Vitamin D inhibition of lung adenocarcinoma cell proliferation in vitro.

Vitamin D has the capability to inhibit tumor cell proliferation and promote tumor cell apoptosis but whether this mechanism exists in lung adenocarcinoma cells remains to be studied. Our objective is to explore whether vitamin D has the capability to inhibit lung adenocarcinoma cell proliferation and synergize with cisplatin. Our method was to explore the effect of different concentrations of 1,25(OH)2D3 with or without cisplatin on lung adenocarcinoma cells by detecting cell proliferation rates at different time points. 1,25(OH)2D3 was capsulated with nanomaterial before acting on lung adenocarcinoma cells, and cell proliferation rates at different time points were detected with the CCK-8 method. When vitamin D was applied at a concentration of 1 × 10(-7) and 1 × 10(-6) mol/L on A549, PC9, SPC-A1, and H1650 cells for 72 h, no inhibition occurred on cell proliferation. Between the concentrations of 1 × 10(-5) and 0.5 × 10(-5) mol/L, inhibition on cell proliferation increased with drug action time. Between the concentration of 2.5 × 10(-5) and 0.03 × 10(-5) mol/L, inhibition on cell proliferation increased with increasing drug concentration. Analysis using bivariate correlations showed that the correlation coefficient of the proliferation inhibition rate and drug content was 0.580 (p < 0.0001). The correlation coefficient of proliferation inhibition rate and the drug action time was 0.379 (p = 0.01). The combined use of vitamin D and dichlorodiammine-platinum(II) (DDP) significantly increased the inhibition rate on A549 cell proliferation, which peaked after culturing for 96 h (Table 4). Further analysis using bivariate correlations showed that the correlation coefficient between proliferation inhibition rate and DDP concentration was 0.319 (p < 0.0001). The correlation coefficient of the proliferation inhibition rate and vitamin D concentration was 0.269 (p < 0.0001). The correlation coefficient of proliferation inhibition and drug action time was 0.221(p = 0.003). Vitamin D capsulated with nanomaterial (5 ng/ml) on PC-9 cells for 72 h did not inhibit cell proliferation, while after 10 days, the content of crystal violet dissolved decreased by 6.3 ± 3.2% for the nonleaded nanomaterial group and decreased by 45.8 ± 10.9% for the nanomaterial-capsulated vitamin D group (p < 0.0001). Vitamin D has the capability to inhibit the proliferation of lung adenocarcinoma cells, synergistically inhibit the proliferation of lung adenocarcinoma cells with DDP, and when capsulated with nanomaterial can significantly inhibit the proliferation of lung adenocarcinoma cells.

Characteristics of Notch2(+) pancreatic cancer stem-like cells and the relationship with centroacinar cells.

Notch2, a surface marker in cell lines, is used to isolate, identify and localise pancreatic cancer stem-like cells and is a target for therapy of these cells. Sphere formation was induced in Panc-1 and Bxpc-3 pancreatic cancer cell lines, and Notch2(+) cells were separated from Bxpc-3 and Panc-1 cell lines by magnetic activated cell sorting (MACS). Expression of stem cell-related markers, OCT4, Nanog and PDX1, were measured by immunofluorescent (IF) staining. Expression of Notch2 was also determined immunohistochemically in pancreatic tissues. Notch2(+) cells were transplanted in subcutaneous of mice. AQP1 and AQP5 were also measured by IF in Bxpc-3 cells. The Notch signal pathway inhibitor, Compound E (CE), was used to treat Notch2(+) Bxpc-3 cells, and their vitalities were subsequently measured by the CCK-8 method. Positive expression of OCT4, Nanog and PDX1 was observed in Notch2(+) cells. Notch2(+) cells at centroacinar cell (CAC) and terminal ductal locations expressed AQP1 and AQP5. They were strongly tumourigenic in mice, and CE inhibited proliferation of Notch2(+) Bxpc-3 cells to some degree. OCT4 and Nanog can be used as markers of self-renewal in pancreatic cancer stem cells. Notch2(+) cells in human pancreatic cancer Bxpc-3 and Panc-1 cell lines had the properties of cancer stem cells. The results suggest that Notch2(+) pancreatic cancer stem-like cells had a close relationship with CAC.

The expression of stem cell-related indicators as a prognostic factor in human lung adenocarcinoma.

INTRODUCTION: The purpose of the present study was to detect the presence of BASC-like stem cell-related indicators, such as clara cell secretory protein (CCSP), Octamer-4 (OCT4) and Bmi-1, and evaluate their implications in the prognosis of patients with lung adenocarcinoma. METHODS: Specimens of 134 cases of lung adenocarcinoma were collected after radical surgery from January 1999 to June 2004. RESULTS: One hundred and twenty-six cases showed cells that were positive for CCSP, 99 cases positive for OCT4, 91 cases simultaneous expression of CCSP and OCT4 and 74 cases positive for Bmi-1. Bmi-1 was significantly higher in patients at stage III compared to patients at stages I and II. The pattern of survival curves showed that Bmi-1 was a significant prognostic factor of poor overall survival in lung adenocarcinoma patients (P = 0.0000), and the patients with OCT4(+) expression showed a greater increase in mortality than OCT4(-) patients (P = 0.0103). The results of univariate and multivariate Cox analysis revealed that the pathological stages of tumor node metastases (P = 0.037), OCT4 (P = 0.046) and Bmi-1 expression (P = 0.001) were independent prognostic factors. CONCLUSIONS: OCT4 and Bmi-1 may be good biomarkers to predict the prognosis of patients with completely resected lung adenocarcinoma.

Prognostic significance of OCT4 expression in adenocarcinoma of the lung.

OBJECTIVE: The purpose of this study was to detect the presence of cancer stem-like cells with bronchioalveolar stem cells (BASCs) properties and investigate the clinicopathological role of expression of OCT4 as well as the correlation with clinical outcomes in adenocarcinoma of the lung. METHODS: Specimens of 112 cases of Stage IB-IIIA lung adenocarcinoma after radical surgery were collected from June 1999 to June 2002. The putative cancer stem cells in tumor sections were visualized immunofluorescently by using the antibodies against three bronchioalveolar stem cells markers: surfactant protein C (SPC), Clara cell secretary protein (CCSP) and Octamer-4 (OCT4). Cancer stem-like cells with bronchioalveolar stem cell properties in human lung adenocarcinoma were subdivided into two phenotypes: OCT4(+)BASC (SPC(+)CCSP(+)OCT4(+)) and OCT4(-)BASC (SPC(+)CCSP(+)OCT4(-)). RESULTS: Cancer cells with CCSP(+)SPC(+)BASC phenotype were detected in 107 cases, 80 cases with OCT4(+)BASC phenotype (SPC(+)CCSP(+)OCT4(+)) and 27 cases with SPC(+)CCSP(+)OCT4(-). There was a correlation between differentiation and OCT4 expression (P = 0.047). The pattern of survival curves shows the expected trend of decreasing survival with increasing stage at diagnosis (P = 0.015) and with OCT4(+)BASC expression (P = 0.019). Multivariate Cox's analysis reveals that pathological stages of TNM (P = 0.008) and bronchioalveolar stem cells phenotypes (P = 0.015) are the independent prognostic factors. CONCLUSIONS: The cancer cells with bronchioalveolar stem cells phenotype are detectable in adenocarcinoma of the lung and the expression of self-renewal regulatory gene OCT4 in these cells indicated the worse clinical outcomes.

LMP1-specific cytotoxic T cells for the treatment of EBV-related post-transplantation lymphoproliferative disorders.

Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) represent a promising treatment option for EBV-associated post-transplantation lymphoproliferative disorders (PTLD). However, production of EBV-CTLs is often complicated and expensive. In the present study, we sought to establish an easy-to-use and economical production protocol for EBV-CTLs. EBV-CTLs were generated using latent membrane protein 1 (LMP1) peptides based on a modified generation protocol of cytokine-induced killer (CIK) cells. After 2-week culture, cells were well expanded (median total cell number: 9.82 × 109; median expansion fold: 107.8) and the median EBV LMP1-specific CD8+ T cell number was 8.94 × 108 (median frequency: 6.7%). However, the EBV-CTL products, unlike CIK cells, did not exhibit NK-like anti-tumor activity. Furthermore, the clinical efficacy of EBV-CTLs was demonstrated with a successful treatment of PTLD on a compassionate use basis in a patient following haploidentical hematopoietic stem cell transplantation. This study indicates the safety and efficacy of EBV LMP1-specific CTLs generated based on a modified generation protocol of CIK cells. Further investigation in a well-designed clinical study is warranted.

[Lung adenocarcinoma stem cell phenotypes and their correlation with patient prognosis].

OBJECTIVE: To detect the cancer stem cells and to evaluate their prognostic implication in patients with lung adenocarcinoma. METHODS: Three phenotypic markers of cancer stem cells (SP-C, CCSP and OCT4) in lung adenocarcinoma were detected by immunofluorecence staining. The correlation among the clinicopathological parameters and phenotypes of cancer stem cells as well as survival were analyzed by Cox proportional hazard method. RESULTS: Of the 57 cases, cancer stem cells were detected in 52, including OCT4(+) bronchioloalveolar stem cell (BASC) phenotype (SP-C(+) CCSP(+) OCT4(+)) in 40 cases and OCT4(-) BASC phenotype (SP-C(+) CCSP(+) OCT4(-)) in 12 cases. Statistical analysis revealed that the phenotype of cancer stem cells was related with the cellular differentiation, i.e. the OCT4(+) BASC phenotype occurred more frequently in the well-differentiated tumors, while the OCT4(-) BASC phenotype usually presented in most of the poorly-differentiated ones. Cox analysis showed that the OCT4(+) BASC phenotype was one of prognostic factors. CONCLUSION: The lung adenocarcinoma stem cells have phenotypic features of bronchioalveolar stem cells (SP-C(+) CCSP(+)). The expression of self-renewal regulatory gene OCT4 in these cells indicates an aggressive nature and unfavorable prognosis.

Epithelial ovarian cancer stem­like cells are resistant to the cellular lysis of cytokine­induced killer cells via HIF1A­mediated downregulation of ICAM­1.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecologic tumors. Cancer spheroid culture is a widely used model to study cancer stem cells. Previous studies have demonstrated the effectiveness of cytokine­induced killer (CIK) cell­based therapies against cancer and cancer stem cells. However, it is not clear how EOC spheroid cells respond to CIK­mediated cellular lysis, and the mechanisms involved have never been reported before. A flow cytometry­based method was used to evaluate the anti­cancer effects of CIK cells against adherent A2780 cells and A2780 spheroids. To demonstrate the association between hypoxia inducible factor­1α (HIF1A) and intercellular adhesion molecule­1 (ICAM­1), two HIF1A short hairpin RNA (shRNA) stable transfected cell lines were established. Furthermore, the protein expression levels of hypoxia/HIF1A­associated signaling pathways were evaluated, including transforming growth factor­ß1 (TGF­ß1)/mothers against decapentaplegic homologs (SMADs) and nuclear factor­κB (NF­κB) signaling pathways, comparing A2780 adherent cells and cancer spheroids. Flow cytometry revealed that A2780 spheroid cells were more resistant to CIK­mediated cellular lysis, which was partially reversed by an anti­ICAM­1 antibody. HIF1A was significantly upregulated in A2780 spheroids compared with adherent cells. Using HIF1A shRNA stable transfected cell lines and cobalt chloride, it was revealed that hypoxia/HIF1A contributed to downregulation of ICAM­1 in A2780 spheroid cells and adherent cells. Furthermore, hypoxia/HIF1A­associated signaling pathways, TGF­ß1/SMADs and NF­κB, were activated in A2780 spheroid cells by using western blotting. The findings indicate that EOC stem­like cells resist the CIK­mediated cellular lysis via HIF1A­mediated downregulation of ICAM­1, which may be instructive for optimizing and enhancing CIK­based therapies.

[Analysis of EGFR mutations in 176 cases of non-small cell lung cancer].

OBJECTIVE: To analyze the incidence and profile of mutations in epidermal growth factor receptor (EGFR) in Chinese patients with non-small cell lung cancer (NSCLC). METHODS: A total of 176 cases of NSCLC tissue was enrolled in this study, among which 123 normal lung samples were also included. The tissue DNA was extracted and the EGFR gene in exon 19 to 21 was subjected for PCR amplification and direct sequencing. RESULTS: The EGFR gene in exon 19-21 was of wild type in all normal lung tissues detected. Mutations were found in 57 cases of 176 lung cancer samples, with an incidence of 32. 4%. Mutations were mainly detected in the exon 19 (37/57 cases, 64. 9% ) and exon 21 (18/57 cases, 31. 6% ) , while that in the exon 20 was rare (2/57 cases, 3. 5% ). There were 7 types of EGFR mutation in the exon 19, resulting in the deletion of codon 746 to 753. A missense mutation was detected in exon 20, dealing with codon 789 to 793. The mutation in exon 21 belonged to the single missense substitution in codon 858. The EGFR mutations were more frequent in female patients than male ones, in adenocarcinoma and adenosquamous cell carcinoma versus cancer of other histologies. CONCLUSION: EGFR mutation is a tumor-specific somatic abnormality. Some one third of Chinese NSCLC tumors harbor EGFR mutations, especially in exons 19 and 21. These mutations are more frequently detected in female, adenocarcinoma and adenosquamous cell carcinoma.

[A primary study of immunotherapy with carcinoembryonic antigen peptide-pulsed, autologous human cultured dendritic cells in patients with advanced non-small cell lung cancer].

BACKGROUND: Dendritic cell (DC)-based immunotherapy is a new approach and effective for some malignant tumors. The aim of this study is to observe the efficacy and toxicity of immunotherapy with carcinoembryonic antigen (CEA) peptide-pulsed DCs in patients with refractory advanced lung cancer. METHODS: Lung cancer patients with high CEA expression were enrolled into this project. Autologous DCs were generated from patients' plastic-adherent peripheral blood mononuclear cells and loaded with CEA 5 days later. Cytokine-induced killer cells (CIK) were cultured from non-adherent peripheral blood mononuclear cells. DCs and CIK were transfused to patients. Responses and toxicities were observed. RESULTS: A total of 22 patients with lung cancer received DCs immunotherapy. DCs doses were 2.5×106-9.6×107 (5.03×106). CIK doses were 3.4×108-46×108. CD3, CD8, NK and IFN-γ levels obviously increased after treatment (P < 0.05). The 1-year survival rate was 68.2% (15/22). Main toxicities were fever and rash. CONCLUSIONS: DCs-based immunotherapy is feasible and safe to patients with lung cancer.

Two-stage induced differentiation of OCT4+/Nanog+ stem-like cells in lung adenocarcinoma.

Stem-like cells in solid tumors are purported to contribute to cancer development and poor treatment outcome. The abilities to self-renew, differentiate, and resist anticancer therapies are hallmarks of these rare cells, and steering them into lineage commitment may be one strategy to curb cancer development or progression. Vitamin D is a prohormone that can alter cell growth and differentiation and may induce the differentiation cancer stem-like cells. In this study, octamer-binding transcription factor 4 (OCT4)-positive/Nanog homeobox (Nanog)- positive lung adenocarcinoma stem-like cells (LACSCs) were enriched from spheroid cultured SPC-A1 cells and differentiated by a two-stage induction (TSI) method, which involved knockdown of hypoxia-inducible factor 1-alpha (HIF1α) expression (first stage) followed by sequential induction with 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3, VD3) and suberoylanilide hydroxamic acid (SAHA) treatment (second stage). The results showed the HIF1α-knockdowned cells displayed diminished cell invasion and clonogenic activities. Moreover, the TSI cells highly expressed tumor suppressor protein p63 (P63) and forkhead box J1 (FOXJ1) and lost stem cell characteristics, including absent expression of OCT4 and Nanog. These cells regained sensitivity to cisplatin in vitro while losing tumorigenic capacity and decreased tumor cell proliferation in vivo. Our results suggest that induced transdifferentiation of LACSCs by vitamin D and SAHA may become novel therapeutic avenue to alter tumor cell phenotypes and improve patient outcome.The development and progression of lung cancer may involve rare population of stem-like cells that have the ability to grow, differentiate, and resist drug treatment. However, current therapeutic strategies have mostly focused on tumor characteristics and neglected the potential source of cells that may contribute to poor clinical outcome. We generated lung adenocarcinoma stem-like cells from spheroid culture and induced their transdifferentiation by a two-stage method of knocking down HIF1α expression followed by vitamin Dand suberoylanilide hydroxamic acid (VD3/SAHA) treatment. We observed the induced cells lost stem-like characteristics, regained sensitivity to cisplatin, and displayed reduced tumorigenic capacity. These findings suggest that targeting stem-like cells by reverting them to more specialized state may be an approach to treat lung cancer.

Wnt signaling regulation of stem-like properties in human lung adenocarcinoma cell lines.

The refractory pulmonary adenocarcinoma is characterized by its metastasis and resistance to cytotoxic agents. While the underlying molecular mechanism is unclear, the property of chemoresistance may mainly lie in the presence of highly resistant cancer stem cells. We examined the function of Wnt/ß-catenin signaling in maintaining cancer stem cells (CSCs) in lung adenocarcinoma. Lentivirus-mediated knockdown of ß-catenin expression accelerated cell cycle. Subsequently, ß-catenin knockdown PC9 cells improve the sensitivity to chemotherapy. Further focusing on Wnt signal by administrating PP and EGFR-TKIs as Wnt antagonists can decrease metastasis and induce apoptosis. Collectively, these results indicate that Wnt signaling pathway plays an essential role in maintaining highly resistant CSCs, regulation of cell cycle, metastasis and apoptosis in lung adenocarcinoma.

Iodide- and glucose-handling gene expression regulated by sorafenib or cabozantinib in papillary thyroid cancer.

CONTEXT: The aberrant silencing of iodide-handling genes accompanied by up-regulation of glucose metabolism presents a major challenge for radioiodine treatment of papillary thyroid cancer (PTC). OBJECTIVE: This study aimed to evaluate the effect of tyrosine kinase inhibitors on iodide-handling and glucose-handling gene expression in BHP 2-7 cells harboring RET/PTC1 rearrangement. MAIN OUTCOME MEASURES: In this in vitro study, the effects of sorafenib or cabozantinib on cell growth, cycles, and apoptosis were investigated by cell proliferation assay, cell cycle analysis, and Annexin V-FITC apoptosis assay, respectively. The effect of both agents on signal transduction pathways was evaluated using the Western blot. Quantitative real-time PCR, Western blot, immunofluorescence, and radioisotope uptake assays were used to assess iodide-handling and glucose-handling gene expression. RESULTS: Both compounds inhibited cell proliferation in a time-dependent and dose-dependent manner and caused cell cycle arrest in the G0/G1 phase. Sorafenib blocked RET, AKT, and ERK1/2 phosphorylation, whereas cabozantinib blocked RET and AKT phosphorylation. The restoration of iodide-handling gene expression and inhibition of glucose transporter 1 and 3 expression could be induced by either drug. The robust expression of sodium/iodide symporter induced by either agent was confirmed, and (125)I uptake was correspondingly enhanced. (18)F-fluorodeoxyglucose accumulation was significantly decreased after treatment by either sorafenib or cabozantinib. CONCLUSIONS: Sorafenib and cabozantinib had marked effects on cell proliferation, cell cycle arrest, and signal transduction pathways in PTC cells harboring RET/PTC1 rearrangement. Both agents could be potentially used to enhance the expression of iodide-handling genes and inhibit the expression of glucose transporter genes.

Wnt blockers inhibit the proliferation of lung cancer stem cells.

BACKGROUND: Previous study has confirmed that the occurrence of Wnt pathway activation is associated with risk of non-small-cell lung cancer recurrence. However, whether the pharmacologic blocking of the Wnt signaling pathway could provide therapeutic possibility remains unknown. The aim of the present study was to evaluate the therapeutic functions of the Wnt signaling pathway inhibitor pyrvinium pamoate (PP) on lung cancer stem cells (LCSCs) in vitro. METHODS: Colony formation and sphere culture were performed to enrich LCSCs from three lung cancer cell lines: PC9, SPC-A1, and A549. After confirming stemness by immunofluorescence, PP was employed for cell viability assay by comparison with three other kinds of Wnt signaling inhibitor: salinomycin, ICG-001, and silibinin. The effect of PP on LCSCs was further verified by colony formation assay and gene expression analysis. RESULTS: LCSCs were successfully generated by sphere culture from SPC-A1 and PC9 cells, but not A549 cells. Immunofluorescence assay showed that LCSCs could express pluripotent stem cell markers, including NANOG, Oct4, KLF5, and SOX2, and Wnt signaling pathway molecules ß-catenin and MYC. Half-maximal inhibitory concentrations of PP on SPC-A1, PC9, and A549 were 10 nM, 0.44 nM, and 0.21 nM, respectively, which are much lower than those of salinomycin, ICG-001, and silibinin. Moreover, significantly decreased colony formation and downregulation of pluripotent stem cell signaling pathway were observed in lung cancer cells after treatment with PP. CONCLUSION: Wnt signaling inhibitor PP can inhibit proliferation of LCSCs, and the Wnt signaling pathway could be considered a promising therapeutic or interventional target in lung adenocarcinoma.

Wnt signaling regulates the stemness of lung cancer stem cells and its inhibitors exert anticancer effect on lung cancer SPC-A1 cells.

Wnt signaling plays an important role in regulating the activity of cancer stem cells (CSCs) in a variety of cancers. In this study, we explored the role of Wnt signaling in the lung cancer stem cells (LCSCs). LCSCs were obtained by sphere culture, for which human lung adenocarcinoma cell line SPC-A1 was treated with IGF, EGF and FGF-10. The stemness of LCSCs was confirmed by immunofluorescence, and pathway analysis was performed by functional genome screening and RT-PCR. The relationship between the identified signaling pathway and the expression of the stemness genes was explored by agonist/antagonist assay. Moreover, the effects of different signaling molecule inhibitors on sphere formation, cell viability and colony formation were also analyzed. The results showed that LCSCs were successfully generated as they expressed pluripotent stem cell markers Nanog and Oct 4, and lung distal epithelial markers CCSP and SP-C, by which the phenotype characterization of stem cells can be confirmed. The involvement of Wnt pathway in LCSCs was identified by functional genome screening and verified by RT-PCR. The expression of Wnt signaling components was closely related to the expression of the Nanog and Oct 4. Furthermore, targeting Wnt signaling pathway by using different signaling molecule inhibitors can exert anticancer effects. In conclusion, Wnt signaling pathway is involved in the stemness regulation of LCSCs and might be considered as a potential therapeutic target in lung adenocarcinoma.

Hematogenous dissemination of lung cancer cells during surgery: quantitative detection by flow cytometry and prognostic significance.

Shedding of neoplastic cells into the circulation is an essential event for the hematogenous metastasis of solid tumors. Recently, several studies reported that a high frequency of cancer cells could be detected in the bloodstream during surgery. The intraoperative detection of hematogenous dissemination of cancer cells was able to identify a subset of patients with malignant diseases at high risk for postoperative metastasis and to predict a poor prognosis. In order to evaluate the association between intraoperative dissemination of cancer cells and postsurgical survival of patients with non-small cell lung cancer (NSCLC), we developed a flow cytometric assay for specific detection of lung cancer cells in the blood. The monocyte-enriched population in the blood was separated by a modified Ficoll-Hypaque density centrifugation and then labeled with a combination of monoclonal antibodies specific for CD45, cytokeratin (CK) and two antigens expressed on lung cancer cells (2F7 and S5A). The assay could detect quantitatively lung cancer cells (defined as CD45(-1) CK(+) 2F7/S5A(+) cells), with the sensitivity limit of one cancer cell in 10(5) normal leukocytes. The specificity for lung cancer was 97%, which was calculated from the results of healthy subjects (20 cases) and patients affected with benign pulmonary diseases (26 cases) or esophageal cancer (14 cases). Blood samples of 31 NSCLC patients were collected from pulmonary vein during open thoracic surgery. Fifteen of them (48.4%) were found to have positive test results. The average cancer cell counts in these cases were 0.306 x 10(6)/l. Patients under 55 years of age had a significantly higher percentage of positive findings than those over 55 years of age (P < 0.05). The positive rate increased over the stages and lymph node status, but the differences were not statistically significant. Moreover, patients with squamous cell carcinoma at later stages (stages III and IV) had an increased frequency of positive test results than those at earlier stages (stages I and II, P < 0.05). In contrast, no such a difference was found in cases with adenocarcinoma. On the basis of 30-months follow-up date, the median survival time and 2-year survival rate for patients with positive and negative findings were 11 vs. 27 months, and 26.7 vs. 62.5%, respectively. There was a statistically significant difference between overall survival curves that favored the patients with negative test results (P = 0.023). Multivariate analysis indicated the stage of disease and the positive test results as two independent factors that affected survival time (P = 0.017 and 0.027). When a comparison was made within the patients at stages III and IV, the presence of cancer cells in blood was associated with a significantly shorter survival. These data indicate that the hematogenous dissemination of lung cancer cells during surgery would be one of the mechanisms of postoperative tumor metastasis. The detection of these cells may help to identify patients with poor prognosis.

[Vascular endothelial growth factor promotes hematogenous metastasis of cancer cells in patients with non-small cell lung cancer].

OBJECTIVE: To evaluate the effect of vascular endothelia1 growth factor (VEGF) on the hematogenous metastasis of non-small cell lung cancer (NSCLC). METHODS: The identification of lung cancer cells in the peripheral blood were carried out by cytological, immunohistocytologica1 and immunofluorecent stains respectively, following isolation of cytokeratin-expressing cells with magnetic activated cell sorting. The quantification of cancer cells in the blood was performed according to the established flow cytometric assay. The plasma VEGF was measured by commercially available ELISA kit. RESULTS: The lung cancer cells in the blood, showing a remarkable nuclear polymorphism, expressed the epithelial marker cytokeratin and telomerase reverse transcriptase (hTERT). These cells were stained positive by an NSCLC-specific monoclonal antibody S5Al0-2, but negative by antibodies against CD34 and CD45 antigens. Using the flow cytometric assay, 44 cases (28.6%) of l54 NSCLC patients were found to have cancer cells in their blood, with the incidence of positive cases correlated with the stage of disease. The plasma VEGF level was significantly increased in NSCLC patients in comparison with healthy individuals and patients with benign pulmonary diseases. This level was correlated with the stages of disease in patients with adenocarcinoma. In patiens with cancer cells in their blood, a higher level of plasma VEGF was related with an increased number of cancer cells. CONCLUSION: The plasma VEGF level is increased in NSCLC patients with approximate1y one fourth to have cancer cells in the peripheral blood. In these patients, increased VEGF level promotes hematogenous tumor metastasis, as indicated by a much higher number of cancer cells in the blood.

[Implication of micrometastatic cancer cells in the peripheral blood on prognosis of non-small cell lung cancer].

OBJECTIVE: To evaluate the relationship of micrometastatic cancer cells in the blood and prognosis of patients with non-small cell lung cancer (NSCLC). METHODS: Blood samples were collected from peripheral vein perioperatively and from the pulmonary vein intraoperatively in NSCLC patients. Cancer cells were detected by flow cytometry, as described previously. The patients were followed up and analyzed statistically. RESULTS: Cancer cells in blood samples were detected in 20 of 58 patients (34.5%). Patients under 57 years of age or with stage III/IV lesions had higher positive findings than those over 57 years or with stage I/II lesions (P = 0.000 and 0.006, respectively). On the basis of 40 month follow-up data, the 2- and 3-year survival rates of patients with positive and negative results were 30.0% vs 20.0%, and 52.6% vs 50.0%, respectively. There was significant difference between the overall survival curves which favored patients with negative findings (P = 0.0291 and 0.0092, respectively). CONCLUSION: This study indicates that cancer cells can be detected in the blood perioperatively from NSCLC patients which means poor prognosis.

[The relationship between drug sensitivity and expression of drug resistance gene mutations in non-small cell lung cancer].

OBJECTIVE: To study the relationship between the expression of drug resistance genes and the results of drug sensitivity test. METHODS: Surgical or biopsy specimens from 48 patients with non-small cell lung cancer (NSCLC) were measured for drug sensitivity by Annexin V combined with PI using flow cytometry. The drug resistance genes MDR(1), GST-pi and MPR were measured by RT-PCR. The relationship between expression of drug resistance gene mutations and the drug sensitivity of lung cancer was analyzed. RESULTS: The anti-tumor cytotoxicity of MMC, DDP, VDS, NVB, TAX, GEM, VP-16 and VCR were measured, and their respective tumor inhibition rates were (10.3 +/- 17.1)%, (20.7 +/- 22.2)%, (5.6 +/- 14.9)%, (7.9 +/- 16.2)%, (15.7 +/- 21.8)%, (11.2 +/- 13.8)%, (9.7 +/- 20.1)%, and (4.7 +/- 8.7)%. The positive rates of MDR(1), MRP and GST-pi expression were 67% (32/48), 42% (20/48), and 48% (23/48) respectively. There was no association between the expression of drug resistance genes MRP and GST-pi and the pathology or the stage of lung cancer. Interestingly, the over-expression of MRP was related to drug resistance to NVB, VDS and MMC; while the over-expression of GST-pi was related to resistance to DDP. No relationship was found between MDR(1) over-expression and drug resistance. CONCLUSION: The expression of some drug resistance genes is related to drug sensitivity test. The detection of the genes may be clinically useful in the administration of chemotherapy.

[Efficient generation and functional characteristics of dendritic cells from malignant pleural effusion in patients with lung cancer].

OBJECTIVE: To clarify whether functionally competent dendritic cells (DC) can be generated from malignant pleural effusion in patients with lung cancer. METHODS: Malignant effusion-associated monocytes were separated by adherence from malignant effusion-associated mononuclear cells and cultured in medium with granulocyte macrophage colony-stimulating factor (GM-CSF) plus interleukin 4 (IL-4). TNF-alpha was added for the last 24 h before culture termination. Cultured DC were identified by (1) using microscopy, scanning electron microscopy, and immunocytochemistry for the morphological features; (2) phenotypic markers; and (3) functional characteristics including a high stimulatory capacity to activate proliferation of lymphocyte in an allogeneic mixed leukocyte reaction and the ability to produce high levels of IFN-gamma. RESULTS: Cultured DC had the typical morphological features. The phenotype of DCs generated from effusion showed higher expression of CD(86) (84.6 +/- 6.1)%, HLA-DR (81.1 +/- 13.0)%, CD(40) (42.0 +/- 21.7)%, CD(1a) (20.0 +/- 9.5)% and lower expression of CD(14) (4.8 +/- 3.5)% than the control group. There was a significant difference in the stimulatory activity in allogeneic lymphocyte proliferation and the ability to produce high levels of IFN-gamma between DC derived from the malignant effusion and the control group. CONCLUSION: These findings suggest that DC can be generated from malignant pleural effusion, which might be a useful source of DC for immunotherapy.

[Vascular endothelial growth factor inhibits dendritic cells from patients with non-small cell lung carcinoma].

OBJECTIVE: To detect the effect of vascular endothelial growth factor (VEGF) on dendritic cells (DC) in patients with non-small cell lung cancer (NSCLC). METHODS: The measurement of DC in the peripheral blood was performed by a novel flow cytometric assay in 85 patients with NSCLC and 14 healthy volunteers. Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of VEGF(165) in the plasma. CD(14)(+) peripheral blood mononuclear cells (PBMC) were cultured to obtain DC in vitro with cytokines. VEGF(165) was added to evaluate its effect on DC differentiation and survival. The phenotypes and apoptosis of cultured cells were detected by flow cytometry. RESULTS: In comparison with healthy volunteers, the level of VEGF(165) was significantly increased (P < 0.05), while that of DC was significantly decreased (P < 0.01) in patients with NSCLC. No significant correlation was noted between the concentration of VEGF(165) and age, gender, differentiation and histological types in patients with NSCLC, neither was found in the level of DC (P > 0.05). The concentration of VEGF(165) was closely associated with TNM stage and distal metastasis (P < 0.05), while no correlation was found between the concentration of VEGF(165) and lymph node metastasis (P > 0.05). Significant correlations were noted between the level of DC in patients with NSCLC and TNM stage, lymph node metastasis and distal metastasis (P < 0.05). There was a negative correlation between the concentration of VEGF(165) and the level of DC (P < 0.05). Patients with abnormally elevated VEGF(165) showed significantly fewer DC. Cells cultured in vitro in the presence of VEGF(165) exhibited higher expression of CD(+)(14)(P = 0.000) and increased ratio of apoptic cells (P < 0.01), but decreased expression of CD(40), CD(86) and HLA-DR (P < 0.01), as compared to cells cultured without VEGF(165). CONCLUSIONS: The level of DC and the concentration of VEGF in the peripheral blood can reflect the malignancy of NSCLC. NSCLC can over-express VEGF to inhibit DC differentiation and maturation to evade host immune surveillance.