Suchergebnisse

Keratoconjunctivitis sicca (KCS) is characterized by ocular discomfort, conjunctival hyperaemia, and corneal scarring, causing reduced aqueous tear production that can be measured using the standard Schirmer tear test (STT). Canine adipose tissue-derived MSCs (cATMSCs) have been proposed as treatment due to their anti-inflammatory effect, by releasing cytokines and immunomodulatory soluble factors. The aim of this study was to evaluate the effect of the systemic administration of cATMSCs on tear production in dogs with immune-mediated KCS, compared to classical Cyclosporine A (CsA) treatment. Twenty-eight client-owned dogs with spontaneous KCS were allocated in the experimental group (n = 14, treated with systemic cATMSCs or control group (n = 14, treated with CsA). SST values increased significantly at days 15 (p = 0.002), 45 (p = 0.042) and 180 (p = 0.005) with no observed side-effects in the experimental group. Eyes with an initial STT value of 11-14 mm/min maintained significant improvement at day 180, needing only artificial tears as treatment. Eyes with an initial STT value <11 mm/min needed cyclosporin treatment at day 45, so follow-up was stopped. Control animals treated with CsA did not improve their STT at day 180. Systemic allogeneic cATMSCs application appeared to be a feasible and effective therapy with positive outcome in dogs with initial STT between 11-14 mm/min, with a significant improvement in tear production. The STT increment was maintained for at least 180 days, without needing additional medication, thus suggesting it could constitute an alternative therapy to classical immunosuppressive treatments. ; This work was supported by the Madrid Regional Government (Avancell-CM; S2017/BMD-3692) and Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS-ISCIII; RD21/0017/0005) to J.G.-C. ; Sí

Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models. ; This research was funded by Fondo de Investigaciones Sanitarias (FIS: PI11/00377, PI14CIII/00005 and PI17CIII/00013 to J.G.-C., FIS: CP11/00206 to A.A., FIS: PI18/006146 to A.P.-G. and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (P2010/BMD-2420 and B2017/BMD-3692 to J.G.-C.), the Asociación Pablo Ugarte, and AFANION grant support. ; Sí

Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models ; This research was funded by Fondo de Investigaciones Sanitarias (FIS: PI11/00377, PI14CIII/00005 and PI17CIII/00013 to J.G.-C., FIS: CP11/00206 to A.A., FIS: PI18/006146 to A.P.- G. and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (P2010/BMD-2420 and B2017/BMD-3692 to J.G.-C.), the Asociación Pablo Ugarte, and AFANION grant support

Homing is an inherent, complex, multistep process performed by cells such as human bone marrow mesenchymal stem cells (hMSCs) to travel from a distant location to inflamed or damaged tissue and tumors. This ability of hMSCs has been exploited as a tumor-targeting strategy in cell-based cancer therapy. The purpose of this study was to investigate the applicability of 111In-oxine for tracking hMSCs in vivo by combining single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). 111In-labeled hMSCs (106 cells) were infused intraperitoneally in neuroblastoma-bearing mice, whereas a control group received a dose of free 111In-oxine. SPECT and MRI studies were performed 24 and 48 hours afterwards. Initially, the images showed similar activity in the abdomen in both controls and hMSC-injected animals. In general, abdominal activity decreases at 48 hours. hMSC-injected animals showed increased uptake in the tumor area at 48 hours, whereas the control group showed a low level of activity at 24 hours, which decreased at 48 hours. In conclusion, tracking 111In-labeled hMSCs combining SPECT and MRI is feasible and may be transferable to clinical research. The multimodal combination is essential to ensure appropriate interpretation of the images. ; Financial disclosure of authors: This work was funded in part by grants from Ministerio de Economía y Competitividad (PLE2009-0115), Red Tematica de Investigacion Cooperativa en Cancer (RTICC/ISCIII; RD12/0036/0027), the Madrid Regional Government (S-BIO-0204-2006–MesenCAM and P2010/BMD-2420-CellCAM), and the Ministerio de Ciencia e Innovación (CEN-20101014 and TEC-2010-21619-C04-01). ; Sí

Factor de impacto: 14,919 ; Osteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously. ; We thank ISCIII and CNIO flow cytometry and cell sorting units for their participation in our studies. We are thankful to the CCEH-Fred Hutchinson Cancer Research Center for LAM-PCR service. We acknowledge Raquel Pérez Tavarez, María Blázquez Mesa, Alicia Giménez Sánchez, Elena Calvo Cazalilla, and Monserrat Arroyo Correas for useful help on the pathology studies; and Teresa Cejalvo, Isabel Cubillo Moreno, and Miguel Angel Rodríguez-Milla for their contributions in experimental setup. We thank the visual artist Isabella Lacquaniti for her help with drawings and schematics. We are also thankful to the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 and PI14CIII/00005 to J.G.-C., FIS: CP11/00206 to A.A., and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C.), the Asociación Pablo Ugarte, and the Asociación Afanion for grants support. ; Sí

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is char-acterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differ-entially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB ; This study was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029 to J.G.C.), MICINN (PLE2009-0115) and the Madrid Regional Government (S-BIO-0204-2006 and P2010/BMD-2420) in Spain. The experiments were approved by the appropriate committees. ; Sí

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45-CD31-CD34-CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings. ; We thank Iván Gutierrez, Ander Abarrategi, Manuel Masip, Mar Arriero, and Daniel Pérez for his assistance in several techniques. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029), the Madrid Regional Government (S‐BIO‐0204–2006, MesenCAM; P2010/BMD‐2420, CellCAM) in Spain, and Consejería de Salud de la Junta de Andalucía (0027/2006) to J.G.‐C. The experiments were approved by the appropriate committees. ; Sí

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated. ; This work was supported by grands BFU2009-10315, SAF2012-33180, and BFU2010-18250 from the Spanish Ministry of Science and Innovation, Spanish Association Against Cancer 2010 AECC; P2010/BMD-2420-CellCAM from Regional Government of Madrid and RD12/0019/0007-TerCell Network from the Health Institute Carlos III. MNVG contract UCM number 50053735 by Comunidad de Madrid. AE grant number AP2010-0795 of Ministry of Education, Culture and Sports. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ; Sí

Oncolytic virotherapy represents a promising alternative for cancer treatment; however, viral delivery to the tumor represents a major challenge. Mesenchymal stem cells (MSCs) chemotax to tumors, and can serve as a viral delivery tool. Previously, we demonstrated antitumor therapeutic efficacy for mesenchymal stem cells (MSCs) infected with the oncolytic human adenovirus ICOVIR5 (Celyvir) for treatment of neuroblastoma patients. Given the lack of suitable immunocompetent preclinical models, the mechanism underlying Celyvir antitumor activity remains unknown. In this study, we used the syngeneic murine CMT64 cell line as a human adenovirus-semi-permissive tumor model and demonstrate the homing capacity of mouse Celyvir (mCelyvir) to CMT64 tumors. We found that the combined treatment of mCelyvir and intratumoral injections (i.t.) of ICOVIR5 was more effective than treatment with i.t. ICOVIR5 alone. Interestingly, the superior therapeutic effect of the combined therapy was associated with a higher tumor infiltration of CD8+ and CD4+ T cells. Our findings suggest that the use of MSCs as carriers of oncolytic adenovirus can improve the clinical efficacy of anti-cancer virotherapy, not only by driving the adenovirus to tumors, but also through their potential to recruit T cells. ; This work was supported by grants from the Fondo de Investigaciones Sanitarias (PI05/2217; PI08/0029; PI14CIII/00005), RTICC (RD12/0036/0015; RD12/0036/0027) and the Madrid Regional Government (S-BIO-0204-2006, MesenCAM; P2010/BMD-2420, CellCAM) in Spain to JG-C. ER and TC are supported by the "Sara Borrell" program of the Instituto de Salud Carlos III. The experiments were approved by the appropriate committees. ; Sí

Dogs with spontaneous tumors treated in veterinary hospitals offer an excellent opportunity for studying immunotherapies, including oncolytic viruses. Oncolytic viruses have advanced into the clinic as an intratumorally administered therapeutic; however, intravenous delivery has been hindered by neutralization in the blood. To circumvent this hurdle, mesenchymal stem cells have been used as a "Trojan horse." Here, we present the treatment of 27 canine patients with cancer with canine mesenchymal stem cells infected with ICOCAV17, a canine oncolytic adenovirus. No significant adverse effects were found. The response rate was 74%, with 14.8% showing complete responses, including total remissions of lung metastasis. We detected virus infection, stromal degeneration, and immune cell infiltration in tumor biopsies after 4 weeks of treatment. The increased presence of antiadenoviral antibodies in the peripheral blood of treated dogs did not appear to prevent the clinical benefit of this therapy. These data indicate that oncolytic viruses loaded in mesenchymal stem cells represent an effective cancer immunotherapy.Significance: The classical clinical limitations of antitumoral viroimmunotherapy can be overcome by use of mesenchymal stem cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4891/F1.large.jpg Cancer Res; 78(17); 4891-901. ©2018 AACR. ; We want to thank the staff of Veterinary Hospital, the technical staff from the Anatomo-pathological Department, as well as Carolina Jiménez and Giulia Setti for their participation in our studies. We are grateful to A. Gómez Vitores for useful advice on the pathology studies. J. Garcia-Castro was awarded grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377, PI17CIII/00013, RD12/0036/0027), the Madrid Regional Government (CellCAM; P2010/BMD-2420), and the Asociación Pablo Ugarte (G86121019). The experiments were approved by the appropriate committees. The costs of publication of this article were defrayed in part by the payment of ...

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation. ; This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the "Miguel Servet" program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees. ; Sí

BACKGROUND: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. METHODS: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. RESULTS: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. CONCLUSIONS: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings. ; This work has been financially supported by the Plan Nacional de I+D+I 2008–2011/2013–2016/FEDER (PI11/01728 to A.F.F.; PI12/01080 and PI15/00892 to M.F.F.; PI 12/0615 to J.A.R.; PI05/2217 and PI08/0029 to J.G‑C); the ISCIII‑Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); Fundacion Cientifica de la AECC (to R.G.U.); Fundación Ramón Areces (to M.F.F); and FICYT (to E.G.T., M.G.G and A.C.); the Madrid Regional Government (S‑BIO‑0204‑2006 and S2010/BMD‑2420 to J.G‑C) and the Asturias Regional Government (GRUPIN14‑052 to M.F.F.). The IUOPA is supported by the Obra Social Cajastur, Spain.

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. ; We thank Ronnie Lendrum for manuscript preparation and Tim Triche Jr. for his invaluable advice. This work has been financially supported by the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to A.F.F., PI 12/0615 to J.A.R., PI10/0449 to P.M., and PI11/0119 to C.B.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contracts CP11/00131 to A.F.F. and CP07/0059 to C.B.); the Spanish Ministry of Health (PS09/02454 and PI12/01080 to M.F.F.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); IUOPA (to C.F. and G.F.B.); Fundacion Cientifica de la AECC (to R.G.U. and P.M.); Fundación Ramón Areces (to M.F.F.); and FICYT (to E.G.T.). J.G.-C. receives funding from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029) and the Madrid Regional Government (S-BIO-0204-2006 and S2010/BMD-2420). J.A.R. receives funding from the Fondo de Investigaciones Sanitarias (ISCIII-FIS PI 12/0615). P.M. is also supported by MINECO (SAF2013/43065), ERANET E-Rare (PI112/03112), and Fundación Sandra Ibarra. P.M. also acknowledges support from Obra Social "La Caixa/Fundacio Josep Carreras." The IUOPA is supported by the Obra Social Cajastur, Spain. ; Sí