Mechanism And Biological Implications Of Basement Membrane-induced Fibronectin Matrix Assembly By Cancer Cells

OBJECTIVE: To investigate the molecular mechanisms and biological implications of fibronectin distribution along basement membrane in vivo and basement membrane-induced fibronectin matrix assembly by cancer cells and fibroblasts.METHODS:(1)Fibronectin distribution along basement membrane in mouse organs was validated by immunofluorescence.(2)Invasive ability through basement membrane by multiple cancer cells upon blocking fibronectin and its cellular receptor were evaluated by trans-well assays.(3)The pattern,speed and amount of fibronectin matrix assembled by multiple cancer cells growing on basement membrane and its two major components-type IV collagen and laminin,were evaluated and compared to the assembly on other extracellular matrix proteins by immunofluorescence and live cell imaging.(4)Potential proteins with change in phosphorylation status in colon cancer cells growing on basement membrane and its components compared to other extacellullar matrix proteins were detected by phospho-antibody array.(5)The morphology,composition,dynamics of focal adhesion in cells growing on basement membrane and its relationship with fibronectin matrix assembly were evaluated by immunofluorescence and live cell imaging using GFP-Paxillin HCT116 cells.(6)The role of actomyosin system in fibronectin matrix assembly on basement membrane is evaluated by immunofluorescence,inhibitors and CRIPR-Cas9-mediated gene knock-out.(7)The role of?2?1,?3?1,?5?1 and ?V?3 were evaluated by immunofluorescence,functional antibodies and CRIPR-Cas9-mediated gene knock-out.RESULTS:(1)Fibronectin can help cancer cells to invade through basement membrane.(2)Cancer cells and fibroblasts and assemble large amount of fibronectin efficiently on basement membrane.(3)Focal adhesions on basement membrane share characteristics with both classical focal adhesions and fibrillar adhesions.(4)Actomyosin system pulls focal adhesion to slide along actin stress fibers and assemble fibronectin matrix.(5)?3?1 is present in early focal adhesions on basement membrane and participates in early fibronectin deposition.?3?1 is replaced by ?5?1 at later stages when focal adhesions matures and continue to assemble fibronectin matrix.(6)?2?1 can substitute ?3?1 to mediate attachment to basement membrane and initiate fibronectin matrix assembly.CONCLUSION: On basement membrane,cancer cells can form distinct sliding focal adhesions which can efficiently assemble fibronectin and promote cancer cell invading though basement membrane.