Tissue infiltration of macrophages, although critical for innate immunity, is also involved in pathologies, such as chronic inflammation and cancer. In vivo, macrophages migrate mostly in a constrained three-dimensional (3D) environment. However, in vitro studies, mainly focused on two dimensions, do not provide meaningful clues about the mechanisms involved in 3D macrophage migration. In contrast, tumor cell 3D migration is well documented. It comprises a protease-independent and Rho kinase (ROCK)-dependent amoeboid migration mode and a protease-dependent and ROCK-independent mesenchymal migration mode. In this study, we examined the influence of extracellular matrix (composition, architecture, and stiffness) on 3D migration of human macrophages derived from blood monocytes (MDMs). We show that: 1) MDMs use either the amoeboid migration mode in fibrillar collagen I or the mesenchymal migration mode in Matrigel and gelled collagen I, whereas HT1080 tumor cells only perform mesenchymal migration; 2) when MDMs use the mesenchymal migratory mode, they form 3D collagenolytic structures at the tips of cell protrusions that share several markers with podosomes as described in two dimensions; 3) in contrast to tumor cells, matrix metalloproteinase inhibitors do not impair protease-dependent macrophage 3D migration, suggesting the involvement of other proteolytic systems; and 4) MDMs infiltrating matrices of similar composition but with variable stiffness adapt their migration mode primarily to the matrix architecture. In conclusion, although it is admitted that leukocytes 3D migration is restricted to the amoeboid mode, we show that human macrophages also perform the mesenchymal mode but in a distinct manner than tumor cells, and they naturally adapt their migration mode to the environmental constraints.

Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck ؊/؊ mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in Hck ؊/؊ BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain-of-3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocytelimited expression with a role in 3D migration, could be a target for new antiinflammatory and antitumor molecules. IntroductionPhagocytes constitute the first line of host defense against microorganisms. 1,2 To reach an infectious site, they transmigrate through the endothelial wall, basal membranes, and connective tissues and infiltrate the damaged organ to affect host defense and tissue repair. 3 Nevertheless, phagocytes have not only friend but also foe functions. 4,5 In several pathologic states, including chronic inflammatory 6,7 and neurodegenerative diseases 8 or atherosclerosis, 9-11 phagocyte-dependent tissue lesions often occur. In addition, it has been established that the presence of macrophages within tumors is a sign of a poor prognosis as they enhance angiogenesis and metastases (see Mantovani et al 12 ; Condeelis and Pollard 13 ; and Balkwill et al 14 for reviews). In contrast, T-cell infiltration into tumors is often associated with a more positive prognosis. 15 Therefore it is becoming a challenge to specifically control tissue infiltration of macrophages without affecting lymphocyte migration. By targeting macrophage migration-related molecules, new anti-inflammatory and antitumorbased drugs could be developed. 16,17 However, the cellular and molecular mechanisms involved in macrophage migration are poorly understood.Phagocyte migration has been studied mostly in vitro in 2 dimensions (2D) in response to chemotactic factors. In these experiments, cells are plated on either plastic or glass coverslips, coated or not with matrix proteins. However, in vivo, phagocyte transendothelial migration and infiltration through tissue involve primarily 3-dimensional (3D) regulation. Transend...