Journal: Cancer Communications

Article Title: Nicotinamide N‐methyltransferase negatively regulates metastasis‐promoting property of cancer‐associated fibroblasts in lung adenocarcinoma

doi: 10.1002/cac2.12633

Figure Lengend Snippet: Primary culture of fibroblasts from lung adenocarcinoma (LUAD) and validation of NNMT expression. (A) Diagram showed the study process of tissue collection from non‐small cell lung cancers, isolation and primary culture of CAFs, and experiments of validating NNMT expression. Unless otherwise indicated, all subsequent experiments following the primary culture were performed using CAFs derived from LUADs. (B) Immunofluorescence assays were used to validate the expression of canonical biomarkers in primary cultured fibroblasts. (C) Western blotting demonstrated protein expression of FAP, SMA, and NNMT in CAFs isolated from LUADs ( n = 11) and in Human Fetal Lung Fibroblast 1 cells (HFL1, control). Simple linear regression demonstrated a negative correlation between NNMT protein expression in CAFs and the pathological stage or size of LUADs ( n = 11). (D) CAFs were divided into high or low NNMT protein expression groups after adjustment with paired NFs, using the median value as the cutoff. Quantitative analyses are presented. (E) Immunofluorescence assay confirmed differences in NNMT protein expression between CAFs in the established groups. (F) Pathological characteristics of LUADs from which primary CAFs were isolated. (G) Transwell assays and quantification analysis demonstrated increased migration of A549 and H1299 cells under coculture with CAFs exhibiting low NNMT protein expression. Data are representative of three independent biological experiments each containing three technical replicates for a given condition (B‐E and G). Data are presented as mean ± standard deviation (D, E and G). P values were calculated by Pearson correlation analysis (C) or Student's t‐test (D, E and G), * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: ADH1B, alcohol dehydrogenase 1B; CAF, cancer‐associated fibroblast; FAP, fibroblast activation protein; LUAD, lung adenocarcinoma; MYH11, myosin heavy chain 11; NF, normal fibroblast; NNMT, nicotinamide N‐methyl transferase; NSCLC, non‐small cell lung cancer; SMA, alpha‐smooth muscle actin; TNM, the American Joint Committee on Cancer's 8th edition of the tumor‐node‐metastasis classification system.

Article Snippet: Primary antibodies against FAP (Cat. No. sc‐65398, Santa Cruz, Dallas, TX, USA), alpha‐smooth muscle actin (SMA, Cat. No. 67735‐1‐Ig, Proteintech, Rosemont, IL, USA), alcohol dehydrogenase 1B (Cat. No. 66939‐1‐Ig, Proteintech, Rosemont, IL, USA), myosin heavy chain 11 (Cat. No. sc‐6956, Santa Cruz, Dallas, TX, USA), pan‐cytokeratin (Cat. No. ab7753, Abcam, Cambridge, MA, USA), CD31 (Cat. No. ab76533, Abcam, Cambridge, MA, USA), and NNMT (Cat. No. ab119758, Abcam, Cambridge, MA, USA) were used.

Techniques: Biomarker Discovery, Expressing, Isolation, Derivative Assay, Immunofluorescence, Cell Culture, Western Blot, Control, Migration, Standard Deviation, Activation Assay