Level of peripheral blood circulating tumor cells in lung cancer patients detected by CI-101 and its clinical significance / 国际肿瘤学杂志

Objective:To explore the correlations between circulating tumor cells (CTCs) level in peripheral venous blood and clinicopathological characteristics and biomarkers of lung cancer patients using CI-101 cell search immunomagnetic bead enrichment technology combined with fluorescent cytochemical staining.Methods:Blood samples were collected from 100 patients with first-diagnosed lung cancer treated in Department of Thoracic Surgery and Department of Cardiothoracic Surgery of Yunnan Cancer Hospital from March 2014 to September 2014, 40 patients with lung benign tumor (all confirmed by pathological biopsy) and 30 healthy volunteers from the physical examination center. CTCs in peripheral blood were enriched by CI-101 cell search immunomagnetic bead, the morphology of CTCs was analyzed by immunocytofluorescence technique, and tumor cells were identified using HE cell staining method. The recovery rate, sensitivity and specificity of CI-101 cell search instrument for CTCs were detected. The difference of positive rate of CTCs in peripheral blood among lung cancer patients, lung benign tumor patients and healthy volunteers was compared. The relationship between the positive rate of CTCs and the clinicopathological characteristics of patients with lung cancer was analyzed. The correlations between CTCs and serum tumor markers were analyzed by coefficient of contingency in patients with lung cancer and lung benign tumor.Results:The recovery rate of CTCs by CI-101 cell search instrument was 72.0%-89.0%, and there was a significant linear correlation between the number of recovered cells and the number of incorporated cells. The correlation coefficient r=0.998 ( P<0.001), the linear regression equation was y=0.781 x+ 11.307, the sensitivity was 85.0%, and the specificity was 71.4%. The positive rate of CTCs in lung cancer patients (85.0%, 85/100) was higher than that in lung benign tumor patients (15.0%, 6/40) and healthy volunteers (46.7%, 14/30) ( χ2=62.798, P<0.001). The positive rate of CTCs in lung cancer patients was correlated with TNM stage ( χ2=19.059, P<0.001), tumor size ( χ2=13.830, P<0.001) and distant metastasis ( χ2=6.005, P=0.014). Coefficient of contingency analysis showed that the positive of CTCs was positively correlated with serum tumor markers CEA ( φ=0.217, P=0.011), CA125 ( φ=0.198, P=0.020), CA199 ( φ=0.169, P=0.049), CA742 ( φ=0.186, P=0.037) and cytokeratin 19 fragment ( φ=0.461, P<0.001) in patients with lung cancer and lung benign tumor. Conclusion:The application of CI-101 cell search instrument combined with immunomagnetic bead method can successfully enrich CTCs in peripheral venous blood of lung cancer patients. The positive rate of CTCs in patients with lung cancer has obvious correlation with tumor size, TNM stage, distant metastasis, serum tumor markers. It can be used as an auxiliary indicator for monitoring the condition of lung cancer patients.

Interferon alpha-inducible protein 27 and tumor / 国际肿瘤学杂志

Interferon alpha-inducible protein 27 (IFI27) is a newly discovered protein that participates in biological functions such as apoptosis,cell autophagy,oncolytic and immunoregulation.It also plays a major role in promoting tumor development,such as in breast cancer,ovarian cancer,liver cancer,squamous cell carcinoma and so on.Besides,studies have shown that IFI27 can interact with signal transducer and activator of transcription 1,microRNAs,interferon regulatory factor 4 and other genes,which promotes the development of tumors.Therefore,IFI27 is expected to be a marker for the occurrence and development of tumors,providing a new target for cancer therapy.

Association between aquaporin 1 and malignant tumors / 国际肿瘤学杂志

Aquaporin1 (AQP1) is a member of a family of specific channel proteins which could mediate the trans-biofilm transportation of small molecules such as water.Recent studies have shown that AQP1 is highly expressed in cancer tissues.It also has an effect on the proliferation and migration of cancer cells, angiogenesis in cancer and so on.AQP1 is expected to be a marker of screening, diagnosis, treatment and prognosis at tumor early stage.

Experimental Study on 99mTc-MIBI Imaging Evaluating Multidrug Resistance of Mice Lewis Lung Cancer / 昆明医科大学学报

Objectives 99mTc-methoxyisobutylisonitrile (MIBI) SPECT imaging technology was used to observe the condition of tumor cell in-taking imaging agent in the C57BL/6J mice Lewis lung cancer model before and after using Ginsenoside Rg3 (short for Rg3).We aimd so as to discuss the feasibility of using this method to evaluate tumor multidrug resistance (MDR) status.Methods Mice Lewis lung cancer models were randomly divided into Rg3 group and the control group.After applying Rg3,semi-quantitative analysis was made on 99mTc-MIBI SPECT imaging and region of interest (ROI) to observe the multidrug resistance state of tumor and then the results were compared with the detection results of flow cytometry.Results The tumor intake ratio (T/N) difference between the control group and the Rg3 group in imaging,imaging before applying Rg3 and imaging after applying Rg3 were separately 15,60 and 120 min.The differences were statistical significant (P ＜ 0.01).The eliminate indexes (WR) of the control group and Rg3 group were positively related to P-gp protein expression positive cells detected by flow cytometry (P ＜ 0.05).Conclusions 99mTc-MIBI imaging is negatively related to P-glycoprotein (P-gp) expression in mice Lewis lung cancer cells,which can clearly show the multidrug resistance state of tumors and dynamically monitor the effect of Rg3 on multidrug resistance reversion of mice Lewis lung cancer.

microRNAs and lung cancer / 国际肿瘤学杂志

microRNAs (miRNAs) are short non-protein-coding RNAs,which play important roles in the cell proliferation,differentiation,apoptosis,as well as activation of oncogenic and antioncogenic signals.Researches show that the abnormal expressions of miRNAs are closely related to the tumorigenesis,histological type,diagnosis,treatment and prognosis of lung cancer.So miRNAs may be the most potential and promising therapeutic targets for lung cancer.

Targeted Sox4 gene-knock-down suppresses xenograft growth of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of targeted Sox4 gene-knock-down on the growth of xenografts of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice.</p><p><b>METHODS</b>Recombinant plasmid pGFP-V-RS-Sox4 shRNA was constructed and transfected into XWLC-05 cells. Real-time quantitative PCR and Western blot were applied to confirm the effect of Sox4 gene-knock-down. XWLC-05 cells stably transfected with the plasmids were inoculated into nude mice to establish the xenograft model. The nude mouse status, tumor formation and tumor growth were observed, and the tumor inhibition rate was calculated. CT scan was performed to assess the metastasis of xenografts. Immunohistochemical staining was applied to detect Sox4 and ki-67 protein expression.</p><p><b>RESULTS</b>Recombinant plasmid pGFP-V-RS-A-Sox4 shRNA which can effectively knocking-down Sox4 gene was successfully constructed and the stable transfected cells were selected by puromycin-screening. The success rate of tumor cell inoculation was 100% in the mice of all groups except those inoculated with saline. The body weight of all mice inoculated with parental XWLC-05 cells (blank control), pGFP-V-RS-scram shRNA trsfected XWLC-05 cells (negative control), and pGFP-V-RS-Sox4 shRNA transfected XWLC-05 cells was increased to a varying degree, but there was no significant difference among the groups (P > 0.05 ). The growth of xenografts was significantly inhibited after silencing the Sox4 gene expression when compared with that of the blank and negative controls (P < 0.05) . The volume of removed tumors of the Sox4 gene-inhibited mice was (2.30 ± 0.34) cm(3) , significantly smaller than that of the negative control (3.99 ± 0.45) cm(3) and the blank control (4.03 ± 0.42) cm(3) (P < 0.05) . The weight of removed tumors of Sox4 gene-inhibited mice was (0.86 ± 0.14) g, significantly lower than that of the negative control (1.84 ± 0.27) g and blank control (1.86 ± 0.22) g, (P < 0.05). Immunohistochemical staining showed that Sox4 and ki-67 proteins mainly expressed in cell nuclei. The staining was significantly decreased in xenografts of Sox4-inhibited mice when compared with the negative and blank controls (P < 0.05). No distant metastasis was found in any mouse by CT imaging and pathological examination during the observation period.</p><p><b>CONCLUSIONS</b>The xenograft model of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice is successfully established. Knocking-down of Sox4 gene can suppress the xenograft tumor growth.</p>

Mechanism and abnormal expression of sox4 gene in tumor / 国际肿瘤学杂志

Recent studies have revealed that sox4 gene expresses abnormally in many kinds of tumor tissues and it probably involves in tumorigenesis,development and metastasis of cancer.Regulating proliferation,differentiation,apoptosis and other process may participate in the work mechanisms of sox4 gene.Therefore,further studies about the relationship between sox4 gene and tumor would provide new ideas of exploring special diagnosis markers and novel targets for tumor therapy.

Establishment of orthotopic Lewis lung cancer model in mouse / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>The mouse lung cancer orthotopic model includes spontaneous lung cancer model and endotracheal transplanted model, and etc. The spontaneous lung cancer needs longer time and does not ensure the rate of the generation of the tumor; as for endotracheal transplanted model, the position and size of the tumor are instable. In this study, the 3LL cell line was orthotopically transplanted into the lung of the C57BL/6 mice, compare to the heterotopic model, to discuss their stability and transfer-characteristics. And this study was also to optimize the method of establishing lung cancer orthotopic animal model.</p><p><b>METHODS</b>Different quantity of 3LL cells were inoculated into the left oxter of C57BL/6 mice to establish the heterotopic model; or suspended with Matrigel then inoculated into the left lung of C57BL/6 mice to establish orthotopic model. The survival-time of the mice was examined. The tissue was collected for the subsequent histology assay after euthanizing the mice. Microvessels density (MVD) was observed and counted by immunohistological chemistry. CD44v was detected by flow cytometry.</p><p><b>RESULTS</b>TTumor-form-rate of the heterotopic group were 100%, 66.7%, 16.7%, respectively, and had no macroscopic transfer. Tumor-form-rate of the orthotopic group were 100%, 100%, 83.3%, respectively, and had widespread transfer in contralateral chest and the lung. The median survival time of the orthotopic group (38, 35, 23 days) were less than the heterotopic group (82, 72, 50 days). MVD of the orthotopic group (120.2 +/- 9.73) was higher than the heterotopic group (92.6 +/- 7.12). The expression of CD44v of orthotopic (26.46 +/- 1.56)% was higher than the heterotopic group (23.13 +/- 1.02)%.</p><p><b>CONCLUSION</b>The lung cancer orthotopic model which established by 3LL cells transplanted into the lung of the mice is simple, dependable, repeatable and has stronger transfer characteristics than the heterotopic model.</p>

Detection and significance of CK19 mRNA-positive circulating tumor cells in peripheral blood of patients with early breast cancer / 肿瘤研究与临床

Objective To study the clinical and prognostic value of CK19 mRNA-positive circulating tumor cells in early breast cancer patients. Methods We analyzed the peripheral blood in 50 patients with early breast cancer after surgery and before the initiation of any adjuvant treatment for the presence of CK19 mRNA-positive circulating tumor cells using a nest reverse polymerase chain reaction assay. All patients were followed up. Results CK19 mRNA-positive cells were detected in 40.0 %(20/50) of patients with early breast cancer, 12.5 %(3/24) of patients with breast benign lesions, but 5 %(1/20) in healthy individuals (P =0.017,P =0.004); 11 to 20 of them relapsed during the follow-up period (P =0.002). There was no significant association between the detection of CK19 mRNA-positive cell and the patients' menstrual status, tumor stage, tumor size, etc (P ＞0.05). Detection of peripheral-blood CK19 mRNA-positive cells was associated with reduced median relapse-free interval in early breast cancer patients (P =0.007). Conclusion CK19 mRNA is one of the molecular markers for the detection of circulating tumor cells in early breast cancer. Detection of peripheral blood CK19 mRNA-positive cells might be an important predictive value as a marker of relapse in early breast cancer patients.

An oral DNA vaccine against VEGFR2 inhibits angiogenesis of Lewis lung carcinoma in C57BL/6 mice / 中国肺癌杂志

<p><b>BACKGROUND</b>It has been known that vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) play important roles in tumor angiogenesis. The aim of this study is to investigate whether an oral DNA vaccine against VEGFR2 has the inhibition effect on tumor growth and angiogenesis, and explore its mechanism in vivo.</p><p><b>METHODS</b>C57BL/6 mice were respectively given the DNA vaccine encoding VEGFR2 (vaccine group), pcDNA3.1 (plasmid group) and saline (saline group). All the mice were then inoculated with Lewis lung carcinoma 3LL cells. Weight, size and microvessel density (MVD) of transplanted tumors were observed. The levels of CD3+ and CD8+ T cells in peripheral blood of mice were detected by flow cytometry.</p><p><b>RESULTS</b>Weight of transplanted tumors in vaccine group was significantly smaller than those in plasmid and saline groups (P < 0.05), and MVD was significantly lower in vaccine group than that in plasmid and saline groups (P < 0.05). After inoculated with 3LL cells, CD3+ and CD8+ T cell levels of vaccine group were markedly higher than those of plasmid and saline groups (P < 0.05).</p><p><b>CONCLUSIONS</b>The oral DNA vaccine can significantly inhibit angiogenesis and growth of transplanted tumor in mice. It may act through killing endothelial cells of tumor.</p>

Inhibitory effect of Periplaneta Americana extract on 3LL lung cancer in mice / 中国肺癌杂志

<p><b>BACKGROUND</b>It has been known that Kangfuxin, a drug derived from Periplaneta Americana, can induce cell apoptosis of many cancer cell lines in vitro. The aim of this study is to investigate the inhibitory effect and mechanism of Periplaneta Americana extract (PAE) on 3LL lung cancer in mice.</p><p><b>METHODS</b>The C57BL/6J mice transplanted with 3LL lung cancer were divided into normal saline (NS), PAE high dose (PAE-H) and PAE low dose (PAE-L) groups. The body weight changes and inhibitory rate of tumor growth in each group were observed. In addition, the cell cycle, apoptosis index (AI) and the expression of apoptosis associated genes were analysed by flow cytometry (FCM).</p><p><b>RESULTS</b>The body weights were decreased in PAE-L and PAE-H treated group compared with NS group and the inhibitive rate of tumor growth was 41.24% and 81.08% respectively. FCM assay indicated that PAE could induce apoptosis of lung cancer cell, and the apoptosis rate was concentration-dependent. At the same time, the number of S and G2/M phase cells was decreased, most of the cells were arrested in G1/G1 phase. The result of TUNEL showed that there were apoptosis and necrosis associated with upregulated expression of Fas, FasR and p53 genes, and downregulated expression of Bcl-2.</p><p><b>CONCLUSIONS</b>PAE may inhibit the growth of 3LL lung cancer in mice and induce apoptosis of 3LL lung cancer cells. It might be related to its effects on the regulation of apoptotic gene expression.</p>

Inducement of Dendritic Cells Derived from Cord Blood Mononuclear Cells and CTLs Antitumor Immunity in Vitro / 昆明医科大学学报

Objective To investigate the way of inducing dendritic cells from precursor in human cord blood and its role in antitumor immunity.Methods Cord blood was collected under sterile condition and the cord blood mononuclear cells were separated by centrifugal in density gradient.CBMCs were cultured with GM-CSF+IL-4+TNF-?and cell phenotype was analyzed with CD1a、CD83 antibody by using indirect immunofluorescence assays.The effects of DCs pulsed with tumoral antigens on cytotoxic T lymphocytes(CTLs) inducement and growth inhibition of YTMLC cells were assayed.Results Our results indicated that DCs precursors in human cord blood can be induced to differentiate in the medium containing GM-CSF、IL-4 and TNF-?.The cells with typical morphological properties of DCs were observed at the 7th day.At that time,(20.8?1.62)%CD1a+ cells were obtained.After incubation with tumor cytolysis antigen,the DCs can activate the CTLs to become tumor specialized CTLs,which had shown significantly inhibition on growth of YTMLC tumor cell line.Conclusion The precursors in human cord blood can be induced to functional DCs which activate T lymphocyte to become tumor specialized CTLs.

Survival and distribution of CD34~+/CD45~+ cells from bone marrow of transgenic GFP mouse in a completely transected spinal cord rat model / 中国病理生理杂志

AIM: To study the survival, transfer and distribution of bone marrow CD34+/CD45+ cells from transgenic GFP mouse after transplanted into the completed transversional spinal cord rat model. METHODS: The bone marrow cells isolated from transgenic GFP mice were cultured in vitro. The cultured cells were identified by anti-CD34 and anti-CD45 monoclonal antibodies, and were transferred into the end of transection spinal cord. Paraformaldehyde was infused into the left ventricle of the rat model at the 24 h, 48 h, 1 week, 2 weeks, 4 weeks and 8 weeks after cell transplantation. Through sank and frozen, the spinal cord was sectioned at 10 ?m thickness. The green fluorescence positive cells were observed under the fluorescence microscope. CD34+/CD45+ cells were identified by immunohistochemistry staining. RESULTS: Green fluorescence positive cells were found at the head and the end of the completed transection part of spinal cord. Most of the green fluorescence positive cells were distributed in the gray substance of spinal cord. CD34+/CD45+ cells were found by immunohistochemistry staining. CONCLUSION: CD34+/CD45+ cells survived in spinal cord of SD rat, and migrated to the head of the transection part. The distance of migration was extended by the time.