RESUMO
OBJECTIVE: To test the hypothesis that nailfold capillaroscopy can noninvasively detect dysregulated retinal angiogenesis and predict retinopathy of prematurity (ROP) in infants born premature before its development. METHODS: In a cohort of 32 infants born <33 weeks of gestation, 1386 nailfold capillary network images of the 3 middle fingers of each hand were taken during the first month of life. From these, 25 infants had paired data taken 2 weeks apart during the first month of life. Images were analyzed for metrics of peripheral microvascular density using a machine learning-based segmentation approach and a previously validated microvascular quantification platform (REAVER vascular analysis). Results were correlated with subsequent development of ROP based on a published consensus ROP severity scale. RESULTS: In total, 18 of 32 (56%) (entire cohort) and 13 of 25 (52%) (2-time point subgroup) developed ROP. Peripheral vascular density decreased significantly during the first month of life. In the paired time point analysis, vessel length density, a key metric of peripheral vascular density, was significantly greater at both time points among infants who later developed ROP (15â563 and 11â996 µm/mm2, respectively) compared with infants who did not (12â252 and 8845 µm/mm2, respectively) (P < .001, both time points). A vessel length density cutoff of >15â100 at T1 or at T2 correctly detected 3 of 3 infants requiring ROP therapy. In a mixed-effects linear regression model, peripheral vascular density metrics were significantly correlated with ROP severity. CONCLUSIONS: Nailfold microvascular density assessed during the first month of life is a promising, noninvasive biomarker to identify premature infants at highest risk for ROP before detection on eye exam.
Assuntos
Retinopatia da Prematuridade , Recém-Nascido , Lactente , Humanos , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/terapia , Angioscopia Microscópica , Recém-Nascido Prematuro , Retina , Idade Gestacional , Fatores de RiscoRESUMO
Chronic pelvic pain conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) remain clinical and mechanistic enigmas. Microglia are resident immune cells of the central nervous system (CNS) that respond to changes in the gut microbiome, and studies have linked microglial activation to acute and chronic pain in a variety of models, including pelvic pain. We have previously reported that mice deficient for the lipase acyloxyacyl hydrolase (AOAH) develop pelvic allodynia and exhibit symptoms, comorbidities, and gut dysbiosis mimicking IC/BPS. Here, we assessed the role of AOAH in microglial activation and pelvic pain. RNAseq analyses using the ARCHS4 database and confocal microscopy revealed that AOAH is highly expressed in wild type microglia but at low levels in astrocytes, suggesting a functional role for AOAH in microglia. Pharmacologic ablation of CNS microglia with PLX5622 resulted in decreased pelvic allodynia in AOAH-deficient mice and resurgence of pelvic pain upon drug washout. Skeletal analyses revealed that AOAH-deficient mice have an activated microglia morphology in the medial prefrontal cortex and paraventricular nucleus, brain regions associated with pain modulation. Because microglia express Toll-like receptors and respond to microbial components, we also examine the potential role of dysbiosis in microglial activation. Consistent with our hypothesis of microglia activation by leakage of gut microbes, we observed increased serum endotoxins in AOAH-deficient mice and increased activation of cultured BV2 microglial cells by stool of AOAH-deficient mice. Together, these findings demonstrate a role for AOAH in microglial modulation of pelvic pain and thus identify a novel therapeutic target for IC/BPS.
Assuntos
Cistite Intersticial , Animais , Hidrolases de Éster Carboxílico , Disbiose , Hiperalgesia , Camundongos , Camundongos Endogâmicos C57BL , Microglia , Dor PélvicaRESUMO
Dendritic spinules are fine membranous protrusions of neuronal spines that play a role in synaptic plasticity, but their nanoscale requires resolution beyond conventional confocal microscopy, hindering live studies. Here, we describe how to track individual spinules in live dissociated cortical pyramidal neurons utilizing fluorescence labeling, optimized confocal imaging parameters, and post-acquisition iterative 3D deconvolution, employing NIS Elements software. This approach enables investigations of spinule structural dynamics and function without using super-resolution microscopy, which involves special fluorophores and/or high laser power. For complete details on the use and execution of this protocol, please refer to Zaccard et al. (2020).
Assuntos
Espinhas Dendríticas/fisiologia , Microscopia Confocal/métodos , Células Piramidais/citologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Biochemical methods can reveal stable protein-protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein-protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay® (PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein-protein interactions, but also can reveal post-translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin-embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma-Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Proximity ligation assay Support Protocol 1: Antigen retrieval method for formalin-fixed, paraffin-embedded tissues Support Protocol 2: Creation of custom PLA probes using the Duolink™ In Situ Probemaker Kit when commercially available probes are not suitable Basic Protocol 2: Imaging, quantification, and analysis of PLA signals.
Assuntos
Bioensaio/métodos , Células/metabolismo , Especificidade de Órgãos , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Antígenos/metabolismo , Formaldeído , Humanos , Imageamento Tridimensional , Inclusão em Parafina , Fixação de TecidosRESUMO
Purpose: Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal-corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal-corneal epithelial boundary organization. Methods: EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell-cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells. Results: Ephrin-A1-expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1-expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin-mediated adhesion at heterotypic boundaries. Conclusions: Ephrin-A1/EphA2 signaling complexes play a key role in limbal-corneal epithelial compartmentalization and the response of these tissues to injury.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Compartimento Celular/fisiologia , Efrina-A1/fisiologia , Efrina-A2/fisiologia , Epitélio Corneano/citologia , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Comunicação Celular/fisiologia , Células Cultivadas , Epitélio Corneano/metabolismo , Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Humanos , Imuno-Histoquímica , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptor EphA2/fisiologia , Células-Tronco/citologiaRESUMO
Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)-based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Coloração e Rotulagem/métodos , Laranja de Acridina/química , Bioensaio/métodos , Neoplasias Encefálicas/diagnóstico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/diagnóstico , Humanos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologiaRESUMO
Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.
Assuntos
Células Endoteliais/fisiologia , Invasividade Neoplásica/fisiopatologia , Migração Transcelular de Célula/fisiologia , Actomiosina/metabolismo , Análise de Variância , Fenômenos Biomecânicos , Células Endoteliais/ultraestrutura , Recuperação de Fluorescência Após Fotodegradação , Células Endoteliais da Veia Umbilical Humana , Humanos , Terapia a Laser , Microscopia de Fluorescência , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Resistência à TraçãoRESUMO
In the earliest stages of metastasis, breast cancer cells must reorganize the cytoskeleton to affect cell shape change and promote cell invasion and motility. These events require the cytoskeletal regulators Cdc42 and Rho, their effectors such as N-WASp/WAVE, and direct inducers of actin polymerization such as Arp2/3. Little consideration has been given to molecules that shape the cell membrane. The F-BAR proteins CIP4, TOCA-1, and FBP17 generate membrane curvature and act as scaffolding proteins for activated Cdc42 and N-WASp. We found that expression of CIP4, but not TOCA-1 or FBP17, was increased in invasive breast cancer cell lines in comparison with weakly or noninvasive breast cancer cell lines. Endogenous CIP4 localized to the leading edge of migrating cells and to invadopodia in cells invading gelatin. Because CIP4 serves as a scaffolding protein for Cdc42, Src, and N-WASp, we tested whether loss of CIP4 could result in decreased N-WASp function. Interaction between CIP4 and N-WASp was epidermal growth factor responsive, and CIP4 silencing by small interfering RNA caused decreased tyrosine phosphorylation of N-WASp at a Src-dependent activation site (Y256). CIP4 silencing also impaired the migration and invasion of MDA-MB-231 cells and was associated with decreased formation of invadopodia and gelatin degradation. This study presents a new role for CIP4 in the promotion of migration and invasion of MDA-MB-231 breast cancer cells and establishes the contribution of F-BAR proteins to cancer cell motility and invasion.
Assuntos
Neoplasias da Mama/patologia , Extensões da Superfície Celular/patologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a Ácido Graxo , Feminino , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Gelatina/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Camundongos , Camundongos Nus , Antígenos de Histocompatibilidade Menor , Invasividade Neoplásica , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor's response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest and differentiate into mature bone. We reasoned that we could use our model system to develop noninvasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed positron emission tomography (PET) combining the use of both (18)F-fluorodeoxyglucose ((18)FDG) and (18)F-flouride ((18)F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of (18)FDG and a significant increase in the uptake of (18)F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate (18)FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that (18)F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both (18)FDG and (18)F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation.
Assuntos
Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Osso e Ossos/patologia , Radioisótopos de Flúor , Fluordesoxiglucose F18 , Inativação Gênica , Genes myc , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/patologia , Animais , Desenvolvimento Ósseo/efeitos da radiação , Neoplasias Ósseas/genética , Diferenciação Celular , Divisão Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Osteossarcoma/genética , Tomografia por Emissão de Pósitrons , RadiografiaRESUMO
The MYC oncogene has been implicated in the regulation of up to thousands of genes involved in many cellular programs including proliferation, growth, differentiation, self-renewal, and apoptosis. MYC is thought to induce cancer through an exaggerated effect on these physiologic programs. Which of these genes are responsible for the ability of MYC to initiate and/or maintain tumorigenesis is not clear. Previously, we have shown that upon brief MYC inactivation, some tumors undergo sustained regression. Here we demonstrate that upon MYC inactivation there are global permanent changes in gene expression detected by microarray analysis. By applying StepMiner analysis, we identified genes whose expression most strongly correlated with the ability of MYC to induce a neoplastic state. Notably, genes were identified that exhibited permanent changes in mRNA expression upon MYC inactivation. Importantly, permanent changes in gene expression could be shown by chromatin immunoprecipitation (ChIP) to be associated with permanent changes in the ability of MYC to bind to the promoter regions. Our list of candidate genes associated with tumor maintenance was further refined by comparing our analysis with other published results to generate a gene signature associated with MYC-induced tumorigenesis in mice. To validate the role of gene signatures associated with MYC in human tumorigenesis, we examined the expression of human homologs in 273 published human lymphoma microarray datasets in Affymetrix U133A format. One large functional group of these genes included the ribosomal structural proteins. In addition, we identified a group of genes involved in a diverse array of cellular functions including: BZW2, H2AFY, SFRS3, NAP1L1, NOLA2, UBE2D2, CCNG1, LIFR, FABP3, and EDG1. Hence, through our analysis of gene expression in murine tumor models and human lymphomas, we have identified a novel gene signature correlated with the ability of MYC to maintain tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Algoritmos , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Linfoma/genética , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
MYC is one of the most commonly overexpressed oncogenes in human cancer. The targeted inactivation of MYC is a possible therapy for neoplasia. Conditional transgenic mouse model systems are tractable methods to precisely dissect how and when the inactivation of MYC might be effective in the treatment for human cancer. From these model systems, several general principles emerge. MYC inactivation stereotypically results in the proliferative arrest, differentiation and/or apoptosis of tumor cells. The specific consequences of MYC inactivation appear to depend both on the type of cancer as well as the constellation of genetic events unique to a given tumor. Tumors can escape from dependence upon MYC by acquiring compensatory genetic events. MYC inactivation can uncover the stem cell properties of tumor cells that differentiate into normal appearing cells. In some cases, these differentiated cells are actually dormant tumor cells that recover their neoplastic properties upon MYC reactivation. In other cases, even brief MYC inactivation is sufficient to induce sustained tumor regression. Insights from conditional transgenic mouse models will be useful in the development of therapies that target MYC for the treatment of cancer.
Assuntos
Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Camundongos , Camundongos TransgênicosRESUMO
MYC was one of the first oncogenes identified to be associated with chromosomal aberrations and one of the most common oncogenes involved in the pathogenesis of cancer. However, until recently it was not clear if MYC would be a good target for the treatment of cancer. New conditional transgenic models have been used to demonstrate that even the brief inactivation of MYC can reverse tumorigenesis. Here we review results from recent experimental model systems, which demonstrate that the inactivation of MYC may be a specific and effective treatment for many types of cancer.
Assuntos
Genes myc/genética , Camundongos Transgênicos , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Diferenciação Celular , Genes myc/genética , Animais , Apoptose , Ductos Biliares/citologia , Ductos Biliares/metabolismo , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Medições Luminescentes , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Alelos , Animais , Apoptose , Northern Blotting , Western Blotting , Divisão Celular , Núcleo Celular/metabolismo , Proliferação de Células , Separação Celular , Replicação do DNA , Citometria de Fluxo , Hepatócitos/citologia , Hepatócitos/metabolismo , Heterozigoto , Antígeno Ki-67/biossíntese , Fígado/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Microscopia de Fluorescência , Dados de Sequência Molecular , Invasividade Neoplásica , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pharmacological inactivation of oncogenes is being investigated as a possible therapeutic strategy for cancer. One potential drawback is that cessation of such therapy may allow reactivation of the oncogene and tumor regrowth. We used a conditional transgenic mouse model for MYC-induced tumorigenesis to demonstrate that brief inactivation of MYC results in the sustained regression of tumors and the differentiation of osteogenic sarcoma cells into mature osteocytes. Subsequent reactivation of MYC did not restore the cells' malignant properties but instead induced apoptosis. Thus, brief MYC inactivation appears to cause epigenetic changes in tumor cells that render them insensitive to MYC-induced tumorigenesis. These results raise the possibility that transient inactivation of MYC may be an effective therapy for certain cancers.