Combination therapy with PD-1 inhibition plus rapamycin and metformin enhances anti-tumor efficacy in triple negative breast cancer.

Immunotherapy using PD-1/PD-L1 inhibitors has been proved to be effective in triple negative breast cancer (TNBC), albeit only in a fraction of patients. Emerging evidences indicate mTOR blockade and metformin may re-orchestrate the immune system in tumors. Herein, in this study we aimed to evaluate the anti-tumor efficacy of PD-1 monoclonal antibody with mTOR inhibitor rapamycin or with the anti-diabetic drug metformin. The status of PD-1/PD-L1 and mTOR pathway was determined through analyzing the TCGA and CCLE data in TNBCs as well as by detection at mRNA and protein level. The inhibition of tumor growth and metastasis by anti-PD-1 combined with rapamycin or with metformin was evaluated in allograft mouse model of TNBC. The effects of combination therapy on the AMPK, mTOR and PD-1/PD-L1 pathways were also evaluated. The combination treatment with PD-1 McAb and rapamycin/metformin had additive effects on suppression of tumor growth and distant metastasis in mice. Compared with the control group and the monotherapy, combined PD-1 McAb with either rapamycin or metformin exhibited more obvious effects on induction of necrosis, CD8+ T lymphocytes infiltrating and inhibition of PD-L1 expression in TNBC homograft. In vitro study showed either rapamycin or metformin not only decreased PD-L1 expression, but increased p-AMPK expression and therefore led to down-regulation of p-S6. In summary, combination of PD-1 antagonist with either rapamycin or metformin led to more infiltrating TILs and decreased PD-L1 resulting in enhanced antitumor immunity and blockade of PD-1/PD-L1 pathway. Our results suggested such combination therapy may be a potential therapeutic strategy for TNBC patients.

Integrated analysis identifies oxidative stress-related lncRNAs associated with progression and prognosis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers in the world. Oxidative stress reactions have been reportedly associated with oncogenesis and tumor progression. By analyzing mRNA expression data and clinical information from The Cancer Genome Atlas (TCGA), we aimed to construct an oxidative stress-related long noncoding RNA (lncRNA) risk model and identify oxidative stress-related biomarkers to improve the prognosis and treatment of CRC. RESULTS: Differentially expressed oxidative stress-related genes (DEOSGs) and oxidative stress-related lncRNAs were identified by using bioinformatics tools. An oxidative stress-related lncRNA risk model was constructed based on 9 lncRNAs (AC034213.1, AC008124.1, LINC01836, USP30-AS1, AP003555.1, AC083906.3, AC008494.3, AC009549.1, and AP006621.3) by least absolute shrinkage and selection operator (LASSO) analysis. The patients were then divided into high- and low-risk groups based on the median risk score. The high-risk group had a significantly worse overall survival (OS) (p < 0.001). Receiver operating characteristic (ROC) and calibration curves displayed the favorable predictive performance of the risk model. The nomogram successfully quantified the contribution of each metric to survival, and the concordance index and calibration plots demonstrated its excellent predictive capacity. Notably, different risk subgroups showed significant differences in terms of their metabolic activity, mutation landscape, immune microenvironment and drug sensitivity. Specifically, differences in the immune microenvironment implied that CRC patients in certain subgroups might be more responsive to immune checkpoint inhibitors. CONCLUSIONS: Oxidative stress-related lncRNAs can predict the prognosis of CRC patients, which provides new insight for future immunotherapies based on potential oxidative stress targets.

Development and Assessment of Assisted Diagnosis Models Using Machine Learning for Identifying Elderly Patients With Malnutrition: Cohort Study.

BACKGROUND: Older patients are at an increased risk of malnutrition due to many factors related to poor clinical outcomes. OBJECTIVE: This study aims to develop an assisted diagnosis model using machine learning (ML) for identifying older patients with malnutrition and providing the focus of individualized treatment. METHODS: We reanalyzed a multicenter, observational cohort study including 2660 older patients. Baseline malnutrition was defined using the global leadership initiative on malnutrition (GLIM) criteria, and the study population was randomly divided into a derivation group (2128/2660, 80%) and a validation group (532/2660, 20%). We applied 5 ML algorithms and further explored the relationship between features and the risk of malnutrition by using the Shapley additive explanations visualization method. RESULTS: The proposed ML models were capable to identify older patients with malnutrition. In the external validation cohort, the top 3 models by the area under the receiver operating characteristic curve were light gradient boosting machine (92.1%), extreme gradient boosting (91.9%), and the random forest model (91.5%). Additionally, the analysis of the importance of features revealed that BMI, weight loss, and calf circumference were the strongest predictors to affect GLIM. A BMI of below 21 kg/m2 was associated with a higher risk of GLIM in older people. CONCLUSIONS: We developed ML models for assisting diagnosis of malnutrition based on the GLIM criteria. The cutoff values of laboratory tests generated by Shapley additive explanations could provide references for the identification of malnutrition. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR-EPC-14005253; https://www.chictr.org.cn/showproj.aspx?proj=9542.

Malnutrition dynamics according to GLIM criteria in hospitalized elderly.

BACKGROUND AND OBJECTIVES: Assess the different nutritional status between admission and discharged in older adult patients using the GLIM criteria. METHODS AND STUDY DESIGN: A retrospective analysis was conducted on a multicenter study which initiated in 34 hospitals in China with 2734 hospitalized older patients. The dynamic changes of malnutrition according to GLIM criteria were performed between at admission and discharge, and their significance was analyzed using the chi-square test. The association between malnutrition and clinical outcomes was analyzed using the chi-square test, t-test, or rank sum test, and divided into different disease types for further analysis. RESULTS: The incidence of nutritional risk in elderly patients was 51.6% at admission and 48.4% at discharge. The prevalence of malnutrition according to the GLIM criteria was 19.6% at admission and increased to 33.4% at discharge, which was significantly different. Different age and disease type were related with nutrition status. Malnutrition is significantly association with adverse clinical outcomes such as increased risk of complications and prolonged length of hospital stay. CONCLUSIONS: The GLIM criteria can be used in elderly patients to assess malnutrition. The prevalence of malnutrition in elderly inpatients is high, and the prevalence of malnutrition at discharge is higher than that observed at admission. Attention should be paid to the dynamic changes of malnutrition in elderly patients during hospitalization.

Effect of oral nutritional supplementation on the post-discharge nutritional status and quality of life of gastrointestinal cancer patients after surgery: a multi-center study.

BACKGROUND AND OBJECTIVES: To evaluate the effect of oral nutritional supplementation (ONS) on the postdischarge nutritional status and quality of life (QoL) of gastrointestinal cancer patients after surgery. METHODS AND STUDY DESIGN: A multi-center study was conducted on gastrointestinal cancer patients who received surgical treatment from 2013-2015. All patients were screened using the Nutrition Risk Screening 2002 (NRS 2002) to assess nutritional risk. Patients with nutritional risk were randomized into two groups: patients in the study group (n=55) were given dietary guidance and ONS, control group (n=59) received only dietary guidance. Anthropometric measurements, nutrition-related laboratory tests, and gastrointestinal function scores were also collected and analyzed using Student's t test and analysis of variance (ANOVA). In addition, the EQ-5D was used to evaluate patients' QoL. RESULTS: Compared with baseline measurements, the body weight of patients in the study group increased by 1.35±0.53 kg and 1.35±0.73 kg at 60 and 90 days, which were significantly higher than those in the control group (-1.01±0.54 kg, and -1.60±0.81 kg at 60 and 90 days). The results from ANOVA showed that only weight and BMI differed significantly between the study and control groups and also between different measurement times (p<0.01). No differences were found for the other indicators or QoL between the study groups. CONCLUSIONS: ONS may improve the weight and BMI of surgically treated gastrointestinal cancer patients postdischarge. However, these effects had little impact on patients' QoL.

Prevalence and predictors of malnutrition in elderly Chinese adults: results from the China Health and Retirement Longitudinal Study.

OBJECTIVE: China has the largest population of elderly citizens in the world, with 177 million adults aged 60 years or older. However, no national estimate of malnutrition in elderly Chinese adults exists. We estimated the prevalence and predictors of malnutrition in this population. DESIGN: Data from the second wave of the Chinese Health and Retirement Longitudinal Study (CHARLS) include interview and biomarker data for 6450 subjects aged 60 years or older from 448 different communities in twenty-eight provinces, allowing for nationally representative results. Malnutrition was identified based on the ESPEN (European Society of Parenteral and Enteral Nutrition and Metabolism) criteria. We used multivariable regression to investigate the predictors of malnutrition, including demographic factors, marital status, self-reported health status, self-reported standard of living, health insurance status and education. SETTING: China. SUBJECTS: Community-dwelling Chinese adults aged 60 years or older. RESULTS: The prevalence of malnutrition in elderly Chinese adults was 12·6 %. Malnutrition was most common among those who were older (OR=1·09; 95 % CI 1·07, 1·10), male (OR=1·41; 95 % CI 1·10, 1·79), lived in rural areas (v. urban: OR=0·75; 95 % CI 0·57, 1·00) or lacked health insurance (P<0·01). CONCLUSIONS: The burden of malnutrition on elderly Chinese adults is significant. Based on current population estimates, up to 20 million are malnourished. Malnutrition is strongly associated with demographic factors, shows a trend to association with health status and is not strongly associated with standard of living or education. A coordinated effort is needed to address malnutrition in this population.

Nutritional Risk and Nutritional Status at Admission and Discharge among Chinese Hospitalized Patients: A Prospective, Nationwide, Multicenter Study.

OBJECTIVE: The objective of this study was to assess nutritional risk and status of Chinese hospitalized patients at admission and discharge and relations with clinical outcomes. METHODS: A prospective, nationwide, multicenter study was conducted from June to September 2014 in 34 large hospitals in 18 cities in China. Patients ≥ 18 years with a hospital stay of 7-30 days were recruited. Anthropometric and laboratory indicators, nutritional risk screening, and assessment by Nutritional Risk Screening 2002 (NRS 2002) and subjective global assessment (SGA) were performed within 24 hours of admission and discharge. Clinical data during hospitalization were collected. RESULTS: A total of 6,638 patients met the criteria with a male: female ratio of 1.39:1 and an average age of 59.72 ± 15.40 years. At admission, the proportion of patients with nutritional risk, body mass index (BMI) < 18.5 kg/m2, and moderate to severe malnutrition was 40.12%, 8.92%, and 26.45%, respectively, whereas at discharge, these percentages were 42.28%, 8.91%, and 30.57%, respectively. The values of all of these indicators were higher in patients 65 years of age and older. Patients with nutritional risk at admission had a longer average hospital stay (14.02 ± 6.42 vs 13.09 ± 5.703 days), higher incidence of total complications (6.90% vs 1.52%), and greater total medical expenses (3.39 ± 7.50 vs 3.00 ± 3.38 million RMB; all p < 0.01) than patients without nutritional risk. Similar results were obtained for the patients with nutritional risk at discharge. CONCLUSION: The prevalence of nutritional risk and malnutrition, including moderate to severe malnutrition, at discharge is higher than that observed at admission; the clinical outcome of patients with nutritional risk is poor.

Hemostatic Effect of Hemocoagulase Agkistrodon on Surgical Wound in Breast Cancer Surgery.

Objective To evaluate the hemostatic effect of hemocoagulase agkistrodon on surgical wound in breast cancer surgery. Methods Totally 60 patients undergoing breast cancer surgery were enrolled in this prospective,randomized,double-blinded,and controlled study. All the patients met the inclusion and exclusion criteria and signed the informed consent. Hemocoagulase agkistrodon (2 U) was injected 20 minutes before surgery and 4 and 24 hours after surgery in the intervention group (n=30),whereas normal saline was used instead in the control group (n=30). The volume of intraoperative bleeding,wound drainage volume 1-3 days after surgery,and total drainage volume were recorded. Meanwhile,the change of blood coagulation function,treatment safety,and clinical outcomes were observed. Results The intra-operative hemorrhage volume of the intervention group [(95.0±48.3)g] was significantly lower than that of the control group [(144.8±105.4)g] (t=-2.07,P=0.044). The volume of total drainage of the intervention group [(166.7±71.2)g] was significantly lower than that of the control group [(251.4±166.3)g] (t=-2.29,P=0.029). The hemoagglutination indicators were similar in the two groups and no complication such as thrombosis occurred. The length of hospital stay of the intervention group [(15.00±3.53)d] was similar to that of the control group [(15.92±2.32)d] (t=-1.057,P=0.297). No research drug-related adverse event was occurred in our study. Conclusion Hemocoagulase agkistrodon has good hemostatic effect for patients undergoing breast cancer surgery without increasing the risk of thrombosis.

Histone demethylase JARID1C promotes breast cancer metastasis cells via down regulating BRMS1 expression.

Metastasis is the leading cause of death in breast cancer patients. However, until now, the mechanisms of breast cancer metastasis remain elusive. Epigenetic switch, including histone methylation or demethylation, which can either activates or represses transcription. The JARID1C is a histone demethylase that promotes cancer cell growth and is involved in transcriptional regulation and chromatin remodeling, cause X-linked mental retardation. But the pathogenic breadth and mechanistic aspects of this effect relative to breast cancer have not been defined. In this study, we aimed to investigate the role of JARID1C in breast cancer. In clinical breast cancer samples, we found that JARID1C expression was significantly upregulated in cancer lesions compared with paired normal breast tissues and its expression level is positively correlated with metastasis. Silencing JARID1C in breast cancer cells could inhibit cell migration and invasion. Moreover, we also found that the expression of BRMS1 was modulated by JARID1C. Silencing of JARID1C dramatically increased BRMS1 expression both at mRNA and protein level. Mechanistically, we found JARID1C exerts its function through modulation of H3K4me3 at the BRMS1 gene promoter, which was associated with inactive BRMS1 transcription. BRMS1 knockdown reversed shJARID1C-induced migration inhibition. Further, BRMS1 expression in human breast cancer is negatively correlated with JARID1C expression. Our results, for the first time, portray a pivotal role of JARID1C in regulating metastatic behaviors of breast cancer cells.

The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer.

The histone acetyltransferase GCN5 has been suggested to be involved in promoting cancer cell growth. But its role in human colon cancer development remains unknown. Herein we discovered that GCN5 expression is significantly upregulated in human colon adenocarcinoma tissues. We further demonstrate that GCN5 is upregulated in human colon cancer at the mRNA level. Surprisingly, two transcription factors, the oncogenic c-Myc and the proapoptotic E2F1, are responsible for GCN5 mRNA transcription. Knockdown of c-Myc inhibited colon cancer cell proliferation largely through downregulating GCN5 transcription, which can be fully rescued by the ectopic GCN5 expression. In contrast, E2F1 expression induced human colon cancer cell death, and suppression of GCN5 expression in cells with E2F1 overexpression further facilitated cell apoptosis, suggesting that GCN5 expression is induced by E2F1 as a possible negative feedback in suppressing E2F1-mediated cell apoptosis. In addition, suppression of GCN5 with its specific inhibitor CPTH2 inhibited human colon cancer cell growth. Our studies reveal that GCN5 plays a positive role in human colon cancer development, and its suppression holds a great therapeutic potential in antitumor therapy.

Estrogen promotes stemness and invasiveness of ER-positive breast cancer cells through Gli1 activation.

BACKGROUND: Although long-term estrogen (E2) exposure is associated with increased breast cancer (BC) risk, and E2 appears to sustain growth of BC cells that express functional estrogen receptors (ERs), its role in promoting BC stem cells (CSCs) remains unclear. Considering that Gli1, part of the Sonic hedgehog (Shh) developmental pathway, has been shown to mediate CSCs, we investigated whether E2 and Gli1 could promote CSCs and epithelial-mesenchymal transition (EMT) in ER+ BC cell lines. METHODS: We knocked down Gli1 in several BC cells using a doxycycline-controlled vector, and compared Gli1-knockdown cells and Gli1+ cells in behavior and expression of ER, Gli1, ALDH1 (BC-CSC marker), Shh, Ptch1 (Shh receptor) and SOX2, Nanog and Bmi-1 (CSC-associated transcriptions factors), using PCR; tissue microarrays, western blot; chromatin immunoprecipitation q-PCR, confocal immunofluorescence microscopy; fluorescence-activated cell sorting; annexin-flow cytometry (for apoptosis); mammosphere culture; and colony formation, immunohistochemistry, Matrigel and wound-scratch assays. RESULTS: Both mRNA and protein expressions of ER correlated with those of Gli1 and ALDH1. E2 induced Gli1 expression only in ER+ BC cells. E2 promoted CSC renewal, invasiveness and EMT in ER+/Gli1+ cells but not in Gli1-knockdown cells. CONCLUSIONS: Our results indicate that estrogen acts via Gli1 to promote CSC development and EMT in ER+ BC cells. These findings also imply that Gli1 mediates cancer stem cells, and thus could be a target of a novel treatment for ER+ breast cancer.

Perioperative and post-hospital whole-course nutrition management in patients with pancreatoduodenectomy - a single-center prospective randomized controlled trial.

OBJECTIVE: Whole-course nutrition management (WNM) has been proven to improve outcomes and reduce complications. We conducted this randomized controlled trial to validate its effectiveness in patients undergoing pancreatoduodenectomy (PD). METHODS: From 1 December 2020, to 30 November 2023, this single-center randomized clinical trial was conducted at the Department of Hepatobiliopancreatic Surgery in a major hospital in Beijing, China. Participants who were undergoing PD were enrolled and randomly allocated to either the WNM group or the control group. The primary outcome was the incidence of postoperative complications. Subgroup analysis in patients who were at nutritional risk was performed. Finally, a 6-month follow-up was conducted and the economic benefit was evaluated using an incremental cost-effectiveness ratio (ICER). RESULTS: A total of 84 patients were randomly assigned (1:1) into the WNM group and the control group. The incidences of total complications (47.6% vs. 69.0%, P =0.046), total infections (14.3% vs. 33.3%, P =0.040), and abdominal infection (11.9% vs. 31.0%, P =0.033) were significantly lower in the WNM group. In the subgroup analysis of patients at nutritional risk, 66 cases were included (35 cases in the WNM group and 31 cases in the control group). The rate of abdominal infection (11.4% vs. 32.3%, P =0.039) and postoperative length of stay (23.1±10.3 vs. 30.4±17.2, P =0.046) were statistically different between the two subgroups. In the 6-month follow-up, more patients reached the energy target in the WNM group (97.0% vs. 79.4%, P =0.049) and got a higher daily energy intake (1761.3±339.5 vs. 1599.6±321.5, P =0.045). The ICER suggested that WNM saved 31 511 Chinese Yuan (CNY) while reducing the rate of total infections by 1% in the intention-to-treat (ITT) population and saved 117 490 CNY in patients at nutritional risk, while WNM saved 31 511 CNY while reducing the rate of abdominal infections by 1% in the ITT population and saved 101 359 CNY in patients at nutritional risk. CONCLUSION: In this trial, whole-course nutrition management was associated with fewer total postoperative complications, total and abdominal infections, and was cost-effective, especially in patients at nutritional risk. It seems to be a favorable strategy for patients undergoing PD.

The cyclic guanosine monophosphate synthase-stimulator of interferon genes pathway as a potential target for tumor immunotherapy.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self-DNA in the cytoplasm. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein stimulator of interferon genes (STING), which then activates the kinases IKK and TBK1 to induce the secretion of interferons and other cytokines. Recently, a series of studies demonstrated that the cGAS-STING pathway, a vital component of host innate immunity, might play an important role in anticancer immunity, though its mechanism remains to be elucidated. In this review, we highlight the latest understanding of the cGAS-STING pathway in tumor development and the advances in combination therapy of STING agonists and immunotherapy.

[Retracted] Metformin exerts anticancer effects through the inhibition of the Sonic hedgehog signaling pathway in breast cancer.

Following the publication of this paper, it was drawn to the Editor's attention by concerned readers that several of the cellular images shown in Figs. 6A and 8A, the scratch­wound assay images shown in Fig. 5A, the western blotting data in Figs. 2C and 7A and the Matrigel invasion assays in Fig. 5C were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 36: 204­214, 2015; DOI: 10.3892/ijmm.2015.2217].

Relationship between preoperative malnutrition, frailty, sarcopenia, body composition, and anthropometry in elderly patients undergoing major pancreatic and biliary surgery.

Objective: To analyze the correlation between preoperative nutritional status, frailty, sarcopenia, body composition, and anthropometry in geriatric inpatients undergoing major pancreatic and biliary surgery. Methods: This is a cross-sectional study of the database from December 2020 to September 2022 in the department of hepatopancreatobiliary surgery, Beijing Hospital. Basal data, anthropometry, and body composition were recorded. NRS 2002, GLIM, FFP 2001, and AWGS 2019 criteria were performed. The incidence, overlap, and correlation of malnutrition, frailty, sarcopenia, and other nutrition-related variables were investigated. Group comparisons were implemented by stratification of age and malignancy. The present study adhered to the STROBE guidelines for cross-sectional study. Results: A total of 140 consecutive cases were included. The prevalence of nutritional risk, malnutrition, frailty, and sarcopenia was 70.0, 67.1, 20.7, and 36.4%, respectively. The overlaps of malnutrition with sarcopenia, malnutrition with frailty, and sarcopenia with frailty were 36.4, 19.3, and 15.0%. There is a positive correlation between every two of the four diagnostic tools, and all six p-values were below 0.002. Albumin, prealbumin, CC, GS, 6MTW, ASMI, and FFMI showed a significantly negative correlation with the diagnoses of the four tools. Participants with frailty or sarcopenia were significantly more likely to suffer from malnutrition than their control groups with a 5.037 and 3.267 times higher risk, respectively (for frailty, 95% CI: 1.715-14.794, p = 0.003 and for sarcopenia, 95% CI: 2.151-4.963, p<0.001). Summarizing from stratification analysis, most body composition and function variables were worsen in the ≥70 years group than in the younger group, and malignant patients tended to experience more intake reduction and weight loss than the benign group, which affected the nutrition diagnosis. Conclusion: Elderly inpatients undergoing major pancreatic and biliary surgery possessed high prevalence and overlap rates of malnutrition, frailty, and sarcopenia. Body composition and function deteriorated obviously with aging.

Development of the Scientific, Transparent and Applicable Rankings (STAR) tool for clinical practice guidelines.

BACKGROUND: This study aimed to develop a comprehensive instrument for evaluating and ranking clinical practice guidelines, named Scientific, Transparent and Applicable Rankings tool (STAR), and test its reliability, validity, and usability. METHODS: This study set up a multidisciplinary working group including guideline methodologists, statisticians, journal editors, clinicians, and other experts. Scoping review, Delphi methods, and hierarchical analysis were used to develop the STAR tool. We evaluated the instrument's intrinsic and interrater reliability, content and criterion validity, and usability. RESULTS: STAR contained 39 items grouped into 11 domains. The mean intrinsic reliability of the domains, indicated by Cronbach's α coefficient, was 0.588 (95% confidence interval [CI]: 0.414, 0.762). Interrater reliability as assessed with Cohen's kappa coefficient was 0.774 (95% CI: 0.740, 0.807) for methodological evaluators and 0.618 (95% CI: 0.587, 0.648) for clinical evaluators. The overall content validity index was 0.905. Pearson's r correlation for criterion validity was 0.885 (95% CI: 0.804, 0.932). The mean usability score of the items was 4.6 and the median time spent to evaluate each guideline was 20 min. CONCLUSION: The instrument performed well in terms of reliability, validity, and efficiency, and can be used for comprehensively evaluating and ranking guidelines.

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

BACKGROUND: Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. METHODS: Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. RESULTS: After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. CONCLUSION: Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

[Establishment of an albumin and cytokeratin 19 genetically-modified embryonic stem cell line and evaluation of its hepatoblast differentiation capacities].

OBJECTIVE: To establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes, albumin (ALB) and cytokeratin 19 (CK19), labeled to facilitate study of their potential applicability as differentiated hepatoblasts. METHODS: Two expression vectors were constructed, one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and anti-neomycin genes (pAlb-EGFP), and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO). The linearized vectors were electroporated into the E14.1 line, and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin. E14.1-2 hepatoblast differentiation was induced by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation. Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency. RESULTS: The pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 expression. The E14.1-2 cell line with labeled ALB and CK19 was established, and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression, namely Oct4 and SSEA-1. After 22 days of induction, 21.27% of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression. CONCLUSIONS: A gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled. The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify, and will likely aid future studies of hepatoblast characteristics.

Preoperative Anemia Is a Predictor of Worse Postoperative Outcomes Following Open Pancreatoduodenectomy: A Propensity Score-Based Analysis.

Background: Preoperative anemia is a common clinical situation proved to be associated with severe outcomes in major surgeries, but not in pancreatic surgery. We aim to study the impact of preoperative anemia on morbidity and mortality in patients undergoing open pancreatoduodenectomy and use propensity score matching (PSM) to balance the basal data and reduce bias. Methods: We analyzed the data of consecutive patients undergoing open pancreatoduodenectomy with a complete record of preoperative hemoglobin, at two pancreatic centers in China between 2015 and 2019. Anemia is defined as hemoglobin less than 12 g/dl for male and 11 g/dl for female, following Chinese criteria. We compared clinical and economic outcomes before and after PSM and used logistic regression analysis to assess the correlation between variables and anemia. Results: The unmatched initial cohort consisted of 517 patients. A total of 148 cases (28.6%) were diagnosed with anemia at admission, and no case received a preoperative blood transfusion or anti-anemia therapy. After PSM, there were 126 cases in each group. The rate of severe postoperative complications was significantly higher in the anemia group than in the normal group (43.7% vs. 27.0%, p = 0.006), among which the differences in prevalence of clinically relevant postoperative pancreatic fistula (CR-POPF) (31.0% vs. 15.9%, p = 0.005) and cardiac and cerebrovascular events (4.0% vs. 0.0%, p = 0.024) were the most significant. The costs involved were more in the anemia group (26958.2 ± 21671.9 vs. 20987.7 ± 10237.9 USD, p = 0.013). Among anemic patients, receiver operating characteristic (ROC) curve analysis shows the cut-off value of hemoglobin, below which, patients are prone to suffer from major complications (104.5 g/l in male and 90.5 g/l in female). Among all patients, multivariate analysis showed that preoperative obstructive jaundice [odds ratio (OR) = 1.813, 95% confidence interval (CI) (1.206-2.725), p = 0.004] and pancreatic ductal adenocarcinoma [OR = 1.861, 95% CI (1.178-2.939), p = 0.008] were predictors of anemia. Among paired patients, preoperative anemia [OR = 2.593, 95% CI (1.481-5.541), p = 0.001] and malignant pathology [OR = 4.266, 95% CI (1.597-11.395), p = 0.004] were predictors of postoperative severe complications. Conclusion: Preoperative anemia is a predictor of worse postoperative outcomes following open pancreatoduodenectomy and needs to be identified and treated.

Acidic Tumor Microenvironment Promotes Pancreatic Cancer through miR-451a/MEF2D Axis.

Pancreatic cancer (PC), as a highly malignant and aggressive solid tumor, is common in the digestive system. The acidic microenvironment is one of the critical markers of cancer. Nonetheless, there are few studies on how the acidic microenvironment affects the development of PC. This study focused on investigating the specific molecular mechanisms of the acidic microenvironment in PC. In our study, qRT-PCR was conducted for examining microRNA (miR)-451a and myocyte enhancer factor 2D (MEF2D) expressions in PANC-1 cells. Then, detailed functional effects of an acidic environment on miR-451a and MEF2D in PANC-1 cells were detected by CCK-8, colony formation, flow cytometry, wound healing, transwell, mitochondrial functionality measurement, JC-1 staining, DCFH-DA staining, and sphere formation assays. The relationship between miR-451a and MEF2D was confirmed by luciferase reporter analysis. Under acidic conditions, the increase of proliferation, migration, and invasion of PANC-1 cells was observed. Moreover, the mitochondrial oxidative respiration-related gene miR-451a was reduced in acidic conditions. In addition, we found that, in PANC-1 cells under an acidic environment, miR-451a overexpression enhanced oxygen consumption, mitochondrial membrane potential (MMP) loss, and ROS generation and inhibited proliferation, migration, invasion, and stemness via sponging MEF2D. In a word, our results revealed that the acidic microenvironment regulated PC progression by affecting the miR-451a/MEF2D axis, indicating a novel avenue for the future treatment of PC.