A influência de macrófagos M1, M2 e TAM, e do receptor P2X7 na invasividade e resistência de neuroblastoma / The influence of different macrophages' phenotypes, and of the P2X7 receptor, on the invasiveness and chemoresistance of neuroblastoma

O neuroblastoma é um tumor sólido muito comum em crianças. O estágio mais avançado da doença é altamente agressivo e invasivo, além de pouco responsivo à terapia, que é limitada por mecanismos de resistência e reincidência relacionados à metástase. Muitos estudos tem sido feitos para identificar mecanismos de invasão e quimioresistência de células tumorais, afim de aumentar a sobrevida dos pacientes com câncer. Nesse trabalho, nós estudamos o efeito dos macrófagos, as células imunes mais abundantes no microambiente tumoral, os TAMs (do inglês tumor-associated macrophage) e do receptor P2X7, um purinoreceptor acionado por ATP, nesses processos. Os TAMs respondem e atuam de acordo com a miríade de fatores que encontram, podendo gerar populações heterogêneas e com funções distintas, tanto antitumorais, como pró-tumorais. Altos níveis de ATP extracelular são encontrados no microambiente tumoral, podendo então ativar o receptor P2X7. Este receptor tem sido relacionado tanto a funções inflamatórias como funções na resolução da inflamação de macrófagos. Além disso, o receptor P2X7 está envolvido em uma variedade de eventos celulares, incluindo a secreção de mediadores pró-inflamatórios, a proliferação celular e a apoptose de células tumorais. Primeiramente, foi avaliado o papel do receptor P2X7 na polarização de macrófagos da derivados medula óssea de camundongos wild-type e nocaute para o P2X7 na presença e ausência de fatores secretados por células de neuroblastoma, e então foi estudada a influência desses diferentes macrófagos polarizados em eventos celulares de grande relevância clínica para o neuroblastoma: a invasividade e quimiorresistência. Os resultados demonstraram que, apesar do reconhecido envolvimento do receptor P2X7 na inflamação, a ausência deste receptor não atenua a expressão de marcadores característicos do fenótipo inflamatório, M1. O aumento da expressão do receptor P2Y2, também envolvido na inflamação, nessas células, sugere um mecanismo genético de compensação para não atenuação da inflamação em macrófagos que não expressam o receptor P2X7. Contudo, a ausência do receptor P2X7 levou a alterações no fenótipo alternativo, M2, de modo que a expressão de Tnf, marcador de M2, não foi reprimido. TAMs noucates para P2X7 tiveram a expressão de arg1, marcador de M2, suprimida, reforçando a importância do receptor P2X7 no estabelecimento de fenótipos ativados alternativamente. Nossos dados também sugerem que ausência do receptor P2X7 em TAMs permite a aquisição de um fenótipo capaz de tornar as células de neuroblastoma que expressam P2X7 mais invasivas e mais quimioresistentes à vincristina. Por outro lado, TAMs, independentemente da presença ou ausência do receptor P2X7, induziram a proliferação e quimioresistência das células de neuroblastoma silenciadas para o receptor P2X7, o que nos leva a concluir que o receptor P2X7 em TAMs é desfavorável à progressão de tumores expressando P2X7

Neuroblastoma is a highly common childhood solid tumor. The most advanced stage of the disease is highly aggressive and invasive, besides from being poorly responsive to therapies, which are limited by resistance and recurrence mechanisms related to metastasis. Several studies attempt to identify invasion and resistance mechanisms of the tumor cells in order to increase overall survival of the patients. On the present work, we investigated the effect of macrophages, the most abundant immune cells on the tumor microenvironment, called TAMs (tumor-associated macrophages), and of the P2X7 receptor, an ATP-gated purinoceptor, on these processes. TAMs and cancer cells crosstalk, and behave accordingly to a miriad of factors present at the TME, generating heterogeneous populations with distinct functionalities, either pro- or antitumor. High extracellular levels of ATP are found in the TME, being able to activate the P2X7 receptor. This receptor mediates both pro- and anti-inflammatory functions in macrophages. In addition, it is involved in several cellular events, including the secretion of pro-inflammatory mediators, cell proliferation and tumor cell apoptosis. At first, we evaluated the role of the P2X7 receptor on the polarization of bone marrow-derived macrophages (BMDM), either wild-type or knockout for the P2X7 receptor, in presence or absence or factors secreted by neuroblastoma cells. Next, we investigated the influence of the polarized macrophages in highly relevant cellular events for neuroblastoma, such as invasiveness and chemoresistance. Our results showed that, despite the known involvement of P2X7 receptor on inflammation, its absence did not decrease the expression if inflammatory markers of M1 macrophage populations. An increase in the expression of the P2Y2 receptor, also involved in inflammation, on these cells suggest a genetic compensation mechanism for preventing attenuation of inflammation when P2X7 is lacking. However, P2X7 receptor absence did compromise the M2 phenotype, driving the expression of Tnf. TAMs knockout for the P2X7 receptor were not able to express arg1, also an M2 marker, reinforcing a role of the P2X7 receptor on establishing alternative macrophage phenotypes. Our data also suggest that TAMs lacking the P2X7 receptor acquire a phenotype capable of turning P2X7R-expressing neuroblastoma cells more invasive and chemoresistant to vincristine. On the other hand, TAMs, independently on the presence of the P2X7 receptor, induced proliferation and resistance of neuroblastoma cells silenced for P2X7 receptor expression, leading us to the conclusion that the P2X7 receptor in TAMs is unfavorable for the progression of P2X7R-expressing tumors

Virtual cell model for osmotic pressure calculation of charged biomolecules.

The osmotic pressure of dilute electrolyte solutions containing charged macro-ions as well as counterions can be computed directly from the particle distribution via the well-known cell model. Originally derived within the Poisson-Boltzmann mean-field approximation, the cell model considers a single macro-ion centered into a cell, together with counterions needed to neutralize the total cell charge, while it neglects the phenomena due to macro-ion correlations. While extensively applied in coarse-grained Monte Carlo (MC) simulations of continuum solvent systems, the cell model, in its original formulation, neglects the macro-ion shape anisotropy and details of the surface charge distribution. In this paper, by comparing one-body and two-body coarse-grained MC simulations, we first establish an upper limit for the assumption of neglecting correlations between macro-ions, and second, we validate the approximation of using a non-spherical macro-ion. Next, we extend the cell model to all-atom molecular dynamics simulations and show that protein concentration-dependent osmotic pressures can be obtained by confining counterions in a virtual, spherical subspace defining the protein number density. Finally, we show the possibility of using specific interaction parameters for the protein-ion and ion-ion interactions, enabling studies of protein concentration-dependent ion-specific effects using merely a single protein molecule.

Surface Plasmon Resonance-Based Method for Rapid Product Sialylation Assessment in Cell Culture.

To streamline cell culture process development, surface plasmon resonance (SPR) biosensors offer a versatile platform for the rapid quantification and quality analysis of recombinant proteins. As a representative case study, the present chapter details a procedure employing a SPR biosensor for determining the differential sialylation levels of recombinant interferon α2b contained in cell culture samples, using immobilized Sambucus nigra lectin. Of interest, this semiquantitative approach can be adapted to work with other lectins with unique carbohydrate-binding specificities, enabling a wide range of product characterization analysis.

Self-assembling outside equilibrium: emergence of structures mediated by dissipation.

A set of disordered interacting building blocks may form ordered structures by means of a self-assembling process. An external intervention in the system by adding a chemical species or by applying forces leads to different self-assembly scenarios with the appearance of new structures. For instance, the formation of microtubules, gels, virus capsides, cells and living beings among others takes place by self-assembly under nonequilibrium conditions. A general evolution criterion able to account for why nature selects some structures outside equilibrium and not others is lacking. Nevertheless, progress in the understanding of nonequilibrium self-assembly (NESA) mechanisms has been made thanks to the formulation of models that take particular situations into consideration. We review recent efforts devoted to describing self-assembly out of equilibrium and we provide a reference linking several current concepts in order to help in the development of new models and experimental studies. We hope that the knowledge of the intimate mechanisms leading to the formation of structures will make the implementation of re-configurable and bio-inspired materials possible and give a simpler perspective on the understanding of the emergence of life.

Cells, drugs and NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool for investigating cellular structures and their compositions. While in vivo and whole-cell NMR have a long tradition in cell-based approaches, high-resolution in-cell NMR spectroscopy is a new addition to these methods. In recent years, technological advancements in multiple areas provided converging benefits for cellular MR applications, especially in terms of robustness, reproducibility and physiological relevance. Here, we review the use of cellular NMR methods for drug discovery purposes in academia and industry. Specifically, we discuss how developments in NMR technologies such as miniaturized bioreactors and flow-probe perfusion systems have helped to consolidate NMR's role in cell-based drug discovery efforts.

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking.

BACKGROUND: Cryo-electron tomography is an important tool to study structures of macromolecular complexes in close to native states. A whole cell cryo electron tomogram contains structural information of all its macromolecular complexes. However, extracting this information remains challenging, and relies on sophisticated image processing, in particular for template-free particle extraction, classification and averaging. To develop these methods it is crucial to realistically simulate tomograms of crowded cellular environments, which can then serve as ground truth models for assessing and optimizing methods for detection of complexes in cell tomograms. RESULTS: We present a framework to generate crowded mixtures of macromolecular complexes for realistically simulating cryo electron tomograms including noise and image distortions due to the missing-wedge effects. Simulated tomograms are then used for assessing the template-free Difference-of-Gaussian (DoG) particle-picking method to detect complexes of different shapes and sizes under various crowding and noise levels. We identified DoG parameter settings that maximize precision and recall for detecting particles over a wide range of sizes and shapes. We observed that medium sized DoG scaling factors showed the overall best performance. To further improve performance, we propose a combination strategy for integrating results from multiple parameter settings. With increasing macromolecular crowding levels, the precision of particle picking remained relatively high, while the recall was dramatically reduced, which limits the detection of sufficient copy numbers of complexes in a crowded environment. Over a wide range of increasing noise levels, the DoG particle picking performance remained stable, but dramatically reduced beyond a specific noise threshold. CONCLUSIONS: Automatic and reference-free particle picking is an important first step in a visual proteomics analysis of cell tomograms. However, cell cytoplasm is highly crowded, which makes particle detection challenging. It is therefore important to test particle-picking methods in a realistic crowded setting. Here, we present a framework for simulating tomograms of cellular environments at high crowding levels and assess the DoG particle picking method. We determined optimal parameter settings to maximize the performance of the DoG particle-picking method.

Cells on biomaterials--some aspects of elemental analysis by means of electron probes.

Electron probe X-ray microanalysis enables concomitant observation of specimens and analysis of their elemental composition. The method is attractive for engineers developing tissue-compatible biomaterials. Either changes in element composition of cells or biomaterial can be defined according to well-established preparation and quantification procedures. However, the qualitative and quantitative elemental analysis appears more complicated when cells or thin tissue sections are deposited on biomaterials. X-ray spectra generated at the cell/tissue-biomaterial interface are modelled using a Monte Carlo simulation of a cell deposited on borosilicate glass. Enhanced electron backscattering from borosilicate glass was noted until the thickness of the biological layer deposited on the substrate reached 1.25 µm. It resulted in significant increase in X-ray intensities typical for the elements present in the cellular part. In this case, the mean atomic number value of the biomaterial determines the strength of this effect. When elements are present in the cells only, the positive linear relationship appears between X-ray intensities and cell thickness. Then, spatial dimensions of X-ray emission for the particular elements are exclusively in the range of the biological part and the intensities of X-rays become constant. When the elements are present in both the cell and the biomaterial, X-ray intensities are registered for the biological part and the substrate simultaneously leading to a negative linear relationship of X-ray intensities in the function of cell thickness. In the case of the analysis of an element typical for the biomaterial, strong decrease in X-ray emission is observed in the function of cell thickness as the effect of X-ray absorption and the limited excitation range to biological part rather than to the substrate. Correction procedures for calculations of element concentrations in thin films and coatings deposited on substrates are well established in materials science, but little is known about factors that have to be taken into account to accurately quantify bioelements in thin and semi-thick biological samples. Thus thorough tests of currently available quantification procedures are required to verify their applicability to cells or tissues deposited on the biomaterials.

In-silico analysis on biofabricating vascular networks using kinetic Monte Carlo simulations.

We present a computational modeling approach to study the fusion of multicellular aggregate systems in a novel scaffold-less biofabrication process, known as 'bioprinting'. In this novel technology, live multicellular aggregates are used as fundamental building blocks to make tissues or organs (collectively known as the bio-constructs,) via the layer-by-layer deposition technique or other methods; the printed bio-constructs embedded in maturogens, consisting of nutrient-rich bio-compatible hydrogels, are then placed in bioreactors to undergo the cellular aggregate fusion process to form the desired functional bio-structures. Our approach reported here is an agent-based modeling method, which uses the kinetic Monte Carlo (KMC) algorithm to evolve the cellular system on a lattice. In this method, the cells and the hydrogel media, in which cells are embedded, are coarse-grained to material's points on a three-dimensional (3D) lattice, where the cell-cell and cell-medium interactions are quantified by adhesion and cohesion energies. In a multicellular aggregate system with a fixed number of cells and fixed amount of hydrogel media, where the effect of cell differentiation, proliferation and death are tactically neglected, the interaction energy is primarily dictated by the interfacial energy between cell and cell as well as between cell and medium particles on the lattice, respectively, based on the differential adhesion hypothesis. By using the transition state theory to track the time evolution of the multicellular system while minimizing the interfacial energy, KMC is shown to be an efficient time-dependent simulation tool to study the evolution of the multicellular aggregate system. In this study, numerical experiments are presented to simulate fusion and cell sorting during the biofabrication process of vascular networks, in which the bio-constructs are fabricated via engineering designs. The results predict the feasibility of fabricating the vascular structures via the bioprinting technology and demonstrate the morphological development process during cellular aggregate fusion in various engineering designed structures. The study also reveals that cell sorting will perhaps not significantly impact the final fabricated products, should the maturation process be well-controlled in bioprinting.

Template-free detection of macromolecular complexes in cryo electron tomograms.

MOTIVATION: Cryo electron tomography (CryoET) produces 3D density maps of biological specimen in its near native states. Applied to small cells, cryoET produces 3D snapshots of the cellular distributions of large complexes. However, retrieving this information is non-trivial due to the low resolution and low signal-to-noise ratio in tomograms. Current pattern recognition methods identify complexes by matching known structures to the cryo electron tomogram. However, so far only a small fraction of all protein complexes have been structurally resolved. It is, therefore, of great importance to develop template-free methods for the discovery of previously unknown protein complexes in cryo electron tomograms. RESULTS: Here, we have developed an inference method for the template-free discovery of frequently occurring protein complexes in cryo electron tomograms. We provide a first proof-of-principle of the approach and assess its applicability using realistically simulated tomograms, allowing for the inclusion of noise and distortions due to missing wedge and electron optical factors. Our method is a step toward the template-free discovery of the shapes, abundance and spatial distributions of previously unknown macromolecular complexes in whole cell tomograms. CONTACT: alber@usc.edu

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium.

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for (15)N and (13)C,(15)N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.

Microfluidic platforms for single-cell analysis.

Microfluidics, the study and control of the fluidic behavior in microstructures, has emerged as an important enabling tool for single-cell chemical analysis. The complex procedures for chemical cytometry experiments can be integrated into a single microfabricated device. The capability of handling a volume of liquid as small as picoliters can be utilized to manipulate cells, perform controlled cell lysis and chemical reactions, and efficiently minimize sample dilution after lysis. The separation modalities such as chromatography and electrophoresis within microchannels are incorporated to analyze various types of intracellular components quantitatively. The microfluidic approach offers a rapid, accurate, and cost-effective tool for single-cell biology. We present an overview of the recent developments in microfluidic technology for chemical-content analysis of individual cells.

A computational tool for Monte Carlo simulations of biomolecular reaction networks modeled on physical principles.

Deciphering and designing complex biomolecular networks in the cell are the goals of systems and synthetic biology, respectively. The effects of localization, spatial heterogeneity, and molecular fluctuations in biomolecular networks are not well understood. We present a theoretical approach based on physical principles to accurately simulate biomolecular networks using the Monte Carlo method. Incorporating this theory, a computational tool named Monte Carlo biomolecular simulator (MBS) was developed, enabling studies of biomolecular kinetics with both spatial and temporal resolutions. The accuracy of MBS was verified by comparison against the classical deterministic approaches. Furthermore, the effects of localization, spatial heterogeneity, and molecular fluctuations were studied in three simulated model systems, showing their impact on the overall reaction kinetics. This work demonstrates the unique insights that can be discovered by considering the subtle effects that can be created by the spatial and temporal kinetics of biomolecular reaction networks.

Scale-free flow of life: on the biology, economics, and physics of the cell.

The present work is intended to demonstrate that most of the paradoxes, controversies, and contradictions accumulated in molecular and cell biology over many years of research can be readily resolved if the cell and living systems in general are re-interpreted within an alternative paradigm of biological organization that is based on the concepts and empirical laws of nonequilibrium thermodynamics. In addition to resolving paradoxes and controversies, the proposed re-conceptualization of the cell and biological organization reveals hitherto unappreciated connections among many seemingly disparate phenomena and observations, and provides new and powerful insights into the universal principles governing the emergence and organizational dynamics of living systems on each and every scale of biological organizational hierarchy, from proteins and cells to economies and ecologies.

Chapter 12: Nanoscale biological fluorescence imaging: breaking the diffraction barrier.

Biological imaging has been limited by the finite resolution of light microscopy. Recent developments in ultra-high-resolution microscopy methods, many of which are based on fluorescence, are breaking the diffraction barrier; it is becoming possible to image intracellular protein distributions with resolution of tens of nanometers or better. Fluorescence photoactivation localization microscopy (FPALM) is an example of such an ultra-high-resolution method which can image living or fixed cells with demonstrated lateral resolution of better than 20 nm. A detailed description of the methods involved in FPALM imaging of biological samples is presented here, accompanied by comparison with existing methods from the literature.

Immunohistochemical assessment of parafibromin in mouse and human tissues.

Parafibromin is a protein encoded by the HRPT2 oncosuppressor gene, whose mutation causes the hyperparathyroidism-jaw tumour syndrome, characterized by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours, and renal neoplastic and non-neoplastic abnormalities. Non-morphological techniques, such as Northern and Western blotting and reverse transcriptase-PCR, indicate that parafibromin is ubiquitously expressed, but extensive immunohistochemical studies have not been performed. To increase our knowledge of the distribution and patterns of expression of parafibromin, we examined its expression and location in many different mouse and human organs by immunohistochemistry. There were no substantial differences in parafibromin expression between mouse and human. We found widespread expression of parafibromin, except in connective tissue, smooth muscle, endothelium and some other types of epithelia (colonic, urinary, tubaric, uterine, thyroid). Heterogeneity of positivity intensity and subcellular location (nuclear, nucleocytoplasmic, cytoplasmic) was found between tissues and cell types, suggesting differential functional involvement of parafibromin. Moreover, higher parafibromin expression was found in cell types, such as hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis, which are characterized by different proliferative capacity, thus indicating that the cellular function of parafibromin may not be reduced only to its anti-proliferative effect.

Dermal exposure assessment of polycyclic aromatic hydrocarbons: in vitro percutaneous penetration from coal dust.

To understand the dermal uptake of chemicals bound to soil and dust, information on the neat substance is helpful but does not seem sufficient. Because of its content of polycyclic aromatic hydrocarbons (PAHs) coal is suspected to be carcinogenic. However, experimental carcinogenity studies on coal dust never succeeded in demonstrating a higher incidence of cancer in treated animals. The aim of the study was to assess dermal penetration through human skin of PAHs from coal dust. A sample of coal dust was grinded and sieved, using the particle size of < 30 microm. An in vitro static diffusion cell system validated by in vitrolin vivo comparison has been used to study dermal penetration through human skin of PAHs from coal dust compared with their percutaneous absorption as pure compounds. No percutaneous penetration of PAHs was observed in the cells where coal dust was applied, while dermal penetration was demonstrated for PAHs applied in an acetone solution. Results agree with the literature that PAHs are poorly absorbed through the skin from solids. Dermal risk assessment of PAHs should take into account not only the degree and the extent of skin contamination, but also their bioavailability, which is heavily influenced by the physico-chemical characteristics of the matrix.