agar and Carcinoma--Non-Small-Cell-Lung

Programmed death ligand-1 (PD-L1) has a known association with the prognosis of human cancers because of its ability to alter tumor immune surveillance via its interaction with PD-1. We questioned whether expression of PD-L1 in tumor cells could directly promote tumor growth and invasiveness in non-small cell lung cancer (NSCLC).. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate PD-L1 messenger RNA (mRNA) expression in lung tumors. The prognostic value of PD-L1 mRNA was assessed by Cox regression model. Transcriptional regulation of PD-L1 by human papillomavirus (HPV) 16/18 E6 oncoprotein or by epidermal growth factor receptor (EGFR) mutation in lung cancer cells was examined by Western blot and luciferase reporter assay. The cell growth and invasion were evaluated by colony formation, soft agar growth, and Boyden chamber assay.. The PD-L1 mRNA levels showed a positive association with HPV 16/18 E6 oncoprotein and with EGFR mutation in 223 surgically resected NSCLC patients. The prognostic significance of PD-L1 was more commonly observed in patients with high PD-L1/E6 positive and high PD-L1/EGFR mutant tumors. Mechanistically, upregulation of PD-L1 transcription by E6 or mutant EGFR occurred largely through the ERK-C/EBPβ-TLR4-NF-κB cascade. PD-L1 promotes the efficacy of colony formation, soft agar growth, and cell invasion. PD-L1 upregulates BAG-1 to reduce transforming growth factor (TGF)-β1 expression, and the decrease in SMAD4 because of TGF-β1 occurs through the p53/microRNA (miR)-224 axis. The decreases in TGF-β1 and SMAD4 are responsible for PD-L1-mediated cell invasiveness.. Induction of PD-L1 by E6 oncoprotein or mutant EGFR through the ERK-C/EBPβ-TLR4-NF-κB cascade may promote tumor growth and invasiveness in NSCLC because of decreasing TGF-β1 and SMAD4 expression.

Topics: Agar; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Gene Expression Regulation, Neoplastic; Human papillomavirus 16; Human papillomavirus 18; Humans; Lung Neoplasms; NF-kappa B; RNA, Messenger; Smad4 Protein; Toll-Like Receptor 4; Transforming Growth Factor beta1

Kirsten rat sarcoma viral oncogene homolog (KRAS) aberrations frequently occur in patients with lung cancer. Oncogenic KRAS is characterized by excessive reactive oxygen species (ROS) accumulation, thus, ROS detoxification may contribute to KRAS‑driven lung tumorigenesis. In the present study, the influence of glutathione peroxidase 2 (GPX2) on malignant progression and cisplatin resistance of KRAS‑driven lung cancer was explored. The RNA sequencing data from TCGA lung cancer samples and GEO database were downloaded and analyzed. The effects of GPX2 on KRAS‑driven lung tumorigenesis were evaluated by western blotting, cell viability assay, soft agar assay, Transwell assay, tumor xenograft model, flow cytometry, BrdU incorporation assay, transcriptome RNA sequencing, luciferase reporter assay and RNA immunoprecipitation. In the present study, GPX2 was upregulated in patients with non‑small cell lung carcinoma (NSCLC), and positively correlated with poor overall survival. Ectopic GPX2 expression facilitated malignant progression of KRAS

Topics: Agar; Bromodeoxyuridine; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Glutathione Peroxidase; Humans; Lung; Lung Neoplasms; Matrix Metalloproteinase 1; MicroRNAs; Proto-Oncogene Proteins p21(ras); Reactive Oxygen Species

Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding.. The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination.. Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of < 26 could be considered negative. Samples with a total CQ value of > 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant.. Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.

Topics: Adenocarcinoma; Agar; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Fixatives; Formaldehyde; Genes, erbB-1; Genetic Testing; Humans; Lung Neoplasms; Mutation; Paraffin Embedding; Proto-Oncogene Proteins p21(ras); Real-Time Polymerase Chain Reaction; Tissue Fixation

Lung cancers have been distinguished into small-cell lung cancer (SCLC) and non-small cell-lung cancer (NSCLC) types on the basis of their clinical behaviors and their responses to treatment. Moreover, growth of most SCLC cell lines in liquid culture medium is nonadherent, while that of most NSCLC cell lines is adherent. In this study, we examined the effect of matrigel (reconstituted basement membrane components), which is known to have growth-stimulatory activity on various human tumor cell lines in immunodeficient mice, on soft-agar colony formation of a panel of SCLC and NSCLC cell lines to clarify its mechanism of growth stimulation of cancer cells. Matrigel enhanced colony formation of all 9 NSCLC cell lines and 4 of 9 SCLC cell lines. There was a statistically significant difference (P < 0.01) between colony formations with and without matrigel of NSCLC cell lines, but not for SCLC cell lines. In liquid culture medium, all 9 NSCLC lines and 3 of 9 SCLC lines adhered to plastic dishes, whereas the other SCLC lines did not. Matrigel enhanced colony formation of all 3 adherent-type SCLC lines and 1 of 6 nonadherent-type NSCLC lines. Matrigel enhanced colony formation of both of 2 adherent-type non-lung cancer cell lines and 1 of 2 nonadherent-type leukemia cell lines. Neither transforming growth factor beta, collagen type IV, fibronectin, nor laminin, which are components of matrigel, enhanced colony formation of an NSCLC cell line in soft agar. The increase in the colony number of the NSCLC cell line by matrigel was abrogated by the protein kinase inhibitors staurosporine and UCN-01.

Topics: Adult; Agar; Aged; Aged, 80 and over; Biocompatible Materials; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Collagen; Drug Combinations; Female; Humans; Laminin; Lung Neoplasms; Male; Middle Aged; Proteoglycans; Tumor Cells, Cultured

We studied the effects human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant interleukin-3 on the colony formation of three human solid tumor cell lines. Using a modified double-layer soft agar clonogenic assay rhGM-CSF enhanced colony formation of all cell lines tested in a dose dependent manner (up to twofold for the breast cancer cell line BT-20, up to 163% of the control for the hypernephroma cell line C 94 and up to 147% for the non-small cell lung cancer cell line CCL 185 at a concentration of 100 ng/ml). RhIL-3 stimulated colony formation of the cell lines C 94 and BT-20, whereas on the cell line CCL 185 rhIL-3 had no effect even at the highest dose level tested (100 ng/ml). Combinations of growth factors showed subadditive stimulation on two cell lines tested (BT-20, C 94). These data indicate that haematopoietic growth factors exert a growth promoting activity on certain solid tumor cells in vitro at physiological concentrations. Therefore our results suggest that the application of these factors in immuno- and myelosuppressed cancer patients after high dose chemotherapy should be seen in light of a possible co-stimulation of the malignant cells.

Topics: Agar; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Renal Cell; Cell Division; Colony-Stimulating Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Kidney Neoplasms; Lung Neoplasms; Neoplastic Stem Cells; Recombinant Proteins; Statistics as Topic; Tumor Cells, Cultured; Tumor Stem Cell Assay