A single-cell gene expression analysis in an in vitro co-culture model containing fibroblast cells directly mixed with NMU-manipulated lung adenocarcinoma cells

NMU gene expression was known to be over-represented in lung adenocarcinoma and negatively associated with overall survival or progression-free survival of patients. However, the mechanism(s) underlying the oncogenic roles of NMU are still not clear. Based on the transcriptome analysis of bulk-tissue of lung cancer datasets in the public domain, we found that NMU expression was significantly associated with those of some pro-fibrotic genes. Using subcutaneous mice xenograft model of human lung adenocarcinoma cells, we found significant increase of fibrotic phenotypes in xenograft derived from cell lines over-expressing NMU, including expression levels of marker proteins of myofibroblast, activation of fibroblast migration, and number of cells with morphology of fibroblast, while these changes can be reversed by knocking down the NMU expression of lung cancer cell lines. These suggest NMU from lung cancer cells may contribute to fibrosis of tumor microenvironment by recruiting and activating fibroblast and/or myofibroblast. Here we perform single-cell analysis of cell mixture containing co-cultured lung cancer cells and fibroblast cells. Overall design: scRNA profiles on hESC-derived lung cells and lung-macrophage co-cultured cells Lung fibroblast cell line WI-38 was cultured in 10-cm dish for 24 hours. Each of lung cancer cell line H1975 or PC9 (and their stable transfectant cells with overexpressed or knock down NMU) was added to dish of fibroblast with ratio of 1:3 of numbers of lung cancer cell to that of fibroblast cell and co-cultured for 72 hours. Cells mixture was trypsinized and subject to single cell isolation and library construction using mRNA Targeted Library kit (BD Rhapsody System). After paired-end sequencing, fastq files were analyzed by Rhapsody Sequence Analysis Pipeline.