Utilização da morfometria celular no microambiente neoplásico e sua relação com a imunoexpressão da interleucina-4 / Use of cellular morphometry in the neoplastic microenvironment and its relationship with the immunoexpression of interleukin-4

A morfometria celular é um tipo de análise quantitativa que utiliza as medidas geométricas para obter informações acerca da morfologia nuclear, citoplasmática e outras medidas gerais das células, sendo que estes parâmetros podem estar alterados devido a processos fisiológicos e patológicos que modificam a morfologia celular normal, sendo uma análise relevante no prognóstico de diversas lesões. Os ceratinócitos possuem morfologia poliédrica com núcleos ovoides, mas podem sofrer alterações com eventos patológicos e fisiológicos, por exemplo, no núcleo. Essas alterações podem estar presentes em neoplasias malignas, como, no carcinoma epidermoide. A interleucina-4 (IL-4) é uma citocina secretada por diversos tipos de células, estando envolvida no desenvolvimento e diferenciação de células Th2, atuando na resposta antiinflamatória e sua expressão parece estar relacionada com o desenvolvimento de algumas neoplasias, incluindo o câncer oral. O objetivo da pesquisa foi realizar análise morfométrica celular (compartimentos nuclear e citoplasmático), identificação de irregularidades nucleares de ceratinócitos malignos e comparar com a imunoexpressão da IL-4 e profundidade de invasão nos casos de Carcinoma Epidermoide de Lábio Inferior (CELI) e Língua Oral (CELO). Foram analisados 30 casos de cada lesão. Para morfometria celular foram analisados 16 ceratinócitos malignos por caso; a imunoexpressão de IL-4 foi analisada no parênquima e estroma das lesões, utilizando softwares de análises de imagens. Foram aplicados os testes estatísticos de Análise de Variância, Kruskal-Wallis e Correlação de Spearman. A imunoexpressão de IL-4 no parênquima foi maior nos casos de CELI em todos os campos analisados (p<0,05); Houve correlação positiva entre área celular total e profundidade de invasão (p=0,038) e negativa entre imunoexpressão da IL-4 no parênquima superficial e perímetro nuclear superficial (p=0,007). Portanto, sugere-se que exista uma relação protetora da imunoexpressão IL-4 com as lesões analisadas, bem como alterações morfométricas dos ceratinócitos malignos com a imunoexpressão da IL-4 nessas lesões (AU).

Cell morphometry is a type of quantitative analysis that uses geometric measurements of cells to obtain information about the nuclear, cytoplasmic and general morphology of cells. Such parameters may be altered due to physiological and pathological processes that modify normal cell morphology, being a relevant analysis in the prognosis of several injuries. Keratinocytes have a polyhedral morphology with ovoid nuclei, but may undergo changes with pathological and physiological events, for example, in the nucleus. These changes may be present in malignant neoplasms, such as squamous cell carcinoma. Interleukin-4 (IL-4) is a cytokine secreted by several types of cells, being involved in the development and differentiation of Th2 cells, acting in the anti-inflammatory response and its expression seems to be related to the development of some neoplasms, including the oral cancer. The objective of the research was to perform cellular morphometric analysis (nuclear and cytoplasmic compartments), identification of nuclear irregularities of malignant keratinocytes and compare with the immunoexpression of IL-4 and depth of invasion in cases of Epidermoid Carcinoma of the Lower Lip (CELI) and Oral Tongue. (CELLO). Thirty cases of each lesion were analyzed. For cell morphometry, 16 malignant keratinocytes were analyzed per case; IL-4 immunoexpression was analyzed in the parenchyma and stroma of the lesions, using image analysis software. The statistical tests of Analysis of Variance, Kruskal-Wallis and Spearman's Correlation were applied. The immunoexpression of IL-4 in the parenchyma was higher in cases of CELI in all analyzed fields (p<0,05); There was a positive correlation between total cell area and depth of invasion (p=0.038) and a negative correlation between IL-4 immunoexpression in the superficial parenchyma and superficial nuclear perimeter (p=0.007). Therefore, it is suggested that there is a protective relationship between IL-4 immunoexpression and the lesions analyzed, as well as morphometric alterations of malignant keratinocytes with IL-4 immunoexpression in these lesions (AU).

A deep hybrid learning pipeline for accurate diagnosis of ovarian cancer based on nuclear morphology.

Nuclear morphological features are potent determining factors for clinical diagnostic approaches adopted by pathologists to analyze the malignant potential of cancer cells. Considering the structural alteration of the nucleus in cancer cells, various groups have developed machine learning techniques based on variation in nuclear morphometric information like nuclear shape, size, nucleus-cytoplasm ratio and various non-parametric methods like deep learning have also been tested for analyzing immunohistochemistry images of tissue samples for diagnosing various cancers. We aim to correlate the morphometric features of the nucleus along with the distribution of nuclear lamin proteins with classical machine learning to differentiate between normal and ovarian cancer tissues. It has already been elucidated that in ovarian cancer, the extent of alteration in nuclear shape and morphology can modulate genetic changes and thus can be utilized to predict the outcome of low to a high form of serous carcinoma. In this work, we have performed exhaustive imaging of ovarian cancer versus normal tissue and developed a dual pipeline architecture that combines the matrices of morphometric parameters with deep learning techniques of auto feature extraction from pre-processed images. This novel Deep Hybrid Learning model, though derived from classical machine learning algorithms and standard CNN, showed a training and validation AUC score of 0.99 whereas the test AUC score turned out to be 1.00. The improved feature engineering enabled us to differentiate between cancerous and non-cancerous samples successfully from this pilot study.

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness.

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.

Shear stress-induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Mechanical principles of nuclear shaping and positioning.

Positioning and shaping the nucleus represents a mechanical challenge for the migrating cell because of its large size and resistance to deformation. Cells shape and position the nucleus by transmitting forces from the cytoskeleton onto the nuclear surface. This force transfer can occur through specialized linkages between the nuclear envelope and the cytoskeleton. In response, the nucleus can deform and/or it can move. Nuclear movement will occur when there is a net differential in mechanical force across the nucleus, while nuclear deformation will occur when mechanical forces overcome the mechanical resistance of the various structures that comprise the nucleus. In this perspective, we review current literature on the sources and magnitude of cellular forces exerted on the nucleus, the nuclear envelope proteins involved in transferring cellular forces, and the contribution of different nuclear structural components to the mechanical response of the nucleus to these forces.

Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC.

A microfluidic device for characterizing nuclear deformations.

Cell nuclei experience and respond to a wide range of forces, both in vivo and in vitro. In order to characterize the nuclear response to physical stress, we developed a microfluidic chip and used it to apply mechanical stress to live cells and measure their nuclear deformability. The device design is optimized for the detection of both nucleus and cytoplasm, which can then be conveniently quantified using a custom-written Matlab program. We measured nuclear sizes and strains of embryonic stem cells, for which we observed negative Poisson ratios in the nuclei. In addition, we were able to detect changes in the nuclear response after treatment with actin depolymerizing and chromatin decondensing agents. Finally, we showed that the device can be used for biologically relevant high-resolution confocal imaging of cells under compression. Thus, the device presented here allows for accurate physical phenotyping at high throughput and has the potential to be applied to a range of cell types.

A Chemomechanical Model for Nuclear Morphology and Stresses during Cell Transendothelial Migration.

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.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation.

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia.

Carcinoma de mama triple negativo. Heterogeneidad inmunofenotípica y en el comportamiento farmacocinético / Triple-negative breast carcinoma: heterogeneity in immunophenotypes and pharmacokinetic behavior

Objetivos. Valorar, mediante resonancia magnética (RM), las características morfocinéticas, farmacocinéticas y de difusión de las neoplasias de mama con fenotipo triple negativo y analizar si hay relación entre estos parámetros y el tiempo hasta la progresión. Material y métodos. Estudio retrospectivo y observacional de una serie consecutiva de 100 pacientes con diagnóstico histológico de cáncer de mama, fenotipo triple negativo, entre enero de 2005 y diciembre de 2010 en nuestro centro. Se revisaron los estudios de RM de extensión locorregional y se recogieron las características anatomopatológicas y el seguimiento de las pacientes hasta agosto de 2014. Resultados. Las características más frecuentes por RM de estos tumores fueron: lesiones tipo masa de morfología redondeada (47,3%), bordes bien delimitados (53,7%), patrón de captación 'en anillo' (46,2%), curvas de tipo 3 (50,5%), hiperintensidad de señal intratumoral en las secuencias potenciadas en T2, valores altos de ADC (coeficiente de difusión aparente) (1,04 × 10−3 mm2/s) y una permeabilidad capilar aumentada (Kep) (0,94 min−1). No se evidenció ninguna relación estadísticamente significativa entre las características morfocinéticas o farmacocinéticas y el tiempo hasta la progresión. Se halló elevada presencia del componente in situ en las piezas quirúrgicas, aunque su representación era baja. En el seguimiento, un 25% presentaron metástasis, con predilección por órganos viscerales y baja supervivencia. Conclusión. Las neoplasias con fenotipo triple negativo mostraron mayoritariamente en la RM lesiones de tipo masa, de morfología redondeada, bordes bien delimitados y patrón de captación "en anillo". No se evidenció ninguna relación estadísticamente significativa entre las características morfocinéticas o farmacocinéticas y el tiempo hasta la progresión (AU)

Objectives. To evaluate the morphokinetic, pharmacokinetic, and diffusion characteristics of triple-negative breast cancers on magnetic resonance (MR) imaging and to analyze whether there is a relation between these parameters and the time to progression. Material and methods. This was a retrospective observational study of a consecutive series of 100 patients with histologically confirmed triple-negative breast cancer studied at our center between January 2005 and December 2010. We reviewed the findings on MR locoregional extension studies, the histological findings, and the follow-up of patients until August 2014. Results. The most common MR findings for these tumors were a rounded mass (47.3%), well-defined borders (53.7%), ring enhancement (46.2%), type 3 curves (50.5%), hyperintensity within the tumor on T2-weighted sequences, high ADC values (1.04 × 10-3 mm2/s), and increased capillary permeability (Kep) (0.94 min-1). No significant association was observed between the morphokinetic or pharmacokinetic characteristics and the time to progression. The in situ component in the surgical specimens was high, although its expression was low. During follow-up, 25% of patients had metastases, with a predilection for the visceral organs, and survival was low. Conclusion. Tumors with the triple-negative phenotype mostly presented in MR as rounded tumors with well-defined borders and ring enhancement. We found no significant association between the morphokinetic or pharmacokinetic characteristics and the time to progression (AU)

Morfología y morfometría del endotelio corneal / Morphometry and morphology of the corneal endothelium

El estudio de parámetros morfológicos y morfométricos del endotelio corneal en poblaciones de individuos sanos ha sido motivo de diversas investigaciones a nivel internacional en los últimos años. La microscopia especular permite una visión clara de las células endoteliales vivas sin alterar su función ni su morfología. A su vez, se puede realizar un recuento por área de superficie y determinar si existe una alteración en la forma o tamaño de las células endoteliales, parámetros a tener en cuenta para conocer la capacidad funcional del endotelio corneal. Se realizó una búsqueda actualizada de los últimos diez años de diversos artículos publicados, con el objetivo de conocer las características cuantitativas y cualitativas de las células endoteliales en las diferentes poblaciones estudiadas. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

The study of morphological and morphometric parameters of the corneal endothelium in healthy individuals have been the main subject of several research studies at international level in the last few years. The specular microscopy allows us a clear vision of the alive endothelial cells without altering either their function or their morphology; a counting per surface area can be made and to determine whether an alteration exists in the form or the size of the endothelial cell, this is a parameter to be kept in mind to know the functional capacity of the corneal endothelium. An updated search of the published articles in the last ten years was made with the objective of determining the quantitative and qualitative characteristics of the endothelial cells in the different studied populations. Infomed platform, particularly the Virtual Library of Health, was used to this end(AU)

Morfología y morfometría del endotelio corneal / Morphometry and morphology of the corneal endothelium

El estudio de parámetros morfológicos y morfométricos del endotelio corneal en poblaciones de individuos sanos ha sido motivo de diversas investigaciones a nivel internacional en los últimos años. La microscopia especular permite una visión clara de las células endoteliales vivas sin alterar su función ni su morfología. A su vez, se puede realizar un recuento por área de superficie y determinar si existe una alteración en la forma o tamaño de las células endoteliales, parámetros a tener en cuenta para conocer la capacidad funcional del endotelio corneal. Se realizó una búsqueda actualizada de los últimos diez años de diversos artículos publicados, con el objetivo de conocer las características cuantitativas y cualitativas de las células endoteliales en las diferentes poblaciones estudiadas. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

The study of morphological and morphometric parameters of the corneal endothelium in healthy individuals have been the main subject of several research studies at international level in the last few years. The specular microscopy allows us a clear vision of the alive endothelial cells without altering either their function or their morphology; a counting per surface area can be made and to determine whether an alteration exists in the form or the size of the endothelial cell, this is a parameter to be kept in mind to know the functional capacity of the corneal endothelium. An updated search of the published articles in the last ten years was made with the objective of determining the quantitative and qualitative characteristics of the endothelial cells in the different studied populations. Infomed platform, particularly the Virtual Library of Health, was used to this end(AU)

Design of a microfluidic device to quantify dynamic intra-nuclear deformation during cell migration through confining environments.

The ability of cells to migrate through tissues and interstitial spaces is an essential factor during development and tissue homeostasis, immune cell mobility, and in various human diseases. Deformation of the nucleus and its associated lamina during 3-D migration is gathering increasing interest in the context of cancer metastasis, with the underlying hypothesis that a softer nucleus, resulting from reduced levels of lamin A/C, may aid tumour spreading. However, current methods to study the migration of cells in confining three dimensional (3-D) environments are limited by their imprecise control over the confinement, physiological relevance, and/or compatibility with high resolution imaging techniques. We describe the design of a polydimethylsiloxane (PDMS) microfluidic device composed of channels with precisely-defined constrictions mimicking physiological environments that enable high resolution imaging of live and fixed cells. The device promotes easy cell loading and rapid, yet long-lasting (>24 hours) chemotactic gradient formation without the need for continuous perfusion. Using this device, we obtained detailed, quantitative measurements of dynamic nuclear deformation as cells migrate through tight spaces, revealing distinct phases of nuclear translocation through the constriction, buckling of the nuclear lamina, and severe intranuclear strain. Furthermore, we found that lamin A/C-deficient cells exhibited increased and more plastic nuclear deformations compared to wild-type cells but only minimal changes in nuclear volume, implying that low lamin A/C levels facilitate migration through constrictions by increasing nuclear deformability rather than compressibility. The integration of our migration devices with high resolution time-lapse imaging provides a powerful new approach to study intracellular mechanics and dynamics in a variety of physiologically-relevant applications, ranging from cancer cell invasion to immune cell recruitment.

Joint modeling of cell and nuclear shape variation.

Modeling cell shape variation is critical to our understanding of cell biology. Previous work has demonstrated the utility of nonrigid image registration methods for the construction of nonparametric nuclear shape models in which pairwise deformation distances are measured between all shapes and are embedded into a low-dimensional shape space. Using these methods, we explore the relationship between cell shape and nuclear shape. We find that these are frequently dependent on each other and use this as the motivation for the development of combined cell and nuclear shape space models, extending nonparametric cell representations to multiple-component three-dimensional cellular shapes and identifying modes of joint shape variation. We learn a first-order dynamics model to predict cell and nuclear shapes, given shapes at a previous time point. We use this to determine the effects of endogenous protein tags or drugs on the shape dynamics of cell lines and show that tagged C1QBP reduces the correlation between cell and nuclear shape. To reduce the computational cost of learning these models, we demonstrate the ability to reconstruct shape spaces using a fraction of computed pairwise distances. The open-source tools provide a powerful basis for future studies of the molecular basis of cell organization.

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading.

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.

Nucleus deformation of SaOs-2 cells on rhombic µ-pillars.

It has been previously shown that osteosarcoma (SaOs-2) cells respond to micropillared surfaces consisting of poly-L-lactic acid with strong deformation of the cell body and nucleus. Until now, cell nucleus deformation of SaOs-2 cells was only studied by exposing them to square shaped micropillars in an isotropic pattern. Here we report on experiments of the cell nucleus response of such cells to rhombic structures of different topographies generated from a rubbery polymer, namely poly(n-butyacrylate). It is observed that cells orientate themselves perpendicular to the long axis of the rhombi. While their spreading on the surface is not influenced by the opening angle of the structures, rhombic structures with sharper angles induce stronger deformation of the cells and accordingly more elongated nuclei.

Spermatid head elongation with normal nuclear shaping requires ADP-ribosyltransferase PARP11 (ARTD11) in mice.

Sperm are highly differentiated cells characterized by their species-specific nuclear shapes and extremely condensed chromatin. Abnormal head shapes represent a form of teratozoospermia that can impair fertilization capacity. This study shows that poly(ADP-ribose) polymerase-11 (ARTD11/PARP11), a member of the ADP-ribosyltransferase (ARTD) family, is expressed preferentially in spermatids undergoing nuclear condensation and differentiation. Deletion of the Parp11 gene results in teratozoospermia and male infertility in mice due to the formation of abnormally shaped fertilization-incompetent sperm, despite normal testis weights and sperm counts. At the subcellular level, PARP11-deficient elongating spermatids reveal structural defects in the nuclear envelope and chromatin detachment associated with abnormal nuclear shaping, suggesting functional relevance of PARP11 for nuclear envelope stability and nuclear reorganization during spermiogenesis. In vitro, PARP11 exhibits mono(ADP-ribosyl)ation activity with the ability to ADP-ribosylate itself. In transfected somatic cells, PARP11 colocalizes with nuclear pore components, such as NUP153. Amino acids Y77, Q86, and R95 in the N-terminal WWE domain, as well as presence of the catalytic domain, are essential for colocalization of PARP11 with the nuclear envelope, but catalytic activity of the protein is not required for colocalization with NUP153. This study demonstrates that PARP11 is a novel enzyme important for proper sperm head shaping and identifies it as a potential factor involved in idiopathic mammalian teratozoospermia.

Epithelial-mesenchymal transition in prostate cancer is associated with quantifiable changes in nuclear structure.

BACKGROUND: Prostate cancer progression is concomitant with quantifiable nuclear structure and texture changes as compared to non-cancer tissue. Malignant progression is associated with an epithelial-mesenchymal transition (EMT) program whereby epithelial cancer cells take on a mesenchymal phenotype and dissociate from a tumor mass, invade, and disseminate to distant metastatic sites. The objective of this study was to determine if epithelial and mesenchymal prostate cancer cells have different nuclear morphology. METHODS: Murine tibia injections of epithelial PC3 (PC3-Epi) and mesenchymal PC3 (PC3-EMT) prostate cancer cells were processed and stained with H&E. Cancer cell nuclear image data was obtained using commercially available image-processing software. Univariate and multivariate statistical analysis were used to compare the two phenotypes. Several non-parametric classifiers were constructed and permutation-tested at various training set fractions to ensure robustness of classification between PC3-Epi and PC3-EMT cells in vivo. RESULTS: PC3-Epi and PC3-EMT prostate cancer cells were separable at the single cell level in murine tibia injections on the basis of nuclear structure and texture remodeling associated with an EMT. Support vector machine and multinomial logistic regression models based on nuclear architecture features yielded AUC-ROC curves of 0.95 and 0.96, respectively, in separating PC3-Epi and PC3-EMT prostate cancer cells in vivo. CONCLUSIONS: Prostate cancer cells that have undergone an EMT demonstrated an altered nuclear structure. The association of nuclear changes and a mesenchymal phenotype demonstrates quantitative morphometric image analysis may be used to detect cancer cells that have undergone EMT. This morphometric measurement could provide valuable prognostic information in patients regarding the likelihood of [future] metastatic disease.

Simple computational technique to quantify nuclear shape asymmetry.

The nucleus of an eukaryotic cell is a membrane-bound organelle containing a major part of the cellular genome. Nuclear shape is controlled by forces generated in the cytoskeleton, nuclear envelope and matrix of the nucleus and may change when the balance of these forces is disturbed. In certain cases, such changes may be indicative of cell pathology. Nuclear shape feature is being commonly addressed in both experimental research and diagnostics; nevertheless its symmetry-related aspects receive little attention. This article introduces a technique allowing to estimate nuclear shape asymmetry in digital images captured from cyto- or histological preparations. Implemented in a software package, this technique quantifies the asymmetry using two scenarios. The first one presumes the identification of nuclear pixels laying outside the largest inscribed circle. According to the second scenario, the algorithm searches for nuclear pixels lacking pixel-partners symmetric with respect to the nuclear area's centroid. In both cases, the proportion of "asymmetric" pixels is used to estimate the feature of interest. The technique was validated on images of cell nuclei having distinctive shape phenotypes. A conclusion was made that shape asymmetry feature may be useful accessory to the toolbox of nuclear morphometry.