[Show abstract][Hide abstract] ABSTRACT: Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.
Proceedings of the National Academy of Sciences 06/2015; 2(Suppl 3). DOI:10.1073/pnas.1506357112 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.
[Show abstract][Hide abstract] ABSTRACT: The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to −40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10 μm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900–2000 kPa and 19–35 mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Mobile cells discriminate and adapt to mechanosensory input from extracellular matrix (ECM) topographies to undergo actin-based polarization, shape change and migration. We tested 'cell-intrinsic' and adaptive components of actin-based cell migration in response to widely used in vitro collagen-based substrates, including a continuous 2D surface, discontinuous fibril-based surfaces (2.5D) and fibril-based 3D geometries. Migrating B16F1 mouse melanoma cells expressing GFP-actin developed striking diversity and adaptation of cytoskeletal organization and migration efficacy in response to collagen organization. 2D geometry enabled keratinocyte-like cell spreading and lamellipod-driven motility, with barrier-free movement averaging the directional vectors from one or several leading edges. 3D fibrillar collagen imposed spindle-shaped polarity with a single cylindrical actin-rich leading edge and terminal filopod-like protrusions generating a single force vector. As a mixed phenotype, 2.5D environments prompted a broad but fractalized leading lamella, with multiple terminal filopod-like protrusions engaged with collagen fibrils to generate an average directional vector from multiple, often divergent, interactions. The migratory population reached >90% of the cells with high speeds for 2D, but only 10-30% of the cells and a 3-fold lower speed range for 2.5D and 3D substrates, suggesting substrate continuity as a major determinant of efficient induction and maintenance of migration. These findings implicate substrate geometry as an important input for plasticity and adaptation of the actin cytoskeleton to cope with varying ECM topography and highlight striking preference of moving cells for 2D continuous-shaped over more complex-shaped discontinuous 2.5 and 3D substrate geometries.
Biochemical Society Transactions 10/2014; 42(5):1356-1366. DOI:10.1042/BST20140139 · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle trafficking and the cell cycle. The roles of Rho GTPases in single mesenchymal cell migration are well established and rely on Cdc42- and Rac-dependent cell protrusion of a leading edge, coupled to Rho-dependent contractility required to move the cell body forward. In cells migrating collectively, cell-cell junctions are maintained, and migrating leader cells are mechanically coupled to, and coordinate, migration with follower cells. Recent evidence suggests that Rho GTPases provide multifunctional input to collective cell polarization, cell-cell interaction and migration. Here, we discuss the role of Rho GTPases in initiating and maintaining front-rear, apical-basal cell polarization, mechanotransduction, and cell-cell junction stability between leader and follower cells, and how these roles are integrated in collective migration. Thereby, spatiotemporal fine-tuning of Rho GTPases within the same cell and among cells in the cell group are crucial in controlling potentially conflicting, divergent cell adhesion and cytoskeletal functions to achieve supracellular coordination and mechanocoupling.
Small GTPases 05/2014; 5(3). DOI:10.4161/sgtp.28997
[Show abstract][Hide abstract] ABSTRACT: Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop both single-cell and collective invasion types, however, the mechanical and molecular programs underlying such plasticity of mesenchymal invasion programs remain unclear.
To test how tissue anatomy determines invasion mode, spheroids of MV3 melanoma and HT1080 fibrosarcoma cells were embedded into 3D collagen matrices of varying density and stiffness and analyzed for migration type and efficacy in the presence or absence of matrix metalloproteinase (MMP)-dependent collagen degradation.
With increasing collagen density and dependent on proteolytic collagen breakdown and track clearance, but independent of matrix stiffness, cells switched from single-cell to collective invasion modes. Conversion to collective invasion included gain of cell-to-cell junctions, supracellular polarization and joint guidance along migration tracks.
The density of the ECM determines the invasion mode of mesenchymal tumor cells. Whereas fibrillar, high porosity ECM enables single-cell dissemination, dense matrix induces cell-cell interaction, leader-follower cell behavior and collective migration as an obligate protease-dependent process. General significance These findings establish plasticity of cancer invasion programs in response to ECM porosity and confinement, thereby recapitulating invasion patterns of mesenchymal tumors in vivo. The conversion to collective invasion with increasing ECM confinement supports the concept of cell jamming as guiding principle for melanoma and fibrosarcoma cells into dense tissue. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.
[Show abstract][Hide abstract] ABSTRACT: Collective cell migration depends on multicellular mechanocoupling between leader and follower cells to coordinate traction force and position change. Co-registration of Rho GTPase activity and forces in migrating epithelial cell sheets now shows how RhoA controls leader-follower cell hierarchy, multicellular cytoskeletal contractility and mechanocoupling, to prevent ectopic leading edges and to move the cell sheet forward.
[Show abstract][Hide abstract] ABSTRACT: Preclinical microscopy has greatly enhanced our mecha-nistic understanding of cancer invasion and metastasis, the contribution of the tumour microenvironment to meta-static progression, and how invasion and the microenviron-ment jointly support cancer cell survival and resistance. Using organotypic models in vitro, live-cell imaging in three-dimensional (3D) tissue culture has identified how cytoskeletal, adhesion and protease systems drive invasion and metastasis . When altered at the molecular level, these pathways underlie the unexpected diversity of the invasive process . The recent use of intravital microscopy has further suggested that cancer invasion into interstitial stroma in vivo: (1) occurs mostly as collective invasion in which cells remain coupled to neighbouring cancer cells, (2) is guided by and responsive to signals delivered by con-nective tissue structures and (3) that invasion pathways cross-talk with pathways of cancer cell survival and resis-tance to anticancer therapy . 1. Principles of collective cell invasion Collective cell migration is defined as the movement of multi-ple cells that retain cell–cell contacts, coordinate their actin dynamics and intracellular signaling, and thereby form a structural and functional unit for joint translocation [1,4]. In contrast to single-cell migration, moving cell masses remain mechanically coupled by cell–cell adhesion receptors, most notably of the cadherin and integrin families, and form a coordinated cortical structure of the actin cytoskeleton, occa-sionally referred to as a 'super-cell' . Besides cancer inva-sion and metastasis, collective cell movement contributes to cell migration in morphogenesis and tissue repair , sug-gesting homologous underlying mechanisms. As in all known types of actomyosin-based cell migration, collective migration is plastic, i.e. it undergoes modification with altered intracellular signaling or an altered environment . Interference with molecules that maintain or regulate collective cell behaviour can lead to single-cell detachment. Depending on the type of single-cell migration obtained after dissociation, two types of conversion are currently known: the epithelial–mesenchymal transition (EMT) and the collec-tive–amoeboid transition (CAT). EMT is a well established molecular process that leads to the down modulation of cell–cell adhesion, whereby the migration machinery remains intact, which induces cell detachment and scattering from multicellular groups  (and references therein). Mechanisms that enable single-cell detachment include reduced cadherin expression, loss-of-function mutations in cadherin and cate-nin [mit Leerzeichen ersetzen] signaling pathways, and deregulated function of proteases degrading cadherins and other cell–cell adhesion molecules . In vivo, EMT corre-sponds to the loss of differentiated epithelial morphology in usually small regions towards a sarcomatous, stromal and, hence, invasive and likely metastatic phenotype. CAT is the transition from collective invasion to amoeboid single-cell crawling after simultaneous weakening of cell–cell and cell– ECM interactions, such as after EMT-independent down-regu-lation of cadherins (data not shown) or inhibition of b1 inte-grins in collectively invading melanoma explants  and in tumour xenografts in vivo (data not shown). Detached cells then survive, continue to move via amoeboid shape change (similarly to interstitial migration of amoeboid leukocytes ), and eventually cause distant metastasis (S. Alexander, MD Anderson Cancer Center). These findings suggest that collective migration represents an invasion mode of high cel-lular and molecular order that, after loss of function of partic-ular adhesion pathways, interconverts to single-cell dissemination and metastasis. The understanding of the sig-nals maintained by simultaneous cell–cell and cell–matrix communication during collective invasion and secondary plasticity will be important in defining the cross-talk between strategies of invasion and resistance signaling .
[Show abstract][Hide abstract] ABSTRACT: Key steps of cancer progression and therapy response depend upon interactions between cancer cells with the reactive tumour microenvironment. Intravital microscopy enables multi-modal and multi-scale monitoring of cancer progression as a dynamic step-wise process within anatomic and functional niches provided by the microenvironment. These niches deliver cell-derived and matrix-derived signals that enable cell subsets or single cancer cells to survive, migrate, grow, undergo dormancy, and escape immune surveillance. Beyond basic research, intravital microscopy has reached preclinical application to identify mechanisms of tumour-stroma interactions and outcome. We here summarise how n-dimensional 'dynamic histopathology' of tumours by intravital microscopy shapes mechanistic insight into cell-cell and cell-tissue interactions that underlie single-cell and collective cancer invasion, metastatic seeding at distant sites, immune evasion, and therapy responses.
Current opinion in cell biology 07/2013; 25(5). DOI:10.1016/j.ceb.2013.07.001 · 8.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal cell migration in interstitial tissue is a cyclic process of coordinated leading edge protrusion, adhesive interaction with extracellular matrix (ECM) ligands, cell contraction followed by retraction and movement of the cell rear. During migration through 3D tissue, the force fields generated by moving cells are non-isotropic and polarized between leading and trailing edge, however the integration of protrusion formation, cell-substrate adhesion, traction force generation and cell translocation in time and space remain unclear. Using high-resolution 3D confocal reflectance and fluorescence microscopy in GFP/actin expressing melanoma cells, we here employ time-resolved subcellular coregistration of cell morphology, interaction and alignment of actin-rich protrusions engaged with individual collagen fibrils. Using single fibril displacement as sensitive measure for force generated by the leading edge, we show how a dominant protrusion generates extension-retraction cycles are transmitted through multiple actin-rich filopods that move along the scaffold in a hand-over-hand manner. The resulting traction force is oscillatory, occurs in parallel to cell elongation and, with maximum elongation reached, is followed by rear retraction and movement of the cell body. Combined live-cell fluorescence and reflection microscopy of the leading edge thus reveals step-wise caterpillar-like extension-retraction cycles that underlie mesenchymal migration in 3D tissue.
Experimental Cell Research 07/2013; 319(16). DOI:10.1016/j.yexcr.2013.04.003 · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm(2); T cells, 4 µm(2); neutrophils, 2 µm(2)). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.
The Journal of Cell Biology 06/2013; 201(7):1069-1084. DOI:10.1083/jcb.201210152 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies of cell migration in three-dimensional (3D) cell culture systems and in vivo have revealed several differences when compared with cell migration in two dimensions, including their morphology and mechanical and signalling control. Here, researchers assess the contribution of 3D models to our understanding of cell migration, both in terms of the mechanisms used to drive single cell and collective cell migration and how migrating cells respond to a changing environment in vivo.
[Show abstract][Hide abstract] ABSTRACT: Adoptive transfer of cells for therapeutic purposes requires efficient and precise delivery to the target organ whilst preserving cell function. Therefore, therapeutically applied cells need to migrate and integrate within their target tissues after delivery, e.g. dendritic cells (DCs) need to migrate to lymph nodes to elicit an antigen-specific immune response. Previous studies have shown that inappropriate cell delivery can hinder DC migration and result in insufficient immune induction. As migration can be extremely difficult to study quantitatively in vivo, we propose an in vitro assay that reproduces key in vivo conditions to optimize cell delivery and migration in vivo. Using DC migration along a chemokine gradient, we describe here a novel (19)F MR-based, large-scale, quantitative assay to measure cell migration in a three-dimensional collagen scaffold. Unlike conventional migration assays, this set-up is amenable to both large and small cell numbers, as well as opaque tissue samples and the inclusion of chemokines or other factors. We labeled primary human DCs with a (19)F label suitable for clinical use; (0.5-15) × 10(6) cells in the scaffolds were imaged sequentially, and migration was assessed using two independent methods. We found no migration with larger numbers of cells, but up to 3% with less than one million cells. Hence, we show that the cell density in cell bolus injections has a decisive impact on migration, and this may explain the limited migration observed using large cell numbers in the clinic.
NMR in Biomedicine 09/2012; 25(9):1095-103. DOI:10.1002/nbm.2774 · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most invasive solid tumours display predominantly collective invasion, in which groups of cells invade the peritumoral stroma while maintaining cell-cell contacts. As the concepts and experimental models for functional analysis of collective cancer cell invasion are rapidly developing, we propose a framework for addressing potential mechanisms, experimental strategies and technical challenges to study this process.