Proteolysis-free amoeboid migration of melanoma cells through crowded environments via bleb-driven worrying.

In crowded microenvironments, migrating cells must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet, some amoeboid cells seem to maintain a near-spherical morphology as they move. To examine how they do so, we visualized amoeboid human melanoma cells in dense environments and found that they carve tunnels via bleb-driven degradation of extracellular matrix components without the need for proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology, and polarization that enables both path generation and persistent movement.

Subcutaneous Administration of Monoclonal Antibodies: Pharmacology, Delivery, Immunogenicity, and Learnings From Applications to Clinical Development.

Subcutaneous (s.c.) administration of monoclonal antibodies (mAbs) can reduce treatment burden for patients and healthcare systems compared with intravenous (i.v.) infusion through shorter administration times, made possible by convenient, patient-centric devices. A deeper understanding of clinical pharmacology principles related to efficacy and safety of s.c.-administered mAbs over the past decade has streamlined s.c. product development. This review presents learnings from key constituents of the s.c. mAb development pathway, including pharmacology, administration variables, immunogenicity, and delivery devices. Restricted mAb transportation through the hypodermis explains their incomplete absorption at a relatively slow rate (pharmacokinetic (PK)) and may impact mAb-cellular interactions and/or onset and magnitude of physiological responses (pharmacodynamic). Injection volumes, formulation, rate and site of injection, and needle attributes may affect PKs and the occurrence/severity of adverse events like injection-site reactions or pain, with important consequences for treatment adherence. A review of immunogenicity data for numerous compounds reveals that incidence of anti-drug antibodies (ADAs) is generally comparable across i.v. and s.c. routes, and complementary factors including response magnitude (ADA titer), persistence over time, and neutralizing antibody presence are needed to assess clinical impact. Finally, four case studies showcase how s.c. biologics have been clinically developed: (i) by implementation of i.v./s.c. bridging strategies to streamline PD-1/PD-L1 inhibitor development, (ii) through co-development with i.v. presentations for anti-severe acute respiratory syndrome-coronavirus 2 antibodies to support rapid deployment of both formulations, (iii) as the lead route for bispecific T cell engagers (BTCEs) to mitigate BTCE-mediated cytokine release syndrome, and (iv) for pediatric patients in the case of dupilumab.

u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions.

We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Interpretable deep learning uncovers cellular properties in label-free live cell images that are predictive of highly metastatic melanoma.

Deep learning has emerged as the technique of choice for identifying hidden patterns in cell imaging data but is often criticized as "black box." Here, we employ a generative neural network in combination with supervised machine learning to classify patient-derived melanoma xenografts as "efficient" or "inefficient" metastatic, validate predictions regarding melanoma cell lines with unknown metastatic efficiency in mouse xenografts, and use the network to generate in silico cell images that amplify the critical predictive cell properties. These exaggerated images unveiled pseudopodial extensions and increased light scattering as hallmark properties of metastatic cells. We validated this interpretation using live cells spontaneously transitioning between states indicative of low and high metastatic efficiency. This study illustrates how the application of artificial intelligence can support the identification of cellular properties that are predictive of complex phenotypes and integrated cell functions but are too subtle to be identified in the raw imagery by a human expert. A record of this paper's transparent peer review process is included in the supplemental information. VIDEO ABSTRACT.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Evaluating Melanoma Viability and Proliferation in 3D Microenvironments.

Researchers often aim to incorporate microenvironmental variables such as the dimensionality and composition of the extracellular matrix into their cell-based assays. A technical challenge created by introduction of these variables is quantification of single-cell measurements and control of environmental reproducibility. Here, we detail a methodology to quantify viability and proliferation of melanoma cells in 3D collagen-based culture platforms by automated microscopy and 3D image analysis to yield robust, high-throughput results of single-cell responses to drug treatment.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1P29S Melanoma Cells.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

BACKGROUND: Every biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput. METHODS: Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput. RESULTS: We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. CONCLUSION: In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.

Nuclear positioning facilitates amoeboid migration along the path of least resistance.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1-3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some-but not all-cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion7, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.

Universal light-sheet generation with field synthesis.

We introduce field synthesis, a theorem and method that can be used to synthesize any scanned or dithered light sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. Compared to LLSM, this user-friendly and modular approach offers a simplified optical design, higher light throughput and simultaneous multicolor illumination. Further, field synthesis achieves lower rates of photobleaching than light sheets generated by lateral beam scanning.

Imaging Subcellular Dynamics with Fast and Light-Efficient Volumetrically Parallelized Microscopy.

In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells. We demonstrate 3D imaging of intracellular processes, including cytoskeletal dynamics in single cell migration and collective wound healing for 1500 and 1000 time points, respectively. Further, we capture rapid biological processes, including trafficking of early endosomes with velocities exceeding 10 microns per second and calcium signaling in primary neurons.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells.

In subcellular light-sheet fluorescence microscopy (LSFM) of adherent cells, glass substrates are advantageously rotated relative to the excitation and emission light paths to avoid glass-induced optical aberrations. Because cells are spread across the sample volume, three-dimensional imaging requires a light-sheet with a long propagation length, or rapid sample scanning. However, the former degrades axial resolution and/or optical sectioning, while the latter mechanically perturbs sensitive biological specimens on pliant biomimetic substrates (e.g., collagen and basement membrane). Here, we use aberration-free remote focusing to diagonally sweep a narrow light-sheet along the sample surface, enabling multicolor imaging with high spatiotemporal resolution. Further, we implement a dithered Gaussian lattice to minimize sample-induced illumination heterogeneities, significantly improving signal uniformity. Compared with mechanical sample scanning, we drastically reduce sample oscillations, allowing us to achieve volumetric imaging at speeds of up to 3.5 Hz for thousands of Z-stacks. We demonstrate the optical performance with live-cell imaging of microtubule and actin cytoskeletal dynamics, phosphoinositide signaling, clathrin-mediated endocytosis, polarized blebbing, and endocytic vesicle sorting. We achieve three-dimensional particle tracking of clathrin-associated structures with velocities up to 4.5 µm/s in a dense intracellular environment, and show that such dynamics cannot be recovered reliably at lower volumetric image acquisition rates using experimental data, numerical simulations, and theoretical modeling.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases.

Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here, we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detected an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrated that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Reciprocally, SNX9 knockdown impairs the process. In vitro studies using several cancer cell lines derived from a variety of human tumors revealed a role for SNX9 in cell invasion and identified mechanisms responsible for this novel function. We showed that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we have discovered that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity, can functionally substitute to the bona fide Rho GAP, GRAF1 (GTPase Regulator Associated with Focal Adhesion Kinase). Together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis.

Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments.

The microenvironment determines cell behavior, but the underlying molecular mechanisms are poorly understood because quantitative studies of cell signaling and behavior have been challenging due to insufficient spatial and/or temporal resolution and limitations on microenvironmental control. Here we introduce microenvironmental selective plane illumination microscopy (meSPIM) for imaging and quantification of intracellular signaling and submicrometer cellular structures as well as large-scale cell morphological and environmental features. We demonstrate the utility of this approach by showing that the mechanical properties of the microenvironment regulate the transition of melanoma cells from actin-driven protrusion to blebbing, and we present tools to quantify how cells manipulate individual collagen fibers. We leverage the nearly isotropic resolution of meSPIM to quantify the local concentration of actin and phosphatidylinositol 3-kinase signaling on the surfaces of cells deep within 3D collagen matrices and track the many small membrane protrusions that appear in these more physiologically relevant environments.

Seeds of Locally Aligned Motion and Stress Coordinate a Collective Cell Migration.

We find how collective migration emerges from mechanical information transfer between cells. Local alignment of cell velocity and mechanical stress orientation-a phenomenon dubbed "plithotaxis"-plays a crucial role in inducing coordinated migration. Leader cells at the monolayer edge better align velocity and stress to migrate faster toward the open space. Local seeds of enhanced motion then generate stress on neighboring cells to guide their migration. Stress-induced motion propagates into the monolayer as well as along the monolayer boundary to generate increasingly larger clusters of coordinately migrating cells that move faster with enhanced alignment of velocity and stress. Together, our analysis provides a model of long-range mechanical communication between cells, in which plithotaxis translates local mechanical fluctuations into globally collective migration of entire tissues.