A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal.

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Development and validation of artificial intelligence-based prescreening of large-bowel biopsies taken in the UK and Portugal: a retrospective cohort study.

BACKGROUND: Histopathological examination is a crucial step in the diagnosis and treatment of many major diseases. Aiming to facilitate diagnostic decision making and improve the workload of pathologists, we developed an artificial intelligence (AI)-based prescreening tool that analyses whole-slide images (WSIs) of large-bowel biopsies to identify typical, non-neoplastic, and neoplastic biopsies. METHODS: This retrospective cohort study was conducted with an internal development cohort of slides acquired from a hospital in the UK and three external validation cohorts of WSIs acquired from two hospitals in the UK and one clinical laboratory in Portugal. To learn the differential histological patterns from digitised WSIs of large-bowel biopsy slides, our proposed weakly supervised deep-learning model (Colorectal AI Model for Abnormality Detection [CAIMAN]) used slide-level diagnostic labels and no detailed cell or region-level annotations. The method was developed with an internal development cohort of 5054 biopsy slides from 2080 patients that were labelled with corresponding diagnostic categories assigned by pathologists. The three external validation cohorts, with a total of 1536 slides, were used for independent validation of CAIMAN. Each WSI was classified into one of three classes (ie, typical, atypical non-neoplastic, and atypical neoplastic). Prediction scores of image tiles were aggregated into three prediction scores for the whole slide, one for its likelihood of being typical, one for its likelihood of being non-neoplastic, and one for its likelihood of being neoplastic. The assessment of the external validation cohorts was conducted by the trained and frozen CAIMAN model. To evaluate model performance, we calculated area under the convex hull of the receiver operating characteristic curve (AUROC), area under the precision-recall curve, and specificity compared with our previously published iterative draw and rank sampling (IDaRS) algorithm. We also generated heat maps and saliency maps to analyse and visualise the relationship between the WSI diagnostic labels and spatial features of the tissue microenvironment. The main outcome of this study was the ability of CAIMAN to accurately identify typical and atypical WSIs of colon biopsies, which could potentially facilitate automatic removing of typical biopsies from the diagnostic workload in clinics. FINDINGS: A randomly selected subset of all large bowel biopsies was obtained between Jan 1, 2012, and Dec 31, 2017. The AI training, validation, and assessments were done between Jan 1, 2021, and Sept 30, 2022. WSIs with diagnostic labels were collected between Jan 1 and Sept 30, 2022. Our analysis showed no statistically significant differences across prediction scores from CAIMAN for typical and atypical classes based on anatomical sites of the biopsy. At 0·99 sensitivity, CAIMAN (specificity 0·5592) was more accurate than an IDaRS-based weakly supervised WSI-classification pipeline (0·4629) in identifying typical and atypical biopsies on cross-validation in the internal development cohort (p<0·0001). At 0·99 sensitivity, CAIMAN was also more accurate than IDaRS for two external validation cohorts (p<0·0001), but not for a third external validation cohort (p=0·10). CAIMAN provided higher specificity than IDaRS at some high-sensitivity thresholds (0·7763 vs 0·6222 for 0·95 sensitivity, 0·7126 vs 0·5407 for 0·97 sensitivity, and 0·5615 vs 0·3970 for 0·99 sensitivity on one of the external validation cohorts) and showed high classification performance in distinguishing between neoplastic biopsies (AUROC 0·9928, 95% CI 0·9927-0·9929), inflammatory biopsies (0·9658, 0·9655-0·9661), and atypical biopsies (0·9789, 0·9786-0·9792). On the three external validation cohorts, CAIMAN had AUROC values of 0·9431 (95% CI 0·9165-0·9697), 0·9576 (0·9568-0·9584), and 0·9636 (0·9615-0·9657) for the detection of atypical biopsies. Saliency maps supported the representation of disease heterogeneity in model predictions and its association with relevant histological features. INTERPRETATION: CAIMAN, with its high sensitivity in detecting atypical large-bowel biopsies, might be a promising improvement in clinical workflow efficiency and diagnostic decision making in prescreening of typical colorectal biopsies. FUNDING: The Pathology Image Data Lake for Analytics, Knowledge and Education Centre of Excellence; the UK Government's Industrial Strategy Challenge Fund; and Innovate UK on behalf of UK Research and Innovation.

Screening of normal endoscopic large bowel biopsies with interpretable graph learning: a retrospective study.

OBJECTIVE: To develop an interpretable artificial intelligence algorithm to rule out normal large bowel endoscopic biopsies, saving pathologist resources and helping with early diagnosis. DESIGN: A graph neural network was developed incorporating pathologist domain knowledge to classify 6591 whole-slides images (WSIs) of endoscopic large bowel biopsies from 3291 patients (approximately 54% female, 46% male) as normal or abnormal (non-neoplastic and neoplastic) using clinically driven interpretable features. One UK National Health Service (NHS) site was used for model training and internal validation. External validation was conducted on data from two other NHS sites and one Portuguese site. RESULTS: Model training and internal validation were performed on 5054 WSIs of 2080 patients resulting in an area under the curve-receiver operating characteristic (AUC-ROC) of 0.98 (SD=0.004) and AUC-precision-recall (PR) of 0.98 (SD=0.003). The performance of the model, named Interpretable Gland-Graphs using a Neural Aggregator (IGUANA), was consistent in testing over 1537 WSIs of 1211 patients from three independent external datasets with mean AUC-ROC=0.97 (SD=0.007) and AUC-PR=0.97 (SD=0.005). At a high sensitivity threshold of 99%, the proposed model can reduce the number of normal slides to be reviewed by a pathologist by approximately 55%. IGUANA also provides an explainable output highlighting potential abnormalities in a WSI in the form of a heatmap as well as numerical values associating the model prediction with various histological features. CONCLUSION: The model achieved consistently high accuracy showing its potential in optimising increasingly scarce pathologist resources. Explainable predictions can guide pathologists in their diagnostic decision-making and help boost their confidence in the algorithm, paving the way for its future clinical adoption.

High expression of SMARCC1 predicts poor prognosis in gastric cancer patients.

The switching/sucrose non-fermenting (SWI/SNF) chromatin remodeling complexes use the energy of ATP hydrolysis to remodel nucleosomes and modulate transcription, which plays an important role in tumors by regulating epigenetics. SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, Subfamily C, Member 1 (SMARCC1) has dual roles in tumors but its role in gastric cancer remains unclear. This study was aimed to find the role of SMARCC1 in gastric cancer. SMARCC1 expression across various tumors from The Cancer Genome Atlas was analyzed using TIMER 2.0 (http://timer.comp-genomics.org/). SMARCC1 mRNA expression profiles in gastric cell lines and gastric tissues were compared with normal tissues and analyzed in the Cancer Cell Line Encyclopedia, Oncomine, and Gene Expression Omnibus databases. SMARCC1 mRNA and protein were then examined in fresh gastric cancer tissues and compared with adjacent normal tissues using quantitative real-time PCR, western blotting, and immunohistochemistry. Associations between SMARCC1 expression and clinicopathological factors, overall survival, and disease-free survival were further evaluated using 130 gastric cancer samples harvested from patients after radical total gastrectomy or subtotal gastrectomy at the Xiangya Hospital of Central South University (Changsha, China). SMARCC1 was frequently upregulated in gastric cancer cells and tissues. SMARCC1 overexpression was significantly associated with tumor size (P=0.002), differentiation (P=0.006), depth of invasion (P=0.001), lymph node involvement (P=0.016), and TNM stage (P=0.007). Furthermore, univariate and multivariate Cox analysis revealed that high SMARCC1 expression, depth invasion, lymph node involvement, and TNM stage were independent risk factors for both overall and disease-free survival in gastric cancer patients (all P<0.05). Kaplan-Meier survival analysis revealed that high SMARCC1 expression predicted poor prognosis in gastric cancer patients (P<0.01). High SMARCC1 expression contributes to poor prognosis in gastric cancer patients. SMARCC1 may be a prognostic biomarker and therapeutic target in gastric cancer.

Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia.

Resistance to chemotherapy is frequently driven by aberrantly activated kinases in cancer. Herein, we characterized the global phosphoproteomic alterations associated with methotrexate (MTX) resistance in gestational trophoblastic neoplastic (GTN) cells. A total of 1111 phosphosites on 713 proteins were significantly changed, with highly elevated Ribosomal S6 Kinase 2 (RSK2) phosphorylation (pS227) observed in MTX-resistant GTN cells. Activation of RSK2 promoted cell proliferation and survival after MTX treatment in GTN cell models. Interestingly, RSK2 might play an important role in the regulation of reactive oxygen species (ROS) homeostasis, as manipulation of RSK2 activation affected ROS accumulation and SOX8 expression in GTN cells. In addition, overexpression of SOX8 partly rescued cell proliferation and survival in RSK2-depleted MTX-resistant GTN cells, suggesting that SOX8 might serve as a downstream effector of RSK2 to promote MTX resistance in GTN cells. Highly activated RSK2/SOX8 signaling was observed in MTX-resistant GTN specimens. Further, the RSK2 inhibitor BIX02565 effectively reduced SOX8 expression, induced ROS accumulation, and enhanced MTX-induced cytotoxicity in vitro and in vivo. Collectively, our findings suggested that RSK2 activation could promote MTX resistance via upregulating SOX8 and attenuating MTX-induced ROS in GTN cells, which may help to develop experimental therapeutics to treat MTX-resistant GTN.

Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown. METHODS: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. RESULTS: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. CONCLUSIONS: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Video Abstract.

FOXC2 Promotes Oxaliplatin Resistance by Inducing Epithelial-Mesenchymal Transition via MAPK/ERK Signaling in Colorectal Cancer.

BACKGROUND: Chemoresistance is a major obstacle to improving the survival rate of colorectal cancer (CRC) patients. Forkhead box protein C2 (FOXC2), a member of the forkhead box (Fox) transcription factor family, is reported to be an important regulator of epithelial-to-mesenchymal transition (EMT) and plays a key role in tumor progression. However, little is known about the effects of FOXC2 on oxaliplatin (OXA) resistance in CRC. METHODS: OXA-resistant cells were generated from HCT116 cells. CCK-8, colony formation, flow cytometry and Transwell assays were used to compare the characteristics of OXA-resistant HCT116/OXA cells and the corresponding parental HCT116 cells. The expression of FOXC2 was confirmed by qRT-PCR and Western blotting in HCT116/OXA and HCT116 cells. Gain- and loss-of-function assays were performed to evaluate the effects of FOXC2 on OXA sensitivity and EMT in HCT116/OXA and HCT116 cells both in vitro and in vivo, and the possible molecular mechanisms were investigated. RESULTS: The relative expression of FOXC2 was significantly increased in HCT116/OXA cells compared with the parental HCT116 cells. Upregulation of FOXC2 in HCT116 cells reduced OXA sensitivity and promoted EMT. However, knockdown of FOXC2 in HCT116/OXA cells markedly increased the in vitro and in vivo sensitivity of HCT116/OXA cells to OXA by regulating EMT progression. Furthermore, FOXC2 activated MAPK/ERK signaling, and blockade of ERK attenuated FOXC2-induced EMT and FOXC2-enhanced OXA resistance. CONCLUSION: FOXC2 induced EMT to promote oxaliplatin resistance by activating the MAPK/ERK signaling pathway. FOXC2 may be a potential therapeutic target for overcoming OXA resistance in human CRC.

Gene expression and methylation profiles identified CXCL3 and CXCL8 as key genes for diagnosis and prognosis of colon adenocarcinoma.

Colon adenocarcinoma (COAD) is one of the most common malignant tumors with high morbidity and mortality rates worldwide. Due to the poor clinical outcomes, it is indispensable to investigate novel biomarkers for the diagnosis and prognosis of COAD. The aim of this study is to explore key genes as potential biomarkers for the diagnosis and prognosis of COAD for clinical utility. Gene expression profiles (GSE44076 and GSE44861) and gene methylation profile (GSE29490) were analyzed to identify the aberrantly methylated-differentially expressed genes by R language and Perl software. Function enrichments were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Moreover, hub genes were identified through protein-protein interaction (PPI) network. Besides, key genes were found by the module analysis and The Cancer Genome Atlas (TCGA) survival analysis. Finally, TCGA data and quantitative real-time polymerase chain reaction (RT-qPCR) was used to validate the key genes involved in COAD. Our study found two hypomethylation-high-expression genes (CXCL3 and CXCL8) in COAD tissues compared with the adjacent normal tissues. These results were also confirmed by RT-qPCR with 25 pairs of COAD and adjacent normal tissues. Meanwhile, low expression of the two genes was associated with poor survival in patients with COAD. CXCL3 and CXCL8 may serve as key genes in the diagnosis and prognosis for COAD.

Diagnostic potential of ionomic profile in the plasma of cervical cancer patients receiving neoadjuvant chemoradiotherapy.

BACKGROUND AND AIM: Major and trace elements play an important role in human body, and it has been reported that ionomic distribution differ greatly in tumor patients. The aim of the present study was to investigate the effects of cisplatin-based neoadjuvant chemoradiotherapy on the ionomic profile in human plasma as a potential biomarker for the therapeutic effects of cervical cancer. METHOD: Thirty-seven patients with cervical cancer receiving neoadjuvant chemoradiotherapy were included in this study, pretherapy and post-treatment blood samples were collected and concentrations of 24 ions were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The results showed that after cisplatin chemotherapy and radiotherapy, patients' plasma Pt level significantly increased, Na, Mg, P, K, Ca, Se, Cu, Zn, Se, Sr, Ba levels significantly decreased (P < 0.01), and Al, Cu ions were significantly correlated with the treatment effect (P < 0.05). In addition, the pattern of elemental correlations changed dramatically after the neoadjuvant chemoradiotherapy. CONCLUSION: The results indicated that the plasma ionomic profile may serve as a quick and convenient tool to reflect the therapeutic effect of cisplatin-based chemoradiotherapy in cervical cancer patients, and supplement of certain essential elements may be of great importance for the maintenance of ion homeostasis in human body and for the reduction of adverse effect of chemotherapy and radiotherapy.

Vps4A mediates the localization and exosome release of ß-catenin to inhibit epithelial-mesenchymal transition in hepatocellular carcinoma.

We previously reported that Vps4A acted as a tumor suppressor by influencing the microRNA profiles of exosomes and their parental cells in hepatocellular carcinoma (HCC). However, the underlying mechanism and if Vps4A contributes to sorting proteins into exosomes are not well known. Here, we performed mass spectrometry analysis of the immunoprecipitated Vps4A complex and confirmed that Vps4A was associated with ß-catenin and CHMP4B. Through this interaction, Vps4A promoted the plasma membrane (PM) localization and exosome release of ß-catenin. Silencing Vps4A or CHMP4B decreased the PM localization and exosome sorting of ß-catenin. Vps4A overexpression decreased ß-catenin signaling pathway and inhibited epithelial-mesenchymal transition (EMT) and motility of HCC cells. And, silencing Vps4A or CHMP4B promoted EMT in HCC. Furthermore, the expression of Vps4A was significantly related to that of several EMT markers in HCC tissues and the level of exosomal ß-catenin in patients with metastatic HCC was significantly lower compared to that of control patients. In conclusion, through the interaction with CHMP4B and ß-catenin, Vps4A regulates the PM localization and exosome sorting of ß-catenin, consequently decreases ß-catenin signaling, and thereby inhibits EMT and metastasis in HCC.

Multiple Comparisons of the Efficacy and Safety for Seven Treatments in Tibia Shaft Fracture Patients.

Background: A tibia shaft fracture is one of the most common long bone fractures, with two general types, open fracture and close fracture. However, there is no universally accepted guideline suggesting which treatment to use under certain circumstances. Therefore, a comprehensive network meta-analysis (NMA) is needed to summarize existing studies and to provide more credible data-based medical guidelines. Methods: Available literature was identified by searching medical databases with relevant key terms. Studies that met the inclusion and exclusion criteria, baseline, intervention, and the outcome of treatments, were extracted. A comparative connection of these studies was demonstrated through net plots. Continuous variables and binary variables were reported as mean difference (MD) and odds ratio (OR) with a 95% credible interval (CrI), respectively. The comparison of direct and indirect outcome and their P-value were listed in the node-splitting table. Treatments for each endpoint were ranked by their surface under the cumulative ranking curve (SUCRA) value. A heat plot was created to illustrate the contribution of raw data and the inconsistency between direct and indirect comparisons. Results: According to the search strategy, 697 publications were identified, and 25 records were included, involving 3,032 patients with tibia shaft fractures. Seven common surgical or non-surgical treatments, including reamed intramedullary nailing (RIN), un-reamed intramedullary nailing (UIN), minimally reamed intramedullary nailing (MIN), ender nailing (EN), external fixation (EF), plate, and cast, were compared, in terms of time to union, reoperation, non-union, malunion, infection and implant failure. Plate performed relatively better for time to union, while cast might be the best choice in close cases to reduce the risks of reoperation, non-union, malunion, and infection. To prevent implant failure, EN seemed to be better. Conclusion: Cast might have the highest probability of the most optimal choice for tibia shaft fracture in close cases, while reamed intramedullary nailing ranked second.

Potential Prognostic and Diagnostic Values of CDC6, CDC45, ORC6 and SNHG7 in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is a common human malignancy. The aims of this study are to investigate the gene expression profile of CRC and to explore potential strategy for CRC diagnosis, therapy and prognosis. METHODS: We use affy and Limma package of Bioconductor R to do differential expression genes (DEGs) and differential expression lncRNAs (DELs) analysis from the gene datasets (GSE8671, GSE21510, GSE32323, GSE39582 and TCGA) respectively. Then, DEGs were analyzed by GO and KEGG pathway and Kaplan-Meier survival curve and Cox regression analyses were used to find aberrantly expressed genes associated with survival outcome of CRC patients. Real-time PCR assay was used to verify the aberrantly expressed genes expression in CRC samples. RESULTS: 306 up-regulation and 213 down-regulation common DEGs were found. A total of 485 DELs were identified, of which 241 up-regulated and 244 down-regulated. Then, GO and KEGG pathway analyses showed that DEGs were involved in cell cycle, mineral absorption, DNA replication, and Nitrogen metabolism. Among them, Kaplan-Meier survival curve and Cox regression analyses revealed that CDC6, CDC45, ORC6 and SNHG7 levels were significantly associated with survival outcome of CRC patients. Finally, real-time PCR assay was used to verify that the CDC6, CDC45, ORC6 and SNHG7 expression were up-regulated in 198 CRC samples compared with the expression levels in individual-matched adjacent mucosa samples. CONCLUSION: CDC6, CDC45, ORC6 and SNHG7 are implicated in CRC initiation and progression and could be explored as potential diagnosis, therapy and prognosis targets for CRC.

Primary Gastric ALK-negative EBV-negative Anaplastic Large Cell Lymphoma Presenting with Iron Deficiency Anemia.

Anaplastic large cell lymphoma (ALCL) is a rare subtype of non-Hodgkin lymphoma (NHL). Primary gastric anaplastic lymphoma kinase (ALK) negative ALCL is extremely rare. Diagnosis of primary gastric ALK-negative ALCL is difficult to establish and prognosis is worse than ALK-positive ALCL. Here, we report a case of an 82-year-old man with a history of cerebrovascular disease presented with weakness and iron deficiency anemia. He denied any abdominal discomforts. The esophagogastroduodenoscopy revealed a large ulcerated, friable mass in the gastric body which encompassed about 80% of entire stomach. Biopsy showed a high grade malignant tumor composed of undifferentiated epithelioid atypical cells, making it difficult to determine the cell of origin. Immunostains for lymphoma, carcinoma, and sarcoma were performed. The tumor cells were positive for CD30, CD4, and CD43, negative for CD20, CD3, ALK-1 and Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) in situ hybridization, establishing the diagnosis of primary gastric ALK-negative ALCL. The patient is currently undergoing chemotherapy with clinical improvement. To the best of our knowledge, this is the first reported case of primary gastric ALK-negative and EBV-negative anaplastic large T-cell lymphoma that presented without gastroenterological symptoms.

Vasoactive intestinal peptide represses activation of tumor-associated macrophages in gastric cancer via regulation of TNFα, IL-6, IL-12 and iNOS.

Vasoactive intestinal peptide (VIP) has been regarded as deactivator for macrophages. However, the depressive effect of VIP on tumor-associated macrophages (TAM) has not been recognized. In the present study, we investigated the effect of VIP on gastric cancer via TAM by suppressing expression levels of TNFα, IL-6, IL-12 and iNOS. Real-time PCR was carried out to examine the expression of CD68 to determine the levels of TAM. The effect of VIP on cell activities was assayed by proliferation assay, colony formation and flow cytometry analysis. The co-culture of TAM and human gastric cancer cell line MKN-45 were performed to understand whether the VIP affects the gastric cancer cells via TAM. Further, the tumor formation in a nude mouse model and VIP injection were performed to illustrate the effect on tumor progression in vivo. CD68 was high expressed in gastric cancer indicating high level of TAM in gastric cancer. Treatment with VIP significantly depressed TAM activation. Moreover, the expression of TNFα, IL-6, IL-12 and iNOS in TAM were depressed by VIP treatment, and the VIP treated TAM depressed gastric cancer cells. The experiment in the nude mouse model also suggested that by injection with TAM+VIP, the tumor volume and tumor weight were both decreased significantly. These data suggest that treatment with VIP inhibits gastric cancer.

[Sensitivity study on preoperative individual concomitant radiochemotherapy for rectal cancer].

OBJECTIVE: To explore the predictive factors of sensitivity to preoperative concomitant radiochemotherapy for the local mid-low advanced rectal cancer in order to guide the individualized therapy. METHODS: Clinicopathologic data of 44 patients with local mid-low advanced rectal cancer receiving preoperational concomitant radiochemotherapy were retrospectively analyzed. Expression of epidemical growth factor receptor (EGFR) in biopsy specimen was detected with SP immunohistochemistry (IHC). Downstaging of tumor TNM stage and tumor regression grade (TRG) after radiochemotherapy were used as the standards of sensitivity to preoperational concomitant radiochemotherapy. Association of EGFR expression and pathological change with clinicopathological data before radiochemotherapy (gender, age, pathological type, tumor TNM stage, serum CEA, CA199, radiation method, etc) was analyzed. RESULTS: Percentage of downstaging of tumor TNM stage and 3-4 TRG in patients with negative or weak positive EGFR expression was significantly higher as compared to those with strong and moderate positive EGFR expression [86.7% (13/15) vs. 30.4% (7/23), P<0.01; 80.0% (12/15) vs. 8.7% (2/23), P<0.01]. Percentage of downstaging of tumor TNM stage and 3-4 TRG in patients with tubular adenocarcinoma was significantly higher as compared to those with mucous adenocarcinoma [61.8% (21/34) vs. 10.0% (1/10), P<0.01; 47.1 (16/34) vs. 0 (0/10), P<0.01]. EGFR expression was not associated with pathological type (P>0.05). Sensitivity to preoperative concomitant radiochemotherapy was not associated with age, gender, tumor stage, tumor differentiation, serum CEA, serum CA199 and radiation method (all P>0.05). CONCLUSIONS: Pathological type and EGFR expression level may be two independent predictive markers of sensitivity to preoperative concomitant radiochemotherapy for patients with rectal cancer. Patients with tubular adenocarcinoma or low EGFR expression in tumor tissue may be more sensitive to concomitant radiochemotherapy.

Predictive factors for the sensitivity of radiotherapy and prognosis of esophageal squamous cell carcinoma.

PURPOSE: To identify predictive biomarkers for radiosensitization and prognosis of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: A total of 150 advanced stage ESCC patients were treated with preoperative radiotherapy. The protein levels of Dicer 1, DNA methyltransferase 1 (Dnmt1), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the mRNA levels of Dicer 1, Dnmt1, and let-7b microRNA (miRNA) were measured in ESCC tumor tissues before and after radiotherapy. Global DNA methylation was measured and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed. RESULTS: Negative Dicer 1, Dnmt1, and DNA-PKcs protein expression were observed in 72%, 67.3%, and 50.7% of ESCC patients, respectively. Primary Dicer 1 and Dnmt1 expression positively correlated with radiation sensitization and longer survival of ESCC patients, while increased Dicer 1 and Dnmt1 expression after radiation correlated with increased apoptosis in residual tumor tissues. Dicer 1 and Dnmt1 expression correlated with let-7b miRNA expression and global DNA methylation levels, respectively. In contrast, positive DNA-PKcs expression negatively correlated with radiation-induced pathological reactions, and increased DNA-PKcs expression correlated with increased apoptosis after radiation. CONCLUSION: Global DNA hypomethylation and low miRNA expression are involved in the sensitization of ESCC to radiotherapy and prognosis of patients with ESCC.

Autophagy of cancer stem cells is involved with chemoresistance of colon cancer cells.

BACKGROUND: Chemoresistance is a major cause of treatment failure in colon cancer, and cancer stem cells have been found to be involved in the chemoresistance of colon cancer. However, the mechanisms driving the chemoresistance of colon cancer stem cells have not been addressed. METHODS: In this study, we investigated the cytotoxicity of paclitaxel in CD44(+)CD24(+) SW1222 colon cancer cells expressing Cdx1 (CD44(+)CD24(+)Cdx1(+) stem cells) and CD44(+)CD24(+) HCT116 colon cancer cells expressing wild-type p53 (CD44(+)CD24(+)p53wt stem cells). RESULTS: SW1222 cells were more resistant to paclitaxel-induced cytotoxicity than HCT116 cells. Conversely, HCT-116 cells had higher matrigel colony formation ability than SW1222 cells. The isolated CD44(+)CD24(+)Cdx1(+) cells showed higher resistance to paclitaxel-induced cytotoxicity than CD44(+)CD24(+)p53wt cells. The resistance of CD44(+)CD24(+)Cdx1(+) cells to paclitaxel is associated with upregulation of Cdx1 and Bcl-2 expression, caspase-3 activity, and the ratio of LC3-II/LC3-I. The sensitivity of CD44(+)CD24(+)p53wt cells to paclitaxel is associated with the downregulation of Bcl-2 expression, upregulation of Bax levels, and upregulation of caspase-3 activity. Silencing of Cdx1 expression and treatment with lysosomal inhibitor bafilomycin A increased paclitaxel-induced cytotoxicity in CD44(+)CD24(+)Cdx1(+) cells. Conversely, overexpression of Cdx1 decreased cell death in CD44(+)CD24(+)p53wt cells. Intratumoral injection of Cdx1 siRNA significantly inhibited tumor growth in a xenograft tumor model inoculated with CD44(+)CD24(+)Cdx1(+) cancer cells. CONCLUSION: Cdx1 exerts a protective role in colon cancer stem cells, which play a crucial role in chemoresistance to paclitaxel through activation of autophagy. Autophagy is activated though the Cdx1-Bcl-2-LC3 pathway. In contrast, p53 exerts a major role in apoptosis and inhibits autophagy in colon cancer stem cells.

Overexpression of EphA2 correlates with epithelial-mesenchymal transition-related proteins in gastric cancer and their prognostic importance for postoperative patients.

The expression of EphA2 and three epithelial-mesenchymal transition-related proteins (E-cadherin, ß-catenin and vimentin) was detected by immunohistochemistry in human gastric cancer and normal gastric mucosa. The expression of EphA2 and vimentin was significantly higher in gastric cancer tissues than in normal gastric mucosa tissues, and similar results were found for negative E-cadherin expression and ectopic ß-catenin expression. Further analysis showed that the expression of EphA2 was closely correlated with the depth of tumor invasion, tumor-node-metastasis (TNM) stages and lymph node metastasis. Down-regulated expression of the epithelial protein E-cadherin, overexpression of the mesenchymal protein vimentin and ectopic expression of ß-catenin were associated with the depth of tumor invasion, tumor differentiation, TNM stages and lymph node metastasis. The Spearman rank test indicated that the positive expression of EphA2 was negatively associated with E-cadherin expression and was positively correlated with ß-catenin ectopic expression and vimentin expression. In addition, the Kaplan-Meier survival analysis showed that the overexpression of EphA2 and vimentin, ectopic expression of ß-catenin and down-regulation of E-cadherin indicate a poor outcome. Moreover, multivariate Cox analysis showed that TNM stages, lymph node metastasis, EphA2 expression, E-cadherin expression and ß-catenin ectopic expression were independent prognostic factors for postoperative gastric cancer. These findings indicate that the overexpression of EphA2 correlates with the loss of epithelial proteins and the appearance of mesenchymal proteins. Therefore, EphA2 may play a role in epithelial-mesenchymal transition in gastric cancer.

Overexpression of G6PD is associated with poor clinical outcome in gastric cancer.

This study aims to investigate the expression and significance of glucose-6-phosphate dehydrogenase (G6PD) in human gastric cancer progression and prognosis. Using immunohistochemistry and real-time RT-PCR assay, we identified abnormally elevated expression of G6PD in gastric cancer tissues compared to paired normal stomach mucosa tissues in 24 patients (p < 0.05). In order to investigate the correlations between G6PD and the clinicopathological features of gastric cancer, the expression of G6PD in 167 patients with gastric cancer were detected by immunohistochemistry, and the results showed that overexpression of G6PD was associated with the size of tumor (p = 0.039), depth of invasion (p = 0.039), lymph node metastasis (p = 0.044), distant metastasis (p = 0.003), TNM stage (p = 0.030), and survival rate (p = 0.010). Further, Cox multivariates analysis indicated that G6PD expression level was an independent prognostic factor for patients after radical resection (p = 0.013). In conclusion, overexpression of G6PD is closely related to progression of gastric cancer, and might be regarded as an independent predictor of poor prognosis for gastric cancer.

Silencing of TKTL1 by siRNA inhibits proliferation of human gastric cancer cells in vitro and in vivo.

Tumorigenesis requires energy production via aerobic glycolysis (Warburg effect) in malignant tumors. Recent research has demonstrated that the pentose phosphate pathway (PPP) is augmented in some tumors, especially the non-oxidative part of the PPP which is controlled by transketolase (TKT) enzyme reactions. One TKT isoform, transketolase-like protein 1 (TKTL1), is specifically upregulated in different human cancers, and its overexpression predicts poor patient survival. To further define the function of in malignant progression, we employed the small interference RNA (siRNA) technique to knockdown gene expression of TKTL1 in the gastric cancer cell line AGS. We used TKTL1 siRNA to observe the effect of reduced TKTL1 expression on gastric cancer tumorigenesis in a nude mice xenograft model and on proliferation in vitro. Our results showed that the expression of double stranded RNA led to the efficient and specific inhibition of endogenous TKTL1 expression in AGS cells. In addition, the TKT activity was significantly deceased in the TKTL1 siRNA-treated AGS cells. TKTL1 suppression resulted in delayed cell proliferation in vitro. Furthermore, loss of TKTL1 inhibited the growth of AGS tumor xenografts. Altogether, our findings indicate that the specific inhibition of TKTL1 may be important therapeutically.