Cancer-Associated Fibroblasts (CAFs) were isolated from specimens taken from primary human hepatocellular carcinoma (HCC) nodules (4 patients in total), characterized for expression of typical stromal markers (including vimentin and alpha smooth muscle actin), and then incubated in complete medium (Iscove's Modified Dulbecco Medium, IMDM + 20% fetal bovine serum + antibiotic/antimycotic) without any treatment, or in the presence of Transforming Growth Factor Beta 1 (TGF beta 1) at a concentration of 5 ng/ml. The medium and the treatment were renewed every 2 days until the 14th day. At that time point, cells were washed 4 times with serum-free IMDM medium and then incubated with serum free IMDM medium for 48 hours to allow for enrichment with secreted CAFs proteins. The CAFs conditioned medium was then collected, concentrated using a centricon device (10 kDa cutoff), assayed for protein concentration, and finally subjected to Liquid Chromatography with tandem mass spectrometry (LC-MS/MS).