Multiphoton fluorescence microscopy for in vivo imaging.

Multiphoton fluorescence microscopy (MPFM) has been a game-changer for optical imaging, particularly for studying biological tissues deep within living organisms. MPFM overcomes the strong scattering of light in heterogeneous tissue by utilizing nonlinear excitation that confines fluorescence emission mostly to the microscope focal volume. This enables high-resolution imaging deep within intact tissue and has opened new avenues for structural and functional studies. MPFM has found widespread applications and has led to numerous scientific discoveries and insights into complex biological processes. Today, MPFM is an indispensable tool in many research communities. Its versatility and effectiveness make it a go-to technique for researchers investigating biological phenomena at the cellular and subcellular levels in their native environments. In this Review, the principles, implementations, capabilities, and limitations of MPFM are presented. Three application areas of MPFM, neuroscience, cancer biology, and immunology, are reviewed in detail and serve as examples for applying MPFM to biological research.

Plasticity of Cell Migration In Vivo and In Silico.

Cell migration results from stepwise mechanical and chemical interactions between cells and their extracellular environment. Mechanistic principles that determine single-cell and collective migration modes and their interconversions depend upon the polarization, adhesion, deformability, contractility, and proteolytic ability of cells. Cellular determinants of cell migration respond to extracellular cues, including tissue composition, topography, alignment, and tissue-associated growth factors and cytokines. Both cellular determinants and tissue determinants are interdependent; undergo reciprocal adjustment; and jointly impact cell decision making, navigation, and migration outcome in complex environments. We here review the variability, decision making, and adaptation of cell migration approached by live-cell, in vivo, and in silico strategies, with a focus on cell movements in morphogenesis, repair, immune surveillance, and cancer metastasis.

Cancer invasion and the microenvironment: plasticity and reciprocity.

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 jamming in a collagen-based interface assay is tuned by collagen density and proteolysis.

Tumor cell invasion into heterogenous interstitial tissues consisting of network-, channel- or rift-like architectures involves both matrix metalloproteinase (MMP)-mediated tissue remodeling and cell shape adaptation to tissue geometry. Three-dimensional (3D) models composed of either porous or linearly aligned architectures have added to the understanding of how physical spacing principles affect migration efficacy; however, the relative contribution of each architecture to decision making in the presence of varying MMP availability is not known. Here, we developed an interface assay containing a cleft between two high-density collagen lattices, and we used this assay to probe tumor cell invasion efficacy, invasion mode and MMP dependence in concert. In silico modeling predicted facilitated cell migration into confining clefts independently of MMP activity, whereas migration into dense porous matrix was predicted to require matrix degradation. This prediction was verified experimentally, where inhibition of collagen degradation was found to strongly compromise migration into 3D collagen in a density-dependent manner, but interface-guided migration remained effective, occurring by cell jamming. The 3D interface assay reported here may serve as a suitable model to better understand the impact of in vivo-relevant interstitial tissue topologies on tumor invasion patterning and responses to molecular interventions.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

New dimensions in cell migration.

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.

A Swiss Army knife for CTLs.

Granzyme B released by leukocytes cleaves multiple intracellular substrates required for target cell lysis. In this issue of Immunity, Prakash et al. (2014) demonstrate that granzyme B cleaves basement membrane proteins and promotes cytotoxic T cell diapedesis into inflamed tissue.

Actomyosin contractility requirements and reciprocal cell-tissue mechanics for cancer cell invasion through collagen-based channels.

The interstitial tumor microenvironment is composed of heterogeneously organized collagen-rich porous networks as well as channel-like structures and interfaces which provide both barriers and guidance for invading cells. Tumor cells invading 3D random porous collagen networks depend upon actomyosin contractility to deform and translocate the nucleus, whereas Rho/Rho-associated kinase-dependent contractility is largely dispensable for migration in stiff capillary-like confining microtracks. To investigate whether this dichotomy of actomyosin contractility dependence also applies to physiological, deformable linear collagen environments, we developed nearly barrier-free collagen-scaffold microtracks of varying cross section using two-photon laser ablation. Both very narrow and wide tracks supported single-cell migration by either outward pushing of collagen up to four times when tracks were narrow, or cell pulling on collagen walls down to 50% of the original diameter by traction forces of up to 40 nN when tracks were wide, resulting in track widths optimized to single-cell diameter. Targeting actomyosin contractility by synthetic inhibitors increased cell elongation and nuclear shape change in narrow tracks and abolished cell-mediated deformation of both wide and narrow tracks. Accordingly, migration speeds in all channel widths reduced, with migration rates of around 45-65% of the original speed persisting. Together, the data suggest that cells engage actomyosin contraction to reciprocally adjust both own morphology and linear track width to optimal size for effective cellular locomotion.

Cancer invasion into musculature: Mechanics, molecules and implications.

Tumor invasion along structural interphases of surrounding tumor-free tissue represents a key process during tumor progression. Much attention has been devoted to mechanisms of tumor cell migration within extracellular matrix (ECM)-rich connective tissue, however a comprehensive understanding of tumor invasion into tissue of higher structural complexity, such as muscle tissue, is lacking. Muscle invasion in cancer patients is often associated with destructive growth and worsened prognosis. Here, we review biochemical, geometrical and mechanical cues of smooth and skeletal muscle tissues and their relevance for guided invasion of cancer cells. As integrating concept, muscle-organizing ECM-rich surfaces of the epi-, peri- and endomysium provide cleft-like confined spaces along interfaces between dynamic muscle cells, which provide molecular and physical cues that guide migrating cancer cells, forming a possible contribution to cancer progression.

Collective cell migration in morphogenesis, regeneration and cancer.

The collective migration of cells as a cohesive group is a hallmark of the tissue remodelling events that underlie embryonic morphogenesis, wound repair and cancer invasion. In such migration, cells move as sheets, strands, clusters or ducts rather than individually, and use similar actin- and myosin-mediated protrusions and guidance by extrinsic chemotactic and mechanical cues as used by single migratory cells. However, cadherin-based junctions between cells additionally maintain 'supracellular' properties, such as collective polarization, force generation, decision making and, eventually, complex tissue organization. Comparing different types of collective migration at the molecular and cellular level reveals a common mechanistic theme between developmental and cancer research.

Adaptive adhesion systems mediate glioma cell invasion in complex environments.

Diffuse brain invasion by glioma cells prevents effective surgical or molecular-targeted therapy and underlies a detrimental outcome. Migrating glioma cells are guided by complex anatomical brain structures but the exact mechanisms remain poorly defined. To identify adhesion receptor systems and matrix structures supporting glioma cell invasion into brain-like environments we used 2D and 3D organotypic invasion assays in combination with antibody-, peptide- and RNA-based interference. Combined interference with ß1 and αV integrins abolished the migration of U-251 and E-98 glioma cells on reconstituted basement membrane; however, invasion into primary brain slices or 3D astrocyte-based scaffolds and migration on astrocyte-deposited matrix was only partly inhibited. Any residual invasion was supported by vascular structures, as well as laminin 511, a central constituent of basement membrane of brain blood vessels. Multi-targeted interference against ß1, αV and α6 integrins expressed by U-251 and E-98 cells proved insufficient to achieve complete migration arrest. These data suggest that mechanocoupling by integrins is relatively resistant to antibody- or peptide-based targeting, and cooperates with additional, as yet unidentified adhesion systems in mediating glioma cell invasion in complex brain stroma.

Interstitial leukocyte migration and immune function.

The trafficking of leukocytes into and within lymphoid and peripheral tissues is central to immune cell development, immunosurveillance and effector function. Interstitial leukocyte trafficking is the result of amoeboid polarization and migration, guided by soluble or tissue-bound chemoattractant signals for positioning and local arrest. In contrast to other migration modes, amoeboid movement is particularly suited for scanning cellular networks and tissues. Here, we review mechanisms of leukocyte migration and sensing involved in diapedesis, tissue-based interstitial migration and egress, immune cell positioning in inflammation, and emerging therapeutic interference strategies.

Third harmonic generation microscopy of cells and tissue organization.

The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra- and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in three-dimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice.

Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture.

Organotypic in vitro culture of 3D spheroids in an extracellular matrix represent a promising cancer therapy prediction model for personalized medicine screens due to their controlled experimental conditions and physiological similarities to in vivo conditions. As in tumors in vivo, 3D invasion cultures identify intratumor heterogeneity of growth, invasion and apoptosis induction by cytotoxic therapy. We here combine in vitro 3D spheroid invasion culture with irradiation and automated nucleus-based segmentation for single cell analysis to quantify growth, survival, apoptosis and invasion response during experimental radiation therapy. As output, multi-parameter histogram-based representations deliver an integrated insight into therapy response and resistance. This workflow may be suited for high-throughput screening and identification of invasive and therapy-resistant tumor sub-populations.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb.

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.

Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.

Diffuse invasion of glioma cells into the brain parenchyma leads to nonresectable brain tumors and poor prognosis of glioma disease. In vivo, glioma cells can adopt a range of invasion strategies and routes, by moving as single cells, collective strands and multicellular networks along perivascular, perineuronal and interstitial guidance cues. Current in vitro assays to probe glioma cell invasion, however, are limited in recapitulating the modes and adaptability of glioma invasion observed in brain parenchyma, including collective behaviours. To mimic in vivo-like glioma cell invasion in vitro, we here applied three tissue-inspired 3D environments combining multicellular glioma spheroids and reconstituted microanatomic features of vascular and interstitial brain structures. Radial migration from multicellular glioma spheroids of human cell lines and patient-derived xenograft cells was monitored using (1) reconstituted basement membrane/hyaluronan interfaces representing the space along brain vessels; (2) 3D scaffolds generated by multi-layered mouse astrocytes to reflect brain interstitium; and (3) freshly isolated mouse brain slice culture ex vivo. The invasion patterns in vitro were validated using histological analysis of brain sections from glioblastoma patients and glioma xenografts infiltrating the mouse brain. Each 3D assay recapitulated distinct aspects of major glioma invasion patterns identified in mouse xenografts and patient brain samples, including individually migrating cells, collective strands extending along blood vessels, and multicellular networks of interconnected glioma cells infiltrating the neuropil. In conjunction, these organotypic assays enable a range of invasion modes used by glioma cells and will be applicable for mechanistic analysis and targeting of glioma cell dissemination.

Plasticity of tumor cell invasion: governance by growth factors and cytokines.

Tumor cell migration, the basis for metastatic dissemination, is an adaptive process which depends upon coordinated cell interaction with the environment, influencing cell-matrix and cell-cell adhesion, cytoskeletal dynamics and extracellular matrix remodeling. Growth factors and cytokines, released within the reactive tumor microenvironment and their intracellular effector signals strongly impact mechanocoupling functions in tumor cells and thereby control the mode and extent of tumor invasion, including collective and single-cell migration and their interconversions. Besides their role in controlling tumor cell growth and survival, cytokines and growth factors thus provide complex orchestration of the metastatic cascade and tumor cell adaptation to environmental challenge. We here review the mechanisms by which growth factors and cytokines control the reciprocal interactions between tumor cells and their microenvironment, and the consequences for the efficacy and plasticity of invasion programs and metastasis.

Anti-CD137 monoclonal antibodies and adoptive T cell therapy: a perfect marriage?

CD137(4-1BB) costimulation and adoptive T cell therapy strongly synergize in terms of achieving maximal efficacy against experimental cancers. These costimulatory biological functions of CD137 have been exploited by means of introducing the CD137 signaling domain in clinically successful chimeric antigen receptors and to more efficiently expand T cells in culture. In addition, immunomagnetic sorting of CD137-positive T cells among tumor-infiltrating lymphocytes selects for the fittest antitumor T lymphocytes for subsequent cultures. In mouse models, co-infusion of both agonist antibodies and T cells attains marked synergistic effects that result from more focused and intense cytolytic activity visualized under in vivo microscopy and from more efficient entrance of T cells into the tumor through the vasculature. These several levels of dynamic interaction between adoptive T cell therapy and CD137 offer much opportunity to raise the efficacy of current cancer immunotherapies.