Purpose: Lysyl oxidase (LOX) increases extracellular matrix stiffness by cross-linking collagen. A stiffer matrix drives integrin activation, and therefore LOX is believed to promote cancer cell invasion through enhanced integrin signaling. However increased matrix stiffness does not necessarily lead to a more permissive environment for invasion. Fibrillar collagen matrix orientation, density and porosity also determine how well cells can invade. Assays based on collagen gel-covered transwell systems have previously been used to show that LOX is required for invasion. These systems are driven by a chemoattractant gradient, and thus a role for LOX in chemotaxis could not be excluded. In this study we aimed to clarify whether LOX was required for matrix invasion or chemotaxis.
Experimental procedures: To set up a 3D invasion assay that was not driven by a chemoattractant gradient, spheroids from multiple LOX expressing cancer cell lines were embedded into a thick 3D collagen gel. Cells were then allowed to randomly invade into the surrounding collagen. Alternatively, cancer cells were allowed to invade a fibroblast-modified 3D collagen gel in an organotypic assay. In this assay the invasion was driven by a strong chemoattractant gradient through a dense collagen gel, with cancer cells cultured atop the collagen gel at the air-medium interface. A Dunn chemotaxis chamber or a transwell tissue culture insert was used to investigate chemotaxis towards serum or EGF. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) specific to human LOX was used to deplete LOX expression in the cancer cells, to test potential LOX functions in the above assays.
Results: LOX depletion in parental MDA-MB-231 cells, MDA-MB-231 brain or bone metastatic sub-population cells, U87 and U118 cells did not impact 3D collagen gel invasion. LOX inhibition by BAPN also did not impact collagen gel invasion in MDA-MB-231, LN229, U87, U118 and U138 cells. The data indicated that LOX was not required for invasion through a thick 3D collagen gel in vitro. However, in the chemotactic-driven organotypic assay, loss of LOX in MDA-MB-231 cells completely abolished invasion. When the chemotactic potential towards serum and EGF of LOX depleted MDA-MB-231 and U87 cells was tested using Dunn chemotaxis chambers, we observed strong chemotactic defects in both cell lines. The chemotactic defects of these cells were also observed when a transwell based chemotaxis assay was used. Random cell migration of MDA-MB-231 and U87 cells were otherwise not affected by LOX depletion.
Conclusions: LOX is not required for collagen matrix invasion per se. Instead LOX is important for cells to sense chemoattractants and to maintain directional cell migration. The molecular mechanisms underlying these observations are now the subject of further investigation.
Citation Format: Haoran Tang, Nathalie Dhomen, Richard Marais. Lysyl oxidase is required for chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2017-905