Requirement of Pax6 for the integration of guidance cues in cell migration

Data accessibility. Cell trajectories data and a summary of directedness and angle values are deposited at Dryad: http://dx.doi.org/10.5061/dryad.53512. Funding MA was funded by an Alban International Research Studentship (code: E07D400602UY).Peer reviewedPublisher PD

Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids

Significance: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart. Aim: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study. Approach: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain. Results: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates. Conclusions: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup

The Tissue Architecture of Oral Squamous Cell Carcinoma Visualized by Staining Patterns of Wheat Germ Agglutinin and Structural Proteins Using Confocal Microscopy

Abstract: Objectives: Tissue architecture and cell morphology suffer profound alterations during oral cancer and are important markers for its progression and outcome. For precise visualization of tissue architecture in oral cancer, we used confocal microscopy to examine the staining pattern of wheat germ agglutinin, a lectin that binds membrane glycoproteins, and the staining patterns of structural proteins. Materials and Methods: Paraffin sections of oral squamous cell carcinoma were stained with fluorescently labeled wheat germ agglutinin and with antibodies against structural proteins, which were revealed by immunohistochemistry with tyramide signal amplification. Results: Membrane localization of wheat germ agglutinin was markedly decreased in the basal layers and in regions of tumor invasion, accompanied by cytoplasmic redistribution of E-cadherin, -actin and syndecan-1. Wheat germ agglutinin staining clearly identified tumor clusters within the surrounding stroma, and tumor cells with elongated morphology. Conclusions: Our results suggest that the wheat germ agglutinin staining pattern is indicative of the degree of cell cohesion in oral squamous cell carcinoma, which decreases in basal layers and invasive tumor clusters with more migratory morphologies. Wheat germ agglutinin staining in combination with confocal microscopy could constitute, therefore, a valuable tool for the study of tissue architecture in oral cancer

Requirement of Pax6 for the integration of guidance cues in cell migration

The intricate patterns of cell migration that are found throughout development are generated through a vast array of guidance cues. Responding integratively to distinct, often conflicting, migratory signals is probably crucial for cells to reach their correct destination. Pax6 is a master transcription factor with key roles in neural development that include the control of cell migration. In this study, we have investigated the ability of cells derived from cortical neurospheres from wildtype (WT) and Pax6-/- mouse embryos to integrate diverging guidance cues. We used two different cues, either separately or in combination: substratum nanogrooves to induce contact guidance, and electric fields (EFs) to induce electrotaxis. In the absence of an EF, both WT and Pax6-/- cells aligned and migrated parallel to grooves, and on a flat substrate both showed marked electrotaxis towards the cathode. When an EF was applied in a perpendicular orientation to grooves, WT cells responded significantly to both cues, migrating in highly oblique trajectories in the general direction of the cathode. However, Pax6-/- cells had an impaired response to both cues simultaneously. Our results demonstrate that these neurosphere derived cells have the capacity to integrate diverging guidance cues, which requires Pax6 function

Hypoxia, acidification and oxidative stress in cells cultured at large distances from an oxygen source

Hypoxia is a condition frequently encountered by cells in tissues, whether as a normal feature of their microenvironment or subsequent to deregulated growth. Hypoxia can lead to acidifcation and increased oxidative stress, with profound consequences for cell physiology and tumorigenesis. Therefore, the interplay between hypoxia and oxidative stress is an important aspect for understanding the efects of hypoxic microenvironments on cells. We have used a previously developed variant of the method of coverslip-induced hypoxia to study the process of acidifcation in a hypoxic microenvironment and to simultaneously visualize intracellular levels of hypoxia and oxidative stress. We observed high accumulation of CO2 in hypoxic conditions, which we show is the main contributor to acidifcation in our model. Also, increased levels of oxidative stress were observed in moderately hypoxic cells close to the oxygen source, where the mitochondrial membrane potential was preserved. Conversely, cells at large distances from the oxygen source showed higher levels of hypoxia, milder oxidative stress and reduced mitochondrial membrane potential. Our results contribute to characterize the interplay between reduced oxygen levels, acidifcation and oxidative stress in a simple in vitro setting, which can be used to model cell responses to an altered environment, such as the early tumor microenvironment

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid‐containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO). Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO. Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método “Hipoxia inducida por cubreobjetos”. Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO. Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral

E-Cadherin gene expression in oral cancer : clinical and prospective data

Low protein expression of E-cadherin in oral squamous cell carcinoma (OSCC) has been associated with clinical and histopathological traits such as metastases, recurrence, low survival and poor tumor differentiation, and it is considered a high-risk marker of malignancy. However, it is still unknown whether low expression of E-cadherin is also present at the mRNA level in OSCC cases. Objective: The aim of this study was to compare E-cadherin mRNA expression in OSCC patients and controls and to correlate the expression with clinical and prospective characteristics. Forty patients and 40 controls were enrolled. E-cadherin mRNA expression was evaluated by quantitative real-time polymerase chain reaction using TaqMan probes. E-cadherin mRNA expression was significantly decreased in OSCC patients compared to that of controls (p<0.001). Whereas no significant association between clinical parameters and E-cadherin expression levels was observed, we noted lower E-cadherin expression levels in patients with positive lymph node metastasis. E-cadherin mRNA expression was markedly diminished in OSCC, in agreement with previous results that examined E-cadherin expression at the protein level. E-cadherin is downregulated in the early clinical stages of OSCC, and its mRNA levels do not change significantly in the advanced stages, suggesting that there is limited usefulness of this parameter for predicting disease progression

Consumo de Marihuana: consecuencias del llamado “uso recreativo”

Frente a la tolerancia social que existe en la actualidad hacia el consumo de diferentes drogas legales e ilegales, se propuso investigar si se puede sostener sin consecuencias para la salud física, psicológica y social el llamado “uso recreativo” de marihuana. Se comprobó que existe una tendencia generalizada entre los jóvenes a considerar la marihuana como una droga que no produce daños a las personas, si se la mantiene dentro de un uso “social - recreativo”. El aporte de esta investigación, al analizar los conocimientos actuales en el imaginario social de las consecuencias del consumo de marihuana con el aporte científico de la neurobiología, la psicología y la sociología permite la formulación de la siguiente hipótesis: el consumo de marihuana es una necesidad creada socialmente. El adolescente recibe un mensaje constante de la sociedad de consumo: para ser y pertenecer se le imponen determinados modismos que van, desde escapar a los malestares de la cotidianeidad al uso de determinados objetos, incluida la marihuana. Entre las conclusiones más relevantes se constata que la familia en sí misma no es predictora de consumo de marihuana sino que son preponderantes factores del contexto, tales como: la presión social, la búsqueda de identidad grupal, la curiosidad y el alivio del estrés negativo De las respuestas obtenidas de los consumidores se evidencia la necesidad de integración al grupo de pares, imitando e interactuando en situaciones de consumo, y allí la sustancia funciona como un facilitador de las relaciones sociales. El análisis de las trayectorias de los pacientes, sus motivos de inicio, la forma y contexto en que tal situación se produjo, cómo fue vivida la primera experiencia son elementos que permitirán argumentar frente a la banalización y justificación del consumo de marihuana que se produce en la actualidad.Facing the social tolerance that exists today towards consumption of different legal and illegal drugs, our goal was to investigate whether the so-called “recreational use” of marijuana can be sustained without consequences for the physical, psychological and social health. There is now a widespread tendency among young people to consider marijuana as a drug that produces no harmto people if maintained within a “social - recreational” use. The contribution of this research whileanalyzing current knowledge of social representations as regards the consequences of marijuanaconsumption as well as with the scientific contribution of neurobiology, psychology and sociologydevelops the following hypothesis: marijuana consumption is a socially created need. The teenagerreceives a constant message of consumption society : in order to be in it and to belong to it certainways of acting are imposed to him such as to escape from the discomfort of everyday life and theuse of certain objects such as marijuana. Relevant conclusions of this research are that the familyitself is not predictive of marijuana consumption while there are more predominant factors suchas social pressure, search for group identity, curiosity and relief from negative stress. The answersobtained from the consumers make evident the need for integration in the group through imitatingand interacting within situations of consumption and there the substance acts as a facilitator forsocial relations. The analysis of the background of patients including why they started consuming,how and where was their first experience are elements that will allow to argue against trivializationand justification of marijuana consumption nowadays

Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the evelopment of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.PEDECIBA. ANII