Subtype-based analysis of cell-in-cell structures in non-small cell lung cancer.

Lung cancer is ranked as the leading cause of cancer-related death worldwide, and the development of novel biomarkers is helpful to improve the prognosis of non-small cell lung cancer (NSCLC). Cell-in-cell structures (CICs), a novel functional surrogate of complicated cell behaviors, have shown promise in predicting the prognosis of cancer patients. However, the CIC profiling and its prognostic value remain unclear in NSCLC. In this study, we retrospectively explored the CIC profiling in a cohort of NSCLC tissues by using the "Epithelium-Macrophage-Leukocyte" (EML) method. The distribution of CICs was examined by the Chi-square test, and univariate and multivariate analyses were performed for survival analysis. Four types of CICs were identified in lung cancer tissues, namely, tumor-in-tumor (TiT), tumor-in-macrophage (TiM), lymphocyte-in-tumor (LiT), and macrophage-in-tumor (MiT). Among them, the latter three constituted the heterotypic CICs (heCICs). Overall, CICs were more frequently present in adenocarcinoma than in squamous cell carcinoma (P = 0.009), and LiT was more common in the upper lobe of the lung compared with other lobes (P = 0.020). In univariate analysis, the presence of TiM, heCIC density, TNM stage, T stage, and N stage showed association with the overall survival (OS) of NSCLC patients. Multivariate analysis revealed that heCICs (HR = 2.6, 95% CI 1.25-5.6) and lymph node invasion (HR = 2.6, 95% CI 1.33-5.1) were independent factors associated with the OS of NSCLC. Taken together, we profiled the CIC subtypes in NSCLC for the first time and demonstrated the prognostic value of heCICs, which may serve as a type of novel functional markers along with classical pathological factors in improving prognosis prediction for patients with NSCLC.

Subtype-Based Analysis of Cell-in-Cell Structures in Esophageal Squamous Cell Carcinoma.

Cell-in-cell (CIC) structures are defined as the special structures with one or more cells enclosed inside another one. Increasing data indicated that CIC structures were functional surrogates of complicated cell behaviors and prognosis predictor in heterogeneous cancers. However, the CIC structure profiling and its prognostic value have not been reported in human esophageal squamous cell Carcinoma (ESCC). We conducted the analysis of subtyped CIC-based profiling in ESCC using "epithelium-macrophage-leukocyte" (EML) multiplex staining and examined the prognostic value of CIC structure profiling through Kaplan-Meier plotting and Cox regression model. Totally, five CIC structure subtypes were identified in ESCC tissue and the majority of them was homotypic CIC (hoCIC) with tumor cells inside tumor cells (TiT). By univariate and multivariate analyses, TiT was shown to be an independent prognostic factor for resectable ESCC, and patients with higher density of TiT tended to have longer post-operational survival time. Furthermore, in subpopulation analysis stratified by TNM stage, high TiT density was associated with longer overall survival (OS) in patients of TNM stages III and IV as compared with patients with low TiT density (mean OS: 51 vs 15 months, P = 0.04) and T3 stage (mean OS: 57 vs 17 months, P=0.024). Together, we reported the first CIC structure profiling in ESCC and explored the prognostic value of subtyped CIC structures, which supported the notion that functional pathology with CIC structure profiling is an emerging prognostic factor for human cancers, such as ESCC.

Accurate Classification of Non-small Cell Lung Cancer (NSCLC) Pathology and Mapping of EGFR Mutation Spatial Distribution by Ambient Mass Spectrometry Imaging.

Objectives: Tumor pathology examination especially epidermal growth factor receptor (EGFR) mutations molecular testing has been integral part of lung cancer clinical practices. However, the EGFR mutations spatial distribution characteristics remains poorly investigated, which is critical to tumor heterogeneity analysis and precision diagnosis. Here, we conducted an exploratory study for label-free lung cancer pathology diagnosis and mapping of EGFR mutation spatial distribution using ambient mass spectrometry imaging (MSI). Materials and Methods: MSI analysis were performed in 55 post-operative non-small cell lung cancer (NSCLC) tumor and paired normal tissues to distinguish tumor from normal and classify pathology. We then compared diagnostic sensitivity of MSI and ADx-amplification refractory mutation system (ARMS) for the detection of EGFR mutation in pathological confirmed lung adenocarcinoma (AC) and explored EGFR mutations associated biomarkers to depict EGFR spatial distribution base on ambient MSI. Results: Of 55 pathological confirmed NSCLC, MSI achieved a diagnostic sensitivity of 85.2% (23/27) and 82.1% (23/28) for AC and squamous cell carcinoma (SCC), respectively. Among 27 AC, there were 17 EGFR-wild-type and 10 EGFR-mutated-positive samples detected by ARMS, and MSI achieved a diagnostic sensitivity of 82.3% (14/17) and 80% (8/10) for these two groups. Several phospholipids were specially enriched in AC compared with SCC tissues, with the higher ions intensity of phospholipids in EGFR-mutated-positive compared with EGFR-wild-type AC tissues. We also found EGFR mutations distribution was heterogeneous in different regions of same tumor by multi-regions ARMS detection, and only the regions with higher ions intensity of phospholipids were EGFR-mutated-positive. Conclusion: MSI method could accurately distinguish tumor pathology and subtypes, and phospholipids were reliable EGFR mutations associated biomarkers, phospholipids imaging could intuitively visualize EGFR mutations spatial distribution, may facilitate our understanding of tumor heterogeneity.

Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli.

Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl ß-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.

Interleukin-24 Concentrations Not Related with Age, but Affect Pro- and Anti-Inflammatory Cytokines Differently in Healthy Donors.

BACKGROUND: Interleukin 24 (IL-24) is expressed at different levels in a variety of tumor tissues and matched normal tissues and is regarded as a potential tumor biomarker as its expression levels in tumor tissues are associated with tumor patient prognosis. At present, the expression level of IL-24 in healthy human peripheral blood is unknown. METHODS: In this study, 1940 blood samples were collected using different processing methods from healthy donors. ELISA was used to detect IL-24 concentrations. RESULTS: The results showed that processing methods had the greatest influence on test results, with the highest IL24 concentration in EDTA plasma and the lowest in sodium citrate plasma. Lengths of storage time at 4°C had no obvious effect on IL-24 test results, and IL-24 in peripheral blood was stable for 15 days. IL-24 concentration in the sera of healthy donors showed no associations with age, blood glucose, hemoglobin, total cholesterol, carcinoembryonic antigen, absolute lymphocyte counts, alpha fetoprotein, white blood cells, thyroid stimulating hormone, or cereal third transaminase. We also confirmed that IL-24 expression level in the blood of healthy subjects was positively correlated with pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), but negatively correlated with anti-inflammatory cytokine, IL-10. CONCLUSIONS: We observed that sample processing methods influence the detection of IL-24 levels as EDTA plasma had the highest IL-24 concentration, and citric acid sodium, the lowest. Age, gender, and physical and chemical indicators were not related to IL-24 concentrations. We confirmed the IL-24 concentration was positively related to IL-6 and TNF-α and negatively to IL-10.

A novel 5-fluorouracil-resistant human esophageal squamous cell carcinoma cell line Eca-109/5-FU with significant drug resistance-related characteristics.

5-Fluorouracil (5-FU) is used for the clinical treatment of esophageal squamous cell carcinomas (ESCCs), yet it also induces chemoresistant cancer cells during treatment, which leads to the failure of the therapy. To further explore the resistance mechanism of 5-FU in ESCC, we established the 5-FU-resistant ESCC cell line Eca-109/5-FU, which was prepared by the stepwise exposure to increasing 5-FU concentrations. MTT assay and nude mouse xenograft models were used to test the drug resistance and proliferation of Eca-109 and Eca-109/5-FU cells in vitro and in vivo. Apoptosis and cell cycle distribution were determined using flow cytometry. Drug resistance-related proteins were detected by western blotting. Metabolomic profiles were obtained from nuclear magnetic resonance (NMR) tests. In regards to Eca-109/5-FU, the decreased susceptibility to 5-FU was determined in vitro and in vivo with slower rate of proliferation. Drug resistance-related proteins (multidrug resistance-associated protein 1 and ATP-binding cassette superfamily G member 2), epithelial-to-mesenchymal transition (EMT)-related proteins (E-cadherin and vimentin) and cancer stem cell-related proteins (prominin-1 and hyaluronate receptor) exhibited significant differences between the two cell lines. The 5-FU-resistant cell line Eca-109/5-FU achieved the ability to tolerate 5-FU, which may depend on significant drug resistance-related characteristics, such as EMT and cancer stem cell-like properties. The metabolism of Eca-109/5-FU was altered, and more than 15 metabolites was found to contribute to the difference in the metabolite profile, such as lactate, glutamate, taurine, glutamine, proline, aspartate, methanol, cystine, glycine and uracil. Our results identified that the resistant cell line Eca-109/5-FU showed quite different characteristics compared with the parental Eca-109 cells. The Eca-109/5-FU cell line provides an experimental model for further steps to select chemotherapeutic sensitizers.

Secreted recombinant human IL-24 protein inhibits the proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro and in vivo.

Interleukin-24 (IL-24) displays cancer-specific apoptosis-inducing properties in a broad spectrum of human tumors without harmful effects on normal cells. The human IL-24 protein is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and a potent antiangiogenic molecule. However, the function of secreted recombinant human IL-24 (srhIL-24) protein in esophageal squamous cell carcinoma (ESCC) cells has not been studied. In the present study, we prepared a stable site-specific-integrated cell line, Flp-InTMCHO/IL-24 (FCHO/IL-24), which secreted rhIL-24 at a higher level than three random-integrated cell lines. In vitro, we identified that the purified srhIL-24 inhibited proliferation and induced the apoptosis of ESCC Eca-109 cells and activated STAT3, which was related with the IL-20 receptors. In vivo, the tumorigenicity of Eca-109 cells was significantly inhibited by s.c. injection of FCHO/IL-24 cells. Decreased tumor microvessel density and an increased number of TUNEL-positive tumor cells were associated with tumor growth inhibition, indicating the presence of antiangiogenic activity and induction of apoptotic activity. In summary, the present study demonstrated that srhIL-24 induced growth inhibition and apoptosis in ESCC Eca-109 cells in vitro and in vivo, which may be mediated by the receptor pathway.

A novel human interleukin-24 peptide created by computer-guided design contributes to suppression of proliferation in esophageal squamous cell carcinoma Eca-109 cells.

Based on the three-dimensional modeling structure of human interleukin-24 (hIL-24) and its most likely active position predicted by solvent accessibility and apparent electrostatic properties, a novel hIL-24 peptide M1 was created by computer-guided molecular design. The cytotoxicity and cell selectivity of M1 were examined in three human carcinoma cell lines and one normal human embryo lung fibroblast cell line (HEL). MTT assay showed that M1 induced growth arrest in two IL-20 receptor complex-positive cancer cell lines (the esophageal squamous cell carcinoma cell line Eca-109 and the melanoma cell line A375), and antibodies against IL-24 or IL-20 receptor complexes significantly neutralized the inhibitory activity. Moreover, M1 had almost no cytotoxicity on the lung cancer A549 cell line, which lacks a full complement of the IL-20 receptor complexes, or on HEL cells that express the IL-20 receptor complexes. These findings demonstrate that M1 could act as an excellent candidate for the induction of growth arrest on receptor complex-positive cancer cells. In summary, the M1 peptide may represent a novel anticancer agent for esophageal squamous cell carcinoma therapy due to its cancer cell selectivity and its relatively low cytotoxicity to normal cells.

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF.

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression.

The role of microRNA in human lung squamous cell carcinoma.

MicroRNAs (miRNAs) are a group of small noncoding RNAs with modulator activity of gene expression. Deregulation of miRNA genes was found in several types of cancers. To explore the role of the miRNAs in Chinese lung squamous cell carcinoma (SCC), the expression profile of 711 miRNAs in SCC was analyzed. Total RNAs were used for hybridization on a commercially available array (miRCURY LNA array v.10.0), which contains 1,200 probes in tetramer, corresponding to 711 human miRNA genes. The results of miRNA microarray analysis were confirmed with quantitative real-time polymerase chain reaction. Seven human miRNAs (miR-126, miR-193a-3p, miR-30d, miR-30a, miR-101, let-7i, and miR-15a) were found to be significantly downregulated in lung SCC (P < 0.05), compared with normal lung tissues. The miRNAs miR-185 * and miR-125a-5p were significantly upregulated in lung SCC (P < 0.05), compared with normal lung tissues. The miRNA let-7i was downregulated in 9 of the 20 SCC samples, and miR-126 was downregulated in 16 of 20. The deregulation of some miRNAs in lung SCC suggests their possible involvement in the development and progression of SCC.

Repair of massive stent-induced tracheoesophageal fistula.

OBJECTIVE: The purpose of this report was to discuss a new surgical procedure in treating esophageal stent related large tracheoesophageal fistula without tracheal resection. METHODS: Clinical records of 5 patients with esophageal stent-related large tracheoesophageal fistulas treated in this hospital between 1997 and 2006 were reviewed. RESULT: All patients had insertion of a covered self-expanding esophageal stent, 1 for benign esophageal stricture and 4 for esophageal perforation resulting from various causes. A double patch technique, in which the esophageal wall was used as a protective patch repairing the defect on the trachea, was performed with an esophagectomy and gastric replacement. No significant complications occurred in the perioperative period. All patients recovered uneventfully. CONCLUSIONS: Use of the adjacent esophageal wall as a patch to close a defect on the trachea is a safe procedure with a favorable outcome. It should therefore be recommended as a reliable surgical procedure in treating massive stent-induced tracheoesophageal fistulas and other complicated tracheoesophageal fistulas that tracheal resection could not safely address. However, the esophagus was damaged to a certain degree.