The effect of metformin on senescence of T lymphocytes.

BACKGROUND: Immunosenescence occurs as people age, leading to an increased incidence of age-related diseases. The number of senescent T cells also rises with age. T cell senescence and immune response dysfunction can result in a decline in immune function, especially in anti-tumor immune responses. Metformin has been shown to have various beneficial effects on health, such as lowering blood sugar levels, reducing the risk of cancer development, and slowing down the aging process. However, the immunomodulatory effects of metformin on senescent T cells still need to be investigated. METHODS: PBMCs isolation from different age population (n = 88); Flow Cytometry is applied to determine the phenotypic characterization of senescent T lymphocytes; intracellular staining is applied to determine the function of senescent T cells; Enzyme-Linked Immunosorbent Assay (ELISA) is employed to test the telomerase concentration. The RNA-seq analysis of gene expression associated with T cell senescence. RESULTS: The middle-aged group had the highest proportion of senescent T cells. We found that metformin could decrease the number of CD8 + senescent T cells. Metformin affects the secretion of SASP, inhibiting the secretion of IFN-γ in CD8 + senescent T cells. Furthermore, metformin treatment restrained the production of the proinflammatory cytokine IL-6 in lymphocytes. Metformin had minimal effects on Granzyme B secretion in senescent T cells, but it promoted the production of TNF-α in senescent T cells. Additionally, metformin increased the concentration of telomerase and the frequency of undifferentiated T cells. The results of RNA-seq showed that metformin promoted the expression of genes related to stemness and telomerase activity, while inhibiting the expression of DNA damage-associated genes. CONCLUSION: Our findings reveal that metformin could inhibit T cell senescence in terms of cell number, effector function, telomerase content and gene expression in middle-aged individuals, which may serve as a promising approach for preventing age-related diseases in this population.

Methylation of N6 adenosine-related long noncoding RNA: effects on prognosis and treatment in 'driver-gene-negative' lung adenocarcinoma.

The improvement of treatment for patients with 'driver-gene-negative' lung adenocarcinoma (LUAD) remains a critical problem to be solved. We aimed to explore the role of methylation of N6 adenosine (m6A)-related long noncoding RNA (lncRNA) in stratifying 'driver-gene-negative' LUAD risk. Patients negative for mutations in EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1 were identified as 'driver-gene-negative' cases. RNA sequencing was performed in 46 paired tumors and adjacent normal tissues from patients with 'driver-gene-negative' LUAD. Twenty-three m6A regulators and relevant lncRNAs were identified using Pearson's correlation analysis. K-means cluster analysis was used to stratify patients, and a prognostic nomogram was developed. The CIBERSORT and pRRophetic algorithms were employed to quantify the immune microenvironment and chemosensitivity. We identified two clusters highly consistent with the prognosis based on their unique expression profiles for 46 m6AlncRNAs. A risk model constructed from nine m6A lncRNAs could stratify patients into high- and low-risk groups with promising predictive power (C-index = 0.824), and the risk score was an independent prognostic factor. The clusters and risk models were closely related to immune characteristics and chemosensitivity. Additional pan-cancer analysis using the nine m6AlncRNAs showed that the expression of DIO3 opposite strand upstream RNA (DIO3OS) is closely related to the immune/stromal score and tumor stemness in a variety of cancers. Our results show that m6AlncRNAs are a reliable prognostic tool and can aid treatment decision-making in 'driver-gene-negative' LUAD. DIO3OS is associated with the development of various cancers and has potential clinical applications.

A novel signature based on immune-related gene pairs and clinical features to predict prognosis and treatment effect in "driver gene negative" lung adenocarcinoma.

OBJECTIVE: Examining the role of immune-related genes (IRGs) in "driver gene negative" lung adenocarcinoma (LUAD) may provide new ideas for the treatment and study for this LUAD subgroup. We aimed to find the hub immune-related gene pairs can stratify the risk of "driver-gene-negative" LUAD. MATERIALS AND METHODS: IRGs were identified according to ImmPort database based on RNA sequencing results of tumors and normal tissues from 46 patients with "driver gene negative" LUAD at The First Affiliated Hospital of Sun Yat-sen University and cyclically singly paired as immune-related gene pairs (IRGPs). Multivariate Cox analysis was used to construct an immune risk model and a prognostic nomogram combining was also been developed. Immune microenvironment landscape described by CIBERSORT and drug sensitivity calculated by pRRophetic algorithm were used to explore possible treatment improvements. RESULTS: A novel immune risk model with 5-IRGPs (CD1A|CXCL135, CD1A|S100A7L2, IFNA7|CMTM2, IFNA7|CSF3, CAMP|TFR2) can accurately distinguish patients in the high- and low-risk groups. Risk score act as an independent prognostic factor and is related to clinical stage. There are significant differences in tumor immune microenvironment and PD-1/PD-L1/CTLA-4 expression between groups. The low-risk patient may benefit more from the commonly used chemotherapy regimens such as gemcitabine and paclitaxel. CONCLUSION: This study constructed 5-IRGPs as a reliable prognostic tool and may represent genes pairs that are potential rationale for choice of treatment for "driver gene negative" LUAD.

PD-1 Expression and Function of T-Cell Subsets in TILs From Human Lung Cancer.

On the basis of the autologous tumor-infiltrating lymphocytes (TILs) or genetically modified TILs for adoptive cell therapy have received more attention. Programmed cell death protein 1 (PD-1) expression on the T cells exert complex response during the tumor immune response. But the composition and function of PD-1T-cell subsets in TILs from human lung cancer still limited. In blood and TILs from human lung cancer patients, we confirmed that PD-1 is expressed in higher levels in CD4T-cell subsets than in CD8T-cell subsets. To further analyze the function of PD-1T cells in TILs, we observed the cytokine production in different T-cell subsets. We found that higher interferon-γ and granzyme B production in CD4/CD8PD-1T-cell subsets in TILs than in peripheral blood mononuclear cells (PBMCs); except for PD-1Tscm, higher tumor necrosis factor-α production was observed in PD-1T-cell subsets in TILs than in PBMCs; the expression level of interleukin-17 were lower in PD-1T cells in TILs than in PBMCs; and perforin expression was significantly reduced in CD4PD-1T cells subsets in TILs compared with peripheral blood. Clarify elucidating the composition and function of PD-1T-cell subsets in TILs will have great value in clinical application for evaluating the sensitivity to PD-1 blockade and selecting the promising candidate T-cell subsets in TILs for combination immunotherapy in human lung cancer.

Effects of Glycogen Synthase Kinase-3ß Inhibitor TWS119 on Proliferation and Cytokine Production of TILs From Human Lung Cancer.

The canonical Wnt-ß-catenin signaling pathway arrests the differentiation of T cells and plays an important role in phenotypic maintenance of naive T cells and stem cell-like memory T cells in human peripheral blood, but its effect on tumor-infiltrating lymphocytes (TILs) from non-small cell lung cancer is little known. In this study, we showed that glycogen synthase kinase-3ß inhibitor TWS119 has different effects on CD4 and CD8 T cells in TILs. TWS119 preserved the expansion of naive T cell and CD8 stem cell-like memory T cells, and induced CD8 effector T-cell proliferation in TILs. To further determine whether TWS119 impaired the effector function of TILs, TILs were stimulated with polyclonal stimulation, IL-2 and IFN-γ production were detected. Our data showed that TWS119 does not affect the production of IFN-γ in TILs compared with the control group; whereas TWS119 inhibited IFN-γ secretion of T cells from healthy donor. IL-2 production in CD4 central memory T cells and CD4 effector memory T cells from TILs was significantly increased with the TWS119 treatment; TWS119 also promoted the secretion of IL-2 in all cell subsets of CD8 TILs. These findings reveal that TWS119 has a distinct effect on the proliferation and cytokine production of TILs, and provide new insights into the clinical application of TILs with TWS119 treatment for the adoptive immunotherapy.

Exogenous H2S promotes cancer progression by activating JAK2/STAT3 signaling pathway in esophageal EC109 cells.

Hydrogen sulfide (H2S) plays an important role in diverse physiological and pathophysiological processes in cancer cells both in vitro and in vivo. We have previously shown that exogenous H2S exerts its biological effects on hepatoma, glioma, and esophageal cancer cells through the activation of NF-κB, p38-MAPK/ERK1/2-COX-2, and HSP90 pathways. However, the role of H2S and the underlying mechanism in esophageal squamous cell carcinoma remain unclear. Here we investigated whether exogenous H2S contributes to the biological behavior of esophageal squamous cancer cell line EC109, through the activation of JAK2/STAT3 signaling pathway. EC109 cells were treated with NaHS (a donor of H2S) and AG490 (a specific inhibitor of JAK2/STAT3 signaling pathway). The expression levels of p-JAK2, p-STAT3, caspase-3/9/12, Bax, Bcl-2, MMP-2/9, and VEGFR were measured by western blot analysis. Cell viability was detected by CCK-8 and quantified by direct counting of cells under a microscope. Cell migration was analyzed by the scratch-wound assay, while the level of VEGF was measured by ELISA. Cells treated with NaHS for 24 h showed significant upregulation of p-JAK2, and p-STAT3 expression, as well as increased cell viability when compared to the control cells. The expression levels of caspase-3/9/12 and Bax decreased, while those of Bcl-2, MMP-2/9, VEGFR, and VEGF increased. NaHS induced the migration of EC109 cells. However, co-treatment with NaHS and AG490 significantly inhibited these effects. Thus, JAK2/STAT3 signaling pathway may contribute to H2S-induced cell proliferation, anti-apoptosis, migration, and angiogenesis in EC109 cells.

The distribution and function of human memory T cell subsets in lung cancer.

The distribution and function of T lymphocytes in human lung cancer remain limited. In this study, we investigated the properties of human T cell subsets in the blood of non-small cell lung cancer (NSCLC) patients. We found a relatively normal level of CD4+ subsets in the blood of NSCLC patients, but CD8+ effector T cells increased and CD8+ effector memory cells declined compared to the healthy donors. To further analyze their properties, we stimulated the peripheral blood mononuclear cells (PBMCs) of NSCLC patients by mitogens to examine cytokine production. Our data suggest that both CD4+ and CD8+ naïve cells in NSCLC patients significantly reduced IFN-γ and TNF-α production. Additionally, fewer CD8+ effector cells produced IFN-γ and TNF-α in NSCLC patients than in healthy subjects. Moreover, similar results were observed for CD4+ or CD8+ memory cells in NSCLC patients for the production of IFN-γ, TNF-α, and IL-17. Therefore, our results strongly suggest that the function of CD4+ and CD8+ T lymphocytes in NSCLC patients is compromised or dysregulated. The development of vaccines and antitumor immunotherapy may be essential for the treatment of lung cancer patients.

The Characteristics of Naive-like T Cells in Tumor-infiltrating Lymphocytes From Human Lung Cancer.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TILs) or genetically modified lymphocytes from TILs is a new effective approach, but the application of TIL immunotherapy is still limited in many solid tumors. Knowledge of the classification and function of TILs is important to develop personalized immunotherapy with TILs in non-small lung cancer (NSCLC). In this study, we show the characteristics of T-cell subsets in TILs isolated from NSCLC. CD3 CD8 CD45RA T cells outnumbered CD3 CD4 CD45RA T cells in CD45RA TILs, but it was the opposite in CD45RO TILs. Effector memory CD4 T cells predominated in CD4 TILs; about 10% of the stem cell-like memory T cells (Tscm) were detected in TILs. To further analyze their functions, we stimulated TILs from NSCLC patients by mitogens to examine cytokine production. Our data demonstrated that naive-phenotype T cells in TILs secret IFN-γ in abundance; TNF-α-producing T cells were significantly increased in TILs; there were more IL-17-expressing CD4 Tscm cells than other subtypes of CD4T cells in TILs. Our findings indicate that the CD4/CD8 naive-phenotype T cells and Tscm cells in TILs from lung cancer exhibit distinct composition and strong cytokine production. Attributes of Tscm cells from a naive-like T-cell population in TILs are the promising cell type for adoptive cell therapy in human lung cancer.

The Distribution of Human Stem Cell-like Memory T Cell in Lung Cancer.

Human stem cell-like memory T (Tscm) cells are long-lived, self-renewing memory lymphocytes that can differentiate into effector cells and mediate strong antitumour response in murine model. The distribution and function of Tscm cells in human lung cancer remain unknown. In this study, we investigated the properties of human Tscm cells in the blood and lymph node of non-small cell lung cancer (NSCLC) patients. There were more CD4 Tscm cells in blood from NSCLC patients than from healthy donors, fewer CD4 and CD8 TSCM cells in blood than in lymph node from NSCLC patients. To further analyze their properties, we stimulated peripheral blood mononuclear cells from NSCLC patients by mitogens to examine cytokine production. Our data suggest that both CD4 and CD8 Tscm cells in blood produced interferon-γ significantly increased in NSCLC patients compare with healthy subjects. In addition, fewer Tscm cells produced interferon-γ in lymph node than in blood from NSCLC patients. Our results strongly suggest that the distribution and function of CD4 Tscm cells in NSCLC patients is upregulated. Understanding of the properties of stem-like memory T cells will supply a good rationale for designing the new adoptive immunotherapy in cancer.

Preoperative serum fibrinogen is an independent prognostic factor in operable esophageal cancer.

In order to fully elucidate the association between serum fibrinogen and prognosis of esophageal cancer, we examined serum fibrinogen concentrations in 1512 patients who underwent esophagectomy by the Clauss method. The impact of fibrinogen on overall survival and disease-free survival was analyzed using the Kaplan-Meier method and Cox proportional hazard models. Hyperfibrinogenemia was significantly associated with older age, male gender, smoking, alcohol consumption, weight loss, advanced pathological T stage and lymph node metastasis. Patients with hyperfibrinogenemia exhibited poor OS (HR=1.20, 95%CI: 1.04-1.38, P=0.012) and DFS (HR=1.18, 95%CI: 1.03-1.35, P=0.019). Subgroup analysis further exhibited an significant association between hyperfibrinogenemia and poor OS (P<0.001), DFS (P<0.001) in esophageal squamous cell carcinoma (P<0.001) and early pathological stage (I-II) (P=0.001). Collectively, this study indicates that preoperative serum fibrinogen is an independent prognostic factor for survival in esophageal cancer.

Effect of mir-16 on proliferation and apoptosis in human A549 lung adenocarcinoma cells.

OBJECTIVE: To investigate the expression of mir-16 in lung adenocarcinoma cancer line and to observe the effect of mir-16 on the biological behaviors of human lung adenocarcinoma cancer A549 cell. Methods the expression of mir-16 in A549 cells was examined by quantitative real-time (qRT)-PCR. mir-16 minics was chemically synthesized and transfected into A549 cells by Lipofectamine 2000. The cell cycle and apoptosis changes were assayed by flow cytometry, the cell proliferation was measured by MTS assay. The wild-type and mutant wip1 3'-UTR luciferase reporter rectors were constructed. The relative activity of renila luciferase was detected to confirm the binding site of mir-16 on wip1 mRNA. Results, the expression of mir-16 is reduced in A549 cell compared with the normal bronchial epithelial cell. Transfection of mir-16 minics significantly suppressed the luciferase reporter containing wild type not mutant wip1 3'-UTR. Furthermore enforced expression of mir-16 lead to reduced A549 cell proliferation and promote apoptosis. Conclusion Therapeutic strategies to resume miRNA-16 expression may be benefit to patients with NSCLC in the feature.

Airway epithelial cell-derived insulin-like growth factor-1 triggers skewed CD8(+) T cell polarization.

Skewed CD8(+) T cell responses are important in airway inflammation. This study investigates the role of the airway epithelial cell-derived insulin-like growth factor 1 (IGF1) in contributing to CD8(+) T cell polarization. Expression of IGF1 in the airway epithelial cell line, RPMI2650 cells, was assessed by quantitative real time RT-PCR and Western blotting. The role of IGF1 in regulating CD8(+) T cell activation was observed by coculture of mite allergen-primed RPMI2650 cells and naïve CD8(+) T cells. CD8(+) T cell polarization was assessed by the carboxyfluorescein succinimidyl ester-dilution assay and the determination of cytotoxic cytokine levels in the culture medium. Exposure to mite allergen, Der p1, increased the expression of IGF1 by RPMI2650 cells. The epithelial cell-derived IGF1 prevented the activation-induced cell death by inducing the p53 gene hypermethylation. Mite allergen-primed RPMI2650 cells induced an antigen-specific CD8(+) T cell polarization. We conclude that mite allergens induce airway epithelial cell line, RPMI2650 cells, to produce IGF1; the latter contributes to antigen-specific CD8(+) T cell polarization.

[Notch1 expression in esophageal squamous cell carcinoma and its relation with microvascular angiogenesis].

OBJECTIVE: To observe Notch1 expression in esophageal squamous cell carcinoma (ESCC) and investigate its relation with microvascular angiogenesis in the tumor. METHODS: Tissue slices of 40 cases ESCC (cancer group) and 8 cases normal esophagus tissues (normal group) were obtained to analyze the expression of Notch1 and vascular endothelial growth factor (VEGF) using immunohistochemistry and estimate the microvessel density (MVD) in the tumor. RESULTS: Notch1 expression was significantly lower in the cancer group than in the normal group (P<0.05). In the cancer group, Notch1 expression was higher in highly differentiated than in poorly differentiated tumors (P<0.05) regardless of tumor infiltration or lymph nodes metastasis (P>0.05). VEGF expression and MVD were significantly higher in cancer group than in normal group, and showed significant differences between tumors with different differentiation degrees, infiltration and lymph node metastasis (P<0.05). Correlation analysis showed that Notch1 expression was inversely correlated to VEGF expression. CONCLUSION: Notch1 may be an anti-oncogene in ESCC and affects cell differentiation in early stage of the malignancy. Abnormally low expression of Notch1 in ESCC may be one of the upstream factors to induce high expression of VEGF and increased MVD. The Notch1 pathway might play a key role in microvascular angiogenesis in ESCC.