Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 µm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

Elevated Expression of NOTCH1 Associates with Lymph Node Metastasis of Gastric Cancer and Knock-Down of NOTCH1 Attenuates Tumor Cell Progression.

BACKGROUND Gastric cancer is the third leading cause of cancer-related death, while its molecular mechanism has not been fully clarified. This study aims to explore the role of Notch signaling in the pathogenesis of gastric cancer. MATERIAL AND METHODS A total of 64 patients with gastric cancer were enrolled. The expressions of NOTCH1 in tumor tissues and adjacent non-tumor tissues were detected by immunohistochemistry staining. The correlation between NOTCH1 expression and clinicopathological features of patients was analyzed. NOTCH1 was knocked down in gastric cancer cells. The effects of NOTCH1 blockade on cell proliferation, migration and cell cycle distribution were analyzed. The expressions of ERK1/2 and phospho-ERK1/2 (p-ERK1/2) were detected using western blotting. RESULTS Gastric cancer tissues expressed higher level of NOTCH1 than adjacent non-tumor tissues (P<0.05). The high level of NOTCH1 was found to be correlated with gender (male) and lymph node metastasis. However, the expression level of NOTCH1 did not affect the overall survival of patients with gastric cancer. NOTCH1 knock-down repressed the migration and proliferation of gastric cancer cells. Moreover, the cell cycle was arrested at G0/G1 phase by NOTCH1 blockade. The expressions of ERK1/2 and p-ERK1/2 decreased with NOTCH1 knock-down. Further inhibition of ERK1/2 signaling by a MEK1/2 inhibitor U0126 reduced the proliferation of AGS cells, which aggravated the inhibition effect of NOTCH1 knock-down on cell proliferation. CONCLUSIONS NOTCH1 may play an oncogenic role in gastric cancer. Inhibition of NOTCH1 can efficiently attenuate gastric cancer cell progression, probably in part through cross-talking with ERK1/2 signaling pathway.

Clinical significance and correlation of miR-200c and P-gp expression in gastric cancer and the effects on multidrug resistance.

Background: Poor prognosis is common in gastric cancer patients due to multidrug resistance (MDR)-induced recurrence and metastasis. In the present study, we investigated the expression of microRNA (miR)-200c in gastric cancer tissues and cell lines and its relationship with the expression of the drug resistant gene ABCB1, which encodes P-glycoprotein (P-gp). Methods: The basic characteristics of 102 patients with gastric cancer were reviewed. Real time-polymerase chain reaction (PCR), immunohistochemistry, and Western blot were employed to detect the expression levels of miR-200c and P-gp in gastric carcinoma tissues and cell lines. The correlation of miR-200c messenger RNA (mRNA) level with clinicopathological characteristics and P-gp protein expression were analyzed. SGC7901/vincristine (VCR) cells were transfected with miR-200c mimics or a specific small interfering RNA (siRNA) targeting the ABCB1 gene. The methyl thiazolyl tetrazolium (MTT) assay and flow cytometry were used to determine the role of miR-200c and ABCB1 on the viability and apoptosis of gastric carcinoma cell lines. Results: The level of miR-200c in carcinoma tissues was significantly lower than that in adjacent tissues, and the expression level of P-gp in carcinoma tissues was obviously higher than that in adjacent tissues (P<0.01, P=0.029). The expression levels of miR-200c and P-gp were associated with the malignant characteristics of gastric cancer, and patients with high expression of miR-200c or negative expression of P-gp had a better prognosis (P=0.006, P=0.022). MiR-200c negatively regulated the ABCB1 gene in gastric cancer cell lines. MiR-200c overexpression and ABCB1 down-regulation increased the sensitivity of SGC7901/VCR cells to VCR and reversed MDR by promoting cell apoptosis. Conclusions: The expression level of miR-200c decreases in gastric carcinoma tissues and drug-resistant gastric cancer SGC7901/VCR cells. Overexpression of miR-200c may enhance the sensitivity of SGC7901/VCR cells to VCR by regulating the expression of P-gp.

Long non-coding RNA ATXN8OS promotes ferroptosis and inhibits the temozolomide-resistance of gliomas through the ADAR/GLS2 pathway.

As the most common malignant tumor, gliomas remain a poor prognosis while chemotherapy resistance is a medical problem for the treatment of glioma. Considering the correlation between drug resistance and ferroptosis, this study aims to explore the mechanism of chemotherapy resistance in glioma from the perspective of epigenetics. Because of the low expression of long non-coding RNA (lncRNA) ATXN8 opposite strand (ATXN8OS) in glioma cell lines, the role of ATXN8OS was explored by the detection on ferrous iron (Fe2+)/lipid reactive oxygen species (ROS), function experiments and assays performed with xenograft model, proving that ATXN8OS inhibited temozolomide (TMZ)-resistance of glioma. After subcellular fractionation and FISH assays revealed that ATXN8OS was mainly located in cytoplasm, we determined the RNA binding protein (RBP) of ATXN8OS as adenosine deaminase acting on RNA (ADAR) via RNA binding protein immunoprecipitation (RIP), RNA pull down and western blot assays. Furthermore, we verified that ATXN8OS stabilized glutaminase 2 (GLS2) mRNA by recruiting ADAR and GLS2 restrained TMZ-resistance of glioma both in vitro and in vivo. Rescue experiments indicated that ATXN8OS modulated TMZ-resistance of glioma through GLS2. In conclusion, ATXN8OS mediated ferroptosis and regulated the TMZ-resistance of glioma via ADAR/GLS2 pathway.

Diffusion kurtosis imaging combined with dynamic susceptibility contrast-enhanced MRI in differentiating high-grade glioma recurrence from pseudoprogression.

OBJECTIVES: To compare the added value of diffusion kurtosis imaging (DKI) with the combination of dynamic susceptibility contrast-enhanced (DSC) MRI in differentiating glioma recurrence from pseudoprogression. METHODS: Thirty-four patients with high-grade gliomas developing new and/or increasing enhanced lesions within six months after surgery and chemoradiotherapy were retrospectively analyzed. All patients were pathologically confirmed to have recurrent glioma (n = 22) or pseudoprogression (n = 12). The DKI and DSC MRI parameters were calculated based on the enhanced lesions on contrast-enhanced T1WI. ROC analysis was performed on significant variables to determine their diagnostic performance. Multivariate logistic regression was used to determine the best prediction model for discrimination. RESULTS: The relative mean kurtosis (rMK), relative axial kurtosis (rKa), relative cerebral blood volume (rCBV), and relative mean transit time (rMTT) of glioma recurrence were higher than those of pseudoprogression (all, P < 0.05). The AUCs and diagnostic accuracy were 0.879 and 82.35% for rMK, 0.723 and 70.59% for rKa, 0.890 and 82.35% for rCBV, 0.765 and 73.53% for rMTT, respectively. A multivariate logistic regression model showed a significant contribution of rMK (P = 0.006) and rCBV (P = 0.009) as independent imaging classifiers for discrimination. The combined use of rMK and rCBV improved the AUC to 0.924 (P < 0.001) and the diagnostic accuracy to 88.24%. CONCLUSION: DKI may be a valuable non-invasive tool in differentiating glioma recurrence from pseudoprogression, and its use in combination with DSC MRI can improve diagnostic performance in assessing treatment response compared with either technique alone.

Conventional MR and DW imaging findings of cerebellar primary CNS lymphoma: comparison with high-grade glioma.

Primary central nervous system lymphomas (PCNSLs) and high-grade gliomas (HGGs) arising in the cerebellum is extremely low, making the differential diagnosis difficult or even impossible. The purpose of this study was to define the MR features of cerebellar PCNSL in immunocompetent patients, and to determine whether a combination of conventional MR and DW imaging can assist in the differentiation of PCNSLs and HGGs. Twelve PCNSLs and 15 HGGs confirmed by pathological analysis were retrospectively identified. The apparent diffusion coefficient (ADC) and conventional MRI parameters were compared for differences between PCNSL and HGG groups using the independent sample t test or chi-square test. Both ADCmin and ADCtotal values were lower in the PCNSL group than those in the HGG group (ADCmin: 0.53 × 10-3 vs. 0.83 × 10-3 mm2/sec, P < 0.001; ADCtotal: 0.66 × 10-3 vs. 0.98 × 10-3 mm2/sec, P = 0.001). As for conventional MR features, there were significant difference in the tumor size, enhancement patterns, the presence of cystic changes, edema degree and streak-like edema (all P < 0.01); but there were no significant difference in lesion type, the presence of bleeding, and involvement of brain surface between two groups (P = 0.554, 0.657 and 0.157, respectively). The results revealed that several conventional MR features, including enhancement patterns, branch-like enhancement and streak-like edema may be useful for the differentiation of PCNSL and HGG in cerebellum and, when combined with ADC values, further improve the discriminating ability.

Characteristics of the isocitrate dehydrogenase gene and telomerase reverse transcriptase promoter mutations in gliomas in Chinese patients.

OBJECTIVES: To explore the characteristics of IDH and TERT promoter mutations in gliomas in Chinese patients. METHODS: A total of 124 Chinese patients with gliomas were enrolled to study the frequencies of mutations in isocitrate dehydrogenase (IDH) and telomerase reverse transcriptase promoter (TERTp). Among the 124 patients, 59 patients were enrolled to study the classification of gliomas based on mutations in IDH and TERTp. RESULTS: Isocitrate dehydrogenase mutations are positively correlated with a good prognosis but mutations in TERTp cannot predict prognoses independently. The combined analysis of the mutations of IDH and TERTp can predict the prognosis more accurately. Patients with IDH and TERTp glioma mutations have the best prognosis, followed by only IDH mutation patients and only TERTp mutation patients, which have the worst prognosis. IDH and TERTp mutations occur frequently in males, younger patients or lower-grade patients. In contrast, only TERTp mutations occur frequently in females, older patients or higher-grade patients. CONCLUSIONS: Patients with IDH and TERTp glioma mutations have the best prognosis, and only IDH mutation patients and only TERTp mutation patients have the worst prognosis. Moreover, the molecular classification of gliomas by mutations of IDH and TERTp is not suitable for pediatric patients.

Immunohistochemical Study of NR2C2, BTG2, TBX19, and CDK2 Expression in 31 Paired Primary/Recurrent Nonfunctioning Pituitary Adenomas.

This study investigated potential markers for predicting nonfunctioning pituitary adenoma (NFPA) invasion and recurrence by high-throughput tissue microarray analyses. We retrospectively studied two groups of patients: 60 nonrecurrent NFPA cases that included noninvasion and invasion subtypes and 43 recurrent cases that included primary NFPA. A total of 31 paired patient samples were evaluated (12 patients with one surgery and 31 who had undergone two operations, with both tumors analyzed). Expressions of nuclear receptor subfamily 2 group C member 2 (NR2C2), B cell translocation gene 2, T-box-19 (TBX19), and cyclin-dependent kinase 2 (CDK2) in surgically resected specimens were assessed by immunohistochemistry. The relationships between marker expression and clinical characteristics including age, sex, tumor volume, and follow-up time were analyzed. Tumor volume and invasion as well as follow-up time were significantly associated with invasion and recurrence (P < 0.01). Of the 60 nonrecurrent samples, 15/41 and 13/19 showed high NR2C2 expression in the noninvasion and invasion groups, respectively (χ 2 =5.287, P = 0.021). NR2C2 was also overexpressed in 43 primary recurrent cases (χ 2 =5.433, P = 0.02), whereas CDK2 (χ 2 = 11.242, P = 0.001) and TBX19 (χ 2 = 4.875, P = 0.027) were downregulated. In the 31 paired samples, NR2C2 was more highly expressed in the recurrent as compared to the primary tumor. High NR2C2 expression was associated with NFPA invasion, recurrence, and progression, while TBX19 and CDK2 were associated with NFPA recurrence.

Assessment of tumor-associated immune cells in laryngeal squamous cell carcinoma.

PURPOSE: This study investigated the characteristics of tumor-associated immune cells (TAICs) in laryngeal squamous cell carcinoma (LSCC) and their correlation with clinicopathological variables. METHODS: The immune cell infiltrates of 71 specimens of stages I-IV LSCC were examined. The density of TAICs expressing CD3, CD4, CD8, CD68, and CD163 was assessed using immunohistochemical staining and image analysis in peritumoral and intratumoral regions. RESULTS: Higher densities of CD3+ and CD8+ cell and lower densities of CD68+ and CD163+ cell infiltrations were found in early tumor stages than in late tumor stages. A higher percentage of patients with strong CD3+ and CD8+ immune cell infiltration and weak CD68+ cell infiltration in both tumor regions presented with T1 stage tumors compared with T4 stage tumors. Further, strong CD68+ cells infiltration in both regions was observed in a greater number of patients who had a relapse, while a weak CD3+ cells infiltration in both regions was found in a greater number of patients with nodal lymphatic metastasis. The univariate analysis showed that a high density of peritumoral CD3+ and CD8+ immune cells in both regions was significantly associated with a favorable overall survival (OS) (P = 0.004; P = 0.006; P = 0.042). In contrast, a high density of intratumoral CD68+ cells and peritumoral CD163+ cells was significantly associated with poor OS durations (P = 0.026; P = 0.030). The multivariate analysis demonstrated that a high density of peritumoral CD163+ cells correlated with poor OS after adjusting for tumor stage, recurrence, and nodal lymphatic metastasis (P = 0.034). This study found different patterns of TAIC infiltration in LSCC. The density and location of TAICs infiltration correlated with the clinicopathological characteristics of LSCC. CONCLUSION: A combined analysis of the density of TAICs and their location may help predict patient survival and response to checkpoint inhibitors.

Glycine protects against high sucrose and high fat-induced non-alcoholic steatohepatitis in rats.

We set out to explore the hypothesis that glycine attenuates non-alcoholic steatohepatitis (NASH) in rats and the possible mechanism by which is it does. Male Sprague-Dawley (SD) rats were fed a diet containing high fat and high sucrose (HSHF) for 24 weeks to induce NASH. Blood and liver tissues were sampled at selected time points throughout the study. Compared with control animals, the content of alanine transaminase (ALT), triglycerides (TGs), and free fatty acids (FFAs) in plasma and the TG and FFA content in the liver was increased from week 4 to 24. The level of TNFα and MCP-1 in plasma, the content of TNFα in the liver, the insulin resistance index, inflammatory cell infiltration, hepatocyte apoptosis, reactive oxygen species (ROS) generation, and endoplasmic stress-associated protein expression were unaltered at 4 weeks. However, these levels were significantly elevated in HSHF fed rats at 12 weeks. At the same time, the level of endotoxin progressively increased from 0.08 ± 0.02 endotoxin EU/ml at week 4 to 0.7 ± 0.19 EU/ml at week 24. Moreover, these rats had elevated blood endotoxin levels, which were positively associated with their NASH indexes. Liver histology progressively worsened over the course of the study. However, we found that with concomitant treatment with glycine, the level of endotoxin decreased, while NASH indexes significantly decreased and liver status markedly improved,. These data support the hypothesis that glycine protects against NASH in rats by decreasing the levels of intestinal endotoxin, alleviating endoplasmic reticulum and oxidative stress.

A model of metabolic syndrome and related diseases with intestinal endotoxemia in rats fed a high fat and high sucrose diet.

AIM: We sought develop and characterize a diet-induced model of metabolic syndrome and its related diseases. METHODS: The experimental animals (Spague-Dawley rats) were randomly divided into two groups, and each group was fed a different feed for 48 weeks as follows: 1) standard control diet (SC), and 2) a high sucrose and high fat diet (HSHF). The blood, small intestine, liver, pancreas, and adipose tissues were sampled for analysis and characterization. RESULTS: Typical metabolic syndrome (MS), non-alcoholic fatty liver disease (NAFLD), and type II diabetes (T2DM) were common in the HSHF group after a 48 week feeding period. The rats fed HSHF exhibited signs of obesity, dyslipidemia, hyperglycaemia, glucose intolerance, and insulin resistance (IR). At the same time, these animals had significantly increased levels of circulating LPS, TNFα, and IL-6 and increased ALP in their intestinal tissue homogenates. These animals also showed a significant reduction in the expression of occluding protein. The HSHF rats showed fatty degeneration, inflammation, fibrosis, cirrhosis, and lipid accumulation when their liver pathologies were examined. The HSHF rats also displayed increased islet diameters from 12 to 24 weeks, while reduced islet diameters occurred from 36 to 48 weeks with inflammatory cell infiltration and islet fat deposition. The morphometry of adipocytes in HSHF rats showed hypertrophy and inflammatory cell infiltration. HSHF CD68 analysis showed macrophage infiltration and significant increases in fat and pancreas size. HSHF Tunel analysis showed significant increases in liver and pancreas cell apoptosis. CONCLUSIONS: This work demonstrated the following: 1) a characteristic rat model of metabolic syndrome (MS) can be induced by a high sucrose and high fat diet, 2) this model can be used to research metabolic syndrome and its related diseases, such as NAFLD and T2DM, and 3) intestinal endotoxemia (IETM) may play an important role in the pathogenesis of MS and related diseases, such as NAFLD and T2DM.

Targeted knockdown of IQGAP1 inhibits the progression of esophageal squamous cell carcinoma in vitro and in vivo.

IQGAP1 is a scaffolding protein that can regulate several distinct signaling pathways. The accumulating evidence has demonstrated that IQGAP1 plays an important role in tumorigenesis and tumor progression. However, the function of IQGAP1 in esophageal squamous cell carcinoma (ESCC) has not been thoroughly investigated. In the present study, we showed that IQGAP1 was overexpressed in ESCC tumor tissues, and its overexpression was correlated with the invasion depth of ESCC. Importantly, by using RNA interference (RNAi) technology we successfully silenced IQGAP1 gene in two ESCC cell lines, EC9706 and KYSE150, and for the first time found that suppressing IQGAP1 expression not only obviously reduced the tumor cell growth, migration and invasion in vitro but also markedly inhibited the tumor growth, invasion, lymph node and lung metastasis in xenograft mice. Furthermore, Knockdown of IQGAP1 expression in ESCC cell lines led to a reversion of epithelial to mesenchymal transition (EMT) progress. These results suggest that IQGAP1 plays crucial roles in regulating ESCC occurrence and progression. IQGAP1 silencing may therefore develop into a promising novel anticancer therapy.