3D Motility Research Articles

Quantitative Analysis of Small Intestinal Motility in 3D Cine-MRI Using Centerline-Aware Motion Estimation.

Currently available tools for noninvasive motility quantification of the small intestine are limited to dynamic 2D MRI scans, which are limited in their ability to differentiate between types of intestinal motility. To develop a method for quantification and characterization of small intestinal motility in 3D, capable of differentiating motile, non-motile and peristaltic motion patterns. Prospective. Fourteen healthy volunteers (127 small intestinal segments) and 10 patients with Crohn's disease (87 small intestinal segments). 3.0 T, 3D balanced fast field echo sequence, 1 volume per second. Using deformable image registration between subsequent volumes, the local velocity within the intestinal lumen was quantified. Average velocity and average absolute velocity along intestinal segments were used with linear classifiers to differentiate motile from non-motile intestines, as well as erratic motility from peristalsis. The mean absolute velocity of small intestinal content was compared between healthy volunteers and Crohn's disease patients, and the discriminative power of the proposed motility metrics for detecting motility and peristalsis was determined. The consensus of two observers was used as referenced standard. Student's t-test to assess differences between groups; area under the receiver operating characteristic curve (AUC) to assess discriminative ability. P < 0.001 was considered significant. A significant difference in the absolute velocity of intestinal content between Crohn's patients and healthy volunteers was observed (median [IQR] 1.06 [0.61, 1.56] mm/s vs. 1.84 [1.37, 2.43] mm/s), which was consistent with manual reference annotations of motile activity. The proposed method had a strong discriminative performance for detecting non-motile intestines (AUC 0.97) and discernible peristalsis (AUC 0.81). Analysis of 3D cine-MRI using centerline-aware motion estimation has the potential to allow noninvasive characterization of small intestinal motility and peristaltic motion in 3D. 3 TECHNICAL EFFICACY: Stage 2.

Contribution of mechanical forces to structural synaptic plasticity: insights from 3D cellular motility mechanisms.

Contribution of mechanical forces to structural synaptic plasticity: insights from 3D cellular motility mechanisms.

Cell morphology best predicts tumorigenicity and metastasis in vivo across multiple TNBC cell lines of different metastatic potential

BackgroundMetastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis.MethodsWe evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell line's cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines.ResultsWe identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo.ConclusionsOur results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo.

Feature-based 3D+t descriptors of hyperactivated human sperm beat patterns

The flagellar movement of the mammalian sperm plays a crucial role in fertilization. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only capacitated spermatozoa may be hyperactivated and only those that transition to hyperactivated motility are capable of fertilizing the egg. Hyperactivated motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, despite the inherently three-dimensional (3D) nature of sperm motion. Recent research has described several 3D beating features of sperm flagella. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main challenges in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, it is worth noting that only a relatively small proportion, approximately 10-20% of sperm incubated under capacitating conditions exhibit hyperactivated motility. In this work, we used a multifocal image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. The classification results obtained using our 3D feature-based descriptors can serve as potential label for future work involving deep neural networks. By using the classification results as labels, it will be possible to train a deep neural network to automatically classify spermatozoa based on their 3D flagellar beating patterns. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately differentiate between non-hyperactivated and hyperactivated 3D motility patterns of the sperm cells. This work contributes to the understanding of 3D flagellar hyperactive motility patterns and provides a framework for research in the fields of human and animal fertility.

The role of ADAM8 in the mechanophenotype of MDA-MB-231 breast cancer cells in 3D extracellular matrices.

The majority of investigations of cancer cells have been performed in an oversimplified 2D in vitro environment. In the last decade there is a trend toward more sophisticated 3D in vitro cell culture model systems that can bridge the existing gap between 2D in vitro and in vivo experiments in the field of biophysical and cell biological cancer cell research. Here, we hypothesize that the bidirectional interplay between breast cancer cells and their tumor microenvironment is critical for the outcome of the disease. Thereby, the tissue remodeling processes evoked by cancer cells are important for cancer cell-driven mechanical probing of their matrix environment and on cancer cell adhesion and motility. When remodeling processes have been explored, the emphasis was placed on matrix metalloproteinases and rather not on a disintegrin and metalloproteases (ADAMs). However, the role of ADAM8 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. Thus, in this study, we focus on the function of ADAM8 in matrix remodeling and migration of 3D extracellular matrix scaffolds. Therefore, human MDA-MB-231 breast carcinoma cells with ADAM8 knocked down, referred to as ADAM8-KD cells, as well as MDA-MB-231 scrambled control cells, referred to as ADAM8-Ctrl cells, have been used to examine their ability to interact with and migrate in dense extracellular 3D matrices. The fiber displacements, as the capacity of cells to deform the environmental 3D matrix scaffold, has been observed. ADAM8-KD cells displace collagen fibers more strongly than ADAM8-Ctrl cells. Moreover, ADAM8-KD cells migrated more numerous in 3D collagen matrices compared to ADAM8-Ctrl cells. The impairment of ADAM8 using the ADAM8 inhibitor BK-1361 led to significantly increased fiber displacements of ADAM8-Ctrl cells to the levels of ADAM8-KD cells. In contrast, the inhibitor had no effect on ADAM8-KD cells in terms of fiber displacements as well as on the quantitative characteristics of cell invasion of ADAM8-Ctrl cells, albeit the cells that were found in the matrix invaded considerably deeper. When matrix remodeling by cells is impaired through GM6001, a broad-band metalloproteinase inhibitor, the fiber displacements of both cell types increased. In fact, ADAM8 is known to degrade fibronectin in a direct and/or indirect manner. The supplementation of fibronectin before polymerization of the 3D collagen matrices caused an enhancement in fiber displacements as well as in cell invasion into fibronectin-collagen matrices of ADAM8-Ctrl cells, whereas the fiber displacements of ADAM8-KD cells did not change. However, fibrinogen and laminin supplementation induced an increase in fiber displacements of both cell types. Thus, the impact of fibronectin on selective increase in fiber displacement of ADAM8-Ctrl cells appears to be ADAM8-dependent. As a consequence, the presence of ADAM8 may provide an explanation for the longstanding controversial results of fibronectin enrichment on malignant progression of cancers such as breast cancer. Finally, ADAM8 is apparently essential for providing cell-driven fiber displacements of the extracellular matrix microenvironment, which fosters 3D motility in a fibronectin-rich environment. Contribution to the field. Currently, the role of ADAM8 has been explored in 2D or at maximum 2.5D in vitro cell culture motility assays. However, the mechanical characteristics of these two cell types have not been examined. In this study, the function of ADAM8 in breast cancer is refined by providing in vitro cell investigations in 3D collagen fiber matrices of various conditions. ADAM8 has been shown to be involved in the reduced generation of fiber displacements and in influencing breast cancer cell migration. However, especially in the presence of fibronectin in 3Dcollagen fiber matrices, the fiber displacements of ADAM8-Ctrl cells are increased.

Changes in cell surface excess are coordinated with protrusion dynamics during 3D motility

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate that, similar to rounded cells in 2D culture, rounded cells in 3D collagen contain large amounts of CSE and use it to cover developing protrusions. Upon retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. We present high-resolution imaging of F-actin and microtubules (MTs) for different cell lines in a 3D environment and demonstrate the correlated changes between CSE and protrusion dynamics. To coordinate CSE storage and release with protrusion formation and motility, we expect cells to have specific mechanisms for regulating CSE, and we hypothesize that MTs play a substantial role in this mechanism by reducing cell surface dynamics and stabilizing CSE. We also suggest that different effects of MT depolymerization on cell motility, such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by this role of MTs in CSE regulation.

3D+t feature-based descriptor for unsupervised flagellar human sperm beat classification.

Human spermatozoa must swim through the female reproductive tract, where they undergo a series of biochemical and biophysical reactions called capacitation, a necessary step to fertilize the egg. Capacitation promotes changes in the motility pattern. Historically, a two-dimensional analysis has been used to classify sperm motility and clinical fertilization studies. Nevertheless, in a natural environment sperm motility is three-dimensional (3D). Imaging flagella of freely swimming sperm is a difficult task due to their high beating frequency of up to 25 Hz. Very recent studies have described several sperm flagellum 3D beating features (curvature, torsion, asymmetries, etc.). However, up to date, the 3D motility pattern of hyperactivated spermatozoa has not been characterized. The main difficulty in classifying these patterns in 3D is the lack of a ground truth reference since differences in flagellar beat patterns are very difficult to assess visually. Moreover, only around 10-20% of induced to capacitate spermatozoa are truly capacitated, i.e., hyperactivated. We used an image acquisition system that can acquire, segment, and track spermatozoa flagella in 3D+t. In this work, we propose an original three-dimensional feature vector formed by ellipses describing the envelope of the 3D+t spatio-temporal flagellar sperm motility patterns. These features allowed compressing an unlabeled 3D+t dataset to separate hyperactivated cells from others (capacitated from non-capacitated cells) using unsupervised hierarchical clustering. Preliminary results show three main clusters of flagellar motility patterns. The first principal component of these 3D flagella measurements correlated with 2D OpenCASA head determinations as a first approach to validate the unsupervised classification, showing a reasonable correlation coefficient near to 0.7. Clinical relevance- The novelty of this work is defining a 3D+t feature-based descriptor consisting of a set of ellipses enveloping the flagellar motion of human sperm for its unsu-pervised classification. This is a new promising tool to determine the viability of human sperm to fertilize the egg.

Abstract 630: Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies

Abstract Immunotherapies (ITx) have revolutionized the oncology landscape. However, predicting patient responses to ITx is difficult based solely on static correlates such as TIL localization and molecular signatures. Anti-tumor immune response depends on motile surveillance by tumor infiltrating lymphocytes (TIL) which recognize antigenic determinants and engage target cells in serial stop-and-go interactions that result in cell killing. However, the hostile tumor microenvironment (TME) can cause TIL dysfunction and lack of cytotoxicity which is manifested as either suppressed or aimless TIL motility. To determine which ITx works best for a given patient, we are developing a diagnostic platform using live tumor fragments (LTF) that preserves the TME and its immune cells. Antibody-based labeling of live tissues is hampered by slow diffusion and function-altering cross-linking. To overcome these limitations, we have used small camelid-derived monovalent antibodies (nanobodies) to monitor TIL motility in LTFs. Human tumor excisions were cut into 300 x 300 x 200 µm LTFs, sorted into glass bottom multi-well plates and cultured. CD8+ cells in LTFs were labeled using an anti-hCD8a camelid VHH nanobody covalently labeled with AF594. The same reagent or a mouse anti-hCD8a whole IgG antibody was used to stain human peripheral blood mononuclear cells, and staining patterns were compared by flow cytometry. Immune responsiveness of LTFs to ITx was ascertained by flow cytometry and secreted protein assays (data shown in a companion abstract). Multiphoton microscopy revealed LTF collagen fibrils and cellular autofluorescence. A fluorescent anti-CD8a nanobody, but not a similarly labeled whole IgG, yielded good contrast and fast staining of two cell subsets. The smaller cells were 12 µm in diameter, cell surface-stained, and lacking autofluorescence, consistent with T cells. The larger cells were elongated, ramified, intracellularly stained, and distinctly autofluorescent, consistent with macrophages. 3D motility tracking revealed characteristic translational motility of the smaller cells at ~10 µm/min along collagenous structures. In contrast, larger cells exhibited only slow motility. Our results show that the motility of human CD8+ T lymphocytes can be revealed in LTF culture using a fluorescent CD8-binding camelid nanobody, likely due to its small size and monovalent binding. Further examination is needed to understand if T cell function is altered. The autofluorescence of larger, immotile cells was consistent with tumor-associated macrophages. Based on this distinction, T cells could be distinguished from the macrophages clearly. Our results support the use of camelid-derived VHH and other small monovalent reagents for live tissue lymphocyte tracking, possibly to evaluate TIL response to ITx in an LTF assay. Citation Format: Leung Kau Tang, Kelsey Tweed, Christina Scribano, David Wahl, Christin Johnson, M. Anna Zal, Jonathan Oliner, Tomasz Zal. Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 630.

PINCH1 Promotes Fibroblast Migration in Extracellular Matrices and Influences Their Mechanophenotype.

Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knock-out cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1−/− cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1−/− cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1−/− cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1−/− cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson’s ratio in PINCH1−/− cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.

Morphodynamics facilitate cancer cells to navigate 3D extracellular matrix

Cell shape is linked to cell function. The significance of cell morphodynamics, namely the temporal fluctuation of cell shape, is much less understood. Here we study the morphodynamics of MDA-MB-231 cells in type I collagen extracellular matrix (ECM). We systematically vary ECM physical properties by tuning collagen concentrations, alignment, and gelation temperatures. We find that morphodynamics of 3D migrating cells are externally controlled by ECM mechanics and internally modulated by Rho/ROCK-signaling. We employ machine learning to classify cell shape into four different morphological phenotypes, each corresponding to a distinct migration mode. As a result, we map cell morphodynamics at mesoscale into the temporal evolution of morphological phenotypes. We characterize the mesoscale dynamics including occurrence probability, dwell time and transition matrix at varying ECM conditions, which demonstrate the complex phenotype landscape and optimal pathways for phenotype transitions. In light of the mesoscale dynamics, we show that 3D cancer cell motility is a hidden Markov process whereby the step size distributions of cell migration are coupled with simultaneous cell morphodynamics. Morphological phenotype transitions also facilitate cancer cells to navigate non-uniform ECM such as traversing the interface between matrices of two distinct microstructures. In conclusion, we demonstrate that 3D migrating cancer cells exhibit rich morphodynamics that is controlled by ECM mechanics, Rho/ROCK-signaling, and regulate cell motility. Our results pave the way to the functional understanding and mechanical programming of cell morphodynamics as a route to predict and control 3D cell motility.

A mathematical framework for modelling 3D cell motility: applications to glioblastoma cell migration.

The collection of 3D cell tracking data from live images of micro-tissues is a recent innovation made possible due to advances in imaging techniques. As such there is increased interest in studying cell motility in 3D in vitro model systems but a lack of rigorous methodology for analysing the resulting data sets. One such instance of the use of these in vitro models is in the study of cancerous tumours. Growing multicellular tumour spheroids in vitro allows for modelling of the tumour microenvironment and the study of tumour cell behaviours, such as migration, which improves understanding of these cells and in turn could potentially improve cancer treatments. In this paper, we present a workflow for the rigorous analysis of 3D cell tracking data, based on the persistent random walk model, but adaptable to other biologically informed mathematical models. We use statistical measures to assess the fit of the model to the motility data and to estimate model parameters and provide confidence intervals for those parameters, to allow for parametrization of the model taking correlation in the data into account. We use in silico simulations to validate the workflow in 3D before testing our method on cell tracking data taken from in vitro experiments on glioblastoma tumour cells, a brain cancer with a very poor prognosis. The presented approach is intended to be accessible to both modellers and experimentalists alike in that it provides tools for uncovering features of the data set that may suggest amendments to future experiments or modelling attempts.

Sp1-Induced FNBP1 Drives Rigorous 3D Cell Motility in EMT-Type Gastric Cancer Cells

Cancer is heterogeneous among patients, requiring a thorough understanding of molecular subtypes and the establishment of therapeutic strategies based on its behavior. Gastric cancer (GC) is adenocarcinoma with marked heterogeneity leading to different prognoses. As an effort, we previously identified a stem-like subtype, which is prone to metastasis, with the worst prognosis. Here, we propose FNBP1 as a key to high-level cell motility, present only in aggressive GC cells. FNBP1 is also up-regulated in both the GS subtype from the TCGA project and the EMT subtype from the ACRG study, which include high portions of diffuse histologic type. Ablation of FNBP1 in the EMT-type GC cell line brought changes in the cell periphery in transcriptomic analysis. Indeed, loss of FNBP1 resulted in the loss of invasive ability, especially in a three-dimensional culture system. Live imaging indicated active movement of actin in FNBP1-overexpressed cells cultured in an extracellular matrix dome. To find the transcription factor which drives FNBP1 expression in an EMT-type GC cell line, the FNBP1 promoter region and DNA binding motifs were analyzed. Interestingly, the Sp1 motif was abundant in the promoter, and pharmacological inhibition and knockdown of Sp1 down-regulated FNBP1 promoter activity and the transcription level, respectively. Taken together, our results propose Sp1-driven FNBP1 as a key molecule explaining aggressiveness in EMT-type GC cells.

PO-1730: Development of a framework to quantify bowel motility in 3D using MRI

PO-1730: Development of a framework to quantify bowel motility in 3D using MRI

Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

Simple SummaryMelanoma, the most aggressive skin cancer, accounts for the majority of deaths due to this disease. Therefore, identification of genes/proteins involved in melanoma genesis and/or progression is urgent. Mutations abrogating expression of Mahogunin Ring Finger 1 (MGRN1) in mice cause complex phenotypes with hyperpigmentation, and known MGRN1 interactors are important regulators of cell shape and movement. This suggests that MGRN1 may modulate the malignant phenotype of melanoma cells. Analysis of MGRN1-KO mouse melanocytes and melanoma cells showed that lack of MGRN1 leads to cell cycle defects and to a more differentiated, less aggressive phenotype, with increased adhesion to various matrices, decreased motility and high genomic instability. The higher aggressivity of MGRN1-expressing melanoma cells was confirmed in an in vivo mouse melanoma model and is consistent with higher survival of human melanoma patients expressing low levels of MGRN1. Therefore, MGRN1 appears an important determinant of the malignant phenotype of melanoma.The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

Mechanical Cues Affect Migration and Invasion of Cells From Three Different Directions.

Cell migration and invasion is a key driving factor for providing essential cellular functions under physiological conditions or the malignant progression of tumors following downward the metastatic cascade. Although there has been plentiful of molecules identified to support the migration and invasion of cells, the mechanical aspects have not yet been explored in a combined and systematic manner. In addition, the cellular environment has been classically and frequently assumed to be homogeneous for reasons of simplicity. However, motility assays have led to various models for migration covering only some aspects and supporting factors that in some cases also include mechanical factors. Instead of specific models, in this review, a more or less holistic model for cell motility in 3D is envisioned covering all these different aspects with a special emphasis on the mechanical cues from a biophysical perspective. After introducing the mechanical aspects of cell migration and invasion and presenting the heterogeneity of extracellular matrices, the three distinct directions of cell motility focusing on the mechanical aspects are presented. These three different directions are as follows: firstly, the commonly used invasion tests using structural and structure-based mechanical environmental signals; secondly, the mechano-invasion assay, in which cells are studied by mechanical forces to migrate and invade; and thirdly, cell mechanics, including cytoskeletal and nuclear mechanics, to influence cell migration and invasion. Since the interaction between the cell and the microenvironment is bi-directional in these assays, these should be accounted in migration and invasion approaches focusing on the mechanical aspects. Beyond this, there is also the interaction between the cytoskeleton of the cell and its other compartments, such as the cell nucleus. In specific, a three-element approach is presented for addressing the effect of mechanics on cell migration and invasion by including the effect of the mechano-phenotype of the cytoskeleton, nucleus and the cell’s microenvironment into the analysis. In precise terms, the combination of these three research approaches including experimental techniques seems to be promising for revealing bi-directional impacts of mechanical alterations of the cellular microenvironment on cells and internal mechanical fluctuations or changes of cells on the surroundings. Finally, different approaches are discussed and thereby a model for the broad impact of mechanics on cell migration and invasion is evolved.

A computational study of amoeboid motility in 3D: the role of extracellular matrix geometry, cell deformability, and cell-matrix adhesion.

Amoeboid cells often migrate using pseudopods, which are membrane protrusions that grow, bifurcate, and retract dynamically, resulting in a net cell displacement. Many cells within the human body, such as immune cells, epithelial cells, and even metastatic cancer cells, can migrate using the amoeboid phenotype. Amoeboid motility is a complex and multiscale process, where cell deformation, biochemistry, and cytosolic and extracellular fluid motions are coupled. Furthermore, the extracellular matrix (ECM) provides a confined, complex, and heterogeneous environment for the cells to navigate through. Amoeboid cells can migrate without significantly remodeling the ECM using weak or no adhesion, instead utilizing their deformability and the microstructure of the ECM to gain enough traction. While a large volume of work exists on cell motility on 2D substrates, amoeboid motility is 3D in nature. Despite recent progress in modeling cellular motility in 3D, there is a lack of systematic evaluations of the role of ECM microstructure, cell deformability, and adhesion on 3D motility. To fill this knowledge gap, here we present a multiscale, multiphysics modeling study of amoeboid motility through 3D-idealized ECM. The model is a coupled fluid‒structure and coarse-grain biochemistry interaction model that accounts for large deformation of cells, pseudopod dynamics, cytoplasmic and extracellular fluid motion, stochastic dynamics of cell-ECM adhesion, and microstructural (pore-scale) geometric details of the ECM. The key finding of the study is that cell deformation and matrix porosity strongly influence amoeboid motility, while weak adhesion and microscale structural details of the ECM have secondary but subtle effects.

Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus xanthus Using Confocal Interference Reflection Microscopy.

The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili.IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope.

Loss of Vimentin Enhances Cell Motility through Small Confining Spaces.

The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.

Vimentin protects cells against nuclear rupture and DNA damage during migration.

Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.

The Small GTPase Rac1 Increases Cell Surface Stiffness and Enhances 3D Migration Into Extracellular Matrices

Membrane ruffling and lamellipodia formation promote the motility of adherent cells in two-dimensional motility assays by mechano-sensing of the microenvironment and initiation of focal adhesions towards their surroundings. Lamellipodium formation is stimulated by small Rho GTPases of the Rac subfamily, since genetic removal of these GTPases abolishes lamellipodium assembly. The relevance of lamellipodial or invadopodial structures for facilitating cellular mechanics and 3D cell motility is still unclear. Here, we hypothesized that Rac1 affects cell mechanics and facilitates 3D invasion. Thus, we explored whether fibroblasts that are genetically deficient for Rac1 (lacking Rac2 and Rac3) harbor altered mechanical properties, such as cellular deformability, intercellular adhesion forces and force exertion, and exhibit alterations in 3D motility. Rac1 knockout and control cells were analyzed for changes in deformability by applying an external force using an optical stretcher. Five Rac1 knockout cell lines were pronouncedly more deformable than Rac1 control cells upon stress application. Using AFM, we found that cell-cell adhesion forces are increased in Rac1 knockout compared to Rac1-expressing fibroblasts. Since mechanical deformability, cell-cell adhesion strength and 3D motility may be functionally connected, we investigated whether increased deformability of Rac1 knockout cells correlates with changes in 3D motility. All five Rac1 knockout clones displayed much lower 3D motility than Rac1-expressing controls. Moreover, force exertion was reduced in Rac1 knockout cells, as assessed by 3D fiber displacement analysis. Interference with cellular stiffness through blocking of actin polymerization by Latrunculin A could not further reduce invasion of Rac1 knockout cells. In contrast, Rac1-expressing controls treated with Latrunculin A were again more deformable and less invasive, suggesting actin polymerization is a major determinant of observed Rac1-dependent effects. Together, we propose that regulation of 3D motility by Rac1 partly involves cellular mechanics such as deformability and exertion of forces.