Nuestros investigadores

Ana Rouzaut Subirá

Bioquímica y Genética
Facultad de Ciencias. Universidad de Navarra
Facultad de Ciencias
Facultad de Ciencias. Universidad de Navarra
Líneas de investigación
Migración y adehsión de células a través de vasos linfáticos, Inmunologia de tumores, Citoesqueleto
Índice H
22, (Scopus, 24/09/2018)

Publicaciones científicas más recientes (desde 2010)

Autores: Garasa, S.; et al.
ISSN 1664-3224  Vol. 9  2018  págs. 2084
The quantity of T-lymphocytes reaching the draining lymph nodes from tumors is likely important to mount effective distant responses and for the establishment of long term systemic memory. Looking into mechanisms behind lymphocyte egress, we directed our attention to leukocyte adhesion mechanisms inside tumors. Here we demonstrate that activated T-cells form intra-tumor aggregates in a LFA-1-ICAM-1-dependent fashion in mouse models of melanoma and breast cancer. We also provide evidence of the presence of T-cell clusters in primary human melanoma. Disruption of LFA-1-ICAM-1 interactions, and thereby T-cell clustering, enhances the arrival of activated CD8+ T-cells to tumor draining lymph nodes in both transplanted and spontaneous cancer models. Interestingly, upon ICAM-1 blockade, the expression of the chemotactic receptor CCR7 augments in tumor in filtrating lymphocytes and in in-vitro de-clustered T cells, as well as their ability to transmigrate across lymphatic endothelial cells. We propose that ICAM-1-mediated homotypic T-lymphocyte aggregation may serve as a tumor-mediated immune retention mechanism entrapping activated CD8+ T cells in the tumor microenvironment. Modulation of T-cell adhesion may be of use to improve the transit of activated lymphocytes toward the lymph nodes and their subsequent recirculation.
Autores: Labiano, S.; Garasa, S.; et al.
ISSN 2326-6066  Vol. 6  Nº 7  2018  págs. 798 - 811
T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of F lymphocytes. herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8(+) T cells. Such mitochondrial changes increased 'T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in rumor-reactive CD8(+) T cells from cancer-hearing mice were invigorated by agonist mAb to CD137, whereas mitochondria) baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD 137 WAS critically dependent on OPA-1 expression in transferred CD8(+) T cells. Moreover, stimulation of CD137 with CD137 mAb in shortterm cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD 117 costimulation of T cells. (C)2018 AACR.
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: Hunter, M. C.; Russo, E.; et al.
ISSN 2211-1247  Vol. 18  Nº 4  2017  págs. 857 - 865
T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process.
Autores: Garasa, S.; Rodriguez, I.; et al.
ISSN 0360-3016  Vol. 97  Nº 2  2017  págs. 389 - 400
Purpose/Objectives: The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods: Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGF beta in this process anti-TGF beta blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGF beta and /or anti-ICAM1 blocking mAb. Results: We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGF beta. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion: Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy. (C) 2016 Elsevier Inc. All rights reserved.
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: Marin-Bejar, O.; Gonzalez, J.; et al.
ISSN 1474-760X  Vol. 18  2017  págs. 202
BACKGROUND: It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long non-coding RNAs (lncRNAs). Many lncRNAs show aberrant expression in cancer, and some of them have been linked to cell transformation. However, the underlying mechanisms remain poorly understood and it is unknown how the sequences of lncRNA dictate their function. RESULTS: Here we characterize the function of the p53-regulated human lncRNA LINC-PINT in cancer. We find that LINC-PINT is downregulated in multiple types of cancer and acts as a tumor suppressor lncRNA by reducing the invasive phenotype of cancer cells. A cross-species analysis identifies a highly conserved sequence element in LINC-PINT that is essential for its function. This sequence mediates a specific interaction with PRC2, necessary for the LINC-PINT-dependent repression of a pro-invasion signature of genes regulated by the transcription factor EGR1. CONCLUSIONS: Our findings support a conserved functional co-dependence between LINC-PINT and PRC2 and lead us to propose a new mechanism where the lncRNA regulates the availability of free PRC2 at the proximity of co-regulated genomic loci.
Autores: Pajares, María Josefa; Ajona, Daniel; Sharma, Ravi Datta; et al.
ISSN 1574-7891  Vol. 10  Nº 9  2016  págs. 1437 - 1449
Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.
Autores: Rodriguez, I.; Garasa, S.; et al.
ISSN 0008-5472  Vol. 76  Nº 20  2016  págs. 5994 - 6005
Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3-dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFN¿ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation.
Autores: Anguiano M; Castilla C; Maska M; et al.
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: Alfaro, Carlos; Echeveste, José Ignacio; et al.
ISSN 2162-4011  Vol. 4  Nº 12  2015  págs. e1054597
CD137 (4-1BB) is a surface marker discovered on activated T lymphocytes. However, its expression pattern is broader and has also been described on activated NK cells, B-cells and myeloid cells including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorates intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated to non-small cell lung cancer showed a similar pattern of immunostaining. Multicolor flow cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes in tonsils and lymph nodes. Short term culture of lymph node cell suspensions in the presence of an agonist anti-CD137 mAb or CD137-ligand results in the functional upregulation of TFH cells, including CD40L surface expression and cytokine production, in three out of six cases. As a consequence, immunostimulatory monoclonal antibodies, anti-CD137 mAb such as urelumab and PF-05082566 should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses.
Autores: Anguiano, M.; Maska, M.; et al.
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: Pajares, María Josefa; Agorreta, J; Behrens, C.; et al.
ISSN 0007-0920  Vol. 110  Nº 6  2014  págs. 1545 - 1551
Background: Transforming growth factor beta-induced protein (TGFBI) is a secreted protein that mediates cell anchoring to the extracellular matrix. This protein is downregulated in lung cancer, and when overexpressed, contributes to apoptotic cell death. Using a small series of stage IV non-small cell lung cancer (NSCLC) patients, we previously suggested the usefulness of TGFBI as a prognostic and predictive factor in chemotherapy-treated late-stage NSCLC. In order to validate and extend these results, we broaden the analysis and studied TGFBI expression in a large series of samples obtained from stage I-IV NSCLC patients. Methods: TGFBI expression was assessed by immunohistochemistry in 364 completely resected primary NSCLC samples: 242 adenocarcinomas (ADCs) and 122 squamous cell carcinomas (SCCs). Kaplan-Meier curves, log-rank tests and the Cox proportional hazards model were used to analyse the association between TGFBI expression and survival. Results: High TGFBI levels were associated with longer overall survival (OS, P < 0.001) and progression-free survival (PFS, P < 0.001) in SCC patients who received adjuvant platinium-based chemotherapy. Moreover, multivariate analysis demonstrated that high TGFBI expression is an independent predictor of better survival in patients (OS: P = 0.030 and PFS: P = 0.026). Conclusions: TGFBI may be useful for the identification of a subset of NSCLC who may benefit from adjuvant therapy.
Autores: Garasa Larraza, S.; Altgevogt, P.; Rouzaut, Ana;
ISSN 1476-4598  Vol. 13  Nº 1  2014  págs. 112
Background Transforming Growth Factor beta (TGF-ß) acts as a tumor suppressor early in carcinogenesis but turns into tumor promoter in later disease stages. In fact, TGF-ß is a known inducer of integrin expression by tumor cells which contributes to cancer metastatic spread and TGF-ß inhibition has been shown to attenuate metastasis in mouse models. However, carcinoma cells often become refractory to TGF-ß-mediated growth inhibition. Therefore identifying patients that may benefit from anti-TGF-ß therapy requires careful selection. Methods We performed in vitro analysis of the effects of exposure to TGF-ß in NSCLC cell chemotaxis and adhesion to lymphatic endothelial cells. We also studied in an orthotopic model of NSCLC the incidence of metastases to the lymph nodes after inhibition of TGF-ß signaling, ß3 integrin expression or both. Results We offer evidences of increased ß3-integrin dependent NSCLC adhesion to lymphatic endothelium after TGF-ß exposure. In vivo experiments show that targeting of TGF-ß and ß3 integrin significantly reduces the incidence of lymph node metastasis. Even more, blockade of ß3 integrin expression in tumors that did not respond to TGF-ß inhibition severely impaired the ability of the tumor to metastasize towards the lymph nodes. Conclusion These findings suggest that lung cancer tumors refractory to TGF-ß monotherapy can be effectively treated using dual therapy that combines the inhibition of tumor cell adhesion to lymphatic vessels with stromal TGF-ß inhibition.
Autores: Melero, Ignacio Javier; Rouzaut, Ana; Motz, G.T.; et al.
ISSN 2159-8274  Vol. 4  Nº 5  2014  págs. 522 - 526
Cancer immunotherapy has great promise, but is limited by diverse mechanisms used by tumors to prevent sustained antitumor immune responses. Tumors disrupt antigen presentation, T/NK-cell activation, and T/NK-cell homing through soluble and cell-surface mediators, the vasculature, and immunosuppressive cells such as myeloid-derived suppressor cells and regulatory T cells. However, many molecular mechanisms preventing the efficacy of antitumor immunity have been identified and can be disrupted by combination immunotherapy. Here, we examine immunosuppressive mechanisms exploited by tumors and provide insights into the therapies under development to overcome them, focusing on lymphocyte traffic.
Autores: Rouzaut, Ana; Melero, Ignacio Javier;
ISSN 1664-3224  Vol. 4  2013  págs. 433
Tissue drains fluid and macromolecules through lymphatic vessels (LVs), which are lined by a specialized endothelium that expresses peculiar differentiation proteins, not found in blood vessels (i.e., LYVE-1, Podoplanin, PROX-1, and VEGFR-3). Lymphatic capillaries are characteristically devoid of a continuous basal membrane and are anchored to the ECM by elastic fibers that act as pulling ropes which open the vessel to avoid edema if tissue volume increases, as it occurs upon inflammation. LVs are also crucial for the transit of T lymphocytes and antigen presenting cells from tissue to draining lymph nodes (LN). Importantly, cell traffic control across lymphatic endothelium is differently regulated under resting and inflammatory conditions. Under steady-state non-inflammatory conditions, leukocytes enter into the lymphatic capillaries through basal membrane gaps (portals). This entrance is integrin-independent and seems to be mainly guided by CCL21 chemokine gradients acting on leukocytes expressing CCR7. In contrast, inflammatory processes in lymphatic capillaries involve a plethora of cytokines, chemokines, leukocyte integrins, and other adhesion molecules. Importantly, under inflammation a role for integrins and their ligands becomes apparent and, as a consequence, the number of leukocytes entering the lymphatic capillaries multiplies several-fold. Enhancing transmigration of dendritic cells en route to LN is conceivably useful for vaccination and cancer immunotherapy, whereas interference with such key mechanisms may ameliorate autoimmunity or excessive inflammation. Recent findings illustrate how, transient cell-to-cell interactions between lymphatic endothelial cells and leukocytes contribute to shape the subsequent behavior of leukocytes and condition the LV for subsequent trans-migratory events.
Autores: Garasa, S.; Pajares, María Josefa; et al.
ISSN 0020-7136  Vol. 132  Nº 9  2013  págs. 1986 - 1995
Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.
Autores: Garasa, S.; Azpilicueta, Arantza; et al.
ISSN 1523-1747  Vol. 133  Nº 9  2013  págs. 2276 - 2285
Dendritic cell (DC) transmigration across the lymphatic endothelium is critical for the initiation and sustenance of immune responses. Under noninflammatory conditions, DC transit across the lymphatic endothelial cell (LEC) has been shown to be integrin independent. In contrast, there is increasing evidence for the participation of integrins and their ligands in DC transit across lymphatic endothelium under inflammation. In this sense, we describe the formation of ICAM-1 (CD54)-enriched three-dimensional structures on LEC/DC contacts, as these DCs adhere to inflamed skin lymphatic vessels and transmigrate into them. In vitro imaging revealed that under inflammation ICAM-1 accumulated on microvilli projections surrounding 60% of adhered DCs. In contrast, these structures were scarcely formed in noninflammatory conditions. Furthermore, ICAM-1-enriched microvilli were important in promoting DC transendothelial migration and DC crawling over the LEC surface. Microvilli formation was dependent on the presence of beta-integrins on the DC side and on integrin conformational affinity to ligand. Finally, we observed that LEC microvilli structures appeared in close vicinity of CCL21 depots and that their assembly was partially inhibited by CCL21-neutralizing antibodies. Therefore, under inflammatory conditions, integrin ligands form three-dimensional membrane projections around DCs. These structures offer docking sites for DC transit from the tissue toward the lymphatic vessel lumen.
Autores: Rodríguez, I.; et al.
ISSN 1078-0432  Vol. 19  Nº 22  2013  págs. 6151 - 6162
Purpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen. Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays. Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy. Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma.
Autores: Danek, O.; Garasa, S.; Rouzaut, Ana; et al.
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: Garasa, S; et al.
ISSN 0892-6638  Vol. 26  Nº 8  2012  págs. 3380 - 3392
Autores: Corrales, L; Ajona, D; Rafail, S; et al.
Revista: The Journal of Immunology
ISSN 0022-1767  Vol. 189  Nº 9  2012  págs. 4674 - 4683
The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.
Autores: Alfaro, Carlos; et al.
Revista: Cancer Discovery
ISSN 2159-8274  Vol. 2  Nº 7  2012  págs. 608 - 623
Autores: Sandra Hervas-Stubbs; Pérez, José Luis; Rouzaut, Ana; et al.
ISSN 1078-0432  Vol. 17  Nº 9  2011  págs. 2619 - 2627
Autores: Alfaro, Carlos; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 6  Nº 3  2011  págs. e17922
IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.
Autores: Alfaro, Carlos; Azpilicueta, Arantza; et al.
Revista: International Journal of Cancer (Print)
ISSN 0020-7136  Vol. 28  Nº 1  2011  págs. 105 - 118
Autores: Sandra Hervas-Stubbs; et al.
Revista: Cancer Research
ISSN 0008-5472  Vol. 71  Nº 3  2011  págs. 801 - 11
Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies
Autores: Rouzaut, Ana; Garasa, S.; et al.
Revista: European Journal of Immunology
ISSN 0014-2980  Vol. 40  Nº 11  2010  págs. 3054 - 3063
Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF-alpha were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-alpha 2b or IFN-alpha 5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro-adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-alpha led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-alpha also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.
Autores: Pajares, María Josefa; Agorreta, J; et al.
Revista: Molecular Cancer
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