Tomás Luque - Academia.edu (original) (raw)
Papers by Tomás Luque
Video S4. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated KO MEFs.
Video S1. MDA-MB231 cells on 3D-ECM generated by Control MEFs.
Video S7. Ela-Myc cells on 3D-ECM generated by KO MEFs.
Video S5. Ela-Myc cells on 3D-ECM generated by Control MEFs.
Video S6. Ela-Myc cells on 3D-ECM generated by TGFβ-treated Control MEFs.
Video S8. Ela-Myc cells on 3D-ECM generated by TGFβ-treated KO MEFs.
Video S3. MDA-MB231 cells on 3D-ECM generated by KO MEFs.
Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells ... more Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells in a Snail1-dependent manner. Supplementary Figure S2. Cell Commitment is Modulated by Fibroblast-derived Matrices in a Snail1-dependent Manner. Supplementary Figure S3. TGF beta controls focal contacts in a Snail1-dependent manner. Supplementary Figure S4. TGF beta regulates N-cadherin intercellular contacts in a Snail1-dependent manner. Supplementary Figure S5. Alignment of MEFs bo-cultured with CAFs. Supplementary Table S1. Baseline Characteristics According to Snail1 Expression in the Stroma Supplementary Table S2. Overall Survival Analysis in Patients with Snail1 Expression in Stroma Supplementary Table S3. Antibodies Used for Protein Analysis
Video S2. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated Control MEFs.
Journal of visualized experiments : JoVE, Nov 29, 2017
Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one o... more Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissu...
Scientific reports, Jan 11, 2017
We present a procedure that allows a reliable determination of the elastic (Young's) modulus ... more We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application redu...
European Respiratory Journal, Sep 1, 2011
Rationale: Exacerbations of COPD (EXCOPD) are characterized by activated pulmonary neutrophils (N... more Rationale: Exacerbations of COPD (EXCOPD) are characterized by activated pulmonary neutrophils (NEUT) with potentially increased cellular stiffness. Objective: The aim of this study was to assess NEUT stiffness in patients with COPD during and after exacerbations. Methods: 12 COPD patients (age, 70±9 yrs; 83% male; all smokers, 71±36 pack-yrs) hospitalized for EXCOPD were included. Stiffness of peripheral NEUT was assessed using the atomic force microscopy technique within the first 72 h of admission and 12 weeks after discharge (Study Group [SG]). If patients suffered a new episode within this period of follow-up, measurements were repeated at this time in point (mean, 7±4 weeks) (Re-exacerbation Group [RG]). The COPD Assessment Test (CAT), the ADO Index and dyspnoea (MMRC) were also assessed. Results: There were no differences on admission between the two subsets of patients. Increased NEUT stiffness in SG patients (n, 6) decreased from admission (890±293 Pa) to discharge (616±226 Pa) (p Conclusions: Changes in peripheral NEUT stiffness during COPD exacerbations and convalescence indicate that the activation of NEUT underlies enhanced pulmonary inflammation. Supported by SEPAR-2010 and CIBERES
European Respiratory Journal, Sep 1, 2011
Background: Recent studies have suggested that bone marrow-derived mesenchymal stem cells (MSC) m... more Background: Recent studies have suggested that bone marrow-derived mesenchymal stem cells (MSC) might have potential therapeutic effects in acute lung injury (ALI), specifically in bleomycin-induced and bacterial LPS-induced lung injury models. However, whether MSC could induce an early anti-inflammatory response in lung injury induced by overventilation remains to be elucidated. Aim: To assess the potential role of MSC in preventing or modulating early inflammation in healthy rats subjected to ventilator induced lung injury (VILI). Methods: Adult male Sprague-Dawley rats (250-300 g) were anaesthetised, tracheotomised, intubated and paralysed by intravenous instillation of pancuronium bromide. Nine rats were ventilated with a high tidal volume of 25 ml/kg (VILI) for 3 h. Five millions of MSC were intravenously injected to 4 of these rats 30 min before starting ventilation. Spontaneously breathing anesthetised rats (N=4) served as controls. After 3 h of VILI/control the animals were sacrificed and broncoalveolar lavage inflammatory cells were assessed. Results: In VILI, MSC significantly (p Conclusion: This preliminary result suggests that infusion of bone marrow-derived MSC prevents early inflammation in VILI. Funding: L. Chimenti was a recipient of a ERS/Marie Curie Postdoctoral Research Fellowships 2010
European Respiratory Journal, Sep 1, 2012
<p>(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-mi... more <p>(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in <i>z</i> as a function of time (<i>t</i>) used to probe the sample mechanoresponse to compression and extension. Note that the <i>z</i> axis was scaled relative to <i>z<sub>c</sub></i>, which is marked with an horizontal dashed line. Although the relative <i>z</i> signal started at 13.5 µm, the <i>z</i> axis was limited to 4 µm above <i>z<sub>c</sub></i> to better visualize the range corresponding to step III and IV. A break in the <i>t</i> axis was introduced for the same purpose. Corresponding <i>F</i> recordings as a function of <i>t</i> on a PAA<sup>+</sup> gel and a single A549 cell are shown in (C) and (D), respectively. A common <i>t</i> axis was used in (B–D). <i>F*</i> was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. <i>E<sub>C</sub></i> and <i>E<sub>T</sub></i> were calculated using signals from step III and IV. <i>F</i> signals from (C) and (D) were plotted against <i>z</i> in (F) and (G), respectively. The parts of the <i>z</i> and <i>F</i> signals obtained in compression were highlighted in gray. All <i>F</i> data were scaled relative to the corresponding zero force (<i>k⋅d<sub>0</sub></i>).</p
Background information: Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injur... more Background information: Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). Question of the study: Whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. Materials and methods: 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 ml/kg). MSCs (5x106) were intravenously or intratracheally administered (N=8 each) 30 min before starting over-ventilation and 8 rats were MSC-untreated. Spontaneously breathing anesthetised rats (N=8) served as controls. After 3 h of over-ventilation/control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. Results: When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI featu...
<p>(A, B) <i>F*</i> data obtained on (A) CCD-19Lu fibroblasts and on (B) A549 c... more <p>(A, B) <i>F*</i> data obtained on (A) CCD-19Lu fibroblasts and on (B) A549 cells. Bar filling patterns shown on the right apply to plots A–B. (C) Plot of <i>F*</i> vs <i>E<sub>T</sub></i> average data collected on all cell types with all tip coatings. Solid line corresponds to a least-squares fitting of a linear polynomial to the data. +<i>P</i><0.05, ++<i>P</i><0.01 and +++<i>P</i><0.005 were determined by unpaired Student's <i>t</i>-test w.r.t. bare.</p
Cells from lung and other tissues are subjected to forces of opposing directions that are largely... more Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with,1 mm 2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent ...
The physical principles underlying the biomechanics of morphogenetic processes are largely unknow... more The physical principles underlying the biomechanics of morphogenetic processes are largely unknown. Epiboly is an essential embryonic event in which three distinct tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly and how these are globally coupled remains elusive. Here we first develop a method, Hydrodynamic Regression (HR), to infer 3D dynamic pressure fields, mechanical power densities and cortical surface tension profiles within living organisms. HR is based on velocity measurements retrieved from 2D+T microscopy time-lapses and their hydrodynamic modeling. We then applied this method to identify biomechanically active structures during epiboly in the zebrafish and the changes in the distribution of cortex local tension as epiboly progresses. Based on these results, we propose a novel simple physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that...
Video S4. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated KO MEFs.
Video S1. MDA-MB231 cells on 3D-ECM generated by Control MEFs.
Video S7. Ela-Myc cells on 3D-ECM generated by KO MEFs.
Video S5. Ela-Myc cells on 3D-ECM generated by Control MEFs.
Video S6. Ela-Myc cells on 3D-ECM generated by TGFβ-treated Control MEFs.
Video S8. Ela-Myc cells on 3D-ECM generated by TGFβ-treated KO MEFs.
Video S3. MDA-MB231 cells on 3D-ECM generated by KO MEFs.
Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells ... more Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells in a Snail1-dependent manner. Supplementary Figure S2. Cell Commitment is Modulated by Fibroblast-derived Matrices in a Snail1-dependent Manner. Supplementary Figure S3. TGF beta controls focal contacts in a Snail1-dependent manner. Supplementary Figure S4. TGF beta regulates N-cadherin intercellular contacts in a Snail1-dependent manner. Supplementary Figure S5. Alignment of MEFs bo-cultured with CAFs. Supplementary Table S1. Baseline Characteristics According to Snail1 Expression in the Stroma Supplementary Table S2. Overall Survival Analysis in Patients with Snail1 Expression in Stroma Supplementary Table S3. Antibodies Used for Protein Analysis
Video S2. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated Control MEFs.
Journal of visualized experiments : JoVE, Nov 29, 2017
Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one o... more Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissu...
Scientific reports, Jan 11, 2017
We present a procedure that allows a reliable determination of the elastic (Young's) modulus ... more We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application redu...
European Respiratory Journal, Sep 1, 2011
Rationale: Exacerbations of COPD (EXCOPD) are characterized by activated pulmonary neutrophils (N... more Rationale: Exacerbations of COPD (EXCOPD) are characterized by activated pulmonary neutrophils (NEUT) with potentially increased cellular stiffness. Objective: The aim of this study was to assess NEUT stiffness in patients with COPD during and after exacerbations. Methods: 12 COPD patients (age, 70±9 yrs; 83% male; all smokers, 71±36 pack-yrs) hospitalized for EXCOPD were included. Stiffness of peripheral NEUT was assessed using the atomic force microscopy technique within the first 72 h of admission and 12 weeks after discharge (Study Group [SG]). If patients suffered a new episode within this period of follow-up, measurements were repeated at this time in point (mean, 7±4 weeks) (Re-exacerbation Group [RG]). The COPD Assessment Test (CAT), the ADO Index and dyspnoea (MMRC) were also assessed. Results: There were no differences on admission between the two subsets of patients. Increased NEUT stiffness in SG patients (n, 6) decreased from admission (890±293 Pa) to discharge (616±226 Pa) (p Conclusions: Changes in peripheral NEUT stiffness during COPD exacerbations and convalescence indicate that the activation of NEUT underlies enhanced pulmonary inflammation. Supported by SEPAR-2010 and CIBERES
European Respiratory Journal, Sep 1, 2011
Background: Recent studies have suggested that bone marrow-derived mesenchymal stem cells (MSC) m... more Background: Recent studies have suggested that bone marrow-derived mesenchymal stem cells (MSC) might have potential therapeutic effects in acute lung injury (ALI), specifically in bleomycin-induced and bacterial LPS-induced lung injury models. However, whether MSC could induce an early anti-inflammatory response in lung injury induced by overventilation remains to be elucidated. Aim: To assess the potential role of MSC in preventing or modulating early inflammation in healthy rats subjected to ventilator induced lung injury (VILI). Methods: Adult male Sprague-Dawley rats (250-300 g) were anaesthetised, tracheotomised, intubated and paralysed by intravenous instillation of pancuronium bromide. Nine rats were ventilated with a high tidal volume of 25 ml/kg (VILI) for 3 h. Five millions of MSC were intravenously injected to 4 of these rats 30 min before starting ventilation. Spontaneously breathing anesthetised rats (N=4) served as controls. After 3 h of VILI/control the animals were sacrificed and broncoalveolar lavage inflammatory cells were assessed. Results: In VILI, MSC significantly (p Conclusion: This preliminary result suggests that infusion of bone marrow-derived MSC prevents early inflammation in VILI. Funding: L. Chimenti was a recipient of a ERS/Marie Curie Postdoctoral Research Fellowships 2010
European Respiratory Journal, Sep 1, 2012
<p>(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-mi... more <p>(A) Representative SEM images of a nanofabricated cylindrical FE-AFM tip. A whole FIB-milled cylindrical tip is shown in the left panel, and detailed lateral and top view images of the tip are shown in the middle and right panels, respectively. (B) Driving signal of the piezotranslator in <i>z</i> as a function of time (<i>t</i>) used to probe the sample mechanoresponse to compression and extension. Note that the <i>z</i> axis was scaled relative to <i>z<sub>c</sub></i>, which is marked with an horizontal dashed line. Although the relative <i>z</i> signal started at 13.5 µm, the <i>z</i> axis was limited to 4 µm above <i>z<sub>c</sub></i> to better visualize the range corresponding to step III and IV. A break in the <i>t</i> axis was introduced for the same purpose. Corresponding <i>F</i> recordings as a function of <i>t</i> on a PAA<sup>+</sup> gel and a single A549 cell are shown in (C) and (D), respectively. A common <i>t</i> axis was used in (B–D). <i>F*</i> was obtained from step IV as illustrated in (C, D). (E) Cartoon describing the tip-sample mechanical interactions corresponding to the 4-steps of the experimental protocol. <i>E<sub>C</sub></i> and <i>E<sub>T</sub></i> were calculated using signals from step III and IV. <i>F</i> signals from (C) and (D) were plotted against <i>z</i> in (F) and (G), respectively. The parts of the <i>z</i> and <i>F</i> signals obtained in compression were highlighted in gray. All <i>F</i> data were scaled relative to the corresponding zero force (<i>k⋅d<sub>0</sub></i>).</p
Background information: Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injur... more Background information: Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). Question of the study: Whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. Materials and methods: 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 ml/kg). MSCs (5x106) were intravenously or intratracheally administered (N=8 each) 30 min before starting over-ventilation and 8 rats were MSC-untreated. Spontaneously breathing anesthetised rats (N=8) served as controls. After 3 h of over-ventilation/control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. Results: When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI featu...
<p>(A, B) <i>F*</i> data obtained on (A) CCD-19Lu fibroblasts and on (B) A549 c... more <p>(A, B) <i>F*</i> data obtained on (A) CCD-19Lu fibroblasts and on (B) A549 cells. Bar filling patterns shown on the right apply to plots A–B. (C) Plot of <i>F*</i> vs <i>E<sub>T</sub></i> average data collected on all cell types with all tip coatings. Solid line corresponds to a least-squares fitting of a linear polynomial to the data. +<i>P</i><0.05, ++<i>P</i><0.01 and +++<i>P</i><0.005 were determined by unpaired Student's <i>t</i>-test w.r.t. bare.</p
Cells from lung and other tissues are subjected to forces of opposing directions that are largely... more Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with,1 mm 2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent ...
The physical principles underlying the biomechanics of morphogenetic processes are largely unknow... more The physical principles underlying the biomechanics of morphogenetic processes are largely unknown. Epiboly is an essential embryonic event in which three distinct tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly and how these are globally coupled remains elusive. Here we first develop a method, Hydrodynamic Regression (HR), to infer 3D dynamic pressure fields, mechanical power densities and cortical surface tension profiles within living organisms. HR is based on velocity measurements retrieved from 2D+T microscopy time-lapses and their hydrodynamic modeling. We then applied this method to identify biomechanically active structures during epiboly in the zebrafish and the changes in the distribution of cortex local tension as epiboly progresses. Based on these results, we propose a novel simple physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that...