Mechanical profile of human keratinocytes expressing HPV-18 oncogenes.

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

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 acidification 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 effects of hypoxic microenvironments on cells. We have used a previously developed variant of the method of coverslip-induced hypoxia to study the process of acidification 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 acidification 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, acidification 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.

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.

A hipóxia é um fator fundamental no processo de gênese tumoral, bem como em patologias precursoras do câncer, como o Líquen Plano Oral (LPO). Objetivo: Determinar se é possível estabelecer uma correlação entre as alterações que os queratinócitos normais sofrem em um microambiente hipóxico in vitro e as alterações que aparecem nos queratinócitos no epitélio da mucosa oral no contexto da patologia do LPO. Métodos: As alterações morfológicas foram estudadas por microscopia de contraste de fase e a detecção de marcadores associados à hipóxia de queratinócitos humanos (HaCaT), como modelo de célula oral, em um microambiente hipóxico gerado pela variante do método "Hipóxia induzida por lamínulas". Resultados: Por microscopia confocal, observou-se a presença dos marcadores de hipóxia GLUT-1 e Hipoxyprobe em culturas de células HaCaT expostas a um microambiente hipóxico. Além disso, a presença do marcador GLUT-1 foi observada por imuno-histoquímica em tecido epitelial humano derivado de biópsias de patologia de LPO. Conclusões: Foi estabelecida uma correlação entre as alterações detectadas em queratinócitos humanos induzidas em um microambiente hipóxico in vitro e as alterações detectadas in vivo no tecido epitelial da mucosa oral.

Hypoxia is a fundamental factor in the process of tumor genesis, as well as in precursor pathologies of cancer, such as Oral Lichen Planus (OLP). Objective: To determine if it is possible to establish a correlation between the alterations that normal keratinocytes suffer in a hypoxic microenvironment in vitro and alterations that appear in the keratinocytes in the epithelium of the oral mucosa in the context of OLP pathology. Methods: Morphological changes were studied by phase contrast microscopy, and the detection of markers associated with hypoxia of human keratinocytes (HaCaT), as an oral cell model, in a hypoxic microenvironment generated by the variant of the method "Hypoxia induced by coverslips". Results: Using confocal microscopy, the presence of hypoxia markers GLUT-1 and Hipoxyprobe was observed in HaCaT cell cultures exposed to a hypoxic microenvironment. In addition, the presence of the GLUT-1 marker was observed by immunohistochemistry in human epithelial tissue derived from biopsies of OLP pathology. Conclusions: A correlation was established between the alterations detected in human keratinocytes induced in a hypoxic microenvironment in vitro and the alterations detected in vivo in epithelial tissue of the oral mucosa.