Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

Collagen linearization is a hallmark of aggressive tumors and a key pathogenic event that promotes cancer cell invasion and metastasis. Cell-generated mechanical tension has been proposed to contribute to collagen linearization in tumors, but it is unknown whether other mechanisms play prominent roles in this process. Here, we show that the secretome of cancer cells is by itself able to induce collagen linearization independently of cell-generated mechanical forces. Among the tumor cell-secreted factors, we find a key role in this process for the matricellular protein WISP1 (CCN4). Specifically, WISP1 directly binds to type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors. Consequently, WISP1-induced type I collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis, without significantly affecting gene expression. Furthermore, higher WISP1 expression in tumors from cancer patients correlates with faster progression to metastatic disease and poor prognosis. Altogether, these findings reveal a conceptually novel mechanism whereby pro-metastatic collagen linearization critically depends on a cancer cell-secreted factor.

Circadian gene variants and the skeletal muscle circadian clock contribute to the evolutionary divergence in longevity across Drosophila populations.

Organisms use endogenous clocks to adapt to the rhythmicity of the environment and to synchronize social activities. Although the circadian cycle is implicated in aging, it is unknown whether natural variation in its function contributes to differences in lifespan between populations and whether the circadian clock of specific tissues is key for longevity. We have sequenced the genomes of Drosophila melanogaster strains with exceptional longevity that were obtained via multiple rounds of selection from a parental strain. Comparison of genomic, transcriptomic, and proteomic data revealed that changes in gene expression due to intergenic polymorphisms are associated with longevity and preservation of skeletal muscle function with aging in these strains. Analysis of transcription factors differentially modulated in long-lived versus parental strains indicates a possible role of circadian clock core components. Specifically, there is higher period and timeless and lower cycle expression in the muscle of strains with delayed aging compared to the parental strain. These changes in the levels of circadian clock transcription factors lead to changes in the muscle circadian transcriptome, which includes genes involved in metabolism, proteolysis, and xenobiotic detoxification. Moreover, a skeletal muscle-specific increase in timeless expression extends lifespan and recapitulates some of the transcriptional and circadian changes that differentiate the long-lived from the parental strains. Altogether, these findings indicate that the muscle circadian clock is important for longevity and that circadian gene variants contribute to the evolutionary divergence in longevity across populations.

Development and characterization of a human orthotopic neuroblastoma xenograft.

Neuroblastoma is a pediatric cancer of the developing sympathoadrenal lineage. The tumors are known to develop from the adrenal gland or paraspinal ganglia and have molecular and cellular features of sympathetic neurons such as dense core vesicles and catecholamine production. Here we present the detailed molecular, cellular, genetic and epigenetic characterization of an orthotopic xenograft derived from a high-risk stage 4 neuroblastoma patient. Overall, the xenografted tumor retained the high risk features of the primary tumor and showed aggressive growth and metastasis in the mouse. Also, the genome was preserved with no additional copy number variations, structural variations or aneuploidy. There were 13 missense mutations identified in the xenograft that were not present in the patient's primary tumor and there were no new nonsense mutations. None of the missense mutations acquired in the xenograft were in known cancer genes. We also demonstrate the feasibility of using the orthotopic neuroblastoma xenograft to test standard of care chemotherapy and molecular targeted therapeutics. Finally, we optimized a new approach to produce primary cultures of the neuroblastoma xenografts for high-throughput drug screening which can be used to test new combinations of therapeutic agents for neuroblastoma.

The cell behavior ontology: describing the intrinsic biological behaviors of real and model cells seen as active agents.

MOTIVATION: Currently, there are no ontologies capable of describing both the spatial organization of groups of cells and the behaviors of those cells. The lack of a formalized method for describing the spatiality and intrinsic biological behaviors of cells makes it difficult to adequately describe cells, tissues and organs as spatial objects in living tissues, in vitro assays and in computational models of tissues. RESULTS: We have developed an OWL-2 ontology to describe the intrinsic physical and biological characteristics of cells and tissues. The Cell Behavior Ontology (CBO) provides a basis for describing the spatial and observable behaviors of cells and extracellular components suitable for describing in vivo, in vitro and in silico multicell systems. Using the CBO, a modeler can create a meta-model of a simulation of a biological model and link that meta-model to experiment or simulation results. Annotation of a multicell model and its computational representation, using the CBO, makes the statement of the underlying biology explicit. The formal representation of such biological abstraction facilitates the validation, falsification, discovery, sharing and reuse of both models and experimental data. AVAILABILITY AND IMPLEMENTATION: The CBO, developed using Protégé 4, is available at http://cbo.biocomplexity.indiana.edu/cbo/ and at BioPortal (http://bioportal.bioontology.org/ontologies/CBO).

3D quantitative analyses of angiogenic sprout growth dynamics.

BACKGROUND: Zebrafish intersegmental vessel (ISV) growth is widely used to study angiogenesis and to screen drugs and toxins that perturb angiogenesis. Most current ISV growth assays observe the presence or absence of ISVs or perturbation of ISV morphology but do not measure growth dynamics. We have developed a four-dimensional (4D, space plus time) quantitative analysis of angiogenic sprout growth dynamics for characterization of both normal and perturbed growth. RESULTS: We tracked the positions of the ISV base and tip for each ISV sprout in 4D. Despite immobilization, zebrafish embryos translocated globally and non-uniformly during development. We used displacement of the ISV base and the angle between the ISV and the dorsal aorta to correct for displacement and rotation during development. From corrected tip cell coordinates, we computed average ISV trajectories. We fitted a quadratic curve to the average ISV trajectories to produce a canonical ISV trajectory for each experimental group, arsenic treated and untreated. From the canonical ISV trajectories, we computed curvature, average directed migration speed and directionality. Canonical trajectories from treated (arsenic exposed) and untreated groups differed in curvature, average directed migration speed and angle between the ISV and dorsal aorta. CONCLUSIONS: 4D analysis of angiogenic sprout growth dynamics: (1) Allows quantitative assessment of ISV growth dynamics and perturbation, and (2) provides critical inputs for computational models of angiogenesis.

Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella.

Sorting maturing neurons into distinct layers is critical for brain development, with disruptions leading to neurological disorders and pediatric cancers. Lamination coordinates where, when, and how cells interact, facilitating events that direct migrating neurons to their destined positions within emerging neural networks and control the wiring of connections in functional circuits. While the role of adhesion molecule expression and presentation in driving adhesive recognition during neuronal migration along glial fibers is recognized, the mechanisms by which the spatial arrangement of these molecules on the cell surface dictates adhesive specificity and translates contact-based external cues into intracellular responses like polarization and cytoskeletal organization remain largely unexplored. We used the cerebellar granule neuron (CGN) system to demonstrate that JAM-C receptor cis-binding on the same cell and trans-binding to neighboring cells controls the recruitment of the Pard3 polarity protein and drebrin microtubule-actin crosslinker at CGN to glial adhesion sites, complementing previous studies that showed Pard3 controls JAM-C exocytic surface presentation. Leveraging advanced imaging techniques, specific probes for cell recognition, and analytical methods to dissect adhesion dynamics, our findings reveal: 1) JAM-C cis or trans mutants result in reduced adhesion formation between CGNs and cerebellar glia, 2) these mutants exhibit delayed recruitment of Pard3 at the adhesion sites, and 3) CGNs with JAM-C mutations experience postponed sorting and entry into the cerebellar molecular layer (ML). By developing a conditional system to image adhesion components from two different cells simultaneously, we made it possible to investigate the dynamics of cell recognition on both sides of neuron-glial contacts and the subsequent recruitment of proteins required for CGN migration. This system and an approach that calculates local correlation based on convolution kernels at the cell adhesions site revealed that CGN to CGN JAM recognition preferentially recruits higher levels of Pard3 and drebrin than CGN to glia JAM recognition. The long latency time of CGNs in the inner external germinal layer (EGL) can be attributed to the combined strength of CGN-CGN contacts and the less efficient Pard3 recruitment by CGN-BG contacts, acting as gatekeepers to ML entry. As CGNs eventually transition to glia binding for radial migration, our research demonstrates that establishing permissive JAM-recognition sites on glia via cis and trans interactions of CGN JAM-C serves as a critical temporal checkpoint for sorting at the EGL to ML boundary. This mechanism integrates intrinsic and extrinsic cellular signals, facilitating heterotypic cell sorting into the ML and dictating the precise spatial organization within the cerebellar architecture.

A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

Adhesion failures determine the pattern of choroidal neovascularization in the eye: a computer simulation study.

Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression.

Progressive development of melanoma-induced cachexia differentially impacts organ systems in mice.

Cachexia is a systemic wasting syndrome that increases cancer-associated mortality. How cachexia progressively and differentially impacts distinct tissues is largely unknown. Here, we find that the heart and skeletal muscle undergo wasting at early stages and are the tissues transcriptionally most impacted by cachexia. We also identify general and organ-specific transcriptional changes that indicate functional derangement by cachexia even in tissues that do not undergo wasting, such as the brain. Secreted factors constitute a top category of cancer-regulated genes in host tissues, and these changes include upregulation of the angiotensin-converting enzyme (ACE). ACE inhibition with the drug lisinopril improves muscle force and partially impedes cachexia-induced transcriptional changes, although wasting is not prevented, suggesting that cancer-induced host-secreted factors can regulate tissue function during cachexia. Altogether, by defining prevalent and temporal and tissue-specific responses to cachexia, this resource highlights biomarkers and possible targets for general and tissue-tailored anti-cachexia therapies.

Modulation of protease expression by the transcription factor Ptx1/PITX regulates protein quality control during aging.

Protein quality control is important for healthy aging and is dysregulated in age-related diseases. The autophagy-lysosome and ubiquitin-proteasome are key for proteostasis, but it remains largely unknown whether other proteolytic systems also contribute to maintain proteostasis during aging. Here, we find that expression of proteolytic enzymes (proteases/peptidases) distinct from the autophagy-lysosome and ubiquitin-proteasome systems declines during skeletal muscle aging in Drosophila. Age-dependent protease downregulation undermines proteostasis, as demonstrated by the increase in detergent-insoluble poly-ubiquitinated proteins and pathogenic huntingtin-polyQ levels in response to protease knockdown. Computational analyses identify the transcription factor Ptx1 (homologous to human PITX1/2/3) as a regulator of protease expression. Consistent with this model, Ptx1 protein levels increase with aging, and Ptx1 RNAi counteracts the age-associated downregulation of protease expression. Moreover, Ptx1 RNAi improves muscle protein quality control in a protease-dependent manner and extends lifespan. These findings indicate that proteases and their transcriptional modulator Ptx1 ensure proteostasis during aging.

Platelet-derived chemokines promote skeletal muscle regeneration by guiding neutrophil recruitment to injured muscles.

Skeletal muscle regeneration involves coordinated interactions between different cell types. Injection of platelet-rich plasma is circumstantially considered an aid to muscle repair but whether platelets promote regeneration beyond their role in hemostasis remains unexplored. Here, we find that signaling via platelet-released chemokines is an early event necessary for muscle repair in mice. Platelet depletion reduces the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7/PPBP. Consequently, early-phase neutrophil infiltration to injured muscles is impaired whereas later inflammation is exacerbated. Consistent with this model, neutrophil infiltration to injured muscles is compromised in male mice with Cxcl7-knockout platelets. Moreover, neo-angiogenesis and the re-establishment of myofiber size and muscle strength occurs optimally in control mice post-injury but not in Cxcl7ko mice and in neutrophil-depleted mice. Altogether, these findings indicate that platelet-secreted CXCL7 promotes regeneration by recruiting neutrophils to injured muscles, and that this signaling axis could be utilized therapeutically to boost muscle regeneration.

Latent Epigenetic Programs in Müller Glia Contribute to Stress, Injury, and Disease Response in the Retina.

Previous studies have demonstrated the dynamic changes in chromatin structure during retinal development that correlate with changes in gene expression. However, a major limitation of those prior studies was the lack of cellular resolution. Here, we integrate single-cell (sc) RNA-seq and scATAC-seq with bulk retinal data sets to identify cell type-specific changes in the chromatin structure during development. Although most genes' promoter activity is strongly correlated with chromatin accessibility, we discovered several hundred genes that were transcriptionally silent but had accessible chromatin at their promoters. Most of those silent/accessible gene promoters were in the Müller glial cells. The Müller cells are radial glia of the retina and perform a variety of essential functions to maintain retinal homeostasis and respond to stress, injury, or disease. The silent/accessible genes in Müller glia are enriched in pathways related to inflammation, angiogenesis, and other types of cell-cell signaling and were rapidly activated when we tested 15 different physiologically relevant conditions to mimic retinal stress, injury, or disease in human and murine retinae. We refer to these as "pliancy genes" because they allow the Müller glia to rapidly change their gene expression and cellular state in response to different types of retinal insults. The Müller glial cell pliancy program is established during development, and we demonstrate that pliancy genes are necessary and sufficient for regulating inflammation in the murine retina in vivo. In zebrafish, Müller glia can de-differentiate and form retinal progenitor cells that replace lost neurons. The pro-inflammatory pliancy gene cascade is not activated in zebrafish Müller glia following injury, and we propose a model in which species-specific pliancy programs underly the differential response to retinal damage in species that can regenerate retinal neurons (zebrafish) versus those that cannot (humans and mice).

An adaptive stress response that confers cellular resilience to decreased ubiquitination.

Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis.

Collagen remodeling contributes to many physiologic and pathologic processes. In primary tumors, the linearization of collagen fibers promotes cancer cell invasion and metastasis and is indicative of poor prognosis. However, it remains unknown whether there are endogenous inhibitors of collagen linearization that could be exploited therapeutically. Here, we show that collagen linearization is controlled by two secreted matricellular proteins with antagonistic functions. Specifically, WISP1 was secreted by cancer cells, bound to type I collagen (Col I), and linearized Col I via its cysteine-rich C-terminal (CT) domain. In contrast, WISP2, which lacks a CT domain, inhibited Col I linearization by preventing WISP1-Col I binding. Analysis of patient data revealed that WISP2 expression is lower in most solid tumors, in comparison with normal tissues. Consequently, genetic or pharmacologic restoration of higher WISP2 levels impaired collagen linearization and prevented tumor cell invasion and metastasis in vivo in models of human and murine breast cancer. Thus, this study uncovers WISP2 as the first inhibitor of collagen linearization ever identified and reveals that collagen architecture can be normalized and metastasis inhibited by therapeutically restoring a high WISP2:WISP1 ratio. SIGNIFICANCE: Two secreted factors, WISP1 and WISP2, antagonistically regulate collagen linearization, and therapeutically increasing the WISP2:WISP1 ratio in tumors limits collagen linearization and inhibits metastasis.See related commentary by Barcus and Longmore, p. 5611.

Oxygen Tension and the VHL-Hif1α Pathway Determine Onset of Neuronal Polarization and Cerebellar Germinal Zone Exit.

Postnatal brain circuit assembly is driven by temporally regulated intrinsic and cell-extrinsic cues that organize neurogenesis, migration, and axo-dendritic specification in post-mitotic neurons. While cell polarity is an intrinsic organizer of morphogenic events, environmental cues in the germinal zone (GZ) instructing neuron polarization and their coupling during postnatal development are unclear. We report that oxygen tension, which rises at birth, and the von Hippel-Lindau (VHL)-hypoxia-inducible factor 1α (Hif1α) pathway regulate polarization and maturation of post-mitotic cerebellar granule neurons (CGNs). At early postnatal stages with low GZ vascularization, Hif1α restrains CGN-progenitor cell-cycle exit. Unexpectedly, cell-intrinsic VHL-Hif1α pathway activation also delays the timing of CGN differentiation, germinal zone exit, and migration initiation through transcriptional repression of the partitioning-defective (Pard) complex. As vascularization proceeds, these inhibitory mechanisms are downregulated, implicating increasing oxygen tension as a critical switch for neuronal polarization and cerebellar GZ exit.

Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

Contact-inhibited chemotaxis in de novo and sprouting blood-vessel growth.

Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier-Graner-Hogeweg model (also called Cellular Potts Model) simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller-Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally observed VE-cadherin-mediated contact inhibition of chemotaxis in the simulation causes randomly distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface-normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. Both mechanisms would also apply if force transmission through the extracellular matrix rather than chemical signaling mediated cell-cell interactions. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.

Interleukin-15 Enhances Anti-GD2 Antibody-Mediated Cytotoxicity in an Orthotopic PDX Model of Neuroblastoma.

PURPOSE: Immunotherapy with IL2, GM-CSF, and an anti-disialoganglioside (GD2) antibody significantly increases event-free survival in children with high-risk neuroblastoma. However, therapy failure in one third of these patients and IL2-related toxicities pose a major challenge. We compared the immunoadjuvant effects of IL15 with those of IL2 for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) in neuroblastoma. EXPERIMENTAL DESIGN: We tested ADCC against neuroblastoma patient-derived xenografts (PDX) in vitro and in vivo and examined the functional and migratory properties of NK cells activated with IL2 and IL15. RESULTS: In cell culture, IL15-activated NK cells induced higher ADCC against two GD+ neuroblastoma PDXs than did IL2-activated NK cells (P < 0.001). This effect was dose-dependent (P < 0.001) and was maintained across several effector-to-tumor ratios. As compared with IL2, IL15 also improved chemotaxis of NK cells, leading to higher numbers of tumorsphere-infiltrating NK cells in vitro (P = 0.002). In an orthotopic PDX model, animals receiving chemoimmunotherapy with an anti-GD2 antibody, GM-CSF, and a soluble IL15/IL15Rα complex had greater tumor regression than did those receiving chemotherapy alone (P = 0.012) or combined with anti-GD2 antibody and GM-CSF with (P = 0.016) or without IL2 (P = 0.035). This was most likely due to lower numbers of immature tumor-infiltrating NK cells (DX5+CD27+) after IL15/IL15Rα administration (P = 0.029) and transcriptional upregulation of Gzmd. CONCLUSIONS: The substitution of IL15 for IL2 leads to significant tumor regression in vitro and in vivo and supports clinical testing of IL15 for immunotherapy in pediatric neuroblastoma.