Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment.

OBJECTIVE: Innate immunity plays important roles in pancreatic ductal adenocarcinoma (PDAC), as non-T-cell-enriched tumour. Neutrophils are major players in innate immune system. Here, we aimed to explore the heterogeneity and pro-tumour mechanisms of neutrophils in PDAC. DESIGN: We analysed single-cell transcriptomes of peripheral blood polymorphonuclear leucocytes (PMNs) and tumour-infiltrating immune cells from five patients with PDAC, and performed immunofluorescence/immunohistochemistry staining, multi-omics analysis and in vitro experiments to validate the discoveries of bioinformatics analysis. RESULTS: Exploration of the heterogeneity of tumour-associated neutrophils (TANs) revealed a terminally differentiated pro-tumour subpopulation (TAN-1) associated with poor prognosis, an inflammatory subpopulation (TAN-2), a population of transitional stage that have just migrated to tumour microenvironment (TAN-3) and a subpopulation preferentially expressing interferon-stimulated genes (TAN-4). Glycolysis signature was upregulated along neutrophil transition trajectory, and TAN-1 was featured with hyperactivated glycolytic activity. The glycolytic switch of TANs was validated by integrative multi-omics approach of transcriptomics, proteomics and metabolomics analysis. Activation of glycolytic activity by LDHA overexpression induced immunosuppression and pro-tumour functions in neutrophil-like differentiated HL-60 (dHL-60) cells. Mechanistic studies revealed BHLHE40, downstream to hypoxia and endoplasmic reticulum stress, was a key regulator in polarisation of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by chromatin immunoprecipitation assay. Pro-tumour and immunosuppression functions were observed in dHL-60 cells overexpressing BHLHE40. Importantly, immunohistochemistry analysis of PDAC tissues revealed the unfavourable prognostic value of BHLHE40+ neutrophils. CONCLUSION: The dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

Using protein microarray to identify and evaluate autoantibodies to tumor-associated antigens in ovarian cancer.

The aim of this study was to develop a noninvasive serological diagnostic approach in identifying and evaluating a panel of candidate autoantibodies to tumor-associated antigens (TAAs) based on protein microarray technology for early detection of ovarian cancer (OC). Protein microarray based on 154 proteins encoded by 138 cancer driver genes was used to screen candidate anti-TAA autoantibodies in a discovery cohort containing 17 OC and 27 normal controls (NC). Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the content of candidate anti-TAA autoantibodies in sera from 140 subjects in the training cohort. Differential anti-TAA autoantibodies were further validated in the validation cohort with 328 subjects. Subsequently, 112 sera from the patients with ovarian benign diseases with 104 OC sera and 104 NC sera together were recruited to identify the specificity of representative autoantibodies to OC among ovarian diseases. Five TAAs (GNAS, NPM1, FUBP1, p53, and KRAS) were screened out in the discovery phase, in which four of them presented higher levels in OC than controls (P < .05) in the training cohort, which was consistent with the result in the subsequent validation cohort. An optimized panel of three anti-TAA (GNAS, p53, and NPM1) autoantibodies was identified to have relatively high sensitivity (51.2%), specificity (86.0%), and accuracy (68.6%), respectively. This panel can identify 51% of OC patients with CA125 negative. This study supports our assumption that anti-TAA autoantibodies can be considered as potential diagnostic biomarkers for detection of OC; especially a panel of three anti-TAA autoantibodies could be a good tool in immunodiagnosis of OC.

Using a panel of multiple tumor-associated antigens to enhance the autoantibody detection in the immunodiagnosis of ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a major malignancy affecting a large population over the world, and a biomarker that holds diagnostic potential is of critical importance. Recently, autoantibodies have been indicated as biomarkers in multiple cancer research. The current study was designed to explore the practice of using autoantibodies in diagnostic settings by the enzyme-linked immunosorbent assay of sera with a panel of tumor-associated antigens (TAAs). METHODS: A panel of 12 TAAs was selected to detect the corresponding autoantibodies in sera sampled from 132 OC patients as case group and 147 normal healthy individuals as the control group. The diagnostic potential of this panel was evaluated by conventional evaluation, receiver operating characteristic (ROC) curve analyses, and classification tree analysis. RESULTS: When the cutoff values were set as mean ± 2 SD for normal healthy individuals, the positive rates of antibodies to any single TAA were less than 20% both in OC and in normal healthy individuals. In a parallel screening approach, a panel of nine TAAs (p53, C-myc, p90, p62, AHSG, 14-3-3zeta, RalA, Koc, and p16), obtained optimal diagnostic performance in OC with the sensitivity of 61.4% at the 85.0% specificity. In addition, when the nine TAAs were combined with CA125, the sensitivity and specificity were improved to 94.7% and 78.2%, respectively. The ROC curve analyses showed that only the area under the receiver operating characteristic curves (AUCs) of antibodies against C-myc, Koc, and RalA was beyond 0.6, which were 0.732, 0.668, and 0.665, respectively. The AUC of the combination was up to 0.914 (P < 0.05). Decision tree analysis showed that C-myc, HCC1.3, RalA, and CA125 held high potential in the detection of OC. The panel of nine TAAs also identified 78.8% of OC patients who had normal CA125 levels in their serum samples, indicating that elevated CA125 and anti-TAA antibodies appeared to be independent but supplementary biomarkers for diagnosing OC. CONCLUSIONS: In summary, the current study further supports that a customized TAA panel can serve as a promising and powerful tool for immunodiagnosis of OC and may be particularly useful in patients with normal CA125 levels.

Using recursive partitioning approach to select tumor-associated antigens in immunodiagnosis of gastric adenocarcinoma.

The present study aimed to select anti-tumor-associated antigen (TAA) autoantibodies as biomarkers in the immunodiagnosis of gastric adenocarcinoma (GAC) by the recursive partitioning approach (RPA) and further construct and evaluate a predictive model. A case-control study was designed including 407 GAC patients as the case group and 407 normal controls. In addition, 67 serial serum samples from 25 GAC patients were collected at different time points before and after gastrectomy treatment. Autoantibodies against 14 TAA were measured in sera from all subjects by enzyme immunoassay. Finally, RPA resulted in the selection of nine-panel TAA (c-Myc, p16, HSPD1, PTEN, p53, NPM1, ENO1, p62, HCC1.4) from all detected TAA in the case-control study; the classification tree based on this nine-TAA panel had area under curve (AUC) of 0.857, sensitivity of 71.5% and specificity of 71.3%; The optimal panel also can identify GAC patients at an early stage from normal individuals, with AUC of 0.737, sensitivity of 64.9% and specificity of 70.5%. However, frequencies of the nine autoantibodies showed no correlation with GAC stage, tumor size, lymphatic metastasis or differentiation. GAC patients positive for more than two autoantibodies in the nine-TAA panel had a worse prognosis than that of the GAC patients positive for no or one antibody. Titers of 10 autoantibodies in serial serum samples were significantly higher in GAC patients after surgical resection than before. In conclusion, this study showed that the panel of nine multiple TAAs could enhance the detection of anti-TAA antibodies in GAC, and may be potential prognostic biomarkers in GAC.

MicroRNA-155 inversely correlates with esophageal cancer progression through regulating tumor-associated macrophage FGF2 expression.

Esophageal cancer (EC) is one of the most common malignancies with high incidence and mortality. Tumor-associated macrophages (TAMs) in the tumor microenvironment have been linked to the accelerated tumor progression. MicroRNAs (miR) are 19-25 nucleotide-long, noncoding RNA molecules, functioning as modulators of gene expression, and mediate a variety of biological functions, including tumor growth. In the present study, the effects and molecular mechanism of miR-155 in TAMs isolated from EC were explored. The expression of miR-155 and fibroblast growth factor-2 (FGF2) in EC tissues and cell lines were analyzed using reverse transcription-quantitative PCR (qRT-PCR) and western blot assays. TAMs were also transfected with the described constructs. Following, the culture medium from TAMs was collected for further analysis. The released FGF2, and inflammatory cytokines were quantified using ELISA. The cell viability, migrated and invaded levels were calculated through Cell Counting kit-8 (CCK8), and transwell analysis. Moreover, human umbilical vein endothelial cells (HUVEC) vasculature formation was determined using matrigel angiogenesis analysis. The results indicated that miR-155 expression was decreased in EC tissues and cell lines, while FGF2 expression was increased in comparison to those in the normal control group. Moreover, miR-155 mimics transfection up-regulated tumor necrosis factor α (TNF-α), interleukin (IL)-12 and inducible nitric oxide synthase (iNOS), while down-regulated IL-10, Arginase-1 (Arg-1) and IL-22 levels in the culture medium from TAMs. And enhancing miR-155 expression in TAMs suppressed the cell viability, migration and invasion of ECA109 cells and reduced the angiogenesis. Nevertheless, over-expressing FGF2 abolished the role of miR-155 in cancer cell survival, migration, invasion as well as angiogenesis. Our findings indicated that miR-155-regulated FGF2 expression from TAMs suppressed EC cell proliferation, migration, invasion and inhibited vasculature formation. Thus, miR-155-modulated FGF2 might be a potential therapeutic target to prevent EC progression.

A panel of autoantibodies against multiple tumor-associated antigens in the immunodiagnosis of esophageal squamous cell cancer.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China with very low 5-year survival rate mostly due to the paucity of effective early diagnostic methods. Serum autoantibodies against 9 tumor-associated antigens (TAAs) from ESCC patients and healthy controls were detected by enzyme-linked immunosorbent assay to evaluate their performances in the immunodiagnosis of ESCC. Logistic regression models were generated to predict the probability of individuals being diagnosed with ESCC in training cohort (648 participants) and further validated in another independent cohort (372 participants). Finally, a panel of four TAAs showed high diagnostic accuracy with areas under the receiver operating characteristic curve of 0.838 in training cohort and 0.872 in validation cohort, respectively. The percentages of individuals correctly classified were 77.01 % in training cohort and 78.49 % in validation cohort, respectively. This model could discriminate early-stage (AJCC stage 0, I and II) ESCC patients from normal controls, with true-positive rate (TPR) of 67.57 % in training cohort and TPR of 63.33 % in validation cohort, and the overall TPR for early-stage ESCC was 66.85 % when the two cohorts were combined. The diagnostic performance of this model showed no significant difference between early-stage and late-stage (AJCC stage III and IV) ESCC patients. In summary, the optimized model with 4 TAAs has a high diagnostic performance for ESCC detection, especially for early-stage ESCC.

BHLHE40 Inhibits Ferroptosis in Pancreatic Cancer Cells via Upregulating SREBF1.

Pancreatic cancer (PCa) is one of the most fatal human malignancies. The enhanced infiltration of stromal tissue into the PCa tumor microenvironment limits the identification of key tumor-specific transcription factors and epigenomic abnormalities in malignant epithelial cells. Integrated transcriptome and epigenetic multiomics analyses of the paired PCa organoids indicate that the basic helix-loop-helix transcription factor 40 (BHLHE40) is significantly upregulated in tumor samples. Increased chromatin accessibility at the promoter region and enhanced mTOR pathway activity contribute to the elevated expression of BHLHE40. Integrated analysis of chromatin immunoprecipitation-seq, RNA-seq, and high-throughput chromosome conformation capture data, together with chromosome conformation capture assays, indicate that BHLHE40 not only regulates sterol regulatory element-binding factor 1 (SREBF1) transcription as a classic transcription factor but also links the enhancer and promoter regions of SREBF1. It is found that the BHLHE40-SREBF1-stearoyl-CoA desaturase axis protects PCa cells from ferroptosis, resulting in the reduced accumulation of lipid peroxidation. Moreover, fatostatin, an SREBF1 inhibitor, significantly suppresses the growth of PCa tumors with high expressions of BHLHE40. This study highlights the important roles of BHLHE40-mediated lipid peroxidation in inducing ferroptosis in PCa cells and provides a novel mechanism underlying SREBF1 overexpression in PCa.

Prospective observational study on biomarkers of response in pancreatic ductal adenocarcinoma.

Adjuvant chemotherapy benefits patients with resected pancreatic ductal adenocarcinoma (PDAC), but the compromised physical state of post-operative patients can hinder compliance. Biomarkers that identify candidates for prompt adjuvant therapy are needed. In this prospective observational study, 1,171 patients with PDAC who underwent pancreatectomy were enrolled and extensively followed-up. Proteomic profiling of 191 patient samples unveiled clinically relevant functional protein modules. A proteomics-level prognostic risk model was established for PDAC, with its utility further validated using a publicly available external cohort. More importantly, through an interaction effect regression analysis leveraging both clinical and proteomic datasets, we discovered two biomarkers (NDUFB8 and CEMIP2), indicative of the overall sensitivity of patients with PDAC to adjuvant chemotherapy. The biomarkers were validated through immunohistochemistry on an internal cohort of 386 patients. Rigorous validation extended to two external multicentic cohorts-a French multicentric cohort (230 patients) and a cohort from two grade-A tertiary hospitals in China (466 patients)-enhancing the robustness and generalizability of our findings. Moreover, experimental validation through functional assays was conducted on PDAC cell lines and patient-derived organoids. In summary, our cohort-scale integration of clinical and proteomic data demonstrates the potential of proteomics-guided prognosis and biomarker-aided adjuvant chemotherapy for PDAC.

Constructing simplified microbial consortia to improve the key flavour compounds during strong aroma-type Baijiu fermentation.

The ester compounds play key roles in maintaining the sensory characteristics of alcoholic beverages. For strong aroma-type Baijiu fermentation, the volatile acids from pit mud microbes are key precursors for ester synthesis. However, the volatile acids can only be efficiently synthesized by the pit mud microbes in grains which attaches to pit mud. Elevating the ester contents in the upper layer's fermented grains is vital to improve the quality of raw liquor. In this study, we applied top-down strategy and aim to simplify and obtain pit mud microbial consortia to efficiently produce caproate but not off-flavour compounds. The simplified consortia with Caproiciproducens spp. as dominant species can use unsterilized fermentation water as sole substrate for caproate production, and stable caproate production was achieved by inoculating these simplified consortia in scaling-up fermentation. The fermented broth was then applied to facilitate the fermentation of upper layer's grains to prompt ester synthesis. Finally, the contents of variety esters such as ethyl caproate, ethyl pentanoate and ethyl octanoate were markedly increased. Together, this study demonstrates that constructing simplified microbial consortia containing key flavour-producing species is feasible to improve the flavour quality of spontaneously fermented foods.

Using Serological Proteome Analysis to Identify and Evaluate Anti-GRP78 Autoantibody as Biomarker in the Detection of Gastric Cancer.

The serological biomarkers as noninvasive tests are the most promising way for diagnosing gastric cancer (GC). Serological proteome analysis (SERPA) has been used to identify tumor-associated antigens (TAAs) and the corresponding autoantibodies in many studies. To explore the relationship between gastric cancer development and serum autoantibody anti-GRP78 response found by the method of SERPA with the GC cell line AGS, we included two cohorts (133 GC and 133 normal individuals in test group; 300 GC and 300 normal individuals in validation group) of patients with newly diagnosed GC for verification. All GC and normal controls were matched by age and gender. The autoantibody levels of the sera in two cohorts were measured by immunoassay. Finally, the results showed that 78-kDa glucose-regulated protein (GRP78) was identified in GC by SERPA and the level of anti-GRP78 antibody in GC was higher than that in normal individuals in the two cohorts. Receiver operating characteristic (ROC) curve analysis showed similar diagnostic value of anti-GRP78 antibody in test group (AUC: 0.718) and validation group (AUC: 0.666) to identify GC patients from normal individuals. The AUCs of anti-GRP78 autoantibody in the diagnosis of GC patients with different clinical characteristic ranged from 0.676 to 0.773 in test group and ranged from 0.645 to 0.707 in validation group. In conclusion, autoantibody against GRP78 might be a potential diagnostic biomarker. Further large-scale studies will be needed to validate and improve its performance of the sensitivity, specificity, and AUC value in distinguishing GC from other diseases.

Establishment and validation of an immunodiagnostic model for prediction of breast cancer.

Serum autoantibodies that react with tumor-associated antigens (TAAs) can be used as potential biomarkers for diagnosis of cancer. This study aims to evaluate the immunodiagnostic value of 11 anti-TAAs autoantibodies for detection of breast cancer (BC) and establish a diagnostic model for distinguishing BC from normal human controls (NHC) and benign breast diseases (BBD). Sera from 10 BC patients and 10 NHC were used to detect 11 anti-TAAs autoantibodies by western blotting. The 11 anti-TAAs autoantibodies were further assessed in 983 sera by relative quantitative enzyme-linked immunosorbent assay (ELISA). Binary logistic regression and Fisher linear discriminant analysis were conducted to establish a prediction model by using 184 BC and 184 NHC (training cohort, n = 568) and validated by leave-one-out cross-validation. Logistic regression model was selected to establish the prediction model. Results were validated using an independent validation cohort (n = 415). The five anti-TAAs (p53, cyclinB1, p16, p62, 14-3-3ξ) autoantibodies were selected to construct the model with the area under the curve (AUC) of 0.943 (95% CI, 0.919-0.967) in training cohort and 0.916 (95% CI, 0.886-0.947) in the validation cohort. In the identification of BC and BBD, AUCs were 0.881 (95% CI, 0.848-0.914) and 0.849 (95% CI, 0.803-0.894) in training and validation cohort, respectively. In summary, our study indicates that the immunodiagnostic model can distinguish BC from NHC and BC from BBD and this model may have a potential application in immunodiagnosis of breast cancer.

Discovery and Validation of Serum Autoantibodies Against Tumor-Associated Antigens as Biomarkers in Gastric Adenocarcinoma Based on the Focused Protein Arrays.

INTRODUCTION: Previous studies have demonstrated that autoantibodies against tumor-associated antigens (TAAs) in patients with cancer can be used as sensitive immunodiagnostic biomarkers for the detection of cancer. Most of these TAAs are involved in the tumorigenesis pathway. Cancer driver genes with intragenic mutations can promote tumorigenesis. This study aims to identify autoantibodies against TAAs encoded by cancer driver genes in sera as potential immunodiagnostic biomarkers for gastric adenocarcinoma (GAC). METHODS: Protein arrays based on cancer driver genes were customized for screening candidate TAAs in 100 GAC sera and 50 normal control (NC) sera. Autoantibodies against candidate TAAs were assessed by enzyme-linked immunosorbent assay in both training group (205 GAC sera and 205 NC sera) and independent validation group (126 GAC sera and 126 NC sera). Moreover, the immunodiagnostic models were respectively established and validated in the training group and validation group. RESULTS: A panel with 5 autoantibodies including anti-TP53, anti-COPB1, anti-GNAS, anti-serine/arginine-rich splicing factor 2, and anti-SMARCB1 was selected by the Fisher linear discriminant analysis model with an areas under receiver operating characteristic curve (AUC) of 0.928 (95% confidence interval [CI]: 0.888-0.967) in the training cohort and an AUC of 0.885 (95% CI: 0.852-0.918) in the validation cohort. Besides, the panel with 5 autoantibodies including anti-TP53, anti-COPB1, anti-GNAS, anti-PBRM1, and anti-ACVR1B which were selected by the binary logistic regression model showed an AUC of 0.885 (95% CI: 0.852-0.919) in the training cohort and 0.884 (95% CI: 0.842-0.925) in the validation cohort. DISCUSSION: Two panels which were selected in this study could boost the detection of anti-TAA autoantibodies in sera as biomarkers for the detection of GAC.

Serological Biomarkers for Early Detection of Hepatocellular Carcinoma: A Focus on Autoantibodies against Tumor-Associated Antigens Encoded by Cancer Driver Genes.

Substantial evidence manifests the occurrence of autoantibodies to tumor-associated antigens (TAAs) in the early stage of hepatocellular carcinoma (HCC), and previous studies have mainly focused on known TAAs. In the present study, protein microarrays based on cancer driver genes were customized to screen TAAs. Subsequently, autoantibodies against selected TAAs in sera were tested by enzyme-linked immunosorbent assays (ELISA) in 1175 subjects of three independent datasets (verification dataset, training dataset, and validation dataset). The verification dataset was used to verify the results from the microarrays. A logistic regression model was constructed within the training dataset; seven TAAs were included in the model and yielded an area under the receiver operating characteristic curve (AUC) of 0.831. The validation dataset further evaluated the model, exhibiting an AUC of 0.789. Remarkably, as the aggravation of HCC increased, the prediction probability (PP) of the model tended to decrease, the trend of which was contrary to alpha-fetoprotein (AFP). For AFP-negative HCC patients, the positive rate of this model reached 67.3% in the training dataset and 50.9% in the validation dataset. Screening TAAs with protein microarrays based on cancer driver genes is the latest, fast, and effective method for finding indicators of HCC. The identified anti-TAA autoantibodies can be potential biomarkers in the early detection of HCC.

Autoantibody against 14-3-3 zeta: a serological marker in detection of gastric cancer.

PURPOSE: Autoantibody to 14-3-3 zeta was identified in gastric cancer (GC) by serological proteome analysis (SERPA) in our previous study. We comprehensively evaluated its ability to detect GC, determined its association with clinical characteristics, and explored its temporal change in GC patients before and after gastrectomy resection in this study. METHODS: Anti-14-3-3 zeta antibody was examined by immunoassay in sera from 465 GC patients and 465 normal individuals, and also in 69 serial sera from 26 GC patients before and after resection. RESULTS: The frequency of anti-14-3-3 zeta were significantly higher in GC group than in control group, with AUC of 0.627. The appearance of anti-14-3-3 zeta showed no difference in different tumor stage, tumor size, tumor differentiation, and lymphatic metastasis, but was higher in GC patients with family tumor history than without family tumor history. When anti-14-3-3 zeta was combined with clinical markers (CEA, CA199 and CA724), the sensitivity increased to 52.7%. In the follow-up analysis, the titer of anti-14-3-3 zeta was higher in post-resection sera than pre-resection sera, and no difference was observed in CEA, CA199 and CA724. Anti-14-3-3 zeta showed an increase from negative status to positive status in six patients after resection, while other three clinical markers presented different change in GC patients after resection. CONCLUSIONS: Autoantibody against 14-3-3 zeta could be a potential diagnostic biomarker and improve the sensitivity of CEA, CA199 and CA724 in diagnosis of GC. Further largescale studies will be needed to validate its performance in GC patients after resection.

Overexpression of p62/IMP2 can Promote Cell Migration in Hepatocellular Carcinoma via Activation of the Wnt/ß-Catenin Pathway.

p62/IMP2 is an oncofetal protein that was first reported as a tumor-associated antigen in hepatocellular carcinoma (HCC). In our previous studies, we demonstrated a high frequency of p62/IMP2 autoantibodies appearing in various types of cancer. Therefore, we hypothesize that p62/IMP2 plays an important role in the progression of HCC, although the mechanism remains to be explored. In this study, we evaluated the expression of p62/IMP2 protein both in human tissues and liver cancer cell lines by immunohistochemistry and western blotting analysis and found that p62/IMP2 protein is overexpressed in human HCC tissue in comparison to normal human liver tissue. To explore the role that p62/IMP2 plays in HCC, p62/IMP2 was knocked out in two p62/IMP2-positive liver cancer cell lines (SNU449 and HepG2). Due to the low expression level of p62/IMP2 in SNU449, we overexpressed p62/IMP2 in this cell line. We subsequently demonstrated that high expression of p62/IMP2 in both cell lines can promote cell migration and invasion abilities in vitro by activating the Wnt/ß-catenin pathway. We also used the Wnt/ß-catenin pathway inhibitor, XAV 939, and a phosphoproteome assay to confirm our findings. Conclusion: Our results suggest that p62/IMP2 is an essential regulator of Wnt signaling pathways and plays an important role in HCC progression and metastasis.

Acid-degradable lactobionic acid-modified soy protein nanogels crosslinked by ortho ester linkage for efficient antitumor in vivo.

It remains a crucial challenge to achieve efficient cellular uptake and intracellular drug release in tumor cells for the nanoscale drug delivery systems. Herein, acid-degradable nanogels were prepared by cross-linking methacrylated soy protein with an acid-labile ortho ester cross-linker (NG1), and then modified with lactobionic acid (LA) to give tumor-targeted nanogels (NG2). Both NG1 and NG2 displayed excellent stability in neutral environment, while showed pH-triggered degradation behaviors under mildly acidic conditions resulting from the breakage of ortho ester bonds. Doxorubicin (DOX) was successfully loaded into nanogels, which exhibited an accelerated release at low pH. In vitro cell studies demonstrated that LA-modified nanogels could effectively improve cellular internalization, show higher cytotoxicity and apoptosis toward asialoglycoprotein receptor (ASGPR) over-expressed HepG2 cells. In vivo antitumor experimentproved that LA modification could significantly enhance the tumor-targeting ability of nanogels and increase DOX concentration in tumor site, leading to better therapeutic efficacy. Histological analysis further demonstrated that soy protein-based nanogels did not cause any damage to normal organs. Overall, these pH-sensitive and tumor-targeting soy protein-based nanogels can be potential drug carriers for efficient tumor treatment.

Circulating plasma microRNAs in the detection of esophageal squamous cell carcinoma.

Circulating microRNAs (miRNAs/miRs) have been reported as diagnostic biomarkers for esophageal cancer (EC) diagnosis. However, contrasting results have been achieved in different studies. In the present study, a meta-analysis was performed, based on the systematic search of PubMed and Web of Science, to evaluate the diagnostic value of circulating miRNAs in the peripheral blood in EC. The top 5 most-studied miRNAs were selected for confirmation by reverse transcription quantitative-polymerase chain reaction using the blood plasma of 125 patients with esophageal squamous cell carcinoma (ESCC) and 125 healthy individuals from Henan, China. A total of 45 studies from 22 articles, regarding 33 miRNAs were considered in the meta-analysis. The pooled sensitivity and specificity were both 0.79 (95% confidence interval, 0.76-0.82 for both). Among the 5 miRNAs considered (miR-21, miR-223, miR-375, miR-25 and miR-100), miR-21 and miR-223 were significantly overexpressed whereas miR-375 expression was reduced in patients with ESCC compared with healthy individuals (all P<0.001). The areas under the curves (AUCs) were 0.80, 0.73, and 0.69 for miR-21, miR-223, and miR-375, respectively. The AUCs increased when discriminating between patients with early ESCC in stage 0-I and the non-invasive carcinoma stage Tis-T1 stage from controls. Thus, it was concluded that plasma miR-21, miR-223 and miR-375 may serve as non-invasive diagnostic biomarkers in patients with ESCC, especially early ESCC in stages 0-I and Tis-T1.

Using a panel of multiple tumor-associated antigens to enhance autoantibody detection for immunodiagnosis of gastric cancer.

Autoantibodies against tumor-associated antigens (TAAs) are attractive non-invasive biomarkers for detection of cancer due to their inherently stable in serum. Serum autoantibodies against 9 TAAs from gastric cancer (GC) patients and healthy controls were measured by enzyme-linked immunosorbent assay (ELISA). A logistic regression model predicting the risk of being diagnosed with GC in the training cohort (n = 558) was generated and then validated in an independent cohort (n = 372). Area under the receiver operating characteristic curve (AUC) was used to assess the diagnostic performance. Finally, an optimal prediction model with 6 TAAs (p62, c-Myc, NPM1, 14-3-3ξ, MDM2 and p16) showed a great diagnostic performance of GC with AUC of 0.841 in the training cohort and 0.856 in the validation cohort. The proportion of subjects being correctly defined were 78.49% in the training cohort and 81.99% in the validation cohort. This prediction model could also differentiate early-stage (stage I-II) GC patients from healthy controls with sensitivity/specificity of 76.60%/72.34% and 80.56%/79.17% in the training and validation cohort, respectively, and the overall sensitivity/specificity for early-stage GC were 78.92%/74.70% when being combined with two cohorts. This prediction model presented no significant difference for the diagnostic accuracy between early-stage and late-stage (stage III - IV) GC patients. The model with 6 TAAs showed a high diagnostic performance for GC detection, particularly for early-stage GC. This study further supported the hypothesis that a customized array of multiple TAAs was able to enhance autoantibody detection in the immunodiagnosis of GC.

pH-sensitive carboxymethyl chitosan hydrogels via acid-labile ortho ester linkage for potential biomedical applications.

Hydrogel systems with favorable biocompatibility and biodegradability are of much interest for application in biomaterials and tissue engineering. In this study, a new ortho ester-based acid-labile crosslink agent with dual-epoxy end groups was synthesized and crosslinked with carboxymethyl chitosan (CMCS) at different molar ratios to prepare a series of pH-sensitive hydrogels for drug delivering. Doxorubicin (DOX) was then readily loaded into the hydrogels and the in vitro release profiles indicated that the release rate increased rapidly while pH decreased from 7.4 to 5.0, which is consistent with the degradation rate of these hydrogels at corresponding pH conditions. In addition, results from MTT assay and flow cytometry demonstrated that these CMCS-based hydrogels and their degradation products have no cytotoxicity against SH-SY5Y and 293T cells. Therefore, the prepared acid-labile hydrogels could be applied in tumorous drug delivery systems and peritumoral implantation therapy by further optimization.

Well-Defined Poly(Ortho Ester Amides) for Potential Drug Carriers: Probing the Effect of Extra- and Intracellular Drug Release on Chemotherapeutic Efficacy.

To compare the chemotherapeutic efficacy determined by extra- and intracellular drug release strategies, poly(ortho ester amide)-based drug carriers (POEAd-C) with well-defined main-chain lengths, are successfully constructed by a facile method. POEAd-C3-doxorubicin (DOX) can be rapidly dissolved to release drug at tumoral extracellular pH (6.5-7.2), while POEAd-C6-DOX can rapidly release drug following gradual swelling at intracellular pH (5.0-6.0). In vitro cytotoxicity shows that POEAd-C3-DOX exhibits more toxic effect on tumor cells than POEAd-C6-DOX at extracellular pH, but POEAd-C6-DOX has stronger tumor penetration and inhibition in vitro and in vivo tumor models. So, POEAd-C6-DOX with the intracellular drug release strategy has stronger overall chemotherapeutic efficacy than POEAd-C3-DOX with extracellular drug release strategy. It is envisioned that these poly(ortho ester amides) can have great potential as drug carriers for efficient chemotherapy with further optimization.