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.

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.

Data science in cell imaging.

Cell imaging has entered the 'Big Data' era. New technologies in light microscopy and molecular biology have led to an explosion in high-content, dynamic and multidimensional imaging data. Similar to the 'omics' fields two decades ago, our current ability to process, visualize, integrate and mine this new generation of cell imaging data is becoming a critical bottleneck in advancing cell biology. Computation, traditionally used to quantitatively test specific hypotheses, must now also enable iterative hypothesis generation and testing by deciphering hidden biologically meaningful patterns in complex, dynamic or high-dimensional cell image data. Data science is uniquely positioned to aid in this process. In this Perspective, we survey the rapidly expanding new field of data science in cell imaging. Specifically, we highlight how data science tools are used within current image analysis pipelines, propose a computation-first approach to derive new hypotheses from cell image data, identify challenges and describe the next frontiers where we believe data science will make an impact. We also outline steps to ensure broad access to these powerful tools - democratizing infrastructure availability, developing sensitive, robust and usable tools, and promoting interdisciplinary training to both familiarize biologists with data science and expose data scientists to cell imaging.

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.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Asymmetric nanotopography biases cytoskeletal dynamics and promotes unidirectional cell guidance.

Many biological and physiological processes depend upon directed migration of cells, which is typically mediated by chemical or physical gradients or by signal relay. Here we show that cells can be guided in a single preferred direction based solely on local asymmetries in nano/microtopography on subcellular scales. These asymmetries can be repeated, and thereby provide directional guidance, over arbitrarily large areas. The direction and strength of the guidance is sensitive to the details of the nano/microtopography, suggesting that this phenomenon plays a context-dependent role in vivo. We demonstrate that appropriate asymmetric nano/microtopography can unidirectionally bias internal actin polymerization waves and that cells move with the same preferred direction as these waves. This phenomenon is observed both for the pseudopod-dominated migration of the amoeboid Dictyostelium discoideum and for the lamellipod-driven migration of human neutrophils. The conservation of this mechanism across cell types and the asymmetric shape of many natural scaffolds suggest that actin-wave-based guidance is important in biology and physiology.

From cellular characteristics to disease diagnosis: uncovering phenotypes with supercells.

Cell heterogeneity and the inherent complexity due to the interplay of multiple molecular processes within the cell pose difficult challenges for current single-cell biology. We introduce an approach that identifies a disease phenotype from multiparameter single-cell measurements, which is based on the concept of "supercell statistics", a single-cell-based averaging procedure followed by a machine learning classification scheme. We are able to assess the optimal tradeoff between the number of single cells averaged and the number of measurements needed to capture phenotypic differences between healthy and diseased patients, as well as between different diseases that are difficult to diagnose otherwise. We apply our approach to two kinds of single-cell datasets, addressing the diagnosis of a premature aging disorder using images of cell nuclei, as well as the phenotypes of two non-infectious uveitides (the ocular manifestations of Behçet's disease and sarcoidosis) based on multicolor flow cytometry. In the former case, one nuclear shape measurement taken over a group of 30 cells is sufficient to classify samples as healthy or diseased, in agreement with usual laboratory practice. In the latter, our method is able to identify a minimal set of 5 markers that accurately predict Behçet's disease and sarcoidosis. This is the first time that a quantitative phenotypic distinction between these two diseases has been achieved. To obtain this clear phenotypic signature, about one hundred CD8(+) T cells need to be measured. Although the molecular markers identified have been reported to be important players in autoimmune disorders, this is the first report pointing out that CD8(+) T cells can be used to distinguish two systemic inflammatory diseases. Beyond these specific cases, the approach proposed here is applicable to datasets generated by other kinds of state-of-the-art and forthcoming single-cell technologies, such as multidimensional mass cytometry, single-cell gene expression, and single-cell full genome sequencing techniques.

Pituitary Adenoma Coexistent with Sellar Clear Cell Meningioma Unattached to the Dura: Case Report and Treatment Considerations.

Collision tumors involving the sella are rare. Intrasellar collision tumors are most commonly composed of a combination of pituitary adenomas and pituitary neuroendocrine tumors; however, collision tumors consisting of a pituitary adenoma and intrasellar meningioma are exceedingly rare. The authors present the case of a 47-year-old man who presented with progressive right eye vision loss. Magnetic resonance imaging showed a large, heterogeneously enhancing sellar mass with suprasellar extension. Using a transcranial approach with a right subfrontal craniotomy, near-total resection of the mass was achieved. Histologic analysis confirmed a diagnosis of a gonadotroph adenoma with concomitant clear cell meningioma (CCM). This patient was discharged with improvement in visual acuity and no signs of diabetes insipidus. Given the indistinguishable radiographic characteristics of pituitary adenoma and CCM, a preoperative diagnosis of a collision tumor was difficult. This case was uniquely challenging since the CCM component lacked the classic dural attachment that is associated with meningiomas on neuroimaging. CCMs are classified as central nervous system (CNS) World Health Organization (WHO) grade 2 tumors and tend to behave more aggressively, therefore warranting close surveillance for signs of tumor recurrence. This is the first case to report a collision tumor consisting of pituitary adenoma and CCM.

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.

Cell shape dynamics: from waves to migration.

We observe and quantify wave-like characteristics of amoeboid migration. Using the amoeba Dictyostelium discoideum, a model system for the study of chemotaxis, we demonstrate that cell shape changes in a wave-like manner. Cells have regions of high boundary curvature that propagate from the leading edge toward the back, usually along alternating sides of the cell. Curvature waves are easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from surfaces or cells that extend over the edge of micro-fabricated cliffs. Without surface contact, curvature waves travel from the leading edge to the back of a cell at -35 µm/min. Non-adherent myosin II null cells do not exhibit these curvature waves. At the leading edge of adherent cells, curvature waves are associated with protrusive activity. Like regions of high curvature, protrusive activity travels along the boundary in a wave-like manner. Upon contact with a surface, the protrusions stop moving relative to the surface, and the boundary shape thus reflects the history of protrusive motion. The wave-like character of protrusions provides a plausible mechanism for the zig-zagging of pseudopods and for the ability of cells both to swim in viscous fluids and to navigate complex three dimensional topography.

Cellular harmonics for the morphology-invariant analysis of molecular organization at the cell surface.

The spatiotemporal organization of membrane-associated molecules is central to the regulation of cellular signals. Powerful new microscopy techniques enable the three-dimensional visualization of localization and activation of these molecules; however, the quantitative interpretation and comparison of molecular organization on the three-dimensional cell surface remains challenging because cells themselves vary greatly in morphology. Here we introduce u-signal3D, a framework to assess the spatial scales of molecular organization at the cell surface in a cell-morphology-invariant manner. We validated the framework by analyzing synthetic signaling patterns painted onto observed cell morphologies, as well as measured distributions of cytoskeletal and signaling molecules. To demonstrate the framework's versatility, we further compared the spatial organization of cell surface signals both within, and between, cell populations, and powered an upstream machine-learning-based analysis of signaling motifs.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals.

Local and global measures of shape dynamics.

The shape and motion of cells can yield significant insights into the internal operation of a cell. We present a simple, yet versatile, framework that provides multiple metrics of cell shape and cell shape dynamics. Analysis of migrating Dictyostelium discoideum cells shows that global and local metrics highlight distinct cellular processes. For example, a global measure of shape shows rhythmic oscillations suggestive of contractions, whereas a local measure of shape shows wave-like dynamics indicative of protrusions. From a local measure of dynamic shape, or boundary motion, we extract the times and locations of protrusions and retractions. We find that protrusions zigzag, while retractions remain roughly stationary along the boundary. We do not observe any temporal relationship between protrusions and retractions. Our analysis framework also provides metrics of the boundary as whole. For example, as the cell speed increases, we find that the cell shape becomes more elongated. We also observe that while extensions and retractions have similar areas, their shapes differ.

Low-Grade Gemistocytic Morphology in H3 G34R-Mutant Gliomas and Concurrent K27M Mutation: Clinicopathologic Findings.

Mutations in histone H3 are key molecular drivers of pediatric and young adult high-grade gliomas. Histone H3 G34R mutations occur in hemispheric high-grade gliomas and H3 K27M mutations occur in aggressive, though histologically diverse, midline gliomas. Here, we report 2 rare cases of histologically low-grade gliomas with gemistocytic morphology and sequencing-confirmed histone H3 G34R mutations. One case is a histologically low-grade gemistocytic astrocytoma with a G34R-mutation in H3F3A. The second case is a histologically low-grade gemistocytic astrocytoma with co-occurring K27M and G34R mutations in HIST1H3B. Review of prior histone H3-mutant gliomas sequenced at our institution shows a divergent clinical and immunohistochemical pattern in the 2 cases. The first case is similar to prior histone H3 G34R-mutant tumors, while the second case most closely resembles prior histone H3 K27M-mutant gliomas. These represent novel cases of sequencing-confirmed histone H3 G34R-mutant gliomas with low-grade histology and add to the known rare cases of G34R-mutant tumors with gemistocytic morphology. Although K27M and G34R mutations are thought to be mutually exclusive, we document combined K27M and G34R mutations in HIST1H3B and present evidence suggesting the K27M-mutation drove tumor phenotype in this dual mutant glioma.

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.

Binary and gray-scale patterning of chemical functionality on polymer films.

We present a facile technique for the gray-scale chemical functionalization of polymer surfaces with high dynamic range. We demonstrate the use of this technique to create amine-functionalized substrates that are used for the patterned binding of fluorophores and the patterned synthesis of peptides. Studies of the behavior of the model organism Dictyostelium discoideum indicate the biocompatibility of the functionalized substrates.

Varicella-Zoster Virus Infection and Osteomyelitis of the Skull.

BACKGROUND: Varicella-zoster virus (VZV) is a common herpesvirus infection that can result in acute varicella/chickenpox, as well as delayed activation in herpes zoster/shingles. Ramsay-Hunt syndrome is a rare presentation of VZV reactivation, involving 1% of cases and resulting in lesion formation along the seventh cranial nerve distribution. We report the first case of a patient who presented with acute calvarial osteomyelitis after VZV reactivation and Propionibacterium acnes suprainfection. CLINICAL PRESENTATION: A 41-year-old man with a history of VZV presented with a 6-month history of chest pain, flulike symptoms, and left-sided headaches. Several concomitant external calvarial lesions were identified, and imaging was concerning for an infectious or neoplastic etiology. The patient underwent surgical debridement, and pathologic samples identified coinfection with P. acnes and VZV. Antibacterial and antiviral therapy resulted in a good outcome. CONCLUSION: Osteomyelitis resulting as a complication of VZV infection is rare, particularly in the calvaria. This is the first reported case of Ramsay-Hunt syndrome-type VZV infection being complicated by osteomyelitis of the calvaria. Our case also demonstrates the diagnosis of VZV osteomyelitis through the use of current pathologic methods.

Preoperative and intraoperative perfusion magnetic resonance imaging in a RELA fusion-positive anaplastic ependymoma: A case report.

BACKGROUND: Ependymomas are rare neuroepithelial tumors thought to arise from radial glial precursor cells lining the walls of the ventricles and central canal of the brain and spinal cord, respectively. Histopathological classification, according to World Health Organization criteria, has only recently defined the RELA-fusion positive ependymoma. These tumors may account for 70% of supratentorial ependymomas in children and represent an aggressive entity distinct from other ependymomas. CASE DESCRIPTION: Here we present the case of a patient with RELA-fusion positive ependymoma of the frontal lobe in whom we used preoperative and intraoperative magnetic resonance (MR) perfusion imaging. In this first demonstrated intraoperative evaluation of MR perfusion in ependymoma, increased peripheral perfusion of the lesion in a ring-like manner with a discrete cutoff around the surgical margin correlated with intraoperative findings of a clear border between the tumor and brain, as well as pathological findings of increased MIB index and hypercellularity-specifically within solid tumor components. An abnormal perfusion pattern also suggested an aggressive lesion, which was later confirmed on pathological analysis. In addition, intraoperative MR perfusion improved detection of tumor tissue in combination with traditional T1-weighted contrast-enhanced methods, which increased extent of resection. CONCLUSIONS: MR perfusion imaging may be a useful method for delineating tumor aggressiveness and borders, which can be prognostic.

Fatal Statin-Induced Rhabdomyolysis by Possible Interaction with Palbociclib.

A 60- to 65-year-old female on prior statin therapy was initiated on palbociclib and fulvestrant for the treatment of metastatic, hormone-receptor positive breast cancer. She subsequently developed sudden progressive muscle weakness that progressed to death within weeks. The patient noticed progressive proximal muscle weakness after two cycles of palbociclib, with no other medication changes in the interim. This rapidly progressed and resulted in death within 7 days of presentation to hospital. There has been one previous report of rhabdmyolysis with palbociclib, occurring in a patient on concomitant statin. In this report, we discuss the possible aetiologies of this progressive rhabdomyolysis including time-dependent inhibition of CYP3A4 or inhibition of hepatic uptake transporters, e.g., OATP1B1.