Nuestros investigadores

Carlos Ortiz de Solórzano Aurusa

Publicaciones científicas más recientes (desde 2010)

Autores: Fernández-Calvet, A.; Rodríguez-Arce, I.; Almagro, G.; et al.
Revista: SCIENTIFIC REPORTS
ISSN 2045-2322  Vol. 8  Nº 1  2018  págs. 6872
Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.
Autores: Paino, T.; Garcia-Gomez, A.; Gonzalez-Mendez, L.; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 23  Nº 1  2017  págs. 225 - 238
Purpose: PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models. Experimental Design: In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies. Results: PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide-dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma. Conclusions: Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma. (C) 2016 AACR.
Autores: Anguiano, M.; Castilla, C.; Maška, M.; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 12  Nº 2  2017  págs. e0171417
Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.
Autores: Peláez, R.; Garasa, S.; Ortiz de Solórzano, Carlos; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 12  Nº 8  2017  págs. e0181579
Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether ß3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-ß induction of ß3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-ß exposure. Invadopodia formation and degradation activity is dependent on ß3 integrin expression since ß3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in ß3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for ß3 integrin in invadopodia. Our results suggest that ß3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. ß3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.
Autores: Ortiz de Solórzano, Carlos; Meijering, E.; Kozubek, M. ;
Revista: IEEE SIGNAL PROCESSING MAGAZINE
ISSN 1053-5888  Vol. 32  Nº 1  2015  págs. 20-29
From a systems biology perspective, the cell is the principal element of information integration. Therefore, understanding the cell in its spatiotemporal context is the key to unraveling many of the still unknown mechanisms of life and disease. This article reviews image processing aspects relevant to the quantification of cell morphology and dynamics. We cover both acquisition (hardware) and analysis (software) related issues, in a multiscale fashion, from the detection of cellular components to the description of the entire cell in relation to its extracellular environment. We then describe ongoing efforts to integrate all this vast and diverse information along with data about the biomechanics of the cell to create a credible model of cell morphology and behavior.
Autores: Jorge-Peñas, A.; de Juan, María Elena; Ortiz de Solórzano, Carlos;
Revista: COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING
ISSN 1025-5842  Vol. 18  Nº 13  2015  págs. 1377 - 1385
Traction force microscopy (TFM) is commonly used to estimate cells' traction forces from the deformation that they cause on their substrate. The accuracy of TFM highly depends on the computational methods used to measure the deformation of the substrate and estimate the forces, and also on the specifics of the experimental set-up. Computer simulations can be used to evaluate the effect of both the computational methods and the experimental set-up without the need to perform numerous experiments. Here, we present one such TFM simulator that addresses several limitations of the existing ones. As a proof of principle, we recreate a TFM experimental set-up, and apply a classic 2D TFM algorithm to recover the forces. In summary, our simulator provides a valuable tool to study the performance, refine experimentally, and guide the extraction of biological conclusions from TFM experiments.
Autores: Jorge-Peñas, A.; de Juan, María Elena; Ortiz de Solórzano, Carlos;
Revista: COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING
ISSN 1025-5842  Vol. 18  Nº 13  2015  págs. 1377 - 1385
Traction force microscopy (TFM) is commonly used to estimate cells' traction forces from the deformation that they cause on their substrate. The accuracy of TFM highly depends on the computational methods used to measure the deformation of the substrate and estimate the forces, and also on the specifics of the experimental set-up. Computer simulations can be used to evaluate the effect of both the computational methods and the experimental set-up without the need to perform numerous experiments. Here, we present one such TFM simulator that addresses several limitations of the existing ones. As a proof of principle, we recreate a TFM experimental set-up, and apply a classic 2D TFM algorithm to recover the forces. In summary, our simulator provides a valuable tool to study the performance, refine experimentally, and guide the extraction of biological conclusions from TFM experiments.
Autores: Castellanos, G.; Fernández-Seara, M. A.; Lorenzo-Betancor, O.; et al.
Revista: MOVEMENT DISORDERS
ISSN 0885-3185  Vol. 30  Nº 7  2015  págs. 945-952
BACKGROUND: We aimed to analyze the diagnostic accuracy of an automated segmentation and quantification method of the SNc and locus coeruleus (LC) volumes based on neuromelanin (NM)-sensitive MRI (NM-MRI) in patients with idiopathic (iPD) and monogenic (iPD) Parkinson's disease (PD). METHODS: Thirty-six patients (23 idiopathic and 13 monogenic PARKIN or LRRK2 mutations) and 37 age-matched healthy controls underwent 3T-NM-MRI. SNc and LC volumetry were performed using fully automated multi-image atlas segmentation. The diagnostic performance to differentiate PD from controls was measured using the area under the curve (AUC) and likelihood ratios based on receiver operating characteristic (ROC) analyses. RESULTS: We found a significant reduction of SNc and LC volumes in patients, when compared to controls. ROC analysis showed better diagnostic accuracy when using SNc volume than LC volume. Significant differences between ipsilateral and contralateral SNc volumes, in relation to the more clinically affected side, were found in patients with iPD (P¿=¿0.007). Contralateral atrophy in the SNc showed the highest power to discriminate PD subjects from controls (AUC, 0.93-0.94; sensitivity, 91%-92%; specificity, 89%; positive likelihood ratio: 8.4-8.5; negative likelihood ratio: 0.09-0.1 at a single cut-off point). Interval likelihood ratios for contralateral SNc volume improved the diagnostic accuracy of volumetric measurements. CONCLUSION: SNc and LC volumetry based on NM-MRI resulting from the automated segmentation and quantification technique can yield high diagnostic accuracy for differentiating PD from health and might be an unbiased disease biomarker. © 2015 International Parkinson and Movement Disorder Society.
Autores: Marcos, J. V. ; Ortiz de Solórzano, Carlos; Cristóbal, G. ;
Revista: ANNALS OF BIOMEDICAL ENGINEERING
ISSN 0090-6964  Vol. 43  Nº 10  2015  págs. 2515 - 2529
Emphysema is a characteristic component of chronic obstructive pulmonary disease (COPD), which has been pointed out as one of the main causes of mortality for the next years. Animal models of emphysema are employed to study the evolution of this disease as well as the effect of treatments. In this context, measures such as the mean linear intercept [Formula: see text] and the equivalent diameter [Formula: see text] have been proposed to quantify the airspace enlargement associated with emphysematous lesions in histological sections. The parameter [Formula: see text], which relates the second and the third moments of the variable [Formula: see text], has recently shown to be a robust descriptor of airspace enlargement. However, the value of [Formula: see text] does not provide a direct evaluation of emphysema severity. In our research, we suggest a Bayesian approach to map [Formula: see text] onto a novel emphysema severity index (SI) reflecting the probability for a lung area to be emphysematous. Additionally, an image segmentation procedure was developed to compute the severity map of a lung section using the SI function. Severity maps corresponding to 54 lung sections from control mice, mice induced with mild emphysema and mice induced with severe emphysema were computed, revealing differences between the distribution of SI in the three groups. The proposed methodology could then assist in the quantification of emphysema severity in animal models of pulmonary disease.
Autores: Jorge-Peñas, A; de Juan, María Elena; Ortiz de Solórzano, Carlos;
Revista: COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING
ISSN 1025-5842  Vol. 18  Nº 13  2015  págs. 1377 - 1385
Traction force microscopy (TFM) is commonly used to estimate cells' traction forces from the deformation that they cause on their substrate. The accuracy of TFM highly depends on the computational methods used to measure the deformation of the substrate and estimate the forces, and also on the specifics of the experimental set-up. Computer simulations can be used to evaluate the effect of both the computational methods and the experimental set-up without the need to perform numerous experiments. Here, we present one such TFM simulator that addresses several limitations of the existing ones. As a proof of principle, we recreate a TFM experimental set-up, and apply a classic 2D TFM algorithm to recover the forces. In summary, our simulator provides a valuable tool to study the performance, refine experimentally, and guide the extraction of biological conclusions from TFM experiments.
Autores: Baumgartner, S.; Rodriguez-Madoz, Juan Roberto; et al.
Revista: STEM CELLS AND DEVELOPMENT
ISSN 1547-3287  Vol. 24  Nº 4  2015  págs. 484 - 496
Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1 beta (NRG-1 beta) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from alpha-MHC-GFP mice were derived and differentiated into CMs through NRG-1 beta and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1 beta and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1 beta and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.
Autores: Anguiano M; Castilla C; Maska M; et al.
Revista: CONFERENCE PROCEEDINGS (IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. CONF.)
ISSN 1557-170X  Vol. 2015  2015  págs. 8139 - 8142
The geometry of 3D collagen networks is a key factor that influences the behavior of live cells within extra-cellular matrices. This paper presents a method for automatic quantification of the 3D collagen network geometry with fiber resolution in confocal reflection microscopy images. The proposed method is based on a smoothing filter and binarization of the collagen network followed by a fiber reconstruction algorithm. The method is validated on 3D collagen gels with various collagen and Matrigel concentrations. The results reveal that Matrigel affects the collagen network geometry by decreasing the network pore size while preserving the fiber length and fiber persistence length. The influence of network composition and geometry, especially pore size, is preliminarily analyzed by quantifying the migration patterns of lung cancer cells within microfluidic devices filled with three different hydrogel types. The experiments reveal that Matrigel, while decreasing pore size, stimulates cell migration. Further studies on this relationship could be instrumental for the study of cancer metastasis and other biological processes involving cell migration.
Autores: Anguiano, M.; Maska, M.; et al.
Revista: CONFERENCE PROCEEDINGS (IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. CONF.)
ISSN 1557-170X  Vol. 2015  2015  págs. 8139 - 8142
The geometry of 3D collagen networks is a key factor that influences the behavior of live cells within extra-cellular matrices. This paper presents a method for automatic quantification of the 3D collagen network geometry with fiber resolution in confocal reflection microscopy images. The proposed method is based on a smoothing filter and binarization of the collagen network followed by a fiber reconstruction algorithm. The method is validated on 3D collagen gels with various collagen and Matrigel concentrations. The results reveal that Matrigel affects the collagen network geometry by decreasing the network pore size while preserving the fiber length and fiber persistence length. The influence of network composition and geometry, especially pore size, is preliminarily analyzed by quantifying the migration patterns of lung cancer cells within microfluidic devices filled with three different hydrogel types. The experiments reveal that Matrigel, while decreasing pore size, stimulates cell migration. Further studies on this relationship could be instrumental for the study of cancer metastasis and other biological processes involving cell migration.
Autores: Chenouard, N. ; Smal, I. ; de Chaumont, F. ; et al.
Revista: NATURE METHODS
ISSN 1548-7091  Vol. 11  Nº 3  2014  págs. 281-289
Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.
Autores: Maška, M. ; Ulman, V. ; Svoboda, D. ; et al.
Revista: BIOINFORMATICS
ISSN 1367-4803  Vol. 30  Nº 11  2014  págs. 1609-1617
MOTIVATION: Automatic tracking of cells in multidimensional time-lapse fluorescence microscopy is an important task in many biomedical applications. A novel framework for objective evaluation of cell tracking algorithms has been established under the auspices of the IEEE International Symposium on Biomedical Imaging 2013 Cell Tracking Challenge. In this article, we present the logistics, datasets, methods and results of the challenge and lay down the principles for future uses of this benchmark. RESULTS: The main contributions of the challenge include the creation of a comprehensive video dataset repository and the definition of objective measures for comparison and ranking of the algorithms. With this benchmark, six algorithms covering a variety of segmentation and tracking paradigms have been compared and ranked based on their performance on both synthetic and real datasets. Given the diversity of the datasets, we do not declare a single winner of the challenge. Instead, we present and discuss the results for each individual dataset separately. AVAILABILITY AND IMPLEMENTATION: The challenge Web site (http://www.codesolorzano.com/celltrackingchallenge) provides access to the training and competition datasets, along with the ground truth of the training videos. It also provides access to Windows and Linux executable files of the evaluation software and most of the algorithms that competed in the challenge.
Autores: Rudyanto, R. D. ; Kerkstra, S.; van Rikxoort, E. M. ; et al.
Revista: MEDICAL IMAGE ANALYSIS
ISSN 1361-8423  Vol. 18  Nº 7  2014  págs. 1217-1232
The VESSEL12 (VESsel SEgmentation in the Lung) challenge objectively compares the performance of different algorithms to identify vessels in thoracic computed tomography (CT) scans. Vessel segmentation is fundamental in computer aided processing of data generated by 3D imaging modalities. As manual vessel segmentation is prohibitively time consuming, any real world application requires some form of automation. Several approaches exist for automated vessel segmentation, but judging their relative merits is difficult due to a lack of standardized evaluation. We present an annotated reference dataset containing 20 CT scans and propose nine categories to perform a comprehensive evaluation of vessel segmentation algorithms from both academia and industry. Twenty algorithms participated in the VESSEL12 challenge, held at International Symposium on Biomedical Imaging (ISBI) 2012. All results have been published at the VESSEL12 website http://vessel12.grand-challenge.org. The challenge remains ongoing and open to new participants. Our three contributions are: (1) an annotated reference dataset available online for evaluation of new algorithms; (2) a quantitative scoring system for objective comparison of algorithms; and (3) performance analysis of the strengths and weaknesses of the various vessel segmentation methods in the presence of various lung diseases.
Autores: Garcia-Gomez, A. ; Quwaider, D. ; Canavese, M. ; et al.
Revista: CLINICAL CANCER RESEARCH
ISSN 1078-0432  Vol. 20  Nº 6  2014  págs. 1542-1554
PURPOSE: MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models. EXPERIMENTAL DESIGN: The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities. RESULTS: Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-¿B ligand)-induced NF-¿B activation, F-actin ring disruption, and diminished expression of ¿Vß3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/ß-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow-disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). CONCLUSION: Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma.
Autores: Ortiz de Solórzano, Carlos;
Revista: IEEE SYSTEMS JOURNAL
ISSN 1932-8184  Vol. 8  Nº 3  2014  págs. 985 - 994
Lung cancer is the deadliest form of cancer mainly because of the absence of reliable early diagnostic protocols. Therefore, there is increasing interest in the development of novel diagnostic noninvasive technologies that may improve the early detection of the disease. Bronchoscope-guided bronchoalveolar lavage (BAL) is a minimally invasive diagnostic technique that is based on the extraction and analysis of cellular material from the bronchial epithelium of patients that present suspicious lung masses on low-dose screening X-ray-computed tomography images. Together with a novel staining technique that combines immunophenotyping of a lung cancer biomarker with fluorescent in situ hybridization of genetically abnormal DNA loci, BAL promises a powerful early diagnostic tool for lung carcinomas. The sensitivity of this method, however, is highly dependent on the pathologist's ability to reliably and repeatedly examine thousands of cells under the microscope. This is an extremely labor-intensive and error-prone task. We have developed a multiscale multidimensional integrated microscopy computer-aided detection platform that autonomously scans and analyzes BAL samples. In this paper, we describe its software architecture and validate the specific image analysis protocols that are developed for this particular application.
Autores: Ortiz de Solórzano, Carlos;
Revista: IEEE SYSTEMS JOURNAL
ISSN 1932-8184  Vol. 8  Nº 3  2014  págs. 985-994
Lung cancer is the deadliest form of cancer mainly because of the absence of reliable early diagnostic protocols. Therefore, there is increasing interest in the development of novel diagnostic noninvasive technologies that may improve the early detection of the disease. Bronchoscope-guided bronchoalveolar lavage (BAL) is a minimally invasive diagnostic technique that is based on the extraction and analysis of cellular material from the bronchial epithelium of patients that present suspicious lung masses on low-dose screening X-ray-computed tomography images. Together with a novel staining technique that combines immunophenotyping of a lung cancer biomarker with fluorescent in situ hybridization of genetically abnormal DNA loci, BAL promises a powerful early diagnostic tool for lung carcinomas. The sensitivity of this method, however, is highly dependent on the pathologist's ability to reliably and repeatedly examine thousands of cells under the microscope. This is an extremely labor-intensive and error-prone task. We have developed a multiscale multidimensional integrated microscopy computer-aided detection platform that autonomously scans and analyzes BAL samples. In this paper, we describe its software architecture and validate the specific image analysis protocols that are developed for this particular application.
Autores: Palacio, J. ; Jorge-Peñas, A. ; Ortiz de Solórzano, Carlos; et al.
Revista: JOURNAL OF BIOMECHANICS
ISSN 0021-9290  Vol. 46  Nº 1  2013  págs. 50-55
The exchange of physical forces in both cell-cell and cell-matrix interactions play a significant role in a variety of physiological and pathological processes, such as cell migration, cancer metastasis, inflammation and wound healing. Therefore, great interest exists in accurately quantifying the forces that cells exert on their substrate during migration. Traction Force Microscopy (TFM) is the most widely used method for measuring cell traction forces. Several mathematical techniques have been developed to estimate forces from TFM experiments. However, certain simplifications are commonly assumed, such as linear elasticity of the materials and/or free geometries, which in some cases may lead to inaccurate results. Here, cellular forces are numerically estimated by solving a minimization problem that combines multiple non-linear FEM solutions. Our simulations, free from constraints on the geometrical and the mechanical conditions, show that forces are predicted with higher accuracy than when using the standard approaches.
Autores: de Torres, Juan Pablo; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 4  2013  págs. e60260
Current or former smokers expressing a well-defined disease characteristic such as emphysema, has a specific plasma cytokine profile. This includes a decrease of cytokines mainly implicated in activation of apoptosis or decrease of immunosurveillance. This information should be taken into account when evaluated patients with tobacco respiratory diseases
Autores: Ortiz de Solórzano, Carlos;
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 11  2013  págs. e78504
The ample variety of labeling dyes and staining methods available in fluorescence microscopy has enabled biologists to advance in the understanding of living organisms at cellular and molecular level. When two or more fluorescent dyes are used in the same preparation, or one dye is used in the presence of autofluorescence, the separation of the fluorescent emissions can become problematic. Various approaches have been recently proposed to solve this problem. Among them, blind non-negative matrix factorization is gaining interest since it requires little assumptions about the spectra and concentration of the fluorochromes. In this paper, we propose a novel algorithm for blind spectral separation that addresses some of the shortcomings of existing solutions: namely, their dependency on the initialization and their slow convergence. We apply this new algorithm to two relevant problems in fluorescence microscopy: autofluorescence elimination and spectral unmixing of multi-labeled samples. Our results show that our new algorithm performs well when compared with the state-of-the-art approaches for a much faster implementation.
Autores: Bastarrika, Gorka; Agorreta, J; et al.
Revista: MEDICAL IMAGE ANALYSIS
ISSN 1361-8423  Vol. 17  Nº 8  2013  págs. 1095-1105
We present and evaluate an automatic and quantitative method for the complex task of characterizing individual nodule volumetric progression in a longitudinal mouse model of lung cancer. Fourteen A/J mice received an intraperitoneal injection of urethane. Respiratory-gated micro-CT images of the lungs were acquired at 8, 22, and 37 weeks after injection. A radiologist identified a total of 196, 585 and 636 nodules, respectively. The three micro-CT image volumes from every animal were then registered and the nodules automatically matched with an average accuracy of 99.5%. All nodules detected at week 8 were tracked all the way to week 37, and volumetrically segmented to measure their growth and doubling rates. 92.5% of all nodules were correctly segmented, ranging from the earliest stage to advanced stage, where nodule segmentation becomes more challenging due to complex anatomy and nodule overlap. Volume segmentation was validated using a foam lung phantom with embedded polyethylene microspheres. We also correlated growth rates with nodule phenotypes based on histology, to conclude that the growth rate of malignant tumors is significantly higher than that of benign lesions. In conclusion, we present a turnkey solution that combines longitudinal imaging with nodule matching and volumetric nodule segmentation resulting in a powerful tool for preclinical research.
Autores: Palacio, J.; Jorge-Penas, A.; Ortiz de Solórzano, Carlos; et al.
Revista: JOURNAL OF BIOMECHANICS
ISSN 0021-9290  Vol. 46  Nº 1  2013  págs. 50 - 55
The exchange of physical forces in both cell-cell and cell-matrix interactions play a significant role in a variety of physiological and pathological processes, such as cell migration, cancer metastasis, inflammation and wound healing. Therefore, great interest exists in accurately quantifying the forces that cells exert on their substrate during migration. Traction Force Microscopy (TFM) is the most widely used method for measuring cell traction forces. Several mathematical techniques have been developed to estimate forces from TFM experiments. However, certain simplifications are commonly assumed, such as linear elasticity of the materials and/or free geometries, which in some cases may lead to inaccurate results. Here, cellular forces are numerically estimated by solving a minimization problem that combines multiple non-linear FEM solutions. Our simulations, free from constraints on the geometrical and the mechanical conditions, show that forces are predicted with higher accuracy than when using the standard approaches. (C) 2012 Elsevier Ltd. All rights reserved.
Autores: Marcos, T. ; Serrano, Diego; et al.
Revista: INTEGRATIVE BIOLOGY
ISSN 1093-4391  Vol. 5  Nº 2  2013  págs. 402-413
Dyskerin is one of the three subunits of the telomerase ribonucleoprotein (RNP) complex. Very little is known about the role of dyskerin in the biology of the telomeres in cancer cells. In this study, we use a quantitative, multiscale 3D image-based in situ method and several molecular techniques to show that dyskerin is overexpressed in lung cancer cell lines. Furthermore, we show that dyskerin expression correlates with telomere length both at the cell population level--cells with higher dyskerin expression have short telomeres--and at the single cell level--the shortest telomeres of the cell are spatially associated with areas of concentration of dyskerin proteins. Using this in vitro model, we also show that exogenous increase in dyskerin expression confers resistance to telomere shortening caused by a telomerase inactivating drug. Finally, we show that resistance is achieved by the recovery of telomerase activity associated with dyskerin. In summary, using a novel multiscale image-based in situ method, we show that, in lung cancer cell lines, dyskerin responds to continuous telomere attrition by increasing the telomerase RNP activity, which in turn provides resistance to telomere shortening.
Autores: Danek, O.; Garasa, S.; Rouzaut, Ana; et al.
Revista: IEEE TRANSACTIONS ON MEDICAL IMAGING
ISSN 0278-0062  Vol. 32  Nº 6  2013  págs. 995 - 1006
We present a fast and robust approach to tracking the evolving shape of whole fluorescent cells in time-lapse series. The proposed tracking scheme involves two steps. First, coherence-enhancing diffusion filtering is applied on each frame to reduce the amount of noise and enhance flow-like structures. Second, the cell boundaries are detected by minimizing the Chan-Vese model in the fast level set-like and graph cut frameworks. To allow simultaneous tracking of multiple cells over time, both frameworks have been integrated with a topological prior exploiting the object indication function. The potential of the proposed tracking scheme and the advantages and disadvantages of both frameworks are demonstrated on 2-D and 3-D time-lapse series of rat adipose-derived mesenchymal stem cells and human lung squamous cell carcinoma cells, respectively.
Autores: Simon-Yarza, T; et al.
Revista: INTERNATIONAL JOURNAL OF PHARMACEUTICS
ISSN 0378-5173  Vol. 454  Nº 2  2013  págs. 784 -790
Autores: Sandra Hervas-Stubbs; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 23  Nº 12  2012  págs. 1258-1268
Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.
Autores: Belza, Idoia; et al.
Revista: ONCOIMMUNOLOGY
ISSN 2162-4011  Vol. 1  Nº 9  2012  págs. 1527-36
Cardiotrophin-1 (CT-1/CTF1) is a member of the interleukin-6 (IL-6) family of cytokines that stimulates STAT-3 phosphorylation in cells bearing the cognate receptor. We report that Ctf1(-/-) mice (hereby referred to as CT-1(-/-) mice) are resistant to the hepatic engraftment of MC38 colon carcinoma cells, while these cells engraft normally in the mouse subcutaneous tissue. Tumor intake in the liver could be enhanced by the systemic delivery of a recombinant adenovirus encoding CT-1, which also partly rescued the resistance of CT-1(-/-) mice to the hepatic engraftment of MC38 cells. Moreover, systemic treatment of wild-type (WT) mice with a novel antibody-neutralizing mouse CT-1 also reduced engraftment of this model. Conversely, experiments with Panc02 pancreatic cancer and B16-OVA melanoma cells in CT-1(-/-) mice revealed rates of hepatic engraftment similar to those observed in WT mice. The mechanism whereby CT-1 renders the liver permissive for MC38 metastasis involves T lymphocytes and natural killer (NK) cells, as shown by selective depletion experiments and in genetically deficient mice. However, no obvious changes in the number or cell killing capacity of liver lymphocytes in CT-1(-/-) animals could be substantiated. These findings demonstrate that the seed and soil concept to understand metastasis can be locally influenced by cytokines as well as by the cellular immune system
Autores: Corbacho, D.; Pelacho, Beatriz; Albiasu, E.; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 7  Nº 7  2012  págs. e41691
Background: The aim of this article is to present an optimized acquisition and analysis protocol for the echocardiographic evaluation of left ventricle (LV) remodeling in a mouse model of myocardial infarction (MI). Methodology: 13 female DBA/2J mice underwent permanent occlusion of the left anterior descending (LAD) coronary artery leading to MI. Mice echocardiography was performed using a Vevo 770 (Visualsonics, Canada) before infarction, and 7, 14, 30, 60, 90 and 120 days after LAD ligation. LV systolic function was evaluated using different parameters, including the fractional area change (FAC%) computed in four high-temporal resolution B-mode short axis images taken at different ventricular levels, and in one parasternal long axis. Pulsed wave and tissue Doppler modes were used to evaluate the diastolic function and Tei Index for global cardiac function. The echocardiographic measurements of infarct size were validated histologically using collagen deposition labeled by Sirius red staining. All data was analyzed using Shapiro-Wilk and Student's t-tests. Principal Findings: Our results reveal LV dilation resulting in marked remodeling an severe systolic dysfunction, starting seven days after MI (LV internal apical diameter, basal = 2.82 +/- 0.24, 7d = 3.49 +/- 0.42; p < 0.001. End-diastolic area, basal = 18.98 +/- 1.81, 7d = 22.04 +/- 2.11; p < 0.001). A strong statistically significant negative correlation exists between the infarct size and long-axis FAC% (r = -0.946; R-2 = 0.90; p < 0.05). Moreover, the measured Tei Index values confirmed significant post-infarction impairment of the global cardiac function (basal = 0.46 +/- 0.07, 7d = 0.55 +/- 0.08, 14 d = 0.57 +/- 0.06, 30 d = 0.54 +/- 0.06, 60 d = 0.54 +/- 0.07, 90 d = 0.57 +/- 0.08; p < 0.01). Conclusions/Significance: In summary, we have performed a complete characterization of LV post-infarction remodeling in a DBA/2J mouse model of MI, using parameters adapted to the particular characteristics of the model In the future, this well characterized model will be used in both investigative and pharmacological studies that require accurate quantitative monitoring of cardiac recovery after myocardial infarction.
Autores: Galarraga M; Campión, Francisco Javier; Boqué, Noemi; et al.
Revista: JOURNAL OF LIPID RESEARCH
ISSN 0022-2275  Vol. 53  Nº 12  2012  págs. 2791 - 2796
The accurate estimation of the number and size of cells provides relevant information on the kinetics of growth and the physiological status of a given tissue or organ. Here, we present Adiposoft, a fully automated open-source software for the analysis of white adipose tissue cellularity in histological sections. First, we describe the sequence of image analysis routines implemented by the program. Then, we evaluate our software by comparing it with other adipose tissue quantification methods, namely, with the manual analysis of cells in histological sections (used as gold standard) and with the automated analysis of cells in suspension, the most commonly used method. Our results show significant concordance between Adiposoft and the other two methods. We also demonstrate the ability of the proposed method to distinguish the cellular composition of three different rat fat depots. Moreover, we found high correlation and low disagreement between Adiposoft and the manual delineation of cells. We conclude that Adiposoft provides accurate results while considerably reducing the amount of time and effort required for the analysis.-Galarraga, M., J. Campion, A. Munoz-Barrutia, N. Boque, H. Moreno, J. A. Martinez, F. Milagro, and C. Ortiz-de-Solorzano. Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections.
Autores: Montuenga, Luis; Ortiz de Solórzano, Carlos;
Revista: INTERNATIONAL JOURNAL OF BIOMEDICAL IMAGING
ISSN 1687-4188  Vol. 2012  2012  págs. 734734
Objective. To define the sensitivity of microcomputed tomography- (micro-CT-) derived descriptors for the quantification of lung damage caused by elastase instillation. Materials and Methods. The lungs of 30 elastase treated and 30 control A/J mice were analyzed 1, 6, 12, and 24 hours and 7 and 17 days after elastase instillation using (i) breath-hold-gated micro-CT, (ii) pulmonary function tests (PFTs), (iii) RT-PCR for RNA cytokine expression, and (iv) histomorphometry. For the latter, an automatic, parallel software toolset was implemented that computes the airspace enlargement descriptors: mean linear intercept (L(m)) and weighted means of airspace diameters (D(0), D(1), and D(2)). A Support Vector Classifier was trained and tested based on three nonhistological descriptors using D(2) as ground truth. Results. D(2) detected statistically significant differences (P < 0.01) between the groups at all time points. Furthermore, D(2) at 1 hour (24 hours) was significantly lower (P < 0.01) than D(2) at 24 hours (7 days). The classifier trained on the micro-CT-derived descriptors achieves an area under the curve (AUC) of 0.95 well above the others (PFTS AUC = 0.71; cytokine AUC = 0.88). Conclusion. Micro-CT-derived descriptors are more sensitive than the other methods compared, to detect in vivo early signs of the disease.
Autores: Serrano, Diego; Bleau, A.M.; Fernandez-Garcia, I.; et al.
Revista: MOLECULAR CANCER
ISSN 1476-4598  Vol. 10  Nº 96  2011  págs. 1 - 15
Background: Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. Results: The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. Conclusions: We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.
Autores: Ceresa, M; Bastarrika, Gorka; de Torres, Juan Pablo; et al.
Revista: Academic Radiology
ISSN 1076-6332  Vol. 18  Nº 11  2011  págs. 1382-1390
Autores: Ceresa, M; et al.
Revista: European Radiology
ISSN 0938-7994  Vol. 21  Nº 5  2011  págs. 954-962
Autores: Arganda-Carreras, I.; Sorzano, C. O. S.; Thevenaz, P.; et al.
Revista: PHYSICS IN MEDICINE AND BIOLOGY
ISSN 0031-9155  Vol. 55  Nº 20  2010  págs. 6215 - 6242
We present a novel algorithm for the registration of 2D image sequences that combines the principles of multiresolution B-spline-based elastic registration and those of bidirectional consistent registration. In our method, consecutive triples of images are iteratively registered to gradually extend the information through the set of images of the entire sequence. The intermediate results are reused for the registration of the following triple. We choose to interpolate the images and model the deformation fields using B-spline multiresolution pyramids. Novel boundary conditions are introduced to better characterize the deformations at the boundaries. In the experimental section, we quantitatively show that our method recovers from barrel/pincushion and fish-eye deformations with subpixel error. Moreover, it is more robust against outliers-occasional strong noise and large rotations-than the state-of-the-art methods. Finally, we show that our method can be used to realign series of histological serial sections, which are often heavily distorted due to folding and tearing of the tissues.
Autores: Pérez-Martín, D; et al.
Revista: European Radiology
ISSN 0938-7994  Vol. 20  Nº 11  2010  págs. 2600 - 2608
Objectives To evaluate the feasibility of using automatic quantitative analysis of breath hold gated micro-CT images to detect and monitor disease in a mouse model of chronic pulmonary inflammation, and to compare image-based measurements with pulmonary function tests and histomorphometry. Material and methods Forty-nine A/J mice were used, divided into control and inflammation groups. Chronic inflammation was induced by silica aspiration. Fourteen animals were imaged at baseline, and 4, 14, and 34 weeks after silica aspiration, using micro-CT synchronized with ventilator-induced breath holds. Lung input impedance was measured as well using forced oscillation techniques. Five additional animals from each group were killed after micro-CT for comparison with histomorphometry. Results At all time points, micro-CT measurements show statistically significant differences between the two groups, while first differences in functional test parameters appear at 14 weeks. Micro-CT measurements correlate well with histomorphometry and discriminate diseased and healthy groups better than functional tests. Conclusion Longitudinal studies using breath hold gated micro-CT are feasible on the silica-induced model of chronic pulmonary inflammation, and automatic measurements from micro-CT images correlate well with histomorphometry, being more sensitive than functional tests to detect lung damage in this model.
Autores: Pelacho, Beatriz; Garbayo, Elisa; et al.
Revista: JOURNAL OF CONTROLLED RELEASE
ISSN 0168-3659  Vol. 147  Nº 1  2010  págs. 30 - 37
The use of pro-angiogenic growth factors in ischemia models has been associated with limited success in the clinical setting, in part owing to the short lived effect of the injected cytokine. The use of a microparticle system could allow localized and sustained cytokine release and consequently a prolonged biological effect with induction of tissue revascularization. To assess the potential of VEGF(165) administered as continuous release in ischemic disease, we compared the effect of delivery of poly(lactic-co-glycolic acid) (PLGA) microparticles (MP) loaded with VEGF(165) with free-VEGF or control empty microparticles in a rat model of ischemia-reperfusion. VEGF165 loaded microparticles could be detected in the myocardium of the infarcted animals for more than a month after transplant and provided sustained delivery of active protein in vitro and in vivo. One month after treatment, an increase in angiogenesis (small caliber caveolin-1 positive vessels) and arteriogenesis (alpha-SMA-positive vessels) was observed in animals treated with VEGF microparticles (p < 0.05), but not in the empty microparticles or free-VEGF groups. Correlating with this data, a positive remodeling of the heart was also detected in the VEGF-microparticle group with a significantly greater LV wall thickness (p < 0.01). In conclusion, PICA microparticle is a feasible and promising cytokine delivery system for treatment of myocardial ischemia. This strategy could be scaled up and explored in pre-clinical and clinical studies. (C) 2010 Elsevier B.V. All rights reserved.
Autores: Ortiz de Solórzano, Carlos;
Revista: IEEE TRANSACTIONS ON IMAGE PROCESSING
ISSN 1057-7149  Vol. 19  Nº 1  2010  págs. 11 - 24
We present an efficient algorithm to compute multidimensional spatially variant convolutions--or inner products--between N-dimensional signals and B-splines--or their derivatives--of any order and arbitrary sizes. The multidimensional B-splines are computed as tensor products of 1-D B-splines, and the input signal is expressed in a B-spline basis. The convolution is then computed by using an adequate combination of integration and scaled finite differences as to have, for moderate and large scale values, a computational complexity that does not depend on the scaling factor. To show in practice the benefit of using our spatially variant convolution approach, we present an adaptive noise filter that adjusts the kernel size to the local image characteristics and a high sensitivity local ridge detector.
Autores: Arganda-Carreras, I.; Ortiz de Solórzano, Carlos;
Revista: MICROSCOPY RESEARCH AND TECHNIQUE
ISSN 1059-910X  Vol. 73  Nº 11  2010  págs. 1019-1029
In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high-content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer-based system integrates image acquisition, annotation, registration, and three-dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high-resolution molecular data.
Autores: Ortiz de Solórzano, Carlos;
Libro:  Lung Imaging and Computer Aided Diagnosis
2012  págs. 137-150
Autores: Fernández-González, R. ; Shelton, D. N.; Welm, B. E.; et al.
Libro:  Methods in Molecular Biology
Vol. 621  2010  págs. 1-28
The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available.
Autores: Ortiz de Solórzano, Carlos;
Libro:  Microscopy: science, technology, applications and education
Vol. 3  Nº 4  2010  págs. 2079 - 2087
The widespread use of fluorescence microscopy along with the vast library of available fluorescent stains and staining methods has been extremely beneficial to researchers in many fields, ranging from material sciences to plant biology. In clinical diagnostics, the ability to combine different markers in a given sample allows the simultaneous detection of the expression of several different molecules, which in turn provides a powerful diagnostic tool for pathologists, allowing a better classification of the sample at hand. The correct detection and separation of multiple stains in a sample is achieved not only by the biochemical and optical properties of the markers, but also by the use of appropriate hardware and software tools. In this chapter, we will review and compare these tools along with their advantages and limitations.
Autores: Shelton, D. N.; Fernández-González, R.; Ortiz de Solórzano, Carlos; et al.
Libro:  Protocols for adult stem cells
Vol. 621  2010  págs. 49 - 55
The regenerative potential of mammary epithelium facilitates assessment of the ¿stemness¿ of any epithelial subpopulation in transplantation assays. Thus, mammary tissue can be dissociated into single cells, stained for cell surface markers of interest and classified using fluorescence-activated cell sorting. The selected cells can then be transplanted into epithelium-devoided fat pads from recipient hosts. Recent publications have described markers that enrich for mammary repopulating potential. Here, we describe the materials and methods necessary to sort cells according to these markers. This approach can be used interchangeably with other cell surface markers with slight variation to the protocol.
Autores: Fernández-González, R.; Shelton, D. N.; Welm, B. E.; et al.
Libro:  Protocols for adult stem cells
Vol. 621  2010  págs. 29 - 47
Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8¿10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps.