RESUMEN
Cell development and behavior are driven by internal genetic programming, but the external microenvironment is increasingly recognized as a significant factor in cell differentiation, migration, and in the case of cancer, metastatic progression. Yet it remains unclear how the microenvironment influences cell processes, especially when examining cell motility. One factor that affects cell motility is cell mechanics, which is known to be related to substrate stiffness. Examining how cells interact with each other in response to mechanically differential substrates would allow an increased understanding of their coordinated cell motility. In order to probe the effect of substrate stiffness on tumor related cells in greater detail, we created hard-soft-hard (HSH) polydimethylsiloxane (PDMS) substrates with alternating regions of different stiffness (200 and 800 kPa). We then cultured WI-38 fibroblasts and A549 epithelial cells to probe their motile response to the substrates. We found that when the 2 cell types were exposed simultaneously to the same substrate, fibroblasts moved at an increased speed over epithelial cells. Furthermore, the HSH substrate allowed us to physically guide and separate the different cell types based on their relative motile speed. We believe that this method and results will be important in a diversity of areas including mechanical microenvironment, cell motility, and cancer biology.
Asunto(s)
Movimiento Celular/fisiología , Elasticidad/fisiología , Microambiente Tumoral/fisiología , Células A549 , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Dimetilpolisiloxanos , Células Epiteliales/fisiología , Fibroblastos/fisiología , HumanosRESUMEN
Stem cell therapy for tissue repair is a rapidly evolving field and the factors that dictate the physiological responsiveness of stem cells remain under intense investigation. In this study we hypothesized that the mechanical loading history of muscle-derived stem cells (MDSCs) would significantly impact MDSC survival, host tissue angiogenesis, and myocardial function after MDSC transplantation into acutely infarcted myocardium. Mice with acute myocardial infarction by permanent left coronary artery ligation were injected with either nonstimulated (NS) or mechanically stimulated (MS) MDSCs. Mechanical stimulation consisted of stretching the cells with equibiaxial stretch with a magnitude of 10% and frequency of 0.5 Hz. MS cell-transplanted hearts showed improved cardiac contractility, increased numbers of host CD31+ cells, and decreased fibrosis, in the peri-infarct region, compared to the hearts treated with NS MDSCs. MS MDSCs displayed higher vascular endothelial growth factor expression than NS cells in vitro. These findings highlight an important role for cyclic mechanical loading preconditioning of donor MDSCs in optimizing MDSC transplantation for myocardial repair.
Asunto(s)
Infarto del Miocardio/terapia , Trasplante de Células Madre , Células Madre/citología , Estrés Mecánico , Animales , Fenómenos Biomecánicos , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Cicatriz/patología , Cicatriz/fisiopatología , Modelos Animales de Enfermedad , Pruebas de Función Cardíaca , Precondicionamiento Isquémico Miocárdico , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Neovascularización Fisiológica , Estrés Oxidativo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cetuximab is a chimeric monoclonal antibody for the epidermal growth factor receptor (EGFR) that may provide benefit to select cancer patients; however, identification of the characteristics of those patients who may benefit from its use is not complete. The ChemoFx® drug response marker (DRM) is an in vitro assay that can provide drug response data on tumor specimens before any patient treatment is initiated. We determined the feasibility of using the ChemoFx DRM to test tumor samples for sensitivity to cetuximab. We exposed four non-small cell lung carcinoma (NSCLC) cell lines (H358, H520, HCC827, and H1666) to cetuximab and determined their sensitivity using the ChemoFx DRM and, in parallel, EGFR status using immunocytochemistry, Western blotting, and In-Cell Western (TM) analysis. We used the ChemoFx DRM to determine cetuximab sensitivity of primary NSCLC and colorectal tumor samples. The ChemoFx DRM distinguished between cetuximab-sensitive and -resistant cell lines. Cetuximab sensitivity was not dependent on EGFR mutational status; H358 cells were non-responsive to cetuximab yet contain wild-type EGFR, whereas H1666 cells were intermediately responsive to cetuximab and contain wild-type EGFR. HCC827 (EGFR-mutant) cells were intermediately responsive and, as expected, H520 cells (EGFR-null) were non-responsive to cetuximab. ChemoFx-determined cetuximab sensitivity of primary NSCLC and colorectal tumor samples was 9.0% and 7.5%, respectively. Use of the ChemoFx DRM is feasible for determining cetuximab sensitivity. The ChemoFx-determined cetuximab responses of primary NSCLC and colorectal tumor specimens were similar to published response rates of patients to treatment with cetuximab monotherapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias/tratamiento farmacológico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma del Pulmón , Anticuerpos Monoclonales/genética , Bioensayo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Transformada , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Receptores ErbB/genética , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Estudios de Factibilidad , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mutación/genética , Neoplasias/genética , Sensibilidad y EspecificidadRESUMEN
Molecular distribution within living cells is organized through multiscaled compartmentalization that enables specialized processes to occur with high efficiency. The ability to control the chemical environment at a subcellular level is limited due to deficient positional control over the aqueous stimulant. Here, a multilayered microfluidic system built from polydimethylsiloxane to separate chemical stimulants over single living cells vertically through aqueous-phase separation under laminar flow is demonstrated. Cells are cultured on top of single micrometer-scale channels inside a larger channel, allowing labeling of the apical domain of single cells through the main channel with simultaneous and distinct labeling of the basal domain via the lower microchannels. The system is transparent, which allows the use of optical microscopy to investigate the spatiotemporal response of labeled components. By employing this technique, the examination of localized subcellular domain responses in polarization, lipid bilayer mobility, and apical-to-basal signal transduction can be explored.
Asunto(s)
Polaridad Celular , Fibroblastos/citología , Microfluídica/métodos , Animales , Supervivencia Celular , Células Cultivadas , Membrana Dobles de Lípidos/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Fracciones Subcelulares/química , Factores de TiempoRESUMEN
We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.
Asunto(s)
Células Endoteliales/citología , Músculo Esquelético/citología , Adolescente , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Antígeno CD56 , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Niño , Células Clonales , Citometría de Flujo , Humanos , Ratones , Ratones SCID , Persona de Mediana Edad , Músculo Esquelético/fisiología , Neoplasias/patología , Regeneración , Factores de TiempoRESUMEN
Mechanical compression has been shown to impact cell activity; however a need for a single device to perform a broader range of parametric studies exists. We have developed an oscillatory displacement controlled device to uniaxially strain cell constructs under both static and dynamic compression and used this device to investigate gene expression in cell constructs. The device has a wide stroke (0.25-4 mm) and frequency range (0.1-3 Hz) and several loading waveforms are possible. Alginate cellular constructs with embedded equine chondrocytes were tested and viability was maintained for the 24 h test period. Off-line mechanical testing is described and a modulus value of 18.2 +/- 1.3 kPa found for alginate disks which indicates the level of stress achieved with this deformation profile. Static (15% strain) and dynamic (15% strain, 1 Hz, triangle waveform) testing of chondrocyte constructs was performed and static compression showed significantly higher collagen II expression than dynamic using quantitative RT-PCR. In contrast, differences in matrix metalloproteinase-3 (MMP-3) expression were statistically insignificant. These studies indicate the utility of our device for studying cell activity in response to compression and suggest further studies regarding how the load and strain spectrum impact chondrocyte activity.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Condrocitos/citología , Condrocitos/fisiología , Mecanotransducción Celular/fisiología , Oscilometría/instrumentación , Estimulación Física/instrumentación , Animales , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Fuerza Compresiva , Diseño de Equipo , Análisis de Falla de Equipo , Caballos , Oscilometría/métodos , Estimulación Física/métodos , PresiónRESUMEN
The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.
Asunto(s)
Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Animales , Sitios de Unión/fisiología , Bovinos , Agregación Celular/fisiología , Línea Celular , Concentración de Iones de Hidrógeno , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Sustancias Macromoleculares/metabolismo , Mutación/genética , Unión Proteica/fisiología , Transducción de Señal/fisiología , Solubilidad , Propiedades de SuperficieRESUMEN
Rebinding of dissociated ligands from cell surface proteins can confound quantitative measurements of dissociation rates important for characterizing the affinity of binding interactions. This can be true also for in vitro techniques such as surface plasmon resonance (SPR). We present experimental results using SPR for the interaction of insulin-like growth factor-I (IGF-I) with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show that the dissociation, even with the addition of soluble heparin in the dissociation phase, does not exhibit the expected exponential decay characteristic of a 1:1 binding reaction. We thus consider the effect of (multiple) rebinding events and, within a self-consistent mean-field approximation, we derive the complete mathematical form for the fraction of bound ligands as a function of time. We show that, except for very low association rate and surface coverage, this function is nonexponential at all times, indicating that multiple rebinding events strongly influence dissociation even at early times. We compare the mean-field results with numerical simulations and find good agreement, although deviations are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data indicates that rebinding is prominent for this system and that the theoretical predictions fit the experimental data well. Our results provide a means for analyzing SPR biosensor data where rebinding is problematic and a methodology to do so is presented.