Figure 2. LOX secreted from hypoxic tumor cells promotes BMDC invasion by cross-linking collagen IV, increasing BMDC adhesion, and enhancing MMP-2 activity of the invading BMDCs.
(A) Matrigel-coated wells were incubated with the indicated additives for 24hr; Matrigel cross-linked with glucose was included for comparison. Solutions were removed and CD11b+ cells isolated from murine whole bone marrow were added. Numbers of CD11b+ cells remaining in solution were quantified after 2.5hr. Data are mean ± SEM. * = p<0.05 relative to control; ** = p<0.05 relative to matrix pre-incubated with LOX.
(B) Naïve mouse lung tissue was excised and a 2cm3 piece was incubated in serum-free media containing either LOX or glucose for 6hr. Media was changed, CD11b+ cells were added, and the numbers of cells remaining in solution after 12hr were quantified. Data are mean ± SEM.
(C) Lung tissue from B was frozen, sectioned, and stained for LOX (green) and CD11b+ cells (red). Scale bar = 300µm.
(D) Gelatin zymography showing MMP-2 activity of monocytes in contact with Matrigel or collagen IV pre-incubated with the indicated CM.
(E) Gelatin zymography showing MMP-2 activity of freshly harvested BMDCs in contact with collagen IV pre-incubated with the indicated CM.
(F) Matrigel filters were incubated with the indicated CM or purified protein for 24hr. The CM was then removed and freshly harvested whole murine bone marrow cells were allowed to invade through the “modified” Matrigel. BMDCs that invaded through the modified Matrigel were also stained for CD11b and c-Kit. Data are mean ± SEM. * = p<0.05 relative to control; ** = p<0.05 relative to matrices pre-incubated with Wt CM.
(G) Matrigel filters were pre-incubated as in 2F, and invasion of isolated CD11b+ cells or c-Kit+ cells through the modified matrix was quantified. Data are mean ± SEM. * = p<0.05 relative to control; ** = p<0.05 relative to matrices pre-incubated with LOX.
(H) Mice with wild-type bone marrow or MMP-2 knockout bone marrow were injected daily with the indicated CM for 3 weeks prior to flow cytometry analysis of lungs for CD11b, c-Kit, or F4/80 positive cells. Data are mean ± SEM. * = p<0.05 relative to no CM mice; ** = p<0.05 relative to mice with Wt bone marrow injected with Wt CM.
(I) Immunofluorescent staining for LOX, CD11b+ cells, and MMP-2 in representative frozen serial sections of lungs from mice with wild-type or MMP-2 knockout bone marrow injected with Wt CM. Scale bar = 75µm.