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Cell migration is a complex physiological process that is important from embryogenesis to senescence. In vivo, the migration of cells is guided by a complex combination of signals and cues. Directed migration is typically observed when one of these cues is presented to cells as a gradient. Several studies have been conducted into directed migration on gradients that are purely mechanical or chemical. Our goal was to investigate cellular migratory behavior when cells are presented with a choice and have to choose between increasing substrate rigidity or higher protein concentration. We chose to focus on this unique environment since it recapitulates several interfacial regions in vivo. We have designed novel hydrogels that exhibit dual and opposing chemical and mechanical profiles using photo-polymerization. Our studies demonstrate that durotaxis, a well-known phenomenon, can be reversed when cells sense a steep protein profile in the opposite direction. Fibroblasts were co-cultured with macrophages to obtain an understanding on how migration occurs when two different cell types are present in the same microenvironment. First, we investigated the migratory behavior of macrophages. These cell types exhibited a statistically significant preference to move towards the rigid/low collagen region of the interface. Interestingly, fibroblasts when co-cultured with macrophages, exhibited a preference for the low modulus-high collagen region of the interface. However, with the current sample size, these trends are statistically insignificant. On the contrary, the presence of fibroblasts in the cellular microenvironment did not result in the reversal of durotaxis exhibited by macrophages. Macrophages secreted significantly higher levels of secreted tumor necrosis factor (TNF-alpha) in mono-cultures in contrast to fibroblast-macrophage co-cultures. This trend could be an indication of macrophage plasticity between mono- and co-cultures. In summary, we have designed dual and opposing rigidity-protein gradients on a hydrogel substrate that can provide new insights into cellular locomotion. These results can be used to design biomimetic interfaces, biomaterial implants and for tissue engineering applications. / Ph. D.

Une réponse immunitaire efficace repose sur un recrutement rapide de leucocytes du sang au tissu enflammé ou endommagé. Pendant ce processus, les leucocytes sont capturés par l'endothélium et migrent le long de la paroi pour atteindre les sites de transmigration. Ces processus sont médiés par des signaux externes parmi lesquels le rôle des molécules d’adhésion reste flou. L’haptotaxie adhésive a été décrite pour les cellules mésenchymateuses qui s’orientent via un mécanisme de tir à la corde - une compétition entre les bords adhérents des cellules. Pour les cellules amiboïdes, l'existence d'une haptotaxie adhésif n'a jamais été observée. Ici, nous avons étudié la migration des lymphocytes T humains sur des substrats dont l’adhérence est spatialement modulée et avons observé une haptotaxie robuste. Mécanistiquement, nous montrons que l'haptotaxie adhésive diffère à la fois de la chimiotaxie, car aucune mécanotransduction n'a été détectée, et du mécanisme de tir à la corde passif, car différentes intégrines induisent des phénotypes opposés. Les cellules ont favorisé des zones plus adhérentes avec VLA-4 et, contre-intuitivement, des zones moins adhérentes avec LFA-1. Ces résultats révèlent que les intégrines contrôlent les comportements différentiels d'haptotaxie adhésive sans mécanotransduction. Nous avons également étudié le mécanisme à l'origine de ce phénotype induit par LFA-1 et avons découvert que la dynamique du lamellipode plutôt que le niveau d'expression de l'intégrine, était impliquée. Les résultats préliminaires avec des lymphocytes T déficients en VASP indiquent également que la protéine VASP pourrait jouer un rôle important dans l'haptotaxie adhésive. / An efficient immune response relies on a rapid recruitment of leukocytes from blood to the inflamed or damaged tissue. During this process, leukocytes are captured by the endothelium and migrate along the vessel wall to reach permissive transmigration sites. These processes are mediated by multiple external cues among which the role of adhesion molecules remains unclear. Adhesive haptotaxis has been described for mesenchymal cells that develop strong pulling forces with their substrates and orient via a tug of war mechanism – a competition between cells’ adherent pulling edges. In the case of amoeboid cells that migrate with minimal interaction with their substrate, the existence of adhesive haptotaxis has yet to be evidenced. Here, we studied the crawling of human T lymphocytes on substrates with spatially modulated adhesion. and observed robust adhesive haptotaxis. Mechanistically, we show that integrin-mediated adhesive haptotaxis of lymphocytes differs both from active chemotaxis, because no mechanotransduction was detected, and from the passive tug of war mechanism, because different integrins support opposite phenotypes. Cells favored more adherent zones with VLA-4 and, counterintuitively, less adherent zones with LFA-1. These results reveal that integrins control differential adhesive haptotaxis behaviors without mechanotransduction. We further investigated the mechanism behind this specific haptotactic phenotype mediated by LFA-1 and find that the lamellipodial dynamics, rather than the integrin expression level, is involved. Preliminary findings with VASP deficient T cells indicate also that VASP protein may play an important role in T cell adhesive haptotaxis.

La migration cellulaire est un processus fondamental au maintien des fonctions physiologiques de l’organisme. Elle est également centrale dans de nombreuses pathologies et entre notamment en jeu lors de la dissémination métastatique. Lorsqu’elles migrent, les cellules utilisent des structures d’adhésion afin de s’appuyer sur leur environnement. Nous avons récemment montré que les puits recouverts de clathrine, plus connus pour leur rôle dans l’endocytose, peuvent également servir de structures d’adhésion. Dans ce manuscrit, je démontre que certains ligands internalisés par la voie d’endocytose clathrine peuvent également se lier à la matrice et orienter la migration cellulaire en régulant les structures adhésives de clathrine.J’ai commencé par montrer que le collagène est associé à plus de structures de clathrine et a plus de protrusions lorsqu’il est recouvert par des ligands. J’ai ensuite montré que les cellules appliquaient plus de forces sur des fibres de collagènes décorées par des ligands et que ce surplus de force nécessite la présence de structures de clathrine. Enfin j’ai montré que les cellules suivent les ligands liés à des réseaux de collagène en 3D et que cette migration dirigée nécessite également la présence de structures de clathrine. Ce mécanisme de migration pourrait notamment permettre aux cellules de suivre des gradients de ligands liés à la matrice in vivo et ainsi de s’orienter dans l’organisme. / Cell migration is a fundamental process in the development and homeostasis of multicellular organisms. It is also central to many pathologies and it is especially important for metastatic dissemination. When migrating, cells use adhesion structures to push on their substrate in order to move forward. We recently showed that clathrin coated structures, primarily known as endocytic structures, can also serve as adhesion structures. In this manuscript, I show that some ligands internalized through clathrin mediated endocytosis can also bind to the extracellular matrix and orient cell migration using adhesive clathrin structures.I first showed that ligand-decorated collagen fibers are associated with more clathrin structures and more protrusions. I then showed that cells applied more forces to the ligand-decorated collagen fibers and this extra amount of forces requires the presence of clathrin structures. Finally, I showed that cells can migrate following collagen-bound ligands in 3D, this directed migration also requiring the presence of clathrin structures. Such migration mechanism could be used by cells to follow in vivo gradient of matrix-bound ligands and thus find their way when migrating inside the body.

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In this thesis, a number of mathematical models of haptotactic cell migration are developed. The modelling of haptotaxis is presented in two distinct parts - the first comprises an investigation of haptotaxis in pre-necrotic avascular tumours, while the second consists of the modelling of adhesion-mediated haptotactic cell migration within tissue, with particular attention paid to the biological appropriateness of the description of cell-extracellular matrix adhesion. A model is developed that describes the effects of passive and haptotactic migration on the cellular dynamics and growth of pre-necrotic avascular tumours. The model includes a description of the extracellular matrix and its effect on cell migration. Questions are posed as to which cell types act as a source of the extracellular matrix, and the model is used to simulate the possible effects of different matrix sources. Simulations in one-dimensional and spherically symmetric geometry are presented, displaying familiar results such as three-phase tumour growth and tumours comprising a rim of proliferating cells surrounding a non-proliferating region. Novel effects are also described such as cell population splitting and tumour shrinkage due to haptotaxis and appropriate extracellular matrix construction. The avascular tumour model is then extended to describe the internalisation of labelled cells and inert microspheres within multicell tumour spheroids. A novel model of adhesion-receptor mediated haptotactic cell migration is presented and specific applications of the model to tumour invasion processes are discussed. This model includes a more biologically realistic description of cell adhesion than has been considered in previous models of cell population haptotaxis. Through assumptions of fast kinetics, the model is simplified with the identification of relationships between the simplified model and previous models of haptotaxis. Further simpli.cations to the model are made and travelling wave solutions of the original model are then investigated. It is noted that the generic numerical solution routine NAG D03PCF is not always appropriate for the solution of the model, and can produce oscillatory and inaccurate solutions. For this reason, a control volume numerical solver with .ux limiting is developed to provide a better method of solving the cell migration models.