Multiplex Profiling of Biomarker and Drug Uptake in Single Cells Using Microfluidic Flow Cytometry and Mass Spectrometry.

To perform multiplex profiling of single cells and eliminate the risk of potential sample loss caused by centrifugation, we developed a microfluidic flow cytometry and mass spectrometry system (µCytoMS) to evaluate the drug uptake and induced protein expression at the single cell level. It involves a microfluidic chip for the alignment and purification of single cells followed by detection with laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). Biofunctionalized nanoprobes (BioNPs), conjugating ∼3000 6-FAM-Sgc8 aptamers on a single gold nanoparticle (AuNP) (Kd = 0.23 nM), were engineered to selectively bind with protein tyrosine kinase 7 (PTK7) on target cells. PTK7 expression induced by oxaliplatin (OXA) uptake was assayed with LIF, while ICP-MS measurement of 195Pt revealed OXA uptake of the drug in individual cells, which provided further in-depth information about the drug in relation to PTK7 expression. At an ultralow flow of ∼0.043 dyn/cm2 (20 µL/min), the chip facilitates the extremely fast focusing of BioNPs labeled single cells without the need for centrifugal purification. It ensures multiplex profiling of single cells at a throughput speed of 500 cells/min as compared to 40 cells/min in previous studies. Using a machine learning algorithm to initially profile drug uptake and marker expression in tumor cell lines, µCytoMS was able to perform in situ profiling of the PTK7 response to the OXA at single-cell resolution for tests done on clinical samples from 10 breast cancer patients. It offers great potential for multiplex single-cell phenotypic analysis and clinical diagnosis.

Dissecting Gut-Microbial Community Interactions using a Gut Microbiome-on-a-Chip.

While the human gut microbiota has a significant impact on gut health and disease, understanding of the roles of gut microbes, interactions, and collective impact of gut microbes on various aspects of human gut health is limited by the lack of suitable in vitro model system that can accurately replicate gut-like environment and enable the close visualization on causal and mechanistic relationships between microbial constitutents and the gut. , In this study, we present a scalable Gut Microbiome-on-a-Chip (GMoC) with great imaging capability and scalability, providing a physiologically relevant dynamic gut-microbes interfaces. This chip features a reproducible 3D stratified gut epithelium derived from Caco-2 cells (µGut), mimicking key intestinal architecture, functions, and cellular complexity, providing a physiolocially relevant gut environment for microbes residing in the gut. Incorporating tumorigenic bacteria, enterotoxigenic Bacteroides fragilis (ETBF), into the GMoC enable the observation of pathogenic behaviors of ETBF, leading to µGut disruption and pro-tumorigenic signaling activations. Pre-treating the µGut with a beneficial gut microbe Lactobacillus spp., effectively prevent ETBF-mediated gut pathogenesis, preserving the healthy state of the µGut through competition-mediated colonization resistance. The GMoC holds potential as a valuable tool for exploring unknown roles of gut microbes in microbe-induced pathogenesis and microbe-based therapeutic development.

Elastocapillarity-driven 2D nano-switches enable zeptoliter-scale liquid encapsulation.

Biological nanostructures change their shape and function in response to external stimuli, and significant efforts have been made to design artificial biomimicking devices operating on similar principles. In this work we demonstrate a programmable nanofluidic switch, driven by elastocapillarity, and based on nanochannels built from layered two-dimensional nanomaterials possessing atomically smooth surfaces and exceptional mechanical properties. We explore operational modes of the nanoswitch and develop a theoretical framework to explain the phenomenon. By predicting the switching-reversibility phase diagram-based on material, interfacial and wetting properties, as well as the geometry of the nanofluidic circuit-we rationally design switchable nano-capsules capable of enclosing zeptoliter volumes of liquid, as small as the volumes enclosed in viruses. The nanoswitch will find useful application as an active element in integrated nanofluidic circuitry and could be used to explore nanoconfined chemistry and biochemistry, or be incorporated into shape-programmable materials.

Enhancing single-cell encapsulation in droplet microfluidics with fine-tunable on-chip sample enrichment.

Single-cell encapsulation in droplet microfluidics is commonly hindered by the tradeoff between cell suspension density and on-chip focusing performance. In this study, we introduce a novel droplet microfluidic chip to overcome this challenge. The chip comprises a double spiral focusing unit, a flow resistance-based sample enrichment module with fine-tunable outlets, and a crossflow droplet generation unit. Utilizing a low-density cell/bead suspension (2 × 106 objects/mL), cells/beads are focused into a near-equidistant linear arrangement within the double spiral microchannel. The excess water phase is diverted while cells/beads remain focused and sequentially encapsulated in individual droplets. Focusing performance was assessed through numerical simulations and experiments at three flow rates (40, 60, 80 µL/min), demonstrating successful focusing at 40 and 80 µL/min for beads and cells, respectively. In addition, both simulation and experimental results revealed that the flow resistance at the sample enrichment module is adjustable by punching different outlets, allowing over 50% of the aqueous phase to be removed. YOLOv8n-based droplet detection algorithms realized the counting of cells/beads in droplets, statistically demonstrating single-cell and bead encapsulation rates of 72.2% and 79.2%, respectively. All the results indicate that this on-chip sample enrichment approach can be further developed and employed as a critical component in single-cell encapsulation in water-in-oil droplets.

Passive microfluidic devices for cell separation.

The separation of specific cell populations is instrumental in gaining insights into cellular processes, elucidating disease mechanisms, and advancing applications in tissue engineering, regenerative medicine, diagnostics, and cell therapies. Microfluidic methods for cell separation have propelled the field forward, benefitting from miniaturization, advanced fabrication technologies, a profound understanding of fluid dynamics governing particle separation mechanisms, and a surge in interdisciplinary investigations focused on diverse applications. Cell separation methodologies can be categorized according to their underlying separation mechanisms. Passive microfluidic separation systems rely on channel structures and fluidic rheology, obviating the necessity for external force fields to facilitate label-free cell separation. These passive approaches offer a compelling combination of cost-effectiveness and scalability when compared to active methods that depend on external fields to manipulate cells. This review delves into the extensive utilization of passive microfluidic techniques for cell separation, encompassing various strategies such as filtration, sedimentation, adhesion-based techniques, pinched flow fractionation (PFF), deterministic lateral displacement (DLD), inertial microfluidics, hydrophoresis, viscoelastic microfluidics, and hybrid microfluidics. Besides, the review provides an in-depth discussion concerning cell types, separation markers, and the commercialization of these technologies. Subsequently, it outlines the current challenges faced in the field and presents a forward-looking perspective on potential future developments. This work hopes to aid in facilitating the dissemination of knowledge in cell separation, guiding future research, and informing practical applications across diverse scientific disciplines.

Mechano-Activated Cell Therapy for Accelerated Diabetic Wound Healing.

Chronic diabetic wounds are a significant global healthcare challenge. Current strategies, such as biomaterials, cell therapies, and medical devices, however, only target a few pathological features and have limited efficacy. A powerful platform technology combining magneto-responsive hydrogel, cells, and wireless magneto-induced dynamic mechanical stimulation (MDMS) is developed to accelerate diabetic wound healing. The hydrogel encapsulates U.S. Food and Drug Administration (FDA)-approved fibroblasts and keratinocytes to achieve ∼3-fold better wound closure in a diabetic mouse model. MDMS acts as a nongenetic mechano-rheostat to activate fibroblasts, resulting in ∼240% better proliferation, ∼220% more collagen deposition, and improved keratinocyte paracrine profiles via the Ras/MEK/ERK pathway to boost angiogenesis. The magneto-responsive property also enables on-demand insulin release for spatiotemporal glucose regulation through increasing network deformation and interstitial flow. By mining scRNAseq data, a mechanosensitive fibroblast subpopulation is identified that can be mechanically tuned for enhanced proliferation and collagen production, maximizing therapeutic impact. The "all-in-one" system addresses major pathological factors associated with diabetic wounds in a single platform, with potential applications for other challenging wound types.

Encapsulation of Human Spinal Cord Progenitor Cells in Hyaluronan-Gelatin Hydrogel for Spinal Cord Injury Treatment.

Transplanting human induced pluripotent stem cells (iPSCs)-derived spinal cord progenitor cells (SCPCs) is a promising approach to treat spinal cord injuries. However, stem cell therapies face challenges in cell survival, cell localization to the targeted site, and the control of cell differentiation. Here, we encapsulated SCPCs in thiol-modified hyaluronan-gelatin hydrogels and optimized scaffold mechanical properties and cell encapsulation density to promote cell viability and neuronal differentiation in vitro and in vivo. Different compositions of hyaluronan-gelatin hydrogels formulated by varying concentrations of poly(ethylene glycol) diacrylate were mechanically characterized by using atomic force microscopy. In vitro SCPC encapsulation study showed higher cell viability and proliferation with lower substrate Young's modulus (200 Pa vs 580 Pa) and cell density. Moreover, the soft hydrogels facilitated a higher degree of neuronal differentiation with extended filament structures in contrast to clumped cellular morphologies obtained in stiff hydrogels (p < 0.01). When transplanted in vivo, the optimized SCPC-encapsulated hydrogels resulted in higher cell survival and localization at the transplanted region as compared to cell delivery without hydrogel encapsulation at 2 weeks postimplantation within the rat spinal cord (p < 0.01). Notably, immunostaining demonstrated that the hydrogel-encapsulated SCPCs differentiated along the neuronal and oligodendroglial lineages in vivo. The lack of pluripotency and proliferation also supported the safety of the SCPC transplantation approach. Overall, the injectable hyaluronan-gelatin hydrogel shows promise in supporting the survival and neural differentiation of human SCPCs after transplantation into the spinal cord.

Modeling periodontal host-microbe interactions using vascularized gingival connective tissue equivalents.

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabeling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly,in silicoanalysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimeticin vitroplatform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.

Recent progress of organ-on-a-chip towards cardiovascular diseases: advanced design, fabrication, and applications.

Cardiovascular diseases (CVDs) are a major cause of death worldwide, leading to increased medical care costs. To turn the scale, it is essential to acquire a more in-depth and comprehensive understanding of CVDs and thus formulate more efficient and reliable treatments. Over the last decade, tremendous effort has been made to develop microfluidic systems to recapitulate native cardiovascular environments because of their unique advantages over conventional 2D culture systems and animal models such as high reproductivity, physiological relevance, and good controllability. These novel microfluidic systems could be extensively adopted for natural organ simulation, disease modeling, drug screening, disease diagnosis and therapy. Here, a brief review of the innovative designs of microfluidic devices for CVDs research is presented, with specific discussions on material selection, critical physiological and physical considerations. In addition, we elaborate on various biomedical applications of these microfluidic systems such as blood-vessel-on-a-chip and heart-on-a-chip, which are conducive to the investigation of the underlying mechanisms of CVDs. This review also provides systematic guidance on the construction of next-generation microfluidic systems for the diagnosis and treatment of CVDs. Finally, the challenges and future directions in this field are highlighted and discussed.

HaptGlove-Untethered Pneumatic Glove for Multimode Haptic Feedback in Reality-Virtuality Continuum.

Novel haptics technologies are urgently needed to bridge the gap between entirely physical world and fully digital environment to render a more realistic and immersive human-computer interaction. Current virtual reality (VR) haptic gloves either deliver limited haptic feedback or are bulky and heavy. The authors develop a haptic glove or HaptGlove, an untethered and lightweight pneumatic glove, that allows users to "physically" interact in a VR environment and enables both kinesthetic and cutaneous sensations naturally and realistically. Integrated with five pairs of haptic feedback modules and fiber sensors, HaptGlove provides variable stiffness force feedback and fingertip force and vibration feedback, allowing users to touch, press, grasp, squeeze, and pull various virtual objects and feel the dynamic haptic changes. Significant improvements in VR realism and immersion are observed in a user study with participants achieving 78.9% accuracy in sorting six virtual balls of different stiffnesses. Importantly, HaptGlove facilitates VR training, education, entertainment, and socialization in a reality-virtuality continuum.

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance.

Cancer cells in secondary tumors are found to form metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, the role of deleterious mechanical stresses in this change of metastatic potential is unclear. Here, by forcing cancer cells to flow through small capillary-sized constrictions, it is demonstrated that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. These results highlight a potential link between the microenvironmental physical stresses and the enhanced malignancy of metastasizing cancer cells which may be utilized as a therapeutic strategy in preventing the metastatic spread of cancer cells.

Dynamic Zero Current Method to Reduce Measurement Error in Low Value Resistive Sensor Array for Wearable Electronics.

One advantage of a resistive sensor array (RSA) with shared rows (M) and shared columns (N) is the reduced number of wires from M × N + 1 to M + N which can greatly lessen the complexity and burden on wearable electronic systems. However, the drawback is the crosstalk current effect between adjacent elements, which will lead to high measurement error. Although several solutions have been reported, they mainly focus on RSAs with high resistance (≥100 Ω). There is a lack of research that addresses RSAs with resistor values below 100 Ω. Here, we introduce a new circuit design named the dynamic zero current method (DZCM) to further decrease the measurement error. From the low value RSA test with ideal resistors, the DZCM exhibits lower error than the zero potential method (ZPM). In the case of the error variation ratio of amplifier offset voltage, the DZCM has a 4%/mV (row) to 7%/mV (column) ratio, while the ZPM has an almost 25%/mV (row) to 45%/mV (column) ratio and it increases with array size.

Mechano-responsive hydrogel for direct stem cell manufacturing to therapy.

Bone marrow-derived mesenchymal stem cell (MSC) is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties. Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers, but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo. To overcome these challenges, in this work, we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy. With our technology, we found that dynamic mechanical stimulation (DMS) enhanced matrix-integrin ß1 interactions which induced MSCs spreading and proliferation. In addition, DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities (e.g., growth factor secretion) through YAP nuclear localization and FAK-ERK pathway. With our magnetic hydrogel, complex procedures from MSC manufacturing to final clinical use, can be integrated into one single platform, and we believe this 'all-in-one' technology could offer a paradigm shift to existing standards in MSC therapy.

Dynamic Magneto-Softening of 3D Hydrogel Reverses Malignant Transformation of Cancer Cells and Enhances Drug Efficacy.

High extracellular matrix stiffness is a prominent feature of malignant tumors associated with poor clinical prognosis. To elucidate mechanistic connections between increased matrix stiffness and tumor progression, a variety of hydrogel scaffolds with dynamic changes in stiffness have been developed. These approaches, however, are not biocompatible at high temperature, strong irradiation, and acidic/basic pH, often lack reversibility (can only stiffen and not soften), and do not allow study on the same cell population longitudinally. In this work, we develop a dynamic 3D magnetic hydrogel whose matrix stiffness can be wirelessly and reversibly stiffened and softened multiple times with different rates of change using an external magnet. With this platform, we found that matrix stiffness increased tumor malignancy including denser cell organization, epithelial-to-mesenchymal transition and hypoxia. More interestingly, these malignant transformations could be halted or reversed with matrix softening (i.e., mechanical rescue), to potentiate drug efficacy attributing to reduced solid stress from matrix and downregulation of cell mechano-transductors including YAP1. We propose that our platform can be used to deepen understanding of the impact of matrix softening on cancer biology, an important but rarely studied phenomenon.

The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

Extracellular matrix mechanobiology in cancer cell migration.

The extracellular matrix (ECM) is pivotal in modulating tumor progression. Besides chemically stimulating tumor cells, it also offers physical support that orchestrates the sequence of events in the metastatic cascade upon dynamically modulating cell mechanosensation. Understanding this translation between matrix biophysical cues and intracellular signaling has led to rapid growth in the interdisciplinary field of cancer mechanobiology in the last decade. Substantial efforts have been made to develop novel in vitro tumor mimicking platforms to visualize and quantify the mechanical forces within the tissue that dictate tumor cell invasion and metastatic growth. This review highlights recent findings on tumor matrix biophysical cues such as fibrillar arrangement, crosslinking density, confinement, rigidity, topography, and non-linear mechanics and their implications on tumor cell behavior. We also emphasize how perturbations in these cues alter cellular mechanisms of mechanotransduction, consequently enhancing malignancy. Finally, we elucidate engineering techniques to individually emulate the mechanical properties of tumors that could help serve as toolkits for developing and testing ECM-targeted therapeutics on novel bioengineered tumor platforms. STATEMENT OF SIGNIFICANCE: Disrupted ECM mechanics is a driving force for transitioning incipient cells to life-threatening malignant variants. Understanding these ECM changes can be crucial as they may aid in developing several efficacious drugs that not only focus on inducing cytotoxic effects but also target specific matrix mechanical cues that support and enhance tumor invasiveness. Designing and implementing an optimal tumor mimic can allow us to predictively map biophysical cue-modulated cell behaviors and facilitate the design of improved lab-grown tumor models with accurately controlled structural features. This review focuses on the abnormal changes within the ECM during tumorigenesis and its implications on tumor cell-matrix mechanoreciprocity. Additionally, it accentuates engineering approaches to produce ECM features of varying levels of complexity which is critical for improving the efficiency of current engineered tumor tissue models.

Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip.

Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva-oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host-microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof-of-principle "gingival crevice-on-chip" microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On-chip co-culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm-like structures at the tissue-microbiome interface, long-term co-culture, and bacterial clearance secondary to simulated GCF (s-GCF) flow. Further, on-chip exposure of the gingival CTEs to the toll-like receptor-2 (TLR-2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s-GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host-microbe interactions in periodontal disease and the development of periodontal therapeutics under near-microphysiological conditions.

Ultrahigh Strain-Insensitive Integrated Hybrid Electronics Using Highly Stretchable Bilayer Liquid Metal Based Conductor.

Human-interfaced electronic systems require strain-resilient circuits. However, present integrated stretchable electronics easily suffer from electrical deterioration and face challenges in forming robust multilayered soft-rigid hybrid configurations. Here, a bilayer liquid-solid conductor (b-LSC) with amphiphilic properties is introduced to reliably interface with both rigid electronics and elastomeric substrates. The top liquid metal can self-solder its interface with rigid electronics at a resistance 30% lower than the traditional tin-soldered rigid interface. The bottom polar composite comprising liquid metal particles and polymers can not only reliably interface with elastomers but also help the b-LSC heal after breakage. The b-LSC can be scalably fabricated by printing and subsequent peeling strategies, showing ultra-high strain-insensitive conductivity (maximum 22 532 S cm-1 ), extreme stretchability (2260%), and negligible resistance change under ultra-high strain (0.34 times increase under 1000% strain). It can act as stretchable vertical interconnect access for connecting multilayered layouts and can be scalably and universally fabricated on various substrates with a resolution of ≈200 µm. It is demonstrated that it can construct stretchable sensor arrays, multi-layered stretchable displays, highly integrated haptic user-interactive optoelectric E-skins, visualized heaters, robot touch sensing systems, and wireless powering for wearable electronics.

The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces.

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order.

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse.

Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints (diagnosis, n=17; post-chemotherapy, n=11; and relapse, n=3), and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes via NanoString, 13 of 17 samples contained CTCs displaying cytologic atypia, TH and PHOX2B expression and/or upregulation of cancer-associated genes. Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state (OLFML2B, STAT1, ARHGDIB, STAB1, TLR2) were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.