Cell Lung Cancer Tumor Tissues Research Articles

Establishment of a 21-color Panel for the Detection of Immune Cell Subsets in Human Non-small Cell Lung Cancer Tumor Tissues with Flow Cytometry

With the rise of multicolor flow cytometry, flow cytometry has become an important means to detect the immune microenvironment of lung cancer, but most of them are used to detect the proportion of cell subsets or the function of major cell subsets, and they cannot be detected at the same time. Therefore, a reliable 21-color flow cytometry protocol was established to detect the immune cell subsets in human non-small cell lung cancer (NSCLC) tumor tissues. Cell membrane surface antibodies cluster of differentiation (CD)45, CD3, CD19, CD4, CD8, programmed cell death 1 (PD-1), CD39, CD103, CD25, CD127, chemokine receptor 8 (CCR8), CD56, CD11c, human leukocyte antigen (HLA)-DR, CD38, CD27, CD69, CD62L, CD45RA, CCR7 and nucleic acid dye L/D were used to develop the protocol. Firstly, antibody titration experiments, voltage optimization, subtraction of one color staining and single color staining experiments were carried out for each antibody, and after the experimental conditions and detection schemes were determined, the feasibility of the scheme was verified by using peripheral blood mononuclear cells (PBMCs) specimens of six healthy adult volunteers. Tumor tissue samples from 6 NSCLC patients were tested and analyzed. The established 21-color flow cytometry protocol was used to detect the tumor tissue samples of 6 NSCLC patients, and the proportion of each cell subset in lung cancer tissue, as well as the immunophenotype and differentiation of the main cell population, were analyzed. The successfully established 21-color flow cytometry protocol is suitable for the detection of PBMCs and NSCLC tissue samples, which provides an effective new idea for monitoring the immune microenvironment status in lung cancer.

The B7-H4 gene induces immune escape partly via upregulating the PD-1/Stat3 pathway in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is associated with high mortality rates worldwide. The costimulatory molecule, B7-H4, a member of the B7 family, plays an important role in immune regulation, mainly by inhibiting the proliferation of T cells to achieve a negative regulatory T cell immune response. The mechanism of action of B7-H4 in non-small cell lung cancer is unknown at present. Tumor tissues from 71 patients subjected to radical pneumonectomy were examined, along with NSCLC cells and BALB/c mice. Among the 71 NSCLC cases, overall and recurrence-free survival rates were significantly lower in those displaying high B7-H4 expression. Mechanistic analyses showed that B7-H4 promoted the growth and metastasis of non-small cell lung cancer tumor tissues in mice through effects on CD8+ T cell apoptosis. Data from western blot experiments further suggested that B7-H4 induced CD8+ T cell death, both in vitro and in vivo, and affecting the PD-1/Stat3 pathway and promoting immune escape of tumor cells. Our collective findings support the potential utility of B7-H4 gene expression as a marker of NSCLC prognosis and provide a novel strategy for targeted therapy.

Downregulation of TCEAL7 expression induces CCND1 expression in non-small cell lung cancer.

Transcription Elongation Factor A-like 7 (TCEAL7) was first reported as a candidate tumor suppressor gene because of its inactivation in ovarian cancer as a result of promoter methylation. Down-regulation of the TCEAL7 gene expression was also associated with other cancers such as endometrial, breast, brain, prostate, gastric cancers, glioblastoma and linked to tumor phenotypes and clinical outcomes. However, there is no report in the literature investigating the role of TCEAL7 in non-small cell lung cancer. Cyclin D1 is an important molecule in the transition from G1 to S phase of the cell cycle, and is frequently deregulated in cancers. Cylin D1 (CCND1) gene is amplified or overexpressed in a variety of tumors. In our previous study we reported that CCND1 over-expression was not associated with amplification in non-small cell lung cancer. Recently, it has been reported that TCEAL7 regulates CCND1 expression through myc-binding E-box sequences. The aim of this study was to investigate the expression of TCEAL7 gene in non-small cell lung cancer and to determine its effect on the CCND1 expression level. For this purpose, expression levels of TCEAL7 and CCND1 genes were investigated in 50 patients with non-small cell lung cancer by quantitative real time polymerase chain reaction (qRT-PCR). TCEAL7 was under-expressed (68%) in non-small cell lung cancer tumor tissues while CCND1 was over-expressed (42%). The TCEAL7 levels negatively correlated with increased CCND1 expression (p = 0.002).

Rapid Detection of Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer Using Real-Time Polymerase Chain Reaction with TaqMan-MGB Probes

We investigated somatic mutations of the epidermal growth factor receptor gene in non-small cell lung cancer tumor tissue and their detection using real-time polymerase chain reaction with TaqMan-MGB probes. The DNA was extracted from surgically resected non-small cell lung cancer tumor specimens. Genes encoding for epidermal growth factor receptor tyrosine (exons 18, 19, and 21) were amplified by nested polymerase chain reaction, sequenced, and analyzed by chromatograms with manual review. TaqMan-MGB probes were designed to detect the epidermal growth factor receptor gene mutations in the tumor tissues using real-time polymerase chain reaction. Somatic point mutations and deletions were identified in the tyrosine kinase domain of the epidermal growth factor receptor gene in 21 of 80 non-small cell lung cancer patients, including 13 patients with deletion mutations occurring in exon 19 and 8 patients with point mutations occurring in codon 858 (exon 21). The results from real-time polymerase chain reaction with TaqMan-MGB probes were completely consistent with sequencing outcomes. Both the sensitivity and specificity for detecting the epidermal growth factor receptor gene mutations using real-time polymerase chain reaction with TaqMan-MGB probes were 100%. The mutation incidence was significantly higher in female patients, nonsmokers, and patients with adenocarcinoma than in male patients, smokers, and those with nonadenocarcinomas (P < 0.05). The mutations were not related to patient's age or tumor nodal metastasis staging. Somatic mutations of the epidermal growth factor receptor gene that develop in non-small cell lung cancer patients are more common in female patients, nonsmokers, and patients with adenocarcinoma. Real-time polymerase chain reaction using TaqMan-MGB probes is effective, simple, and fast in the detection of epidermal growth factor receptor gene mutations.