;and for CD34 in N staging,84.38%and 78.57%,respectively.Specificity and sensitivity of joint diagnosis in T staging were 55.68%and 98.72%,and N staging were 75.15%and 98.47%,respectively,with the area under the curve for diagnosis improving accordingly.According to multivariate analysis,a longer tumor diameter,higher pathological T stage,lower differ-entiation degree,and higher expression of CD34-marked MVD were independent risk factors for lymph node metastasis in patients with GC.CONCLUSION With high accuracy in preoperatively determining the invasion depth and lymph node metastasis of GC,CD34 expression and three-phase dynamic contrast-enhanced CT can provide a reliable basis for surgical resection.

COX-2 expression in gastric cancer and its relationship with angiogenesis using tissue microarray

AIM: To explore the expression and clinicopathological significance of cyclooxygenase-2 (COX-2) and microvessel density (MVD) in gastric carcinogenesis, and to investigate their roles in the invasion and the relationship between biological behaviors and prognosis of gastric cancer. METHODS: Using Envision immunohistochemistry, COX-2 and CD34 expressions in gastric cancer tissue array were examined. MVD was counted and the relationship between the biological behaviors and prognosis was analyzed. RESULTS: The expression of COX-2 in gastric cancer tissue was significantly higher than that in normal mucosa (χ2 = 12.191, P < 0.05). The over-expression of COX-2 in gastric cancer was obviously related to metastasis and depth of invasion (χ2 = 6.315, P < 0.05), but not related to the histological type and Borrmann type (χ2 = 5.391 and χ2 = 2.228, respectively). Moreover, MVD in gastric cancer tissues was significantly higher than that in the normal mucosa (65.49 ± 20.64 vs 36.21 ± 18.47, t/F = 7.53, P < 0. 05). MVD was related to the histologic type and metastasis (t/F = 3.68 and t/F = 4.214, respectively, P < 0. 05), but not related to the depth of invasion and Borrmann type (t/F = 0.583 and t/F = 0.459, respectively). MVD in COX-2-positive tissues was markedly higher compared to COX-2-negative tissues, indicating a positive correlation between COX-2 expression and MVD (t = 13.12, P < 0. 05). CONCLUSION: Tissue microarray (TMA) is a powerful tool for rapid identifi cation of the molecular alterations in gastric cancer. COX-2 expression, via inducingangiogenesis, may play an important role in gastric carcinogenesis. It could be served as a determinant factor for clinical prognosis and curative effect.

CD44v6 in peripheral blood and bone marrow of patients with gastric cancer as micro-metastasis

AIM： To detect the expression of CD44 correlated with the ability of micro-metastasis in peripheral blood and bone marrow of patients with gastric cancer and to deduce its clinical significance. METHODS： Preoperative peripheral blood and bone marrow specimens from 46 patients with gastric cancer and 6 controls were studied by semi-quantitative RTPCR amplification of CD44v6mRNA. Preoperative and postoperative peripheral blood specimens from 40 patients with gastric cancer and 14 controls were studied by quantitative RT-PCR amplification of CD44v6mRNA in the corresponding period. RESULTS： Semi-quantitative RT-PCR amplification showed that CD44v6mRNA expression of peripheral blood and bone marrow was positive in 39 （84.8%） and 40 （86.9%） of 46 patients with gastric cancer, respectively. In peripheral blood, CD44v6mRNA expression was positive for diffuse type in 30 （93.8%） of 32 patients and for intestinal type in 9 （64.3%） of 14 patients. On the other hand, in bone marrow, CD44v6mRNA expression was positive for diffuse type in 31 （96.9%） of 32 patients and for intestinal type in 10 （71.4%） of 14 patients. There was a significant difference between the diffuse type and intestinal type. Quantitative RTopCR amplification demonstrated that CD44v6mRNA was not expressed in the peripheral blood of controls and CD44v6mRNA expression was positive for preoperative peripheral blood in 40 patients with gastric cancer, the expression levels being from 4.9 × 10^2 to 3.2× 10^5 copies/g RNA. The average expression level of CD44v6mRNA in peripheral blood was 3.9 × 10^10 copies/g RNA. The expression levels of CD44v6mRNA in peripheral blood in gastric cancer patients after curative operation increased from 5.5 × 100 to 7.6 × 10 copies/g RNA （P = 0.00496）. After curative operation, the expression level decreased markedly. CONCLUSION： Semi-quantitative and quantitative RTPCR amplification for CD44v6mRNA is a sensitive and specific method for the detection of micro-metastasis in peripheral blood and bone marrow, which might be used as an indicator of tumor burden and therapeutic effect.

Micro RNA-145 exerts tumor-suppressive and chemoresistance lowering effects by targeting CD44 in gastric cancer

AIM To determine the potential roles of CD4 and micro RNA(mi R)-145 in gastric cancer.METHODS The levels of CD44 and mi R-145 were determined in gastric cancer cells. Quantitative real-time polymerase chain reaction was used to measure to the level of CD44 m RNA. A luciferase reporter assay and western blotting were performed to examine the effect of mi R-145 on CD44 expression. Tumor sphere and MTT assays were carried out to evaluate the self-renewal and chemo-resistance properties of gastric cancer cells.RESULTS The expression of CD44 was greatly increased and mi R-145 was decreased in gastric cancer cells that were highly enriched in cancer stem cells(CSCs). The results demonstrated that mi R-145 regulated CD44 by targeting directly the CD44 3'-untranslated region(3'-UTR). In gastric cancer cells, overexpression of mi R-145 repressed the activity of the CD44 3'-UTR, and disruption of mi R-145/CD44 3'-UTR interactions abrogated the silencing effects. In addition, mi R-145 inhibition stimulated CD44 3'-UTR activity and disruption of mi R-145/CD44 3'-UTR interactions abrogated this stimulatory effect. Enforced CD44 expression greatly increased tumor sphere formation and chemoresistance in gastric cancer cells. Furthermore, the inhibition of CSCs and the chemo-sensitivity of gastric cancer cells treated with mi R-145 were significantly abrogated by overexpression of CD44. CONCLUSION mi R-145 targeting of CD44 plays critical roles in the regulation of tumor growth and chemo-resistance in gastric cancer.

Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer

AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their lev- els and clinicopathological parameters in gastric cancer.METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

Effects of angiopoietin-1 on attachment and metastasis of human gastric cancer cell line BGC-823

AIM： To evaluate the effects of angiopoietin-1 （Ang-1） on adhesion of gastric cancer cell line BGC-823 and expression of integrin β1, CD44V6, urokinase-type plasminogen activator （uPA） and matrix metalloproteinase-2 （MMP-2）. METHODS： BGC-823 cells were transfected transiently with adenovirus-Ang-1 （Ad-Ang-1）. Cells transfected transiently with adenovirus-green fluorescent protein （Ad-GFP） and untransfected cells were used as a negative and blank control group, respectively. The cell adhesion rate between cell and extracellular matrix （ECM） was determined by cell adhesion assay. To investigate whether Ang-1 could reinforce gastric carcinoma metastasis, we performed migration and invasion assays in BGC-823 cells. The mRNA and protein expression of integrin β1, CD44V6, uPA and MMP-2 were detected by reverse transcription polymerase chain reaction and Western blotting, respectively. The expression of integrin β1 and CD44V6 was measured by immunohistochemistry. RESULTS： BGC-823 cells were transfected successfully. The adhesion rate increased significantly in the Ad-Ang-1 group （P 〈 0.05）. The Ad-Ang-1-transfected group had a significant increase in migration and invasion compared with that of the mock-transfected and Ad-GFP groups. The mRNA and protein expression of integrin β1, CD44V6, uPA and MMP-2 in the Ad- Ang-1 group was higher than that in the Ad-GFP and blank control groups （P 〈 0.05）. Compared with mocktransfected and Ad-GFP groups, integrin 131 and CD44V6 expression intensity greatly increased （P 〈 0.05）. CONCLUSION： Transfection of Ang-1 into human gastric cancer cell line BGC-823 can significantly increase expression of integrin β1 and CD44V6, by which cell adhesion and metastasis to the ECM are promoted.

Association of CD14/-260 polymorphism with gastric cancer risk in Highland Tibetans

AIM: To investigate the relationship between CD14-260 and -651 polymorphisms and the risk of developing gastric cancer.

Prognostic potential of an immune score based on the density of CD8＋ T cells, CD20＋ B cells, and CD33＋/p-STAT1＋ double-positive cells and HMGB1 expression within cancer nests in stage ⅢA gastric cancer patients

Objoctive： There is heterogeneity in the prognosis of gastric cancers staged according to the tumornodes-metastasis （TNM） system. This study evaluated the prognostic potential of an immune score system to supplement the TNM staging system. Mothodsg An immunohistochemical analysis was conducted to assess the density of T cells, B cells, and myeloid-derived suppressor cells （MDSCs） in cancer tissues from 100 stage IIIA gastric cancer patients; the expression of the high-mobility group protein B1 （HMGB1） was also evaluated in cancer cells. The relationship between the overall survival （OS）, disease-free survival （DFS）, and immunological parameters was analyzed.Results： An immune score system was compiled based on the prognostic role of the density ofT cells, B cells, MDSCs, and the expression of HMGB1 in cancer tissues. The median 5-year survival of this group of patient was 32%. However, the 5-year survival rates of 80.0%, 51.7%, 0%, 5.8%, and 0% varied among the patients with an immune score of 4 to those with an immune score of 0 based on the immune score system, respectively. Similarly, differences in DFS rates were observed among the immune score subgroups. Concluslons： An immune score system could effectively identify the prognostic heterogeneity within stage IliA gastric cancer patients, implying that this immune score system may potentially supplement the TNM staging system, and help in identifying a more homogeneous group of patients who on the basis of prognosis can undergo adjuvant therapy.

CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer

AIM:To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor(MIF)/Toll-like receptor 4(TLR4) in gastric cancer.METHODS:CD74,MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining.Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting.MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8(CCK8) assay and MIF concentration in the culture medium was detected by enzymelinked immunosorbent assay.Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide(LPS) was measured by flow cytometry.MIF,CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.RESULTS:CD74,MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage,and were also associated with lymph node metastasis.Correlation analysis revealed that CD74 was positively correlated with MIF(r = 0.2367,P < 0.01) and both proteins were also associated with TLR4(r = 0.4414,r = 0.5001,respectively,P < 0.01).LPS can significantly promote MKN-45 cell proliferation(3.027 ± 0.388 vs 4.201 ± 0.092,P < 0.05),induce MIF production(54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL,P < 0.01) and cell surface expression of CD74(75.6% ± 4.046%vs 9.4% ± 0.964%,P < 0.01) at LPS concentration of 1 μg/mL compared to medium control.Knockdown of CD74 or using antiCD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation(4.201 ± 0.092 vs 3.337 ± 0.087,4.534 ± 0.222 vs 3.368 ± 0.290,4.058 ± 0.292vs 2.934 ± 0.197,respectively,P < 0.01).MIF,CD74 and TLR4 could co-localize in the MKN-45 cell line.CONCLUSION:Upregulation of MIF,CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.

PEI-PEG as a siRNA Genetic Vector Demonstrating Interference in the Expression of CD44v6 Protein in Gastric Cancer Cells

OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.

CD97 promotes gastric cancer cell proliferation and invasion through exosome-mediated MAPK signaling pathway

AIM: To investigate the mechanism underlying the promoting role of CD97 in gastric cancer cell proliferation and invasion. METHODS: Two types of exosomes released by gastric cancer cells with high(SGC/wt) or low(SGC/kd) CD97 expression were isolated by ultracentrifugation and identified by electron microscopy and western blot analysis. The influences of the two exosomes on gastric cancer cell proliferation and invasion were investigated by proliferation and Matrigel invasion assays. Exosomal mi RNAs were subsequently isolated from the two samples and their mi RNA profiles were compared via microarray assay analysis. Reverse transcriptionquantitative real-time polymerase chain reaction was used to validate the microarray assay. Target genes of the differently expressed micro RNAs were predicted based on five independent algorithms and were then subjected to gene oncology enrichment and Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis. After identifying the pathway that was the most likely altered, tumor cells were treated with the two exosomes at different concentrations, and the pathway activation was identified through western blot analysis.RESULTS: Exosomes isolated from SGC/wt cells significantly promoted tumor cell proliferation in a dose-dependent manner in vitro. SGC/wt exosomesalso significantly elevated the invasiveness of both SGC/wt(129.67 ± 8.327 vs 76.00 ± 5.292, P < 0.001) and SGC/kd(114.52 ± 9.814 vs 45.73 ± 4.835, P < 0.001) cells as compared to the exosomes released by SGC/kd cells. Microarray assay of the two exosomes revealed that 62 mi RNAs were differently regulated with a signal intensity of > 500 and a false discovery rate < 0.05. The following KEGG analysis defined the MAPK signaling pathway as the most likely candidate pathway that regulated tumor cell proliferation and invasion. Through western blot analysis, significant up-regulations of phosphorylated MAPKs, including extracellular signal-regulated kinase, Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase, were detected in a dose-dependent manner in the SGC/wt exosomes treated groups, confirming activation of the MAPK signaling pathway stimulated by SGC/wt exosomes.CONCLUSION: CD97 promotes gastric cancer cell proliferation and invasion in vitro through exosomemediated MAPK signaling pathway, and exosomal mi RNAs are probably involved in activation of the CD97-associated pathway.

Assessment of vascular invasion in gastric cancer: A comparative study

AIM: To evaluate and compare detection of lymphatic and blood vessel invasion (LVI and BVI) by hematox-ylin-eosin (HE) and immunohistochemistry (IHC) in gastric cancer specimens, and to correlate with lymph node status. METHODS: IHC using D2-40 (a lymphatic endothelial marker) and CD34 (a pan-endothelial marker) was performed to study LVI and BVI in surgical specimens froma consecutive series of 95 primary gastric cancer cases. The results of the IHC study were compared with the detection by HE using McNemar test and kappa index. The morphologic features of the tumors and the presence of LVI and BVI were related to the presence of lymph node metastasis. A χ2 test was performed to obtain associations between LVI and BVI and other prognostic factors for gastric cancer. RESULTS: The detection rate of LVI was considerably higher than that of BVI. The IHC study identified eight false-positive cases and 13 false-negative cases for LVI, and 24 false-positive cases and 10 false-negative cases for BVI. The average Kappa value determined was moderate for LVI (k=0.50) and low for BVI (k=0.20). Both LVI and BVI were statistically associated with the presence of lymph node metastasis (HE: P=0.001, P=0.013, and IHC: P=0.001, P=0.019). The mor-phologic features associated with LVI were location of the tumor in the distal third of the stomach (P=0.039), Borrmann's macroscopic type (P=0.001), organ inva-sion (P=0.03) and the depth of tumor invasion (P=0.001). The presence of BVI was related only to the depth of tumor invasion (P=0.003). CONCLUSION: The immunohistochemical identification of lymphatic and blood vessels is useful for increasing the accuracy of the diagnosis of vessel invasion and for predicting lymph node metastasis.

High expression of CD11c indicates favorable prognosis in patients with gastric cancer

AIM: To determine the relationship between CD11 c expression level and prognosis in patients with gastric cancer(GC).METHODS: This retrospective survival study was performed from July 31,2008 to June 30,2014. Our study inclusion criteria included all the patients with GC who underwent surgical resection between January 1998 and December 2009 in the Third Affiliated Hospital of Soochow University. CD11 c expression levels in 140 patients with GC at different UICC stages were evaluated using immunohistochemistry,and GC tissues from 16 cases were further verified by q RTPCR. The χ2 test was used to compare the patientand disease-related factors between the low CD11 c expression group and the high expression group. Univariate probabilities of overall survival(OS) and disease-free survival(DFS) were assessed using the Kaplan-Meier method. The log rank test was used to compare survival curves. Different multivariate COX models were used to estimate the association between CD11 c expression and both death and recurrence riskin GC patients.RESULTS: The average CD11 c expression level was 5.1 ± 1.8/high power field(HPF) in 10 gastritis samples,4.5 ± 2.3/HPF in 10 gastric polyp samples and 9.7 ± 6.3/HPF in 140 gastric cancer samples,respectively. The CD11 c expression level was significantly decreased from UICC stage Ⅰ to stage Ⅳ(stage Ⅰ: 16.0 ± 7.4,stage Ⅱ: 10.4 ± 5.5,stage Ⅲ: 9.4 ± 6.1,stage Ⅳ: 5.3 ± 3.2,P < 0.001). Patients in the high CD11 c expression group had a greater 3- and 5-year OS probability and longer median survival time compared with the low CD11 c expression group,(67.7% vs 39.2%; 51.4% vs 29.0%; 67.0 mo vs 28.0 mo; χ2 = 6.80,P = 0.009),and had a greater 3- and 5-year DFS probability and longer median DFS time(63.7% vs 24.0%; 49.1% vs 11.9%; 64.0 mo vs 18.0 mo; χ2 = 15.39,P < 0.001). Patients with high CD11 c high expression had a reduced risk of death(HR = 0.56,95%CI: 0.33-0.98,P < 0.05) and relapse(HR = 0.39,95%CI: 0.23-0.67,P < 0.01) compared with patients with low CD11 c expression after adjustment of potential confounders,with the exception of tumor size. However,the protective effect related to death(HR = 0.90,95%CI: 0.49-1.67,P = 0.749) and relapse(HR = 0.65,95%CI: 0.36-1.19,P = 0.160) disappeared when tumor size was incorporated into the model.CONCLUSION: High expression of CD11 c decreased the risk of death and relapse,and may be regarded as an alternative indicator of favorable prognosis in patients with GC.

CD24 genetic variants contribute to overall survival inpatients with gastric cancer

AIM: To investigate the role of single nucleotide polymorphisms(SNPs) in CD24 gene in susceptibility and overall survival of gastric cancer(GC).METHODS: We genotyped 3 tagging SNPs of CD24-P-534 in the promoter region, P170 in the coding region of exon 2 and P1527 in the 3′ untranslated region- using polymerase chain reaction-restriction fragment length polymorphism in specimens from 679 histologically-confirmed GC cases, 111 gastric atrophy(GA) cases and 976 tumor-free controls. Serumimmunoglobulin G antibodies to Helicobacter pylori(H. pylori) of all subjects were detected by enzyme-linked immunosorbent assay. CD24 expression was evaluated by immunohistochemistry in 131 GC specimens. Correlations between SNPs and risk of GC or GA were shown by P values and odd ratios(ORs) with 95% confidence intervals(95%CI) compared with the most common genotype of each SNP using the unconditional logistic regression model after adjusting for age, sex and H. pylori infection. Survival within each SNP group was plotted by Kaplan-Meier method and compared by log-rank test(recessive model). Hazard ratios with 95%CIs were computed by Cox regression model after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy.RESULTS: All of the three loci were in Hardy-Weinberg equilibrium in the control group. Median followup time for the 600 GC patients included in the survival analysis was 36.2 mo(range, 2.1-66.7 mo; 95%CI: 34.3-36.5 mo). Patients with the P-534 A/A genotype had significantly shorter survival(HR = 1.38, 95%CI: 1.01-1.88, P = 0.042) than did the C/C or C/A genotype carriers after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy. This trend was more evident in patients who lived longer than 2.5 years(HR = 7.55, 95%CI: 2.16-26.32, P = 0.001). The P170 T/T genotype was associated with a shorter lifespan than the non-T/T genotypes, but not significantly so. None of the three genetic variants was found to be associated with risk of GC(including tumor stage, grade and distant metastasis) or with risk of gastric atrophy. Furthermore, no difference of CD24 expression was found among the genotypes.CONCLUSION: The P-534 site in CD24 gene affects the overall survival of gastric cancer and may serve as a prognostic marker for gastric cancer.

Novel CD9-targeted therapies in gastric cancer

There are 33 human tetraspanin proteins,emerging as key players in malignancy,the immune system,fertilization,cellular signaling,adhesion,morphology,motility,proliferation,and tumor invasion.CD9,a member of the tetraspanin family,associates with and influences a variety of cell-surface molecules.Through these interactions,CD9 modifies multiple cellular events,including adhesion,migration,proliferation,and survival.CD9 is therefore considered to play a role in several stages during cancer development.Reduced CD9 expression is generally related to venous vessel invasion and metastasis as well as poor prognosis.We found that treatment of mice bearing human gastric cancer cells with anti-CD9 antibody successfully inhibited tumor progression via antiproliferative,proapoptotic,and antiangiogenic effects,strongly indicating that CD9 is a possible therapeutic target in patients with gastric cancer.Here,we describe the possibility of CD9 manipulation as a novel therapeutic strategy in gastric cancer,which still shows poor prognosis.