RESUMEN
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.
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Astrocitos , Comunicación Celular , Perfilación de la Expresión Génica , Memoria a Largo Plazo , Neuronas , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/fisiología , Complejo Nuclear Basolateral/citología , Complejo Nuclear Basolateral/metabolismo , Complejo Nuclear Basolateral/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Memoria a Largo Plazo/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Análisis de Secuencia de ARN , Imagen Individual de Molécula , Análisis de Expresión Génica de una Sola Célula , UbiquitinaciónRESUMEN
The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.
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Enfermedad de Alzheimer , Encéfalo , Susceptibilidad a Enfermedades , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/citología , Encéfalo/metabolismo , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Estudio de Asociación del Genoma Completo , Hipocampo/irrigación sanguínea , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones , Microglía/metabolismo , Pericitos/metabolismo , TranscriptomaRESUMEN
The role of gene expression during learning and in short-term memories has been studied extensively1-3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage.
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Regulación de la Expresión Génica , Consolidación de la Memoria , Memoria a Largo Plazo/fisiología , Transcripción Genética , Animales , Astrocitos/metabolismo , Biomarcadores/análisis , Exocitosis , Miedo/fisiología , Masculino , Ratones , Microglía/metabolismo , Neuronas/clasificación , Neuronas/citología , Neuronas/metabolismo , Corteza Prefrontal/citología , Corteza Prefrontal/fisiología , Análisis de la Célula Individual , Factores de TiempoRESUMEN
The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.
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Envejecimiento/metabolismo , Envejecimiento/patología , Barrera Hematoencefálica/metabolismo , Transcitosis , Fosfatasa Alcalina/metabolismo , Animales , Anticuerpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administración & dosificación , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Salud , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Plasma/metabolismo , Proteoma/administración & dosificación , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores de Transferrina/inmunología , Transcripción Genética , Transferrina/metabolismoRESUMEN
Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell-cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil-endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC-PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN-tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor-PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site.
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Quimiocina CXCL1/inmunología , Quimiotaxis/inmunología , Interleucina-8/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Neutrófilos/inmunología , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Quimiotaxis/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Técnicas Analíticas Microfluídicas/métodos , Neoplasias/patología , Neutrófilos/patología , Pez CebraRESUMEN
The formation of gaps in the endothelium is a crucial process underlying both cancer and immune cell extravasation, contributing to the functioning of the immune system during infection, the unfavorable development of chronic inflammation and tumor metastasis. Here, we present a stochastic-mechanical multiscale model of an endothelial cell monolayer and show that the dynamic nature of the endothelium leads to spontaneous gap formation, even without intervention from the transmigrating cells. These gaps preferentially appear at the vertices between three endothelial cells, as opposed to the border between two cells. We quantify the frequency and lifetime of these gaps, and validate our predictions experimentally. Interestingly, we find experimentally that cancer cells also preferentially extravasate at vertices, even when they first arrest on borders. This suggests that extravasating cells, rather than initially signaling to the endothelium, might exploit the autonomously forming gaps in the endothelium to initiate transmigration.
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Células Endoteliales/metabolismo , Endotelio Vascular/patología , Uniones Comunicantes/patología , Fenómenos Biomecánicos , Adhesión Celular , Movimiento Celular/fisiología , Células Endoteliales/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Neoplasias/patología , Migración Transendotelial y TransepitelialRESUMEN
Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types.
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Metástasis de la Neoplasia , Neoplasias/patología , Fenómenos Biomecánicos , Técnicas de Cultivo de Célula , Humanos , Microambiente TumoralRESUMEN
It is now evident that the cell nucleus undergoes dramatic shape changes during important cellular processes such as cell transmigration through extracellular matrix and endothelium. Recent experimental data suggest that during cell transmigration the deformability of the nucleus could be a limiting factor, and the morphological and structural alterations that the nucleus encounters can perturb genomic organization that in turn influences cellular behavior. Despite its importance, a biophysical model that connects the experimentally observed nuclear morphological changes to the underlying biophysical factors during transmigration through small constrictions is still lacking. Here, we developed a universal chemomechanical model that describes nuclear strains and shapes and predicts thresholds for the rupture of the nuclear envelope and for nuclear plastic deformation during transmigration through small constrictions. The model includes actin contraction and cytosolic back pressure that squeeze the nucleus through constrictions and overcome the mechanical resistance from deformation of the nucleus and the constrictions. The nucleus is treated as an elastic shell encompassing a poroelastic material representing the nuclear envelope and inner nucleoplasm, respectively. Tuning the chemomechanical parameters of different components such as cell contractility and nuclear and matrix stiffnesses, our model predicts the lower bounds of constriction size for successful transmigration. Furthermore, treating the chromatin as a plastic material, our model faithfully reproduced the experimentally observed irreversible nuclear deformations after transmigration in lamin-A/C-deficient cells, whereas the wild-type cells show much less plastic deformation. Along with making testable predictions, which are in accord with our experiments and existing literature, our work provides a realistic framework to assess the biophysical modulators of nuclear deformation during cell transmigration.
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Forma del Núcleo Celular/fisiología , Núcleo Celular/química , Núcleo Celular/fisiología , Modelos Biológicos , Estrés Fisiológico/fisiología , Migración Transendotelial y Transepitelial/fisiología , Actinas/química , Actinas/metabolismo , Animales , Simulación por Computador , Citosol/química , Citosol/metabolismo , Elasticidad , Células Endoteliales/química , Células Endoteliales/fisiología , Dispositivos Laboratorio en un Chip , Microscopía de Fuerza Atómica , Permeabilidad , Presión , Estrés Mecánico , Agua/química , Agua/metabolismoRESUMEN
We report a synthesis approach based on stop-flow lithography (SFL) for fabricating colloidal microparticles with any arbitrary 2D-extruded shape. By modulating the degree of oxygen inhibition during synthesis, we achieved previously unattainable particle sizes. Brownian diffusion of colloidal discs in bulk suggests the out-of-plane dimension can be as small as 0.8 µm, which agrees with confocal microscopy measurements. We measured the hindered diffusion of microdiscs near a solid surface and compared our results to theoretical predictions. These colloidal particles can also flow through physiological microvascular networks formed by endothelial cells undergoing vasculogensis under minimal hydrostatic pressure (â¼5 mm H2O). This versatile platform creates future opportunities for on-chip parametric studies of particle geometry effects on particle passage properties, distribution and cellular interactions.
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Coloides/química , Coloides/síntesis química , Modelos Químicos , Oxígeno/química , Polietilenglicoles/químicaRESUMEN
Purpose: Precision targeting is crucial to successful stereotactic radiosurgery for trigeminal neuralgia (TGN). We investigated the impact of intra-fractional 6-dimensional corrections during frameless image-guided radiosurgery (IGRS) for pain outcome in TGN patients. Materials and methods: A total of 41 sets of intra-fractional corrections from 35 patients with TGN treated by frameless IGRS from 2009 to 2013 were retrospectively studied. For each IGRS, the intra-fractional 6-dimensional shifts were conducted at 6 couch angles. Clinical pain outcome was recorded according the Barrow Neurological Institute (BNI) 5-points score. The relationship in 6-dimensional corrections and absolute translational distances between patients with pain relief score points <2 versus ≥2 were analyzed. Results: The absolute mean lateral, longitudinal, and vertical translational shifts were 0.46 ± 0.15 mm, 0.36 ± 0.16 mm and 0.21 ± 0.08 mm, respectively, with 97% of translational shifts being within 0.7 mm. The absolute mean lateral (pitch), longitudinal (roll), and vertical (yaw) rotational corrections are 0.33 ± 0.24°, 0.18 ± 0.09°, and 0.27 ± 0.15°, respectively, with 97% of rotational corrections being within 0.6°. The median follow-up duration for pain outcome was 26 months after IGRS. The average calculated absolute shift for patients with pain relief <2 and ≥2 BNI points, were 0.228 ± 0.008 mm and 0.259 ± 0.007 mm, respectively. There was no statistically significant difference in the translational shifts, rotational corrections or absolute distances between these two patient groups. Conclusions: Our data demonstrate high spatial targeting accuracy of frameless IGRS for TGN with only nominal intra-fraction 6-dimensional corrections.
RESUMEN
The interplay between genetic transformations, biochemical communications, and physical interactions is crucial in cancer progression. Metastasis, a leading cause of cancer-related deaths, involves a series of steps, including invasion, intravasation, circulation survival, and extravasation. Mechanical alterations, such as changes in stiffness and morphology, play a significant role in all stages of cancer initiation and dissemination. Accordingly, a better understanding of cancer mechanobiology can help in the development of novel therapeutic strategies. Targeting the physical properties of tumours and their microenvironment presents opportunities for intervention. Advancements in imaging techniques and lab-on-a-chip systems enable personalized investigations of tumor biomechanics and drug screening. Investigation of the interplay between genetic, biochemical, and mechanical factors, which is of crucial importance in cancer progression, offers insights for personalized medicine and innovative treatment strategies.
RESUMEN
Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.
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Diferenciación Celular , Proteínas Tirosina Quinasas Receptoras , Células Madre , Animales , Ratones , Envejecimiento , Diferenciación Celular/genética , Senescencia Celular/genética , Intestinos/citología , Intestinos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/metabolismo , Vía de Señalización Wnt , Proteínas Señalizadoras YAPRESUMEN
Brain endothelial cells (BECs) are key constituents of the blood-brain barrier (BBB), protecting the brain from pathogens and restricting access of circulatory factors. Yet, because circulatory proteins have prominent age-related effects on adult neurogenesis, neuroinflammation, and cognitive function in mice, we wondered whether BECs receive and potentially relay signals between the blood and brain. Using single-cell RNA sequencing of hippocampal BECs, we discover that capillary BECs-compared with arterial and venous BECs-undergo the greatest transcriptional changes in normal aging, upregulating innate immunity and oxidative stress response pathways. Short-term infusions of aged plasma into young mice recapitulate key aspects of this aging transcriptome, and remarkably, infusions of young plasma into aged mice exert rejuvenation effects on the capillary transcriptome. Together, these findings suggest that the transcriptional age of BECs is exquisitely sensitive to age-related circulatory cues and pinpoint the BBB itself as a promising therapeutic target to treat brain disease.
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Envejecimiento/fisiología , Circulación Sanguínea/fisiología , Encéfalo/citología , Células Endoteliales/metabolismo , Envejecimiento/sangre , Envejecimiento/genética , Animales , Arterias/fisiología , Capilares/fisiología , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Transcripción Genética/efectos de los fármacos , Transcriptoma/genética , Venas/fisiologíaRESUMEN
Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.
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Plaquetas/patología , Trombosis/patología , Animales , Plaquetas/ultraestructura , Línea Celular Tumoral , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Adhesividad Plaquetaria , Agregación Plaquetaria , Conejos , Receptores de Superficie Celular/metabolismoRESUMEN
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.
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Envejecimiento/sangre , Encéfalo/metabolismo , Células-Madre Neurales/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adolescente , Adulto , Anciano , Envejecimiento/inmunología , Envejecimiento/metabolismo , Animales , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Eliminación de Gen , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Microglía/metabolismo , Células-Madre Neurales/citología , Molécula 1 de Adhesión Celular Vascular/sangre , Molécula 1 de Adhesión Celular Vascular/genética , Adulto JovenRESUMEN
The proto-oncogene c-myb (and corresponding nuclear transcription factor, c-Myb) regulates the proliferation and differentiation of hematologic and vascular smooth muscle cells; however, the role of c-Myb in blood pressure regulation is unknown. Here, we show that mice homozygous for a hypomorphic c-myb allele ( c-myb h/h) conferring reduced c-Myb activity manifest reduced peripheral blood and kidney B220+ B-cells and have decreased systolic (104±2 versus 120±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 83±1 mm Hg; P<0.0001) compared with WT (wild type) mice. Additionally, c-myb h/h mice had lower susceptibility to deoxycorticosterone acetate-salt experimental hypertension. Although cardiac (echocardiography) and resistance artery (perfusion myography) functions were normal, metabolic cage studies revealed that c-myb h/h mice had increased 24-hour urine output and sodium excretion versus WT. Reconstitution of WT mice with c-myb h/h bone marrow transplant and chimeric bone marrow transplant using mice lacking B-cells ( J H T; h/h>WT and h/h:J H T>WT, respectively) decreased blood pressure and increased 24-hour urine output compared with controls ( WT>WT; WT:J H T>WT). J H T mice also had decreased systolic (103±2 versus 115±1 mm Hg; P<0.0001) and diastolic blood pressure (71±2 versus 79±1; P<0.01) and increased 24-hour urine output versus WT. Real-time quantitative reverse transcription polymerase chain reaction of kidney medulla revealed reduced V2R (vasopressin receptor 2) expression in c-myb h/h and J H T mice. These data implicate B-cells in the regulation of V2R and its associated effects on salt and water handling and blood pressure homeostasis.
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Linfocitos B/metabolismo , Presión Sanguínea/fisiología , Hipertensión/inmunología , Miocitos del Músculo Liso/metabolismo , Animales , Linfocitos B/patología , Diferenciación Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/patología , Proteínas Proto-Oncogénicas c-myb/biosíntesis , Proteínas Proto-Oncogénicas c-myb/genética , ARN/genéticaRESUMEN
The epithelial-mesenchymal transition (EMT) endows carcinoma cells with traits needed to complete many of the steps leading to metastasis formation, but its contributions specifically to the late step of extravasation remain understudied. We find that breast cancer cells that have undergone an EMT extravasate more efficiently from blood vessels both in vitro and in vivo. Analysis of gene expression changes associated with the EMT program led to the identification of an EMT-induced cell-surface protein, podocalyxin (PODXL), as a key mediator of extravasation in mesenchymal breast and pancreatic carcinoma cells. PODXL promotes extravasation through direct interaction of its intracellular domain with the cytoskeletal linker protein ezrin. Ezrin proceeds to establish dorsal cortical polarity, enabling the transition of cancer cells from a non-polarized, rounded cell morphology to an invasive extravasation-competent shape. Hence, the EMT program can directly enhance the efficiency of extravasation and subsequent metastasis formation through a PODXL-ezrin signaling axis.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas del Citoesqueleto/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Sialoglicoproteínas/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Xenoinjertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/irrigación sanguínea , Sialoglicoproteínas/genética , Transducción de SeñalRESUMEN
Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing protocol describing a microfluidic platform that offers additional applications. This protocol describes an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4-5 d, after which the tumor can be perfused and extravasation events are easily tracked over 72 h via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells, combined with high-resolution imaging, can be easily achieved because of the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly as compared with those in thick tissues. The ability to generate large numbers of devices (â¼36) per experiment further allows for highly parametric studies, which are required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single-cell extravasation events, allowing both tumor and endothelial morphological dynamics to be observed in high detail with a moderate number of data points.
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Movimiento Celular , Microfluídica/métodos , Microvasos/anatomía & histología , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , HumanosRESUMEN
Tumor integrin ß1 (ITGB1) contributes to primary tumor growth and metastasis, but its specific roles in extravasation have not yet been clearly elucidated. In this study, we engineered a three-dimensional microfluidic model of the human microvasculature to recapitulate the environment wherein extravasation takes place and assess the consequences of ß1 depletion in cancer cells. Combined with confocal imaging, these tools allowed us to decipher the detailed morphology of transmigrating tumor cells and associated endothelial cells in vitro at high spatio-temporal resolution not easily achieved in conventional transmigration assays. Dynamic imaging revealed that ß1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast with control cells. Specifically, adhesion via α3ß1 and α6ß1 to subendothelial laminin was a critical prerequisite for successful transmigration. ß1 was required to invade past the endothelial basement membrane, whereas its attenuation in a syngeneic tumor model resulted in reduced metastatic colonization of the lung, an effect not observed upon depletion of other integrin alpha and beta subunits. Collectively, our findings in this novel model of the extravasation microenvironment revealed a critical requirement for ß1 in several steps of extravasation, providing new insights into the mechanisms underlying metastasis. Cancer Res; 76(9); 2513-24. ©2016 AACR.
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Cadenas beta de Integrinas/metabolismo , Invasividad Neoplásica/patología , Migración Transendotelial y Transepitelial/fisiología , Animales , Western Blotting , Línea Celular , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Técnicas Analíticas Microfluídicas , Microscopía Confocal , Reacción en Cadena de la PolimerasaRESUMEN
In recognition of the enormous potential of immunotherapies against cancer, research into the interactions between tumor and immune cells has accelerated, leading to the recent FDA approval of several drugs that reduce cancer progression. Numerous cellular and molecular interactions have been identified by which immune cells can intervene in the metastatic cascade, leading to the development of several in vivo and in vitro model systems that can recapitulate these processes. Among these, microfluidic technologies hold many advantages in terms of their unique ability to capture the essential features of multiple cell type interactions in three-dimensions while allowing tight control of the microenvironment and real-time monitoring. Here, we review current assays and discuss the development of new microfluidic technologies for immunotherapy.