| Objective:To explore the infiltration and function of B lymphocytes in tumor tissues of non-small cell lung cancer and provide a theoretical basis for further improving the effect of immunotherapy.Methods:1.Single-cell transcriptome sequencing was used to explore the infiltration of immune cells in lung cancer tumor tissues from 6 patients diagnosed with stage ?-? NSCLC.2.The gene expression in B lymphocytes of peripheral blood was compared to B lymphoid gene expression in tumor tissues to find the difference between them.3.Evaluating the infiltration level of B cells in the microenvironment of NSCLC using immunohistochemistry and immunofluorescence.4.Another 30 specimens were taken to analyze the infiltration of B cells in tumor tissues and their relationship with tumorigenesis and development by flow cytometry.5.The clinical data and paraffin specimens of 164 patients with non-small cell lung cancer in Shanghai Pulmonary Hospital affiliated Tongji University were collected to explore the distribution of B lymphocytes in different stages of lung cancer and their relationship with the prognosis of patients.6.To further examined the expression level of subtype-specific marker genes of the two B cell types in different stages of lung cancer using the transcriptome datasets from the LUAD TCGA cohorts.7.A549 cell line and H1299 cell line were cultured separately as a control group.A549 cell line and H1299 cell line were co-cultured with B lymphocytes and the supernatants of B lymphocyte as experimental groups to observe the cell growth of the two groups?8.GraphPad Prism 7.0a was used for statistical analysis with the IHC quantification?flow cytometry analysis?OD45 data and survival analysis.P values from unpaired two tailed student's t tests were used for estimating the statistical significance between different groups.P values from Log-rank test were used for survival analysis.P<0.05 is considered statistically significant.Result:1.There are many types of monocyte infiltration in non-small cell lung cancer(NSCLC)tumor tissues,including B lymphocyte infiltration.2.The B cells could be divided into two major groups based on their B lymphocyte gene expression,including plasma-like B cells and naive-like B cells.Clustering of B cells in PBMC is different from that in tumor tissue.3.The CD20 positive cells were mainly located at the tertiary lymphoid structures(TLS)of lung tumors while CD79 positive cells not only located at the TLS but also randomly enriched within tumor tissues.4.The flow cytometry demonstrated the co-existence of the two subtypes of B cells.The naive-like B cells was significantly decreased at the advanced stages of lung cancers with flow cytometry.While the amount of the plasma-like B cells infiltration was not associated with the progression of lung cancer.5.The staining results of 164 samples showed a decrease of the naive-like B cell in the advanced stages of NSCLC,which was positively correlated with the good prognosis of patients.Using the transcriptome datasets from the LUAD TCGA cohorts,we reached the same conclusion.6.From the co-culture experiments of plasma-like B cells or their supernatants from different staging lung cancer with A549,H1299 cell lines,we found that plasma-like B cells or supernatants isolated from advanced NSCLC tumor tissues promote the cell growth,while plasma-like B cells or supernatants isolated from early NSCLC tumor tissues inhibited the cell growth compared to the control group.Conclusion:B lymphocyte infiltration was found in NSCLC tumor tissues.There were different distribution and functions between various kinds of B lymphocyte in different stage of NSCLC patients.Naive-like B cells were related to a prolonged survival for patients.Plasma-like B cells in the advanced stage of NSCLC promote proliferation while those in the early stage of NSCLC partially inhibit the proliferation in NSCLC. |
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