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Neuromodulation technologies are crucial for investigating neuronal connectivity and brain function. Magnetic neuromodulation offers wireless and remote deep brain stimulations that are lacking in optogenetic- and wired-electrode-based tools. However, due to the limited understanding of working principles and poorly designed magnetic operating systems, earlier magnetic approaches have yet to be utilized. Furthermore, despite its importance in neuroscience research, cell-type-specific magnetic neuromodulation has remained elusive. Here we present a nanomaterials-based magnetogenetic toolbox, in conjunction with Cre-loxP technology, to selectively activate genetically encoded Piezo1 ion channels in targeted neuronal populations via torque generated by the nanomagnetic actuators in vitro and in vivo. We demonstrate this cell-type-targeting magnetic approach for remote and spatiotemporal precise control of deep brain neural activity in multiple behavioural models, such as bidirectional feeding control, long-term neuromodulation for weight control in obese mice and wireless modulation of social behaviours in multiple mice in the same physical space. Our study demonstrates the potential of cell-type-specific magnetogenetics as an effective and reliable research tool for life sciences, especially in wireless, long-term and freely behaving animals.
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Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to understand how extracellular cues are converted into biochemical signals and differentially regulate cellular responses. Using single-cell perturbation tools such as optogenetics, specific biochemical cues can be delivered to selective molecules or cells at any desired location and time. By measuring cellular responses to provided perturbations, investigators have decoded and deconstructed the working mechanisms of a variety of neuroelectric and biochemical signaling processes. However, analogous methods for deciphering the working mechanisms of mechanosensitive signaling by regulating mechanical inputs to cell receptors have remained elusive. To address this unmet need, we have recently developed a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. This tool combines a magnetofluorescent nanoparticle (MFN) actuator, which provides precise spatial and mechanical signals to receptors via target-specific one-to-one interaction, with a micromagnetic tweezers that remotely controls the force exerted on a single nanoparticle. This chapter provides comprehensive experimental protocols of mechanogenetics consisting of four stages: (i) chemical synthesis of MFNs, (ii) bio-conjugation and purification of monovalent MFNs, (iii) establishment of cells with genetically encoded mechanosensitive proteins, and (iv) modular targeting and control of cell-surface receptors in live cells. The entire procedure takes up to 1 week. This mechanogenetic tool can be generalized to study many outstanding questions related to the dynamics of cell signaling and transcriptional control, including the mechanism of mechanically activated receptor.
Assuntos
Nanopartículas , Optogenética , Optogenética/métodos , Nanopartículas/química , Transdução de Sinais , Receptores de Superfície Celular/metabolismo , Fenômenos MagnéticosRESUMO
Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (â¼0.004 µm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (â¼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.
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DNA , Nanotecnologia , DNA/química , Nanotecnologia/métodos , Nanoporos , MagnetismoRESUMO
Measuring cellular and tissue mechanics inside intact living organisms is essential for interrogating the roles of force in physiological and disease processes. Current agents for studying the mechanobiology of intact, living organisms are limited by poor light penetration and material stability. Magnetomotive ultrasound is an emerging modality for real-time in vivo imaging of tissue mechanics. Nonetheless, it has poor sensitivity and spatiotemporal resolution. Here we describe magneto-gas vesicles (MGVs), protein nanostructures based on gas vesicles and magnetic nanoparticles that produce differential ultrasound signals in response to varying mechanical properties of surrounding tissues. These hybrid nanomaterials significantly improve signal strength and detection sensitivity. Furthermore, MGVs enable non-invasive, long-term and quantitative measurements of mechanical properties within three-dimensional tissues and in vivo fibrosis models. Using MGVs as novel contrast agents, we demonstrate their potential for non-invasive imaging of tissue elasticity, offering insights into mechanobiology and its application to disease diagnosis and treatment.
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Nanopartículas , Nanoestruturas , Diagnóstico por Imagem/métodos , Proteínas/química , Acústica , Nanopartículas/químicaRESUMO
Here, we introduce the magneto-mechanical-genetic (MMG)-driven wireless deep brain stimulation (DBS) using magnetic nanostructures for therapeutic benefits in the mouse model of Parkinson's disease (PD). Electrical DBS of the subthalamic nucleus (STN) is an effective therapy for mitigating Parkinson's motor symptoms. However, its broader application is hampered by the requirement for implanted electrodes and the lack of anatomical and cellular specificity. Using the nanoscale magnetic force actuators (m-Torquer), which deliver torque force under rotating magnetic fields to activate pre-encoded Piezo1 ion channels on target neurons, our system enables wireless and STN-specific DBS without implants, addressing key unmet challenges in the DBS field. In both late- and early-stage PD mice, MMG-DBS significantly improved locomotor activity and motor balance by 2-fold compared to untreated PD mice. Moreover, MMG-DBS enabled sustained therapeutic effects. This approach provides a non-invasive and implant-free DBS with cellular targeting capability for the effective treatment of Parkinsonian symptoms.
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Estimulação Encefálica Profunda , Doença de Parkinson , Transtornos Parkinsonianos , Núcleo Subtalâmico , Camundongos , Animais , Doença de Parkinson/genética , Doença de Parkinson/terapia , Transtornos Parkinsonianos/terapia , Núcleo Subtalâmico/fisiologia , Neurônios/fisiologia , Canais IônicosRESUMO
As a new enabling nanotechnology tool for wireless, target-specific, and long-distance stimulation of mechanoreceptors in vivo, here we present a hydrogel magnetomechanical actuator (h-MMA) nanoparticle. To allow both deep-tissue penetration of input signals and efficient force generation, h-MMA integrates a two-step transduction mechanism that converts magnetic anisotropic energy to thermal energy within its magnetic core (i.e., Zn0.4Fe2.6O4 nanoparticle cluster) and then to mechanical energy to induce the surrounding polymer (i.e., pNiPMAm) shell contraction, finally delivering forces to activate targeted mechanoreceptors. We show that h-MMAs enable on-demand modulation of Notch signaling in both fluorescence reporter cell lines and a xenograft mouse model, demonstrating its utility as a powerful in vivo perturbation approach for mechanobiology interrogation in a minimally invasive and untethered manner.
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Hidrogéis , Nanopartículas , Humanos , Animais , Camundongos , Fenômenos MecânicosRESUMO
Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.
Assuntos
Secretases da Proteína Precursora do Amiloide , Secretases da Proteína Precursora do Amiloide/genética , LigantesRESUMO
A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.
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Sistemas CRISPR-Cas/genética , Edição de Genes , Técnicas de Transferência de Genes , Lipopeptídeos/metabolismo , Nanopartículas/química , Ribonucleoproteínas/genética , Células HEK293 , Humanos , Hidrodinâmica , Lipossomos , Nanopartículas/ultraestruturaRESUMO
Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.
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Neoplasias da Mama/patologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Neutrófilos/metabolismo , Microambiente Tumoral , Animais , Carcinogênese/metabolismo , Progressão da Doença , Humanos , Pulmão/patologia , Neoplasias Pulmonares/secundário , Células-Tronco Mesenquimais/citologiaRESUMO
Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.
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Actinas/análise , Caderinas/análise , Corantes Fluorescentes/química , Nanopartículas de Magnetita/química , Nanopartículas/química , Polietilenoglicóis/química , Junções Aderentes/ultraestrutura , Linhagem Celular Tumoral , Microambiente Celular , Química Click , Humanos , Micromanipulação , Imagem ÓpticaRESUMO
Measuring multiple omics profiles from the same single cell opens up the opportunity to decode molecular regulation that underlies intercellular heterogeneity in development and disease. Here, we present co-sequencing of microRNAs and mRNAs in the same single cell using a half-cell genomics approach. This method demonstrates good robustness (~95% success rate) and reproducibility (R2 = 0.93 for both microRNAs and mRNAs), yielding paired half-cell microRNA and mRNA profiles, which we can independently validate. By linking the level of microRNAs to the expression of predicted target mRNAs across 19 single cells that are phenotypically identical, we observe that the predicted targets are significantly anti-correlated with the variation of abundantly expressed microRNAs. This suggests that microRNA expression variability alone may lead to non-genetic cell-to-cell heterogeneity. Genome-scale analysis of paired microRNA-mRNA co-profiles further allows us to derive and validate regulatory relationships of cellular pathways controlling microRNA expression and intercellular variability.
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MicroRNAs/metabolismo , RNA/metabolismo , Regulação da Expressão Gênica , Humanos , Células K562 , Células MCF-7 , RNA/genética , TranscriptomaRESUMO
To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.
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Neoplasias da Mama/imunologia , Carcinoma Ductal de Mama/imunologia , Carcinoma Intraductal não Infiltrante/imunologia , Perfilação da Expressão Gênica/métodos , Linfócitos T/imunologia , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Complexo CD3/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Antígenos Comuns de Leucócito/genética , Receptor ErbB-2/genética , Microambiente TumoralRESUMO
Following uncontrolled proliferation, a subset of primary tumour cells acquires additional traits/mutations to trigger phenotypic changes that enhance migration and are hypothesized to be the initiators of metastasis. This study reveals an adaptive mechanism that harnesses synergistic paracrine signalling via IL-6/8, which is amplified by cell proliferation and cell density, to directly promote cell migration. This effect occurs in metastatic human sarcoma and carcinoma cells- but not in normal or non-metastatic cancer cells-, and likely involves the downstream signalling of WASF3 and Arp2/3. The transcriptional phenotype of high-density cells that emerges due to proliferation resembles that of low-density cells treated with a combination of IL-6/8. Simultaneous inhibition of IL-6/8 receptors decreases the expression of WASF3 and Arp2/3 in a mouse xenograft model and reduces metastasis. This study reveals a potential mechanism that promotes tumour cell migration and infers a strategy to decrease metastatic capacity of tumour cells.
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Carcinoma/patologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Comunicação Parácrina/fisiologia , Sarcoma/patologia , Transdução de Sinais/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Humanos , Interleucina-6/genética , Interleucina-8/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular/métodos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Comunicação Parácrina/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Sarcoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report a Mg alloy Mg-2.2Nd-0.1Zn-0.4Zr (wt.%, denoted as JDBM-2) showing great potential in clinical vascular stent application by integrating the advantages of traditional medical stainless steel and polymer. This alloy exhibits high yield strength and elongation of 276 ± 6 MPa and 34.3 ± 3.4% respectively. The JDBM-2 with a stable degradation surface results in a highly homogeneous degradation mechanism and long-term structural and mechanical durability. In vitro cytotoxicity test of the Mg extract via human vascular endothelial cells (HUVECs) indicates that the corrosion products are well tolerated by the tested cells and potentially negligible toxic effect on arterial vessel walls. This alloy also exhibits compromised foreign body response (FBR) determined by human peripheral blood derived macrophage adhesion, foreign body giant cell (FBGC) formation and inflammatory cytokine and chemokine secretion. Finally, vascular stents manufactured from the JDBM-2 were implanted into rabbits for long-term evaluation. The results confirm excellent tissue compatibility and up to 6-month structural and mechanical integrity of the stent in vivo. Thus, the JDBM-2 stent with up to 6-month structural and mechanical integrity and excellent tissue compatibility represents a major breakthrough in this field and a promising alternative to traditional medical stainless steel and polymer for the clinical application.
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Ligas/química , Materiais Biocompatíveis/química , Magnésio/química , Stents , Angiografia , Animais , Sobrevivência Celular , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Teste de Materiais , Fenômenos Mecânicos , Coelhos , Propriedades de SuperfícieRESUMO
Single-cell analysis of cytokine production is increasingly recognized as an important method to understand the inflammatory microenvironment and hematopoietic disease state. Certain cytokines are critical to the regulation of lineage specification, and the aberrant production of these cytokines can contribute to lineage reprogramming. Here, we describe of a platform combining subnanoliter microchambers and a high-density antibody barcode array for the study of single-cell cytokine secretions in hematopoietic cancer cell populations.
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Citocinas/metabolismo , Neoplasias Hematológicas/imunologia , Análise de Célula Única/métodos , Citometria de Fluxo , Humanos , Imunofenotipagem , Análise Serial de TecidosRESUMO
Inorganic nanowires are among the most attractive functional materials, which have emerged in the past two decades. They have demonstrated applications in information technology and energy conversion, but their utility in biological or biomedical research remains relatively under-explored. Although nanowire-based sensors have been frequently reported for biomolecular detection, interfacing nanowire arrays and living mammalian cells for the direct analysis of cellular functions is a very recent endeavor. Cell-penetrating nanowires enabled effective delivery of biomolecules, electrical and optical stimulation and recording of intracellular signals over a long period of time. Non-penetrating, high-density nanowire arrays display rich interactions between the nanostructured substrate and the micro/nanoscale features of cell surfaces. Such interactions enable efficient capture of rare cells including circulating tumor cells and trafficking leukocytes from complex biospecimens. It also serves as a platform for probing cell traction force and neuronal guidance. The most recent advances in the field that exploits nanowire arrays (both penetrating and non-penetrating) to perform rapid analysis of cellular functions potentially for disease diagnosis and monitoring are reviewed.
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Fenômenos Fisiológicos Celulares , Células Eucarióticas/fisiologia , Análise em Microsséries , Nanofios/química , Animais , Adesão Celular , Comunicação Celular/fisiologia , Humanos , Propriedades de Superfície , Alicerces Teciduais/químicaRESUMO
Magnesium (Mg) alloys have revolutionized the application of temporary load-bearing implants as they meet both engineering and medical requirements. However, rapid degradation of Mg alloys under physiological conditions remains the major obstacle hindering the wider use of Mg-based implants. Here we developed a simple method of preparing a nanoscale MgF2 film on Mg-Nd-Zn-Zr (denoted as JDBM) alloy, aiming to reduce the corrosion rate as well as improve the biological response. The corrosion rate of JDBM alloy exposed to artificial plasma is reduced by â¼20% from 0.337 ± 0.021 to 0.269 ± 0.043 mm·y(-1) due to the protective effect of the MgF2 film with a uniform and dense physical structure. The in vitro cytocompatibility test of MgF2-coated JDBM using human umbilical vein endothelial cells indicates enhanced viability, growth, and proliferation as compared to the naked substrate, and the MgF2 film with a nanoscale flakelike feature of â¼200-300 nm presents a much more favorable environment for endothelial cell adhesion, proliferation, and alignment. Furthermore, the animal experiment via implantation of MgF2-coated JDBM stent to rabbit abdominal aorta confirms excellent tissue compatibility of the well re-endothelialized stent with no sign of thrombogenesis and restenosis in the stented vessel.
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Ligas/química , Materiais Biocompatíveis/química , Fluoretos/química , Compostos de Magnésio/química , Stents , Ligas/uso terapêutico , Ligas/toxicidade , Animais , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/patologia , Materiais Biocompatíveis/uso terapêutico , Materiais Biocompatíveis/toxicidade , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas Eletroquímicas , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia de Fluorescência , Próteses e Implantes , Coelhos , Propriedades de Superfície , UltrassonografiaRESUMO
UNLABELLED: The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) has led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis and improves overall survival; however, the mechanism by which JAK inhibitors achieve efficacy has not been delineated. Patients with MPN present with increased levels of circulating proinflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single-cell profiling demonstrated that hematopoietic cells from myelofibrosis models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. In contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and nonmalignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations. SIGNIFICANCE: Our results demonstrate that JAK-STAT3-mediated cytokine production from malignant and nonmalignant cells contributes to MPN pathogenesis and that JAK inhibition in both populations is required for therapeutic efficacy. These findings provide novel insight into the mechanisms by which JAK kinase inhibition achieves therapeutic efficacy in MPNs.
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Transformação Celular Neoplásica/metabolismo , Janus Quinases/metabolismo , Transtornos Mieloproliferativos/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Humanos , Mediadores da Inflamação/metabolismo , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Janus Quinases/genética , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Knockout , Mutação , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição STAT/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of the CSF from patients.