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Objective@#To investigate the application value of cerebrospinal fluid circulating cell-free DNA (cfDNA) in the diagnosis and treatment of leptomeningeal metastases in non-small-cell lung cancer (NSCLC).@*Methods@#Twenty-five patients with leptomeningeal metastases of NCSLC from Fudan University Huashan Hospital North during the period from September 2017 to November 2018 were enrolled. All 25 patients were confirmed leptomeningeal metastases by cerebrospinal fluid cytology and immunocytochemical staining of cytokeratin(CK7), carcinoembryonic antigen(CEA), thyroid transcription factor-1(TTF-1) and Ki67. The cerebrospinal fluid cfDNA was extracted and genetic variation of 12 genes including epidermal growth factor receptor(EGFR), TP53 and anaplastic lymphoma kinase(ALK) was detected by next-generation sequencing [PlasAim TM gene non-invasive detection of lung cancer (12 gene) kit, Singlera Genomics].The application value of cerebrospinal fluid cfDNA in the diagnosis and treatment of leptomeningeal metastases of NSCLC was analyzed with the cfDNA mutation data and the clinical follow-ups.@*Results@#Morphologically typical lung cancer tumor cells with tumor immunochemistry markerCK, CK7 and CEA were found in the cerebrospinal fluid of all 25 patients. Next generation sequencing of cerebrospinal fluid showed that 96% (24/25) patients had at least one single nucleotide variation (SNV) or copy number variation (CNV). The EGFR and TP53 mutations were identified in 80% (20/25) and 48%(12/25) of the patients, respectively. In addition, patients with bone metastases had a higher rate of EGFR mutations than those without bone metastases (100% vs 64%, P<0.05). Changes in the mutant allele frequency of EGFR and TP53 in cerebrospinal fluid were consistent with patients′ disease progression parameters including neurological symptoms, imaging, and tumor biomarkers. The results indicate that genetic alteration of EGFR in cerebrospinal fluid cfDNA is an actionable biomarker for targeted therapy of leptomeningeal metastases of lung cancer.@*Conclusion@#Cerebrospinal fluid cfDNA accurately reveals the unique genetic background of leptomeningeal metastasis in NSCLC, showing great application value in the diagnosis and treatment of the leptomeningeal metastasis of NSCLC.
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Objective To investigate the application value of cerebrospinal fluid circulating cell-free DNA (cfDNA) in the diagnosis and treatment of leptomeningeal metastases in non-small-cell lung cancer (NSCLC). Methods Twenty-five patients with leptomeningeal metastases of NCSLC from Fudan University Huashan Hospital North during the period from September 2017 to November 2018 were enrolled. All 25 patients were confirmed leptomeningeal metastases by cerebrospinal fluid cytology and immunocytochemical staining of cytokeratin(CK7), carcinoembryonic antigen(CEA), thyroid transcription factor-1(TTF-1) and Ki67. The cerebrospinal fluid cfDNA was extracted and genetic variation of 12 genes including epidermal growth factor receptor(EGFR), TP53 and anaplastic lymphoma kinase(ALK) was detected by next-generation sequencing [PlasAim TM gene non-invasive detection of lung cancer (12 gene) kit, Singlera Genomics].The application value of cerebrospinal fluid cfDNA in the diagnosis and treatment of leptomeningeal metastases of NSCLC was analyzed with the cfDNA mutation data and the clinical follow-ups. Results Morphologically typical lung cancer tumor cells with tumor immunochemistry marker CK, CK7 and CEA were found in the cerebrospinal fluid of all 25 patients. Next generation sequencing of cerebrospinal fluid showed that 96％(24/25) patients had at least one single nucleotide variation (SNV) or copy number variation (CNV). The EGFR and TP53 mutations were identified in 80％(20/25) and 48％(12/25) of the patients, respectively. In addition, patients with bone metastases had a higher rate of EGFR mutations than those without bone metastases (100％ vs 64％, P<0.05). Changes in the mutant allele frequency of EGFR and TP53 in cerebrospinal fluid were consistent with patients' disease progression parameters including neurological symptoms, imaging, and tumor biomarkers. The results indicate that genetic alteration of EGFR in cerebrospinal fluid cfDNA is an actionable biomarker for targeted therapy of leptomeningeal metastases of lung cancer. Conclusion Cerebrospinal fluid cfDNA accurately reveals the unique genetic background of leptomeningeal metastasis in NSCLC, showing great application value in the diagnosis and treatment of the leptomeningeal metastasis of NSCLC.
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Objective: To study the effects of deguelin on proliferation and apoptosis of human breast cancer cell line MCF-7 and on phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Methods: After treatment with 0, 1, 5, 10, 15 and 20 μmol/L of deguelin for 6, 24, 48 and 72 hours, the proliferation inhibition rate of MCF-7 cells was measured by cell counting kit-8 assay. Apoptosis rate of MCF-7 cells was detected with Annexin V-fluorescein isothiocyanate/propidium iodide double staining by flow cytometry and the apoptotic morphology was observed under a transmission electron microscope. After treatment with 0, 1 and 5 μmol/L of deguelin for 6 hours, 5 proteins involved in the PI3K/Akt signaling pathway were examined by Western blot analysis. Results: Deguelin at doses of 5, 10, 15 and 20 μmol/L inhibited the proliferation of MCF-7 cells at 6, 24, 48 and 72 hours. There was a significant difference in each group compared with the control group (P0.05). Deguelin at doses of 5, 10, 15 and 20 μmol/L induced apoptosis of MCF-7 cells at 6 hours. There were significant differences (P<0.01) in the early and late apoptosis rate between the treated groups and the control group. The typical apoptotic MCF-7 cells were observed under the transmission electron microscopy. However, 1 μmol/L of deguelin had no apparent effect in inducing apoptosis of MCF-7 cells at 6 hours. After treatment with 5 μmol/L of deguelin for 6 hours the expression of phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (Ser380), phosphorylated 3-phosphoinositide-dependent protein kinase 1 (PDK1) (Ser241), phosphorylated Akt (Thr308) and phosphorylated glycogen synthase kinase-3β (GSK-3β) (Ser9) proteins were significantly reduced in MCF-7 cells, while there was no significant change in the expression of total Akt protein. However, after treatment with 1 μmol/L of deguelin for 6 hours, there was no apparent change in the expression of these 5 proteins. Conclusion: Deguelin can inhibit the phosphorylation of GSK-3β (Ser9) via inhibition of the phosphorylation of PTEN (Ser380) and PDK1 (Ser241) pathway, thus inducing apoptosis and inhibiting proliferation of MCF-7 cells.
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Objective:To establish a method for isolating tumor infiltrating dendritic cells(TIDC)from mice models of Lewis lung cancer by positive selection using anti-CD11c magnetic beads.Methods:A total of 1.0?10~6 Lewis lung cancer cells were subcutaneously injected into a C57BL/6 mice at the lateral abdominal wall to establish the mouse model of lung cancer.TIDC were isolated positively using anti-mouse CD11c magnetic beads;they were labeled by anti-mouse CD11c,and then the purity of the isolated cells was tested by FACScan flow cytometer.The cells were also double labeled by PE-conjugated MHC-ⅡmAb and FITC-conjugated CD83 mAb or FITC-conjugated CD86 mAb to analyze the phenotype of cells by FACScan flow cytometer.Results:The positive selection using anti-CD11 c magnetic beads isolated(1.73?0.31)?10~6 TIDC from each gram of Lewis lung cancer tissue,which accounted for(2.18?0.29)%of the cells in the tumor tissues.The purity of TIDC was 96.49%.Electron microscope showed that the isolated TIDC had the typical character of DC cells.The positive rates of MHC-Ⅱ,CD83 and CD86 molecules in TIDC surface were(51.25?4.21)%, (3.48?0.34)%and(3.07?0.65)%,respectively.Conclusion:The positive selection using anti-CD11c magnetic beads is highly effective,simple,and economic,and is worth popularizing.
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Objective:To explore the expression of B7-1,B7-2 and B7-H1 on tumor-infiltrating dendritic cells(TIDC) and on splenic dendritic cells(SDC),and to investigate TIDC-mediated and SDC-mediated T-cell function after blocking B7-H1 expression in these dendritic cells.Methods: The TIDCs and SDCs were isolated from tumor-bearing mice using anti-mouse CD11c magnetic beads.The expression of B7-1,B7-2 and B7-H1 on TIDC and SDC was analyzed using flow cytometer.T cells were co-cultured with TIDCs or SDCs for the mixed lymphocyte reaction(MLR),and monoclonal antibodies to B7-H1 or the isotype control antibodies were added to the MLR cultures.T-cell proliferation was assessed using XTT method and the secretion of IL-10 was detected using ELISA.Results: B7-1 and B7-2 positive TIDCs were significantly less than SDCs(P0.05).T-cell proliferation stimulated by TIDCs was weaker than that stimulated by SDCs;T cells produced more IL-10 after TIDCs stimulation than after SDCs stimulation(P
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Objective:Investigate the relation between the phosphorylation of FOXO1 and the apoptosis and the proliferation of lymphoma cells and to clarify its specific mechanism.Methods:The lymphoma cells Namalwa and Jurkat were treated with PI3K inhibitor wort mannin or etoposide or Wortmannin plus etoposide for different times-pan and at different concentration.The inhibition rates for cell growth of lymphoma cells were examined by XTT assay.Apoptosis were detected by flow cytometry.The expressions of p-Akt,p-FOXO1,FOXO1 and Bim were determined by Western blot analysis.Results:Wortmannin induced apoptosis of Jurkat cells and Namalwa cells and inhibited their survival effectively.The growth inhibition rate and the apoptosis rate of lymphoma cells induced by Wortmannin plus etoposide were higher than those induced by etoposide alone.After treated with Wortmannin,phosphorylation of FOXO1 remarkably reduced and bim markedly increased.Conclusion:The dephosphorylation of FOXO1 inhibits proliferation of Jurkat cells and Namalwa cells,promotes their apoptosis and enhanced the sensitivity of Non-Hodgkin lymphoma cells to etoposide.Bim activated by FOXO1 promotes cell apoptosis.