RESUMEN

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.

Asunto(s)

Antígenos de Histocompatibilidad Clase I , Neoplasias , Células Presentadoras de Antígenos , Análisis por Conglomerados , Colon , Antígenos de Histocompatibilidad Clase II , Complejo Mayor de Histocompatibilidad

RESUMEN

Metabolomics is an emerging field of cell biology that aims at the comprehensive identification of metabolite levels in biological fluids or cells in a specific functional state. Currently, the major tools for determining metabolite concentrations are mass spectrometry coupled with chromatographic techniques and nuclear magnetic resonance, which are expensive, time consuming and destructive for the samples. Here, we report a time resolved approach to monitor metabolite dynamics in cell cultures, based on Surface Enhanced Raman Scattering (SERS). This method is label-free, easy to use and provides the opportunity to simultaneously study a broad range of molecules, without the need to process the biological samples. As proof of concept, NIH/3T3 cells were cultured in vitro, and the extracellular medium was collected at different time points to be analyzed with our engineered SERS substrates. By identifying individual peaks of the Raman spectra, we showed the simultaneous detection of several components of the conditioned medium, such as L-tyrosine, L-tryptophan, glycine, L-phenylalanine, L-histidine and fetal bovine serum proteins, as well as their intensity changes during time. Furthermore, analyzing the whole Raman data set with the Principal Component Analysis (PCA), we demonstrated that the Raman spectra collected at different days of culture and clustered by similarity, described a well-defined trajectory in the principal component plot. This approach was then utilized to determine indirectly the functional state of the macrophage cell line Raw 264.7, stimulated with the lipopolysaccharide (LPS) for 24 hours. The collected spectra at different time points, clustered by the PCA analysis, followed a well-defined trajectory, corresponding to the functional change of cells toward the activated pro-inflammatory state induced by the LPS. This study suggests that our engineered SERS surfaces can be used as a versatile tool both for the characterization of cell culture conditions and the functional state of cells over time.

Asunto(s)

Espacio Extracelular/metabolismo , Macrófagos/metabolismo , Espectrometría Raman/métodos , Aminoácidos/metabolismo , Animales , Bovinos , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Subunidad p40 de la Interleucina-12/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Nanopartículas del Metal/química , Ratones , Microscopía Electrónica de Rastreo , Células 3T3 NIH , Análisis de Componente Principal , Rodaminas/química , Albúmina Sérica Bovina/metabolismo , Plata/química , Propiedades de Superficie , Factores de Tiempo

RESUMEN

A Universal plasmonic/microfluidic platform for spatial and temporal controlled intracellular delivery is described. The system can inject/transfect the desired amount of molecules with an efficacy close to 100%. Moreover, it is highly scalable from single cells to large ensembles without administering the molecules to an extracellular bath. The latter enables quantitative control over the amount of injected molecules.

Asunto(s)

Sistemas de Liberación de Medicamentos/métodos , Nanotubos , Animales , Sistemas de Liberación de Medicamentos/instrumentación , Espacio Intracelular/metabolismo , Dispositivos Laboratorio en un Chip , Rayos Láser , Ratones , Células 3T3 NIH , Imagen Óptica , Análisis Espacio-Temporal

RESUMEN

Out-of-plane plasmonic nanoantennas protruding from the substrate are exploited to perform very sensitive surface enhanced Raman scattering analysis of living cells. Cells cultured on three-dimensional surfaces exhibit tight adhesion with nanoantenna tips where the plasmonic hot-spot resides. This fact provides observable cell adhesion sites combined with high plasmonic enhancement, resulting in an ideal system for Raman investigation of cell membranes.

Asunto(s)

Nanoestructuras , Espectrometría Raman/métodos , Actinas/química , Animales , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Campos Electromagnéticos , Oro/química , Nanopartículas del Metal , Ratones , Microscopía Confocal , Microscopía Electrónica de Rastreo , Células 3T3 NIH , Nanotecnología , Tamaño de la Partícula , Plata/química , Programas Informáticos , Espectroscopía Infrarroja Corta , Resonancia por Plasmón de Superficie

RESUMEN

Neuronal signaling in brain circuits occurs at multiple scales ranging from molecules and cells to large neuronal assemblies. However, current sensing neurotechnologies are not designed for parallel access of signals at multiple scales. With the aim of combining nanoscale molecular sensing with electrical neural activity recordings within large neuronal assemblies, in this work three-dimensional (3D) plasmonic nanoantennas are integrated with multielectrode arrays (MEA). Nanoantennas are fabricated by fast ion beam milling on optical resist; gold is deposited on the nanoantennas in order to connect them electrically to the MEA microelectrodes and to obtain plasmonic behavior. The optical properties of these 3D nanostructures are studied through finite elements method (FEM) simulations that show a high electromagnetic field enhancement. This plasmonic enhancement is confirmed by surface enhancement Raman spectroscopy of a dye performed in liquid, which presents an enhancement of almost 100 times the incident field amplitude at resonant excitation. Finally, the reported MEA devices are tested on cultured rat hippocampal neurons. Neurons develop by extending branches on the nanostructured electrodes and extracellular action potentials are recorded over multiple days in vitro. Raman spectra of living neurons cultured on the nanoantennas are also acquired. These results highlight that these nanostructures could be potential candidates for combining electrophysiological measures of large networks with simultaneous spectroscopic investigations at the molecular level.

Asunto(s)

Técnicas Biosensibles/métodos , Nanotecnología/métodos , Potenciales de Acción , Animales , Células Cultivadas , Simulación por Computador , Electrodos , Análisis de Elementos Finitos , Oro/química , Hipocampo/patología , Microscopía Electrónica de Rastreo , Nanoestructuras/química , Neuronas/metabolismo , Neuronas/patología , Óptica y Fotónica , Ratas , Espectrometría Raman/métodos , Resonancia por Plasmón de Superficie/métodos

RESUMEN

The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized.

Asunto(s)

Modelos Neurológicos , Red Nerviosa/citología , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología , Red Nerviosa/fisiología , Neuroblastoma/patología , Neuronas/citología , Neuronas/fisiología , Porosidad , Silicio , Propiedades de Superficie

RESUMEN

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Asunto(s)

Neoplasias Colorrectales/patología , Células Madre Neoplásicas/patología , Espectrometría Raman/métodos , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Humanos , Gotas Lipídicas , Ratones , Células Madre Neoplásicas/metabolismo , Vía de Señalización Wnt

RESUMEN

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1), indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

Asunto(s)

Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Membrana Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fenotipo , Espectrometría Raman , Estrés Mecánico , Escape del Tumor

RESUMEN

We have fabricated anodic porous alumina from thin films (100/500 nm) of aluminium deposited on technological substrates of silicon/glass, and investigated the feasibility of this material as a surface for the development of analytical biosensors aiming to assess the status of living cells. To this goal, porous alumina surfaces with fixed pitch and variable pore size were analyzed for various functionalities. Gold coated (about 25 nm) alumina revealed surface enhanced Raman scattering increasing with the decrease in wall thickness, with factor up to values of approximately 10(4) with respect to the flat gold surface. Bare porous alumina was employed for micro-patterning and observation via fluorescence images of dye molecules, which demonstrated the surface capability for a drug-loading device. NIH-3T3 fibroblast cells were cultured in vitro and examined after 2 days since seeding, and no significant (P > 0.05) differences in their proliferation were observed on porous and non-porous materials. The effect on cell cultures of pore size in the range of 50-130 nm--with pore pitch of about 250 nm--showed no significant differences in cell viability and similar levels in all cases as on a control substrate. Future work will address combination of all above capabilities into a single device.

Asunto(s)

Óxido de Aluminio/química , Técnicas Biosensibles , Técnicas de Cultivo de Célula/instrumentación , Animales , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Adhesión Celular , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/química , Estudios de Factibilidad , Oro/química , Ensayo de Materiales , Ratones , Células 3T3 NIH , Nanoestructuras/química , Porosidad , Propiedades de Superficie

RESUMEN

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 µL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

Asunto(s)

Técnicas Citológicas/instrumentación , Técnicas Citológicas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Anticuerpos/química , Línea Celular Tumoral , Supervivencia Celular/fisiología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Fenotipo , Propiedades de Superficie

RESUMEN

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Asunto(s)

Adenocarcinoma/inmunología , Neoplasias del Colon/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Células Madre Neoplásicas/inmunología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Linaje de la Célula/inmunología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Citotoxicidad Inmunológica , Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Confocal , Receptor 2 Gatillante de la Citotoxidad Natural/genética , Receptor 3 Gatillante de la Citotoxidad Natural/genética , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Especificidad de Órganos , Células Tumorales Cultivadas

RESUMEN

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Células Asesinas Naturales/inmunología , Técnicas Analíticas Microfluídicas/instrumentación , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Unión Proteica

RESUMEN

Porous silicon (PSi) is a promising material in several biomedical applications because of its biocompatibility and biodegradability. Despite the plethora of studies focusing on the interaction of cells with micrometer and submicro geometrical features, limited information is available on the response of cells to substrates with a quasi-regular distribution of nanoscopic pores. Here, the behavior of four different cell types is analyzed on two mesoporous (MeP) silicon substrates, with an average pore size of ∼5 (MeP1) and ∼20 nm (MeP2), respectively. On both MeP substrates, cells are observed to spread and adhere in a larger number as compared to flat silicon wafers. At all considered time points, the surface density of the adhering cells nd is larger on the PSi substrate with the smaller average pore size (MeP1). At 60 h, nd is from ∼1.5 to 5 times larger on MeP1 than on MeP2 substrates, depending on the cell type. The higher rates of proliferation are observed for the two neuronal cell types, the mouse neuroblastoma cells (N2A) and the immortalized human cortical neuronal cells (HCN1A). It is speculated that the higher adhesion on MeP1 could be attributed to a preferential matching of the substrate topography with the recently observed multiscale molecular architecture of focal adhesions. These results have implications in the rational development of PSi substrates for supporting cell adhesion and controlling drug release in implants and scaffolds for tissue engineering applications.

Asunto(s)

Silicio/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Microscopía Confocal , Porosidad , Ingeniería de Tejidos , Vinculina/metabolismo

RESUMEN

Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (alpha1, alpha2, and alpha3) once folded by peptide and beta(2)-microglobulin show the presence of two alpha-helix streams and one beta-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+beta(2)-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm(-1), the intensity variation of cells associated with HLA class I molecules could be measured.

Asunto(s)

Antígenos de Histocompatibilidad Clase I/sangre , Leucocitos/inmunología , Espectrometría Raman/métodos , Células Cultivadas , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells.

Asunto(s)

Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Animales , Presentación de Antígeno/inmunología , Línea Celular , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Ligandos , Neoplasias/genética , Transcripción Genética/inmunología , Transgenes/genética , Regulación hacia Arriba