Effects of dynamic environments on extracellular morphogen gradients

Tissue morphogenesis and remodeling is often orchestrated by cell-secreted proteins, referred to as morphogens that can trigger cellular responses in addition to modifying the local extracellular matrix (ECM). Of specific interest is the distribution of cell secreted proteins in the cell microenvironment and how this distribution can lead to varied responses. Morphogens often provide directional cues, and as such their specific distribution is critical to the signaling that they induce, in other words cells are sensitive to morphogen gradients and not just absolute amounts. Natural cellular environments in native tissue involve three dimensional ECM and are typically subjected to dynamic conditions such as interstitial flow (IF). Despite the importance of the extracellular environment in signaling, very little is known regarding the effects of mechanical stresses and subtle interstitial flows. This thesis examines the role of IF on morphogen gradients using mass transport models to elucidate mechanisms responsible for several specific biological phenomena including in vitro capillary formation and lymphatic metastasis of tumor cells. In vitro capillary formation had been shown in our lab to be separately enhanced by bound VEGF alone and slow IF alone, with the combination of these two conditions resulting in marked increase in organization. Using our computational model we were able to propose a novel mechanistic model to explain this synergy. Specifically showed how, through the combined effects of IF and matrix-bound growth factors, cells could create their own biased chemokine gradients without recourse to internal amplification methods. This process, which we have termed "autologous chemotaxis," drives directional signaling and migration in the direction of flow. This can occur even at very low Peclet numbers, when diffusion still dominates the overall transport, but convection changes the shape of the gradient, which is what a cell responds to. We then examined the effects of IF on tumor cell migration to explore a novel mechanism of lymphatic metastasis. Tumors often produce high amounts of proteoglycans creating a microenvironment is rich in morphogen binding sites, and tumors are also a source of interstitial flow due to their leaky vasculature. The lymphatic system drains interstitial fluid, therefore IF is always directed toward lymphatic capillaries. The lymphatic system is also a common route of tumor metastasis, which is what lead us to study the role of IF mediated chemotaxis. A computational model of an in vitro tumor invasion assay was constructed that predicted tumor cell migration that was in very good agreement with the in vitro experimental results. This phenomenon was further studied by modeling an in vivo geometry and examining the factors controlling autologous gradient formation in a physiological setting. In conclusion, we have demonstrated the importance of convective mass transport at very low flow velocities in a biological context. While these velocities are low enough that the convective effects would typically be ignored, their subtle effects on pericellular mass transport can be translated into relevant and observable morphogenic responses.

In vitro lymphatic endothelial morphogenesis

Carolyn Yong Pullin

Cell organization into functional multicellular three-dimensional structures is a long-standing challenge. In particular, engineering of vascularized tissues requires both blood and lymphatic neovascular network formation in a simultaneous and coordinated fashion. While extensive in vitro investigations in blood and lymphangiogenesis have identified a vast array of cellular phenomena and key cellular and extracellular bioactive substances, the dynamic natural cellular environment and its regulatory role in governing cellular response and interactions remain largely unclear. Furthermore, the wealth of existing knowledge describing the molecular regulation of vessel morphogenesis is heavily biased towards the angiogenic growth of blood capillaries, despite the importance of the lymphatic system. This thesis first addresses the differential gene regulation of isolated microvascular lymphatic endothelial cells (LECs) in response to the lymphatic growth factor VEGF-C and to fluid shear stress using gene arrays and QRT-PCR. First, characteristic of a response to a growth factor stimulus, the largest numbers of differentially expressed genes in response to VEGF-C stimulation were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C. The ability of lymphatic endothelium to sense and actively regulate drainage function is poorly understood, and shear stress is likely a key indicator of lymphatic drainage function. Thus, LECs were exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. Important adaptive responses to both low and high shear stress conditions that were correlated to lymphatic function, including changes in gene expression patterns related to water and solute transport, cell-cell and cell-matrix adhesion, inflammatory cytokines and cell cycle were found. These changes were consistent with increased transport function, both active and passive, in high shear conditions and indicate that during lymphedema, lymphatic endothelium shuts down transport functions. These data demonstrate a functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their drainage function. It has been previously shown in our lab that in vitro blood and lymphatic morphogenesis are differentially enhanced by interstitial flow. Using a three-dimensional tissue culture model, the molecular mechanisms orchestrating endothelial cell morphogenesis, specifically gene regulation of the lymphatic microvascular system was investigated, focusing on genes implicated in both cellular morphogenesis and lymphatic development. Finally, concurrent angiogenesis and lymphangiogenesis were studied in vitro to examine the influences and crosstalk between blood and lymphatic microvascular development. The data suggest that both blood endothelial cell (BEC) and LEC in vitro capillary morphogenesis are enhanced by the presence of BEC-secreted factors, and these effects are, in part, mediated through proliferative and migratory responses of which the latter was induced by VEGFR-2 and VEGFR-3 signaling for BECs and LECs, respectively. When maintained in mixed three-dimensional cultures, both homotypic and heterotypic interactions between LECs and BECs were observed. Furthermore, capillary morphogenesis and interactions were enhanced and distinct when under the additional influence of interstitial flow. In conclusion, the novelties of the work presented in this dissertation include studies of 1) overall LEC gene response to its primary growth factor VEGF-C and biophysical factor, fluid flow and 2) the genetic regulation underlying the morphogenetic processes of LECs under interstitial flow in vitro, by using unique approaches which combine bioengineering principles to address fundamental biological questions and thereby contribute to this novel niche which has so far been neglected. Furthermore, the importance of biophysical processes along with crucial biochemical signal regulation is demonstrated in the concurrent induction of angiogenesis and lymphangiogenesis. Such are the requisites for the understanding of microvascular development and the determination of useful design principles for the in vitro vascularization of engineered tissues.

EPFL2011

Dendritic cell chemotaxis in 3D

Ulrike Haessler

Dendritic cells (DCs) are crucial for the onset of adaptive immunity. Their outstanding migratory capacities in combination with their antigen presenting properties make them perfectly suited to pick up antigens in the periphery and transport them to the lymph nodes, where antigen presentation to T-cells is most efficient [1]. Their migration from the periphery to the lymph nodes, called DCs cell homing, is involved in both mounting effective adaptive immune responses as well as maintaining tolerance to self-antigens [2]. Both of them need to be balanced to maintain a healthy organism. So far two alternative mechanisms have been proposed to guide DCs from the interstitium through the extracellular matrix to the proximal lymphatic vessel [3]. One of these mechanisms is based on paracrine chemotaxis towards a CCL21 gradient created by the proximal lymphatic vessel. The second mechanism, called autologous chemotaxis, describes how cells can follow an autocrine gradient, created by the biasing of self-secreted chemokine by interstitial flow [4, 5]. To answer the question of how these two mechanisms contribute to DC homing, a quantitative understanding of how cells respond to gradients and interstitial flow present in their physiological microenvironment is needed [6]. To date, appropriate tools to study DC migration under defined gradients and in the presence of tunable interstitial flow rates in a 3D environment have been lacking. Within this thesis, we present two micro-scale devices that allow live cell observation on an inverted microscope. The first was optimized to expose cells to a steady-state chemokine gradient within a 3D microenvironment and is based on a microfluidic agarose scaffold which functions as a gradient buffer (Chapter 3). The novelty of a gradient buffer allows us to quickly establish stable gradients, which is an important feature in order to create stable and precisely defined gradients during tracking of fast-moving cells like DCs. We then performed an in-depth study of DC cell chemotactic responses towards the CCR7 ligands (CCL19 and CCL21) in 3D using this new agarose chamber (Chapter 4). With the help of an integrated computational model, we could precisely predict the gradient formation of matrix-bound CCL21 versus soluble CCL19 in the microcellular environment and found that DC answer differently to the two chemokines in response to gradient steepness. Furthermore, CCL21 was more chemoattractive than CCL19 when cells were exposed to competing gradients of both chemokines, and this was independent of the CCL21-binding properties. This represents the first quantitative study of CCR7-mediated DC chemotaxis in 3D in response to well-defined ligand gradients. The second device we developed for studying the effects of the physiological flow environment, which cells experience in capillary-fed tissues, on cell migration [6]. A 2-gel strategy allowed us to flexibly fine tune interstitial flow velocities and to precisely determine the actual flow rate for each position imaged on a microscope. We exposed DCs to a wide range of flow rates and observed an increase in persistence under flow compared to static (Chapter 6). In conclusion, this work presents quantitative insights into DC migration within precisely controlled 3D-microenvironments using new devices. It also highlights the role of a quantitative understanding of biological processes to distinguish between chemotactic mechanisms of DC cells.

EPFL2010

Dendritic cell chemotaxis in 3D under defined chemokine gradients reveals differential response to ligands CCL21 and CCL19

Ulrike Haessler, Marco Pisano, Melody Swartz, Mingming Wu

Dendritic cell (DC) homing to the lymphatics and positioning within the lymph node is important for adaptive immunity, and is regulated by gradients of CCL19 and CCL21, ligands for CCR7. Despite the importance of DC chemotaxis, it is not well understood how DCs interpret gradients of these chemokines in a complex 3D microenvironment. Here, we use a microfluidic device that allows rapid establishment of stable gradients in 3D matrices to show that DC chemotaxis in 3D can respond to CCR7 ligand gradients as small as 0.4%, which helps explain how DCs sense lymphatic vessels in an environment where broadcast distance for chemokine diffusion is hindered by convective flows into the vessel. Interestingly, DCs displayed similar sensitivities to both chemokines at small gradients (≤ 60 nM/mm), but migrated more efficiently towards higher gradients of CCL21, which unlike CCL19 binds strongly to matrix proteoglycans and signals without the need for internalization. Furthermore, cells preferentially migrated towards CCL21 when exposed to equal and opposite gradients of CCL21 and CCL19 simultaneously, even when matrix-binding of CCL21 was prevented. Although these ligands have similar binding affinity to CCR7, our results demonstrate that, in a 3D environment, CCL21 is a more potent directional cue for DC migration than CCL19. These findings provide new quantitative insight into DC chemotaxis in a physiological 3D environment and suggest how CCL19 and CCL21 may signal differently to fine-tune DC homing and positioning within the lymphatic system. These results also have broad relevance to other systems of cell chemotaxis, which remain poorly understood in the 3D context.