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ABSTRACT: Cancer cell invasion is an adaptive process based on cell-intrinsic properties to migrate individually or collectively, and their adaptation to encountered tissue structure acting as barrier or providing guidance. Whereas molecular and physical mechanisms of cancer invasion are well-studied in 3D in vitro models, their topographic relevance, classification and validation toward interstitial tissue organization in vivo remain incomplete. Using combined intravital third and second harmonic generation (THG, SHG), and three-channel fluorescence microscopy in live tumors, we here map B16F10 melanoma invasion into the dermis with up to 600 µm penetration depth and reconstruct both invasion mode and tissue tracks to establish invasion routes and outcome. B16F10 cells preferentially develop adaptive invasion patterns along preformed tracks of complex, multi-interface topography, combining single-cell and collective migration modes, without immediate anatomic tissue remodeling or destruction. The data suggest that the dimensionality (1D, 2D, 3D) of tissue interfaces determines the microanatomy exploited by invading tumor cells, emphasizing non-destructive migration along microchannels coupled to contact guidance as key invasion mechanisms. THG imaging further detected the presence and interstitial dynamics of tumor-associated microparticles with submicron resolution, revealing tumor-imposed conditioning of the microenvironment. These topographic findings establish combined THG, SHG and fluorescence microscopy in intravital tumor biology and provide a template for rational in vitro model development and context-dependent molecular classification of invasion modes and routes.
IntraVital. 07/2012; 1(1-1):1-12.
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ABSTRACT: The cross-disciplinary focus of the meeting highlighted recent progress in physical and genetic analysis and engineering of cancer disease models. As the central theme, mechanical forces affecting cell signaling, growth, differentiation, and metastasis were discussed with emphasis on the tumor microenvironment and cellular immunity, taking into account novel nanotechnology, biosensing, and intravital microscopy tools to monitor animal cancer models and human cancer. Emerging themes were the role of extracellular matrix imposing mechanical mechanisms on tumor cell function, including microenvironmental cues controlling the movement of tumor and immune cells, advanced genetic animal models for cancer that better recapitulate human disease, and preclinical and clinical molecular imaging of tumor architecture and stiffness, as well as novel nanotechnologies for anticancer drug delivery.
Cancer Research 02/2012; 72(4):841-4. · 7.86 Impact Factor
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ABSTRACT: Fluorescence lifetime imaging microscopy (FLIM) enables detection of complex molecular assemblies within a single voxel for studies of cell function and communication with subcellular resolution in optically transparent tissue. We describe a fast FLIM technique consisting of a novel time-correlated single-photon counting (TCSPC) detector that features 80 MHz average count rate and the phasor analysis for efficient data acquisition and evaluation. This method in combination with multiphoton microscopy enables acquisition of a lifetime image every 1-2 s in 3D live organotypic tissue culture. 3D time-lapse fluorescence lifetime data were acquired over up to 20 h and analyzed by using exponential fitting and phasor analysis. By correlating specific areas in the phasor plot to the actual image, we obtained direct insight into cancer-cell invasion into a 3D collagen matrix, the differential uptake of doxorubicin by cells, and the consequences on cell invasion and apoptosis induction. Based on the fast acquisition and simplified image postprocessing and quantification, time-lapse 3D FLIM is a versatile approach for monitoring the 3D topography, kinetics, and biological output of structurally and spectrally complex cell and tissue models.
Methods in enzymology 01/2012; 504:109-25. · 1.90 Impact Factor
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ABSTRACT: Cancer cell invasion into healthy tissues develops preferentially along pre-existing tracks of least resistance, followed by secondary tissue remodelling and destruction. The tissue scaffolds supporting or preventing guidance of invasion vary in structure and molecular composition between organs. In the brain, the guidance is provided by myelinated axons, astrocyte processes, and blood vessels which are used as invasion routes by glioma cells. In the human breast, containing interstitial collagen-rich connective tissue, disseminating breast cancer cells preferentially invade along bundled collagen fibrils and the surface of adipocytes. In both invasion types, physical guidance prompted by interfaces and space is complemented by molecular guidance. Generic mechanisms shared by most, if not all, tissues include (i) guidance by integrins towards fibrillar interstitial collagen and/or laminins and type IV collagen in basement membranes decorating vessels and adipocytes, and, likely, CD44 engaging with hyaluronan; (ii) haptotactic guidance by chemokines and growth factors; and likely (iii) physical pushing mechanisms. Tissue-specific, resticted guidance cues include ECM proteins with restricted expression (tenascins, lecticans), cell-cell interfaces, and newly secreted matrix molecules decorating ECM fibres (laminin-332, thrombospondin-1, osteopontin, periostin). We here review physical and molecular guidance mechanisms in interstitial tissue and brain parenchyma and explore shared principles and organ-specific differences, and their implications for experimental model design and therapeutic targeting of tumour cell invasion.
The Journal of Pathology 01/2012; 226(2):185-99. · 6.32 Impact Factor
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ABSTRACT: Cancer progression and outcome depend upon two key functions executed by tumor cells: the growth and survival capability leading to resistance to therapy and the invasion into host tissues resulting in local and metastatic dissemination. Although both processes are widely studied separately, the underlying cell-intrinsic and microenvironmentally controlled signaling pathways reveal substantial overlap in mechanism. Candidate signaling hubs that serve both tumor invasion and resistance include growth factor and chemokine signaling, integrin engagement, and components of the Ras/MAPKs, PI3K, and mTOR signaling pathways. In this review, we summarize these and other mechanisms controlled by the microenvironment that jointly support cancer cell survival and resistance, as well as the invasion machinery. We also discuss their interdependencies and the implications for therapeutic dual- or multi-pathway targeting.
Trends in Molecular Medicine 12/2011; 18(1):13-26. · 10.35 Impact Factor
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ABSTRACT: Cell invasion into the 3D extracellular matrix (ECM) is a multistep biophysical process involved in inflammation, tissue repair, and metastatic cancer invasion. Migrating cells navigate through tissue structures of complex and often varying physicochemical properties, including molecular composition, porosity, alignment and stiffness, by adopting strategies that involve deformation of the cell and engagement of matrix-degrading proteases. We review how the ECM determines whether or not pericellular proteolysis is required for cell migration, ranging from protease-driven invasion and secondary tissue destruction, to non-proteolytic, non-destructive movement that solely depends on cell deformability and available tissue space. These concepts call for therapeutic targeting of proteases to prevent invasion-associated tissue destruction rather than the migration process per se.
Trends in cell biology 12/2011; 21(12):736-44. · 12.12 Impact Factor
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ABSTRACT: Cancer invasion is a cell- and tissue-driven process for which the physical, cellular, and molecular determinants adapt and react throughout the progression of the disease. Cancer invasion is initiated and maintained by signaling pathways that control cytoskeletal dynamics in tumor cells and the turnover of cell-matrix and cell-cell junctions, followed by cell migration into the adjacent tissue. Here, we describe the cell-matrix and cell-cell adhesion, protease, and cytokine systems that underlie tissue invasion by cancer cells. We explain how the reciprocal reprogramming of both the tumor cells and the surrounding tissue structures not only guides invasion, but also generates diverse modes of dissemination. The resulting "plasticity" contributes to the generation of diverse cancer invasion routes and programs, enhanced tumor heterogeneity, and ultimately sustained metastatic dissemination.
Cell 11/2011; 147(5):992-1009. · 32.40 Impact Factor
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ABSTRACT: During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics at the leading edge and trailing end, and, as a result, undergoes complex changes in position and shape, which in turn affects cell polarity, shape, and migration efficiency. We here describe the steps of nuclear positioning and deformation during cell polarization and migration, focusing on migration through three-dimensional matrices. We discuss molecular components that govern nuclear shape and stiffness, and review how nuclear dynamics are connected to and controlled by the actin, tubulin and intermediate cytoskeleton-based migration machinery and how this regulation is altered in pathological conditions. Understanding the regulation of nuclear biomechanics has important implications for cell migration during tissue regeneration, immune defence and cancer.
Current opinion in cell biology 02/2011; 23(1):55-64. · 14.15 Impact Factor
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ABSTRACT: Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 µm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.
Physical Biology 02/2011; 8(1):015010. · 2.60 Impact Factor
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ABSTRACT: Immunological control of cancer lesions requires local uptake of tumor-specific antigen followed by the activation and expansion of tumor specific cytotoxic T-lymphocytes (CTL). An efficient effector phase further depends upon the entry of activated CTL into the tumor microenvironment and scanning of tumor tissue, which leads to direct interaction of the CTL with target cells followed by apoptosis induction and shrinkage of the tumor lesion. Whereas the antigens and pathways that lead to efficient activation of tumor-specific CTL are well established, the local mechanisms that enable efficient - or deficient - CTL function in the tumor tissue are poorly understood. Firstly, effector T lymphocytes need to be mobile to reach the tumor lesion. Next, they must physically interact with and scan tumor cells for antigenic MHC/peptide complexes. Lastly, CTLs must undergo activation and functional conjugation with target cells to induce apoptosis either by the release of perforins or the engagement of Fas/FasL. All these steps of effector function are interdependent and require the amoeboid migration of CTL through tissue to reach, engage with and leave encountered cells.
Immunology letters 02/2011; 138(1):19-21. · 2.91 Impact Factor
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ABSTRACT: Cytotoxic T lymphocytes (CTL) mediate antigen- and cell-cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix assay to monitor CTL migration together with CTL-mediated killing of target cells. The assay supports both, time-lapse microscopy of killing dynamics as well as population analysis of killing after matrix digestion and flow cytometry. The assay was used to assess the detrimental effect of cyclosporine A (CsA) present during CTL activation, which caused an inhibition of CTL-target cell conjugation and strongly impaired CTL-mediated killing, particularly at low effector-target ratios. Thus, the organotypic assay is useful to monitor spatiotemporal control mechanisms of cytotoxic immune effector functions.
Biochemical pharmacology 12/2010; 80(12):2087-91. · 4.25 Impact Factor
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Angewandte Chemie International Edition 12/2010; 49(49):9422-5. · 13.45 Impact Factor
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Current opinion in cell biology 10/2010; 22(5):557-9. · 14.15 Impact Factor
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ABSTRACT: Collective migration is a basic mechanism of cell translocation during morphogenesis, wound repair and cancer invasion. Collective movement requires cells to retain cell-cell contacts, exhibit group polarization with defined front-rear asymmetry, and consequently move as one multicellular unit. Depending on the cell type, morphology of the group and the tissue context, distinct mechanisms control the leading edge dynamics and guidance. Leading edge migration may either result from adhesion to ECM and contractile pulling, or from forward pushing. The leading edge consists of either one or few dedicated tip cells or a multicellular leading row that generate adhesion and traction towards the tissue substrate. Alternatively, a multicellular bud consisting of many cells protrudes collectively by proliferation and growth thereby mechanically expanding and pushing towards the tissue stroma. Each type of collective guidance engages distinct spatiotemporal molecular control and feedback towards rearward cells and the adjacent tissue microenvironment; these include intrinsic polarity mechanisms regulated by the interplay between cell-cell and cell-ECM interactions; or the heterotypic integration of stromal cells that adopt leader cell functions. We here classify molecular and mechanical mechanisms of leading function in collective cell migration during morphogenesis and wound repair and discuss how these are recapitulated during collective invasion of cancer cells.
Integrative Biology 10/2010; 2(11-12):568-74. · 4.51 Impact Factor
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ABSTRACT: For homeostasis, T cells integrate non-cognate TCR-dependent and -independent signals to survive and weakly proliferate. In contrast to antigen-specific, stable, and long-lived contacts, signaling in short-lived homeostatic interactions depends upon the coordination of ongoing T-cell migration on the surface of DC and signaling at the cell-cell junction. To mimic peripheral tissues and analyze how T-cell migration and cell-cell signaling are integrated, we used live-cell imaging and 3-D reconstruction of fixed conjugates between DO11.10 T cells and DC in 3-D low-density collagen matrices. T cells simultaneously maintained amoeboid migration and polarized towards the DC, leading to a fully dynamic interaction plane that delivered signals for homeostatic T-cell survival and proliferation. The contact plane comprised three zones, the actin-rich leading edge poor in signal but driving migration, a mid-zone mediating TCR/MHC-induced signal associated with proliferation, and the rear uropod mediating predominantly MHC-independent signals. Thus a dynamic immunological synapse with distinct signaling sectors enables moving T cells to serially sample resident tissue cells and acquire molecular information "en passant".
European Journal of Immunology 10/2010; 40(10):2741-50. · 5.10 Impact Factor
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ABSTRACT: Riboflavin/ultraviolet A (UVA) cross-linking (CXL) of corneal collagen is a novel method of stabilizing corneal mechanical properties and preventing progression of keratectasias. This study was conducted to investigate whether CXL influences ablation rate, flap thickness, and refractive results of excimer laser procedures ex vivo.
Corneal epithelium was removed from enucleated porcine eyes, and CXL was performed with riboflavin 0.1% and UVA radiation (365 nm, 3 mW/cm(2)) for 30 minutes. Control eyes received epithelial abrasion only. Diffusion of riboflavin through the cornea was assessed by using infrared-excited, two-photon microscopy of riboflavin autofluorescence, combined with second-harmonic generation of fibrillar collagen. During phototherapeutic keratectomy, corneal thickness was measured by optical coherence pachymetry. During LASIK for myopia, the flap thickness of microkeratome cuts was measured and the induced refractive change assessed by Placido topography. Data were analyzed by Shapiro-Wilk test and Student's t-test.
Multiphoton imaging showed a rapid (30-minute) and even distribution of riboflavin throughout the corneal stroma. No difference in ablation rate was measured in treated and untreated corneas (P = 0.90). Mean flap thickness was increased by 44% in cross-linked corneas (P < 0.01). After LASIK for myopia of 4 to 25 D, the mean corneal refractive change was reduced in CXL-treated eyes by 20.1% (P < 0.05). This effect was less pronounced in thinner flaps.
CXL reduces the amount of refractive change after LASIK for myopia. Although the laser ablation rate is unaffected, CXL results in an increased flap thickness. This study suggests the need for adjustment of microkeratome and laser parameters for LASIK after CXL and indirectly endorses the theory of a direct stiffening effect of CXL.
Investigative ophthalmology & visual science 03/2010; 51(8):3929-34. · 3.43 Impact Factor
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Barbara Belletti,
Ilenia Pellizzari,
Stefania Berton,
Linda Fabris,
Katarina Wolf,
Francesca Lovat,
Monica Schiappacassi,
Sara D'Andrea,
Milena S Nicoloso,
Sara Lovisa,
Maura Sonego,
Paola Defilippi,
Andrea Vecchione,
Alfonso Colombatti, Peter Friedl,
Gustavo Baldassarre
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ABSTRACT: p27(kip1) (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.
Molecular and cellular biology 03/2010; 30(9):2229-40. · 6.06 Impact Factor
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Peter Friedl
Nature Reviews Molecular Cell Biology 01/2010; 11(1):3. · 39.12 Impact Factor
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ABSTRACT: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells.
Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes.
Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.
Molecular Cancer 01/2010; 9:201. · 3.99 Impact Factor
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ABSTRACT: Cell migration underlies tissue formation, maintenance, and regeneration as well as pathological conditions such as cancer invasion. Structural and molecular determinants of both tissue environment and cell behavior define whether cells migrate individually (through amoeboid or mesenchymal modes) or collectively. Using a multiparameter tuning model, we describe how dimension, density, stiffness, and orientation of the extracellular matrix together with cell determinants--including cell-cell and cell-matrix adhesion, cytoskeletal polarity and stiffness, and pericellular proteolysis--interdependently control migration mode and efficiency. Motile cells integrate variable inputs to adjust interactions among themselves and with the matrix to dictate the migration mode. The tuning model provides a matrix of parameters that control cell movement as an adaptive and interconvertible process with relevance to different physiological and pathological contexts.
Journal of Experimental Medicine 01/2010; 207(1):11-9. · 13.85 Impact Factor
