Fig. 2

Fibronectin is a key architectural component in tumor biofilm in vitro and in vivo. a Top panel- Schematic of 3D sample preparation where cells were embedded in laminin-rich ECM (lrECM) and cultured for 10 days to recapitulate growth of melanoma with a basement membrane. Bottom panel- Immunoblots showing an increase in the levels of FN in conditioned media (CM) and cell lysates (CL) when cells are cultured in 2D vs. 3D. The A375 clone shows a threefold increase in FN production, whereas the A375.S2 clone shows a modest increase. Numbers next to the immunoblots represent fold of increase in fibronectin expression. b Micrographs showing the differences in assembly of secreted FN (green) between A375 and A375.S2 in both 2D and 3D culture. Green arrows show the locations of FN. Scale bars for 2D represent 5 μm and for 3D, 20 μm, respectively. c Schematic of 3D sample preparation for anchorage-independent growth where cells were embedded in soft agar and cultured for 28 days. Micrographs show that all FN is tumor-generated in this assay. Scale bars represent 50 μm. d Top panel- micrographs of human tissue arrays of normal skin as well as malignant and metastatic melanoma show that there is an increase in FN deposition within the tumor mass as a function of increasing metastatic potential, whereas FN deposition is restricted to the dermis in normal skin epithelium. Micrographs show increased FN deposition within the metastatic lung lesion compared to normal adjacent lung epithelium in the mouse following tail vein injection of melanoma cells. Scale bars represent 50 μm and 20 μm for the inset