Journal: Cells

Article Title: AREG Upregulation in Cancer Cells via Direct Interaction with Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression Through EGFR-Erk/p38 MAPK Signaling

doi: 10.3390/cells13201733

Figure Lengend Snippet: Amphiregulin (AREG) in esophageal squamous cell carcinoma (ESCC) cells induced by direct co-culture with cancer-associated fibroblast (CAF)-like cells promotes survival and migration of ESCC cells: ( A ) A schematic representation of the experimental design of the direct co-culture and cDNA microarray analysis. ESCC cell lines (TE-9, -10, and -15) and mesenchymal stem cells (MSCs) were co-cultured in the same dish for 4 d. Individually cultured ESCC cell lines and MSCs were prepared as control monocultures. Following monoculture or co-culture, cells were separated using epithelial cell adhesion molecule (EpCAM) microbeads. EpCAM-positive and EpCAM-negative cells after co-culture were defined as TE co (TE-9, -10, and -15 co) and CAF-like cells (CAF9, 10, and 15), respectively. Similarly, ESCC cell lines and MSCs after monoculture were defined as TE mono (TE-9, -10, and -15 mono) and MSC mono, respectively. While a previous study analyzed gene expression between MSC mono and CAF9, this study focuses on gene expression changes between TE-9 mono and TE-9 co using cDNA microarray analysis. ( B ) Double immunofluorescence staining for EpCAM (red) and FAP (green) was performed on a direct co-culture of ESCC cells and MSCs. The nucleus of each cell was counterstained with DAPI (blue). ( C ) Venn diagram depicting the overlap between genes exhibiting a global normalization threshold of TE-9 co > 100 and TE-9 co/TE-9 mono ratio > 4 in the cDNA microarray analysis as well as genes displaying a global normalization threshold of MSC mono and CAF9 < 100 in the previous analysis. Five genes were identified that overlapped between the two groups, with AREG showing the highest fold change. ( D , E ) The mRNA expression and secreted protein levels of AREG in TE mono and TE co were compared using qRT-PCR ( D ) and enzyme-linked immunosorbent assay ( E ). ( F , G ) The effects of recombinant human AREG (rhAREG) (10 and 100 ng/mL) on the survival ( F ) and growth ( G ) of ESCC cells were evaluated using the MTS assay. ( H ) The effect of rhAREG (1, 10, and 100 ng/mL) on the migration of ESCC cells was evaluated using the transwell migration assay. ESCC cells were seeded in the upper chamber, and migrated cells were counted in five representative fields of view using a microscope after 48 h. Representative images for each condition are presented below the graphs. The data are presented as the mean ± standard error of the mean (SEM) of three independent experiments ( D – H ). N.S., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 50 μm ( B ); and 100 μm ( H ).

Article Snippet: After co-culture, cells were fixed with 4% paraformaldehyde for 10 min at 25 °C, then incubated overnight at 4 °C with primary antibodies against EpCAM (1:150; #2929; CST) and FAP (1:300; #AF3715; R&D Systems).

Techniques: Co-Culture Assay, Migration, Microarray, Cell Culture, Control, Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, MTS Assay, Transwell Migration Assay, Microscopy