Publications by authors named "Pilar Martin-Duque"

28 Publications

  • Page 1 of 1

Combination Chemotherapy with Cisplatin and Chloroquine: Effect of Encapsulation in Micelles Formed by Self-Assembling Hybrid Dendritic-Linear-Dendritic Block Copolymers.

Int J Mol Sci 2021 May 14;22(10). Epub 2021 May 14.

Grupo de Terapia Génica y Celular, Instituto Aragonés de Ciencias de la Salud (IACS/IIS-Aragon), 50009 Zaragoza, Spain.

Clinical outcomes of conventional drug combinations are not ideal due to high toxicity to healthy tissues. Cisplatin (CDDP) is the standard component for many cancer treatments, yet its principal dose-limiting side effect is nephrotoxicity. Thus, CDDP is commonly used in combination with other drugs, such as the autophagy inhibitor chloroquine (CQ), to enhance tumor cell killing efficacy and prevent the development of chemoresistance. In addition, nanocarrier-based drug delivery systems can overcome chemotherapy limitations, decreasing side effects and increasing tumor accumulation. The aim of this study was to evaluate the toxicity of CQ and CDDP against tumor and non-tumor cells when used in a combined treatment. For this purpose, two types of micelles based on Pluronic F127 hybrid dendritic-linear-dendritic block copolymers (HDLDBCs) modified with polyester or poly(esteramide) dendrons derived from 2,2'-bis(hydroxymethyl)propionic acid (HDLDBC-bMPA) or 2,2'-bis(glycyloxymethyl)propionic acid (HDLDBC-bGMPA) were explored as delivery nanocarriers. Our results indicated that the combined treatment with HDLDBC-bMPA(CQ) or HDLDBC-bGMPA(CQ) and CDDP increased cytotoxicity in tumor cells compared to the single treatment with CDDP. Encapsulations demonstrated less short-term cytotoxicity individually or when used in combination compared to the free drugs. However, and more importantly, a low degree of cytotoxicity against non-tumor cells was maintained, even when drugs were given simultaneously.
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http://dx.doi.org/10.3390/ijms22105223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156097PMC
May 2021

Albumin-based nanostructures for uveal melanoma treatment.

Nanomedicine 2021 Jul 29;35:102391. Epub 2021 Mar 29.

IMDEA Nanociencia & Nanobiotecnología (IMDEA-Nanociencia) Unidad Asociada al Centro Nacional de Biotecnología (CSIC), Madrid, Spain. Electronic address:

Uveal melanoma (UM) is an intraocular tumor which is almost lethal at the metastatic stage due to the lack of effective treatments. In this regard, we have developed an albumin-based nanostructure (ABN) containing AZD8055 (ABN-AZD), which is a potent mTOR kinase inhibitor, for its efficient delivery to the tumors. The drug has been conjugated to ABN using tailored linkers that have a disulfide moiety, allowing its release selectively and effectively in the presence of an elevated concentration of glutathione, such as inside the tumoral cells. Our therapeutic approach induced significant cellular toxicity in uveal melanoma cells, but not in non-tumoral keratinocytes, highlighting the excellent selectivity of the system. In addition, these nanostructures showed excellent activity in vivo, decreasing the tumor surface compared to the free AZD8055 in mice models. Remarkably, the results obtained were achieved employing a dose 23 times lower than those used in previous reports.
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http://dx.doi.org/10.1016/j.nano.2021.102391DOI Listing
July 2021

Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis.

Nat Protoc 2021 01 27;16(1):131-163. Epub 2020 Nov 27.

Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain.

The use of exosomes as selective delivery vehicles of therapeutic agents, such as drugs or hyperthermia-capable nanoparticles, is being intensely investigated on account of their preferential tropism toward their parental cells. However, the methods used to introduce a therapeutic load inside exosomes often involve disruption of their membrane, which may jeopardize their targeting capabilities, attributed to their surface integrins. On the other hand, in recent years bio-orthogonal catalysis has emerged as a new tool with a myriad of potential applications in medicine. These bio-orthogonal processes, often based on Pd-catalyzed chemistry, would benefit from systems capable of delivering the catalyst to target cells. It is therefore highly attractive to combine the targeting capabilities of exosomes and the bio-orthogonal potential of Pd nanoparticles to create new therapeutic vectors. In this protocol, we provide detailed information on an efficient procedure to achieve a high load of catalytically active Pd nanosheets inside exosomes, without disrupting their membranes. The protocol involves a multistage process in which exosomes are first harvested, subjected to impregnation with a Pd salt precursor followed by a mild reduction process using gas-phase CO, which acts as both a reducing and growth-directing agent to produce the desired nanosheets. The technology is scalable, and the protocol can be conducted by any researcher having basic biology and chemistry skills in ~3 d.
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http://dx.doi.org/10.1038/s41596-020-00406-zDOI Listing
January 2021

Isolation of exosomes from whole blood by a new microfluidic device: proof of concept application in the diagnosis and monitoring of pancreatic cancer.

J Nanobiotechnology 2020 Oct 22;18(1):150. Epub 2020 Oct 22.

Department of Chemical Engineering, University of Zaragoza, 50018, Zaragoza, Spain.

Background: Exosomes are endocytic-extracellular vesicles with a diameter around 100 nm that play an essential role on the communication between cells. In fact, they have been proposed as candidates for the diagnosis and the monitoring of different pathologies (such as Parkinson, Alzheimer, diabetes, cardiac damage, infection diseases or cancer).

Results: In this study, magnetic nanoparticles (FeONPs) were successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. This was carried out under flow in a 1.6 mm (outer diameter) microchannel whose wall was in contact with a set of NdFeB permanent magnets, giving a high magnetic field across the channel diameter that allowed exosome separation with a high yield. To show the usefulness of the method, the direct capture of exosomes from whole blood of patients with pancreatic cancer (PC) was performed, as a proof of concept. The captured exosomes were then subjected to analysis of CA19-9, a protein often used to monitor PC patients.

Conclusions: Here, we describe a new microfluidic device and the procedure for the isolation of exosomes from whole blood, without any need of previous isolation steps, thereby facilitating translation to the clinic. The results show that, for the cases analyzed, the evaluation of CA19-9 in exosomes was highly sensitive, compared to serum samples.
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http://dx.doi.org/10.1186/s12951-020-00701-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579907PMC
October 2020

Radioactive Labeling of Milk-Derived Exosomes with Tc and In Vivo Tracking by SPECT Imaging.

Nanomaterials (Basel) 2020 May 30;10(6). Epub 2020 May 30.

Unidad de Medicina y Cirugía Experimental, Instituto de Investigación Sanitaria Gregorio Marañón, 28007 Madrid, Spain.

Over the last decade, exosomes from diverse biological sources have been proposed as new natural platforms in drug delivery. Translation of these nanometric tools to clinical practice requires deep knowledge of their pharmacokinetic properties and biodistribution. The pharmacokinetic properties of exosomes are sometimes evaluated using biochemical and histological techniques that are considerably invasive. As an alternative, we present radiochemical labeling of milk-derived exosomes based on reduced Tc (IV) without modifying biological and physicochemical properties. This approach enables longitudinal tracking of natural exosomes by non-invasive single photon emission computed tomography (SPECT) imaging and the evaluation of their pharmacokinetic properties according to the route of administration.
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http://dx.doi.org/10.3390/nano10061062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352469PMC
May 2020

Cancer-derived exosomes loaded with ultrathin palladium nanosheets for targeted bioorthogonal catalysis.

Nat Catal 2019 Oct 9;2(10):864-872. Epub 2019 Sep 9.

Cancer Research UK Edinburgh Centre, MRC Institute of Genetics & Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, UK.

The transformational impact of bioorthogonal chemistries has inspired new strategies for the synthesis of bioactive agents through non-natural means. Among these, palladium (Pd) catalysts have played a prominent role in the growing subfield of bioorthogonal catalysis by producing xenobiotics and uncaging biomolecules in living systems. However, delivering catalysts selectively to specific cell types still lags behind catalyst development. Here we have developed a bio-artificial device consisting of cancer-derived exosomes loaded with Pd catalysts by a method that enables the controlled assembly of Pd nanosheets directly inside the vesicles. This hybrid system mediates Pd-triggered dealkylation reactions and inside cells and displays preferential tropism for their progenitor cells. The use of Trojan exosomes to deliver abiotic catalysts into designated cancer cells creates the opportunity for a new targeted therapy modality: exosome-directed catalyst prodrug therapy, whose first steps are presented herein with the cell-specific release of the anticancer drug panobinostat.
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http://dx.doi.org/10.1038/s41929-019-0333-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795537PMC
October 2019

Efficient encapsulation of theranostic nanoparticles in cell-derived exosomes: leveraging the exosomal biogenesis pathway to obtain hollow gold nanoparticle-hybrids.

Nanoscale 2019 Oct 9;11(40):18825-18836. Epub 2019 Oct 9.

Networking Research Center of Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, 28029-Madrid, Spain and Instituto Aragonés de Ciencias de la Salud (IACS), Centro de Investigación Biomédica de Aragón (CIBA), 50009-Zaragoza, Spain and IIS Aragón(IISA), Centro de Investigación Biomédica de Aragón (CIBA), 50009-Zaragoza, Spain and Fundación ARAID. Avda. Ranillas, 1-D, planta 2ª, oficina b, 50018-Zaragoza, Spain.

Exosomes can be considered natural targeted delivery systems able to carry exogenous payloads, drugs or theranostic nanoparticles (NPs). This work aims to combine the therapeutic capabilities of hollow gold nanoparticles (HGNs) with the unique tumor targeting properties provided by exosomes. Here, we tested different methods to encapsulate HGNs (capable of absorbing light in the NIR region for selective thermal ablation) into murine melanoma cells derived exosomes (B16-F10-exos), including electroporation, passive loading by diffusion, thermal shock, sonication and saponin-assisted loading. These methods gave less than satisfactory results: although internalization of relatively large NPs into B16-F10-exos was achieved by almost all the physicochemical methods tested, only about 15% of the exosomes were loaded with NPs and several of those processes had a negative effect regarding the morphology and integrity of the loaded exosomes. In a different approach, B16-F10 cells were pre-incubated with PEGylated HGNs (PEG-HGNs) in an attempt to incorporate the NPs into the exosomal biogenesis pathway. The results were highly successful: exosomes recovered from the supernatant of the cell culture showed up to 50% of HGNs internalization. The obtained hybrid HGN-exosome vectors were characterized with a battery of techniques to make sure that internalization of HGNs did not affect exosome characteristics compared with other strategies. PEG-HGNs were released through the endosomal-exosome biogenesis pathway confirming that the isolated vesicles were exosomes.
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http://dx.doi.org/10.1039/c9nr06183eDOI Listing
October 2019

Exosome origin determines cell targeting and the transfer of therapeutic nanoparticles towards target cells.

J Nanobiotechnology 2019 Jan 25;17(1):16. Epub 2019 Jan 25.

Department of Chemical Engineering, Aragon Institute of Nanoscience (INA), University of Zaragoza, Campus Río Ebro-Edificio I+D, C/ Mariano Esquillor S/N, 50018, Zaragoza, Spain.

Background: Exosomes are considered key elements for communication between cells, but very little is known about the mechanisms and selectivity of the transference processes involving exosomes released from different cells.

Results: In this study we have investigated the transfer of hollow gold nanoparticles (HGNs) between different cells when these HGNs were loaded within exosomes secreted by human placental mesenchymal stem cells (MSCs). These HGNs were successfully incorporated in the MSCs exosome biogenesis pathway and released as HGNs-loaded exosomes. Time-lapse microscopy and atomic emission spectroscopy allowed us to demonstrate the selective transfer of the secreted exosomes only to the cell type of origin when studying different cell types including cancer, metastatic, stem or immunological cells.

Conclusions: In this study we demonstrate the selectivity of in vitro exosomal transfer between certain cell types and how this phenomenon can be exploited to develop new specific vectors for advanced therapies. Specifically, we show how this preferential uptake can be leveraged to selectively induce cell death by light-induced hyperthermia only in cells of the same type as those producing the corresponding loaded exosomes. We describe how the exosomes are preferentially transferred to some cell types but not to others, thus providing a better understanding to design selective therapies for different diseases.
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http://dx.doi.org/10.1186/s12951-018-0437-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346572PMC
January 2019

Cationic poly(ester amide) dendrimers: alluring materials for biomedical applications.

J Mater Chem B 2018 Jun 31;6(23):3956-3968. Epub 2018 May 31.

Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain.

Novel cationic poly(ester amide) dendrimers have been synthesized by copper(i) azide-alkyne cycloaddition (CuAAC) of a tripropargylamine core and azide-terminated dendrons, in turn prepared by iterative amide coupling of the new monomer 2,2'-bis(glycyloxymethyl)propionic acid (bis-GMPA). The alternation of ester and amide groups provided a dendritic scaffold that was totally biocompatible and degradable in aqueous media at physiological and acidic pH. The tripodal dendrimers naturally formed rounded aggregates with a drug that exhibited low water solubility, camptothecin, thus improving its cell viability and anti-Hepatitis C virus (anti-HCV) activity. The presence of numerous peripheral cationic groups enabled these dendrimers to form dendriplexes with both pDNA and siRNA and they showed effective in vitro siRNA transfection in tumoral and non-tumoral cell lines.
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http://dx.doi.org/10.1039/c8tb00639cDOI Listing
June 2018

The effect of PEGylated hollow gold nanoparticles on stem cell migration: potential application in tissue regeneration.

Nanoscale 2017 Jul;9(28):9848-9858

Department of Chemical Engineering, Aragon Institute of Nanoscience (INA), University of Zaragoza, Campus Río Ebro-Edificio I+D, C/Poeta Mariano Esquillor S/N, 50018-Zaragoza, Spain.

Mesenchymal stem cells (MSCs) not only can be differentiated into different cell types but also have tropism towards injured or inflamed tissues serving as repair cells. Here we have demonstrated that MSCs containing gold nanoparticles (GNPs) whose surface has been functionalized with PEG show accelerated cell migration, successful scaffold colonization and regeneration. We report the impact of GNPs on the migration (by the wound healing assay), and proliferation (by flow cytometry analysis and by the detection of metabolic mitochondrial activity) on the behaviour of different cell lines including MSCs, HeLa cells, and human dermal fibroblasts. We conclude that GNPs are easily internalized by MSCs causing an increase in their migration rate, mediated by actin and tubulin with a 4-fold increased expression level of those proteins. We also demonstrate that MSCs containing GNPs are able to successfully colonize fibrin and PCL-based scaffolds and that an enhanced osteoblastic differentiation is reached when using the nanoparticle-laden cells compared to untreated cells used as a control. These results highlight the potential use of MSCs as therapeutic nanoparticle-carriers in regenerative medicine.
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http://dx.doi.org/10.1039/c7nr01853cDOI Listing
July 2017

Polymer functionalized gold nanoparticles as nonviral gene delivery reagents.

J Gene Med 2017 Jun;19(6-7)

Facultad de Ciencias Biosanitarias, Carretera Pozuelo a Majadahonda, Universidad Francisco de Vitoria, Madrid, Spain.

Background: In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes.

Methods: Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase.

Results: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector.

Conclusions: Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.
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http://dx.doi.org/10.1002/jgm.2964DOI Listing
June 2017

DNA Transfection to Mesenchymal Stem Cells Using a Novel Type of Pseudodendrimer Based on 2,2-Bis(hydroxymethyl)propionic Acid.

Bioconjug Chem 2017 04 13;28(4):1135-1150. Epub 2017 Mar 13.

Departamento de Quı́mica Orgánica, Facultad de Ciencias, Instituto de Nanociencia de Aragón, Universidad de Zaragoza , Zaragoza 50009, Spain.

In the search for effective vehicles to carry genetic material into cells, we present here new pseudodendrimers that consist of a hyperbranched polyester core surrounded by amino-terminated 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) dendrons. The pseudodendrimers are readily synthesized from commercial hyperbranched bis-MPA polyesters of the second, third, and fourth generations and third-generation bis-MPA dendrons, bearing eight peripheral glycine moieties, coupled by the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This approach provides globular macromolecular structures bearing 128, 256, and 512 terminal amino groups, and these can complex pDNA. The toxicity of the three pseudodendrimers was studied on two cell lines, mesenchymal stem cells, and HeLa, and it was demonstrated that these compounds do not affect negatively cell viability up to 72 h. The complexation with DNA was investigated in terms of N-to-P ratio and dendriplex stability. The three generations were found to promote internalizing of pDNA into mesenchymal stem cells (MSCs), and their transfection capacity was compared with two nonviral commercial transfection agents, Lipofectamine and TransIT-X2. The highest generations were able to transfect these cells at levels comparable to both commercial reagents.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00037DOI Listing
April 2017

New Ionic bis-MPA and PAMAM Dendrimers: A Study of Their Biocompatibility and DNA-Complexation.

Macromol Biosci 2015 May 28;15(5):657-67. Epub 2015 Jan 28.

Departamento de Química Orgánica-Instituto de Nanociencia de Aragón (INA), University of Zaragoza, C/Pedro Cerbuna 12, 50009, Zaragoza, Spain.

Herein, the synthesis of five novel ionic dendrimers and their evaluation as biological carriers is reported. The compounds include an ionic bis-MPA dendrimer and four PAMAM dendrimers of different generations decorated with negatively charged hydrophilic chains of 2-[2-(2-methoxyethoxy)ethoxy]acetic acid as counter ions in order to increase their biocompatibility. The ionic dendrimers derived from bis-MPA have a low cytotoxicity at 0.5 mg · mL(-1) against U251MG and are even less toxic against mesenchymal stem cells; however, the PAMAM derivatives show high cytotoxicity at the same concentration. The five compounds are able to form complexes with plasmid DNA at different N/P ratios. The cytotoxicity and complexation ability of the new dendrimers were also compared with jetPEI, a linear polyethylenimine derivative commercially available as transfection reagent. The results indicate that the cytotoxicity of the ionic PAMAM dendrimers remains as an important drawback, whereas the ionic I-bis-MPA compound exhibits a high ability to complex pDNA and very low toxicity compared with jetPEI.
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http://dx.doi.org/10.1002/mabi.201400422DOI Listing
May 2015

Calcium homeostasis in myogenic differentiation factor 1 (MyoD)-transformed, virally-transduced, skin-derived equine myotubes.

PLoS One 2014 22;9(8):e105971. Epub 2014 Aug 22.

Comparative Neuromuscular Diseases Laboratory, Department of Clinical Sciences and Services, Royal Veterinary College, London, United Kingdom.

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells' calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105971PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141859PMC
March 2016

Adenovirus-mediated expression of myogenic differentiation factor 1 (MyoD) in equine and human dermal fibroblasts enables their conversion to caffeine-sensitive myotubes.

Neuromuscul Disord 2014 Mar 23;24(3):250-8. Epub 2013 Nov 23.

Department of Clinical Sciences and Services, Royal Veterinary College, Royal College Street, London NW1 0TU, UK. Electronic address:

Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.
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http://dx.doi.org/10.1016/j.nmd.2013.11.009DOI Listing
March 2014

Tissue-derived mesenchymal stromal cells used as vehicles for anti-tumor therapy exert different in vivo effects on migration capacity and tumor growth.

BMC Med 2013 May 28;11:139. Epub 2013 May 28.

Instituto Aragones de Ciencias de la Salud-IIS Aragon, Zaragoza, Spain.

Background: Mesenchymal stem cells (MSCs) have been promoted as an attractive option to use as cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines. Multiple isolated populations have been described as MSCs, but despite extensive in vitro characterization, little is known about their in vivo behavior.The aim of this study was to investigate the efficacy and efficiency of different MSC lineages derived from five different sources (bone marrow, adipose tissue, epithelial endometrium, stroma endometrium, and amniotic membrane), in order to assess their adequacy for cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurate targeted therapeutic applications.

Methods: We first measured the in vivo migration capacity and effect on tumor growth of the different MSCs using two imaging techniques: (i) single-photon emission computed tomography combined with computed tomography (SPECT-CT), using the human sodium iodine symporter gene (hNIS) and (ii) magnetic resonance imaging using superparamagnetic iron oxide. We then sought correlations between these parameters and expression of pluripotency-related or migration-related genes.

Results: Our results show that migration of human bone marrow-derived MSCs was significantly reduced and slower than that obtained with the other MSCs assayed and also with human induced pluripotent stem cells (hiPSCs). The qPCR data clearly show that MSCs and hiPSCs exert a very different pluripotency pattern, which correlates with the differences observed in their engraftment capacity and with their effects on tumor growth.

Conclusion: This study reveals differences in MSC recruitment/migration toward the tumor site and the corresponding effects on tumor growth. Three observations stand out: 1) tracking of the stem cell is essential to check the safety and efficacy of cell therapies; 2) the MSC lineage to be used in the cell therapy needs to be carefully chosen to balance efficacy and safety for a particular tumor type; and 3) different pluripotency and mobility patterns can be linked to the engraftment capacity of the MSCs, and should be checked as part of the clinical characterization of the lineage.
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http://dx.doi.org/10.1186/1741-7015-11-139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670996PMC
May 2013

Telomerase-driven expression of the sodium iodide symporter (NIS) for in vivo radioiodide treatment of cancer: a new broad-spectrum NIS-mediated antitumor approach.

J Clin Endocrinol Metab 2011 Sep 22;96(9):E1435-43. Epub 2011 Jun 22.

Molecular Endocrinology, Instituto de Investigaciones Biomedicas, Arturo Duperier 4, Madrid, Spain 28029.

Context: Telomerase promoters (hTERT and hTR) are useful for transcriptional targeting in gene therapy models of cancer. Telomerase-driven expression of the sodium iodide symporter (NIS) in tumor cells has been successfully used as a reporter gene in vivo using positron emission tomography (PET) imaging.

Objective: The aim of this study was to investigate the NIS-mediated therapeutic effect of telomerase promoters in a wide variety of human cancer cell lines.

Design And Methods: Promoter fragments from either hTERT or hTR were used to drive the expression of NIS in cell lines derived from melanoma (M14), breast (MDA-MB-231), colon (HT-29), lung (H460), ovarian (OVCAR-3), and thyroid (TPC-1) carcinomas. Iodide uptake assays, protein immunodetection, and clonigenic assays were used to confirm NIS functional expression and the (131)I-mediated cytopathic effect. Tumor xenografts in mice were infected with hTERT and hTR and then treated using radioiodide.

Results: Both promoters were selectively active in cancer cells that were effectively killed by exposure to (131)I. One single dose of 1 mCi (131)I markedly suppressed tumor growth of melanoma-derived tumor xenografts compared with controls. This effect was more modest in colon cancer-derived xenografts in part due to the reduced infectivity and the tumor cystic nature. The therapeutic effect of hTR promoter was found to be stronger than that of hTERT promoter.

Conclusions: These results demonstrate that telomerase-driven expression of NIS could potentially have applications for (131)I therapy of a wide variety of cancers. Additionally, this is the first study to report NIS-mediated (131)I therapy of melanoma tumors in vivo.
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http://dx.doi.org/10.1210/jc.2010-2373DOI Listing
September 2011

Using living cells to transport therapeutic genes for cancer treatment.

Clin Transl Oncol 2011 Jan;13(1):10-7

Instituto de Investigación Sanitaria de Aragón, Zaragoza, Spain.

One of the key problems in cancer gene therapy is the inefficient delivery of therapeutic transgenes to tumour sites, after the systemic injection of the viral vector. Hence, new vector discovery is extremely important for the improvement of gene therapy results. Previously, mammalian cells were proposed as new vector systems; however with recent advances in stem cell research this modality makes them more suitable candidates. Tumours are composed of both malignant and benign cells. As "benign" cell types are able to form blood vessels, and stroma, it has been hypothesised that exogenously administrated cells of a different kind would preferentially engraft at the stromal tumour site and could deliver cancer gene therapy vectors to tumours.
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http://dx.doi.org/10.1007/s12094-011-0611-3DOI Listing
January 2011

Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours.

Eur J Nucl Med Mol Imaging 2010 Jul 6;37(7):1377-85. Epub 2010 Feb 6.

Centre for Molecular Oncology, Institute of Cancer, Queen Mary's School of Medicine and Dentistry, London, UK.

Purpose: In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma.

Methods: We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated.

Results: In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart.

Conclusion: This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects.
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http://dx.doi.org/10.1007/s00259-009-1379-3DOI Listing
July 2010

Targeted radionuclide therapy using a Wnt-targeted replicating adenovirus encoding the Na/I symporter.

Clin Cancer Res 2009 Nov 27;15(21):6595-601. Epub 2009 Oct 27.

Centre for Molecular Oncology, Institute of Cancer, Queen Mary's School of Medicine and Dentistry, London, United Kingdom.

Purpose: The Na/I symporter (hNIS) promotes concentration of iodine in cells. In cancer gene therapy, this transgene has potential as a reporter gene for molecular imaging of viral biodistribution and as a therapeutic protein promoting (131)I-mediated radiotherapy. Here, we combined the imaging and therapeutic potential of hNIS in an oncolytic adenoviruses targeting colorectal cancer cells.

Experimental Design: We generated an adenovirus (AdIP2) encoding hNIS and capable of selective replication in colorectal carcinoma cells. The selectivity of this virus was verified in vitro and in vivo. Its spread in tumors was monitored in vivo using single-photon emission computed tomography/CT imaging upon (99m)TcO(4)(-) injection and confirmed by immunohistochemistry. Metabolic radiotherapy was done through injection of therapeutic doses of (131)I(-).

Results: We showed in vitro and in vivo the selectivity of AdIP2 and that hNIS expression is restricted to the target cells. Imaging and immunohistochemical data showed that viral spread is limited and that the point of maximal hNIS expression is reached 48 hours after a single intratumoral injection. Administration of a single therapeutic dose of (131)I at this time point led to a dramatic reduction in tumor size not observed in hNIS-negative viruses.

Conclusions: This report showed for the first time that the combination of the imaging and therapeutic potentials of hNIS can be applied to oncolytic adenoviruses in experimental models of cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-0262DOI Listing
November 2009

Visualization of gene expression in the live subject using the Na/I symporter as a reporter gene: applications in biotherapy.

Br J Pharmacol 2010 Feb 8;159(4):761-71. Epub 2009 Oct 8.

Inserm U948, Université de Nantes, Nantes Atlantique Universités, EA4274, Institut des Maladies de l'Appareil Digestif, CHU Hôtel Dieu, Nantes, France.

Biotherapies involve the utilization of antibodies, genetically modified viruses, bacteria or cells for therapeutic purposes. Molecular imaging has the potential to provide unique information that will guarantee their biosafety in humans and provide a rationale for the future development of new generations of reagents. In this context, non-invasive imaging of gene expression is an attractive prospect, allowing precise, spacio-temporal measurements of gene expression in longitudinal studies involving gene transfer vectors. With the emergence of cell therapies in regenerative medicine, it is also possible to track cells injected into subjects. In this context, the Na/I symporter (NIS) has been used in preclinical studies. Associated with a relevant radiotracer ((123)I(-), (124)I(-), (99m)TcO4(-)), NIS can be used to monitor gene transfer and the spread of selectively replicative viruses in tumours as well as in cells with a therapeutic potential. In addition to its imaging potential, NIS can be used as a therapeutic transgene through its ability to concentrate therapeutic doses of radionuclides in target cells. This dual property has applications in cancer treatment and could also be used to eradicate cells with therapeutic potential in the case of adverse events. Through experience acquired in preclinical studies, we can expect that non-invasive molecular imaging using NIS as a transgene will be pivotal for monitoring in vivo the exact distribution and pharmacodynamics of gene expression in a precise and quantitative way. This review highlights the applications of NIS in biotherapy, with a particular emphasis on image-guided radiotherapy, monitoring of gene and vector biodistribution and trafficking of stem cells.
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http://dx.doi.org/10.1111/j.1476-5381.2009.00412.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829202PMC
February 2010

Cancer-specific transgene expression mediated by systemic injection of nanoparticles.

Cancer Res 2009 Mar 3;69(6):2655-62. Epub 2009 Mar 3.

Centre for Molecular Oncology, Queen Mary's School of Medicine and Dentistry, London, United Kingdom.

The lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na/I symporter (NIS) as reporter gene shows unique and highly specific tumor targeting with no detection of gene transfer in any of the other tissues of tumor-bearing mice. Tumor-selective transgene expression was confirmed by quantitative reverse transcription-PCR at autopsy of scanned animals, whereas genomic PCR showed that the tumor sites are the predominant sites of nanoparticle accumulation. Considering that NIS imaging of transgene expression has been recently validated in humans, our data highlight the potential of these nanoparticles as a new formulation for cancer gene therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-2657DOI Listing
March 2009

The genetics of malignant melanoma.

Front Biosci 2008 May 1;13:5071-93. Epub 2008 May 1.

Instituto de Investigaciones Biomedicas Alberto Sols, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid, Spain.

Melanoma probably is the most aggressive cancer in humans and remains one of the leading causes of cancer death in developed countries. This review summarizes the most important alterations in protooncogenes and tumor suppressor genes that contribute to the pathogenesis of malignant melanoma, with a special emphasis on the involved signaling pathways. Our knowledge of the molecular biology of melanoma has been benefited from recent advances on high-throughput technologies analyzing wide genomic and gene expression profiles that have uncovered unknown candidate genes. To test the interactions between distinct pathways and of those with the environment a wealth of genetically modified animal models has been generated over the past years. Other studies have focused on the isolation of melanoma stem cells and on the characterization of signaling pathways that contribute to their survival and maintenance. A consequence of all these studies is the emergence of potential new strategies that could improve the still inadequate arsenal of therapeutic tools to fight against this fatal disease.
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http://dx.doi.org/10.2741/3065DOI Listing
May 2008

Molecular biology of malignant melanoma.

Adv Exp Med Biol 2008 ;624:252-64

Instituto de Investigaciones Biomédicas Alberto Sols, Departamento de Biotecnologia, Universidad Francisco de Vitoria, CSIC-UAM, Arturo Duperier 4, 28029-Madrid, Spain.

The incidence of melanoma has increased more rapidly than any other type of cancer. In this review, we summarize the most important genetic alterations that contribute to the development of malignant melanoma. Our knowledge of the genetic and biological events involved in the genesis and progression of this disease has been benefited from the evolvement of a wealth of genetically engineered animal models. Hopefully, the understanding generated by all these studies will contribute to develop new therapeutic strategies to handle this fatal malignancy.
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http://dx.doi.org/10.1007/978-0-387-77574-6_20DOI Listing
April 2008

Uveal vs. cutaneous melanoma. Origins and causes of the differences.

Clin Transl Oncol 2008 Mar;10(3):137-42

Universidad Francisco de Vitoria, Facultad de Ciencias Biosanitarias, Dpto. Biotecnología, Pozuelo de Alarcón, Madrid, Spain.

Melanoma is a malignant tumour derived from melanocytes (dendritic cells originated from the neural crest and capable to produce melanin synthesis) that could be established on the skin or less frequently on the uvea. The cellular origin from both kind of melanoma seems to be the same but the melanocytes migrates to the epithelia for cutaneous melanoma, while for uveal melanoma, they migrate to mesodermic tissues. Despite the common origin, both melanomas show extreme differences in their metastatic potential, clinical response to treatments, immune response and genetic alterations. We will describe some of those differences in this review.
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http://dx.doi.org/10.1007/s12094-008-0170-4DOI Listing
March 2008

Caspase-1 as a radio- and chemo-sensitiser in vitro and in vivo.

Int J Mol Med 2006 May;17(5):841-7

Cancer Research-UK Molecular Oncology Unit, Bart's and The London School of Medicine and Dentistry, John Vane Science Centre, UK.

The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. This observation raises the possibility that factors affecting caspase activation might be important determinants of anticancer drug sensitivity. Ectopic expression of caspase-1 has been shown to trigger apoptosis. However, the role of caspase-1 in apoptosis is now considered as minor compared to other caspases. In patients, high levels of caspase-1 expression may be associated with spontaneous regression in neuroblastomas and with a good clinical response to chemotherapy in acute myeloid leukemia and osteosarcoma. In experimental therapeutics for cancer, caspase-1 has been related to some anticancer activity. These observations led us to examine the effect of over-expression on the response to chemotherapy and radiotherapy in vitro and in vivo. Caspase-1 expression mediated by an adenoviral vector was able to kill directly cells and to sensitise the remaining cells to cisplatin or gamma-radiation in vitro. In HeLa cells stably transfected with caspase-1, sensitisation to cisplatin was due to an amplification of the cisplatin-induced mitochondrial apoptotic pathway activation. Caspase-1 mediated sensitisation to cisplatin and gamma-radiation was also observed in vivo. Altogether, we conclude that caspase-1 can act as a radio- and chemo-sensitiser, in vitro and in vivo.
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May 2006

Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes.

Hum Gene Ther 2004 Oct;15(10):995-1002

Molecular Oncology Unit, Cancer Research UK Clinical Centre, Barts and the London School of Medicine and Dentistry, John Vane Science Centre, London EC1M 6BQ, United Kingdom.

Targeted gene expression can be achieved through the use of cell-selective promoters. However, when the expression cassette is delivered by an adenovirus, "promoter interference," resulting in the loss of specificity, has been reported. To overcome this problem, insulator elements (the bovine growth hormone transcription stop signal or HS4 chromatin insulators of the chicken beta-globin locus) have been used. The present study examines the efficacy of these insulators elements, when two independent expression cassettes (one in which a strong, ubiquitous promoter drives the expression of the green fluorescent protein and another in which the "cancer-selective" ERBB2 promoter drives the expression of the herpes simplex virus thymidine kinase [HSVtk] gene) are incorporated into the same recombinant adenovirus. As expected, the presence of either insulator does not alter the expression of HSVtk in ERBB2-positive cells, measured through sensitization of the cells to ganciclovir. When ERBB2-negative cells were infected at a multiplicity of infection (MOI) of 10, the uninsulated virus sensitized ERBB2-negative cells to the same extent as it did for ERBB2-positive cells; partial sensitization was observed when transcriptional terminators were used, indicating a partial insulating effect; and complete insulation (no sensitization to ganciclovir) was observed when HS4 chromatin insulators were used. However, this complete insulation was lost when the MOI of virus was increased to 100. Our study demonstrates the possibility of insulating a conditionally expressed transgene in the vicinity of another expression cassette, but this insulating effect is lost when the multiplicity of infection is increased.
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http://dx.doi.org/10.1089/hum.2004.15.995DOI Listing
October 2004

Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes.

Hum Gene Ther 2004 Oct;15(10):995-1002

Molecular Oncology Unit, Cancer Research UK Clinical Centre, Barts and the London School of Medicine and Dentistry, John Vane Science Centre, London EC1M 6BQ, United Kingdom.

Targeted gene expression can be achieved through the use of cell-selective promoters. However, when the expression cassette is delivered by an adenovirus, "promoter interference," resulting in the loss of specificity, has been reported. To overcome this problem, insulator elements (the bovine growth hormone transcription stop signal or HS4 chromatin insulators of the chicken beta-globin locus) have been used. The present study examines the efficacy of these insulators elements, when two independent expression cassettes (one in which a strong, ubiquitous promoter drives the expression of the green fluorescent protein and another in which the "cancer-selective" ERBB2 promoter drives the expression of the herpes simplex virus thymidine kinase [HSVtk] gene) are incorporated into the same recombinant adenovirus. As expected, the presence of either insulator does not alter the expression of HSVtk in ERBB2-positive cells, measured through sensitization of the cells to ganciclovir. When ERBB2-negative cells were infected at a multiplicity of infection (MOI) of 10, the uninsulated virus sensitized ERBB2-negative cells to the same extent as it did for ERBB2-positive cells; partial sensitization was observed when transcriptional terminators were used, indicating a partial insulating effect; and complete insulation (no sensitization to ganciclovir) was observed when HS4 chromatin insulators were used. However, this complete insulation was lost when the MOI of virus was increased to 100. Our study demonstrates the possibility of insulating a conditionally expressed transgene in the vicinity of another expression cassette, but this insulating effect is lost when the multiplicity of infection is increased.
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http://dx.doi.org/10.1089/hum.2004.15.995DOI Listing
October 2004
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