Pablo Iglesias

Fluorescent drug-loaded, polymeric-based, branched gold nanoshells for localized multimodal therapy and imaging of tumoral cells.

Here we report the synthesis of PLGA/DOXO-core Au-branched shell nanostructures (BGNSHs) functionalized with a human serum albumin/indocyanine green/folic acid complex (HSA-ICG-FA) to configure a multifunctional nanotheranostic platform. First, branched gold nanoshells (BGNSHs) were obtained through a seeded-growth surfactant-less method. These BGNSHs were loaded during the synthetic process with the chemotherapeutic drug doxorubicin, a DNA intercalating agent and topoisomerase II inhibitior. In parallel, the fluorescent near-infrared (NIR) dye indocyanine green (ICG) was conjugated to the protein human serum albumin (HSA) by electrostatic and hydrophobic interactions. Subsequently, folic acid was covalently attached to the HSA-ICG complex. In this way, we created a protein complex with targeting specificity and fluorescent imaging capability. The resulting HSA-ICG-FA complex was adsorbed to the gold nanostructures surface (BGNSH-HSA-ICG-FA) in a straightforward incubation process thanks to the high affinity of HSA to gold surface. In this manner, BGNSH-HSA-ICG-FA platforms were featured with multifunctional abilities: the possibility of fluorescence imaging for diagnosis and therapy monitoring by exploiting the inherent fluorescence of the dye, and a multimodal therapy approach consisting of the simultaneous combination of chemotherapy, provided by the loaded drug, and the potential cytotoxic effect of photodynamic and photothermal therapies provided by the dye and the gold nanolayer of the hybrid structure, respectively, upon NIR light irradiation of suitable wavelength. The combination of this trimodal approach was observed to exert a synergistic effect on the cytotoxicity of tumoral cells in vitro. Furthermore, FA was proved to enhance the internalization of nanoplatform. The ability of the nanoplatforms as fluorescence imaging contrast agents was tested by preliminary analyzing their biodistribution in vivo in a tumor-bearing mice model.

Retinoblastoma Loss Modulates DNA Damage Response Favoring Tumor Progression

Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRasV12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

A novel BRET-based genetically encoded biosensor for functional imaging of hypoxia.

Optical imaging methods, such as fluorescence, have greatly increased its impact as a monitoring technique with the development of a broad range of fluorescent proteins used to visualize many types of biological processes, such as cancer biology. Although the most popular of these proteins is the green fluorescent protein (GFP), autofluorescence due to the absorption of the exciting radiation by endogenous fluorophores and signal dispersion raises doubts about its suitability as an in vivo tracer. In the last years a number of groups have developed several NIR fluorescent proteins that enables real-time imaging to take place without interference from autofluorescence events allowing at the same time to take a deep view into the tissues. We therefore have outlined fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1alpha, which is upregulated in growing tumors. At the same time, by fusing a fluorescent to a bioluminescent protein, we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.

NIR-light active hybrid nanoparticles for combined imaging and bimodal therapy of cancerous cells

We report the synthesis of a multifunctional biocompatible theranostic nanoplatform consisting of a biodegradable PLGA matrix surface-functionalized with indocyanine green (ICG), a near-IR fluorescent dye, and co-loaded with superparamagnetic iron oxide nanoparticles (SPIONs) and the anticancer drug doxorubicin (DOXO). Combination of chemo- and photothermal therapeutic efficacy as well as magnetic resonance and optical fluorescence imaging performance were successfully tested in vitro on a tumoral cervical HeLa cell line. Magnetic in vitro guided targeting of these nanoplatforms was also proven. These nanoconstructs also enabled to monitor their in vivo biodistribution by fluorescence imaging in a mice model, which revealed their effective accumulation in the tumor and, unexpectedly, in the brain area. A lower presence of nanoplatforms was noted in the reticulo-endothelial system. The present observations suggest the nanoplatforms ability to possibly overcome the blood brain barrier. These results open up new possibilities to use our multifunctional nanoplatforms to treat brain-located diseases.

Defining hypoxic microenvironments by non-invasive functional optical imaging

Functional imaging has become an important tool in oncology by informing about localisation and size of the tumour as well as the pathophysiological features of tumoural cells. One of the most characteristic features of some tumour types is the activation of the neoangiogenic programme which is specifically mediated by the transcription factor hypoxia-inducible factor (HIF)-1α, an important player in regulating this process and a prognostic marker of tumoural aggressiveness. Here we report a non-invasive in vivo detection of lung micrometastases in a mouse model of breast cancer using self-illuminating genetically encoded tracers responsive to intracellular HIF-1α levels and a preliminary analysis of the contribution of the tumoural masses to the metastatic niche. This model lays the foundations for novel hypoxia sensing probes able to detect micrometastatic disease with high sensitivity and specificity. Thus, optical functional imaging shows promise in the understanding of disease, drug development or image-guided therapy.

The maintenance of mitochondrial genetic stability is crucial during the oncogenic process

The main energetic resources of the cell are the mitochondria. As such, these organelles control a number of processes related to the life and death of the cell and also have a prominent function in the maintenance of tumor cells. In the last years, several authors have proposed an active role for mitochondria in tumorigenesis, more specifically concerning somatic mutations in mitochondrial DNA (mtDNA). Here, we wanted to evaluate this hypothesis based on the conclusions obtained in a model of gliomagenesis with elevated levels of ROS (reactive oxygen species), a toxic by-product of tumor metabolism. According to our findings, none of the mtDNA variants were found relevant to the tumoral process or suggest the involvement of mitochondria in tumorigenesis beyond the metabolic requirements of the tumoral cell. We conclude that there is not enough evidence to support the claim that mitochondrial instability holds any relevant role in the tumoral process.

The antimitotic potential of PARP inhibitors, an unexplored therapeutic alternative.

ADP-ribosylation or PARsylation is one of the most abundant modifications of proteins and DNA. Although the usual context for PARsylation involves the detection and repair of DNA damage in the cell, poly(ADP-ribose) polymerases are known to regulate a number of biological processes besides maintaining genome integrity. One of these processes is the assembly and maintenance of the mitotic spindle where the presence of PARP-1 and tankyrase 1 (TNKS1), two of the best-characterized members of the PARP superfamily, is of critical importance. Here, we recapitulate the biological implications of the absence of poly(ADP-ribose) polymerases and depletion of PARsylation occurrence in mitosis in order to better understand the antimitotic effects of PARP inhibitors. In this regard, we also present an overview of the existing and more relevant molecules, with a special attention to the historical development of their pharmacological properties and structures, as well as a brief summary of clinical trials involving PARP inhibitors.

Abstract 2893: PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo

Although PARP-1 has been traditionally related to DNA repair, in the last years an increasing number of studies have linked this polymerase to other cellular processes such as metabolism and cell mitosis. In this study we wanted to investigate the biological importance of the interaction between PARP-1 and the transcription factor E2F-1, and more specifically in scenarios where the activity, or hyperactivity, of E2F-1 is of critical importance such as embryonic development or oncogenesis. In this regard, we have found that the treatment either with enzymatic inhibitors of PARP-1 has an effect on the transcriptional activity of E2F-1 as well as the proliferative rate of treated cells. This effect is significantly increased with the treatment with gossypol, a specific inhibitor of PARP-1 protein-protein interactions occurring through the BRCT domain, as in the case of E2F-1. This effect is also observed in vivo since the severity of histological malformations in Rb-/- embryos is significantly reduced in Parp-1-/- Rb-/- embryos, which phenotype closely resembles that of E2f-1-/- Rb-/- mice. Finally, we also found that the deletion or inhibition of PARP-1 in an animal model of gliomagenesis helps the cell to block oncogenic stimuli derived from E2F-1 hyperactivity, by reactivating critical signalling pathways involved in oncogene-induced senescence. In summary, the importance of the relationship between PARP-1 and E2F-1 in different biological contexts leads us to believe that the search for ways of altering or disrupting this interaction could be a novel strategy for the development of molecules of therapeutic potential. Citation Format: Pablo Iglesias, Marcos Seoane, Isabel Castro-Piedras, Victor M. Arce, Jose A. Costoya. PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2893.

Molecular Imaging of Hypoxia Using Genetic Biosensors

In the last years, the need for visualization of tumoral processes has become a high-top priority in molecular imaging. This is especially true for those methods dedicated to functional imaging that focus on revealing phenomena associated with biological processes such as hypoxia to cancer. Among them, optical imaging methods such as fluorescence are provided with a broad range of proteins and dyes used to visualize many types of these biological processes widely used in cell biology studies. Although the most popular of these proteins is the green fluorescent protein (GFP), autofluorescence due to the absorption of the exciting radiation by endogenous fluorophores and signal dispersion raises doubts about its suitability as an in vivotracer. In the last years a number of groups have developed several NIR fluorescent proteins that enables real-time imaging to take place without interference from autofluorescence events allowing at the same time to take a deep view into the tissues. With all of this in mind, we devised a novel fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1α, which appears upregulated in growing tumors. At the same time, by fusing a NIR emitting flurochrome (mCherry) and the firefly luciferase together we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.

Abstract 469: Functional imaging of the hypoxic tumor microenvironment in metastatic progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In the last years, the need for visualization of tumoral processes has become a high-top priority in molecular imaging. This is especially true for those methods dedicated to functional imaging that focus on revealing phenomena associated with biological processes such as hypoxia to cancer. Among them, optical imaging methods such as fluorescence are provided with a broad range of proteins and dyes used to visualize many types of these biological processes widely used in cell biology studies. Although the best-known example of these fluorochromes is the green fluorescent protein (GFP), tissue autofluorescence and signal dispersion raise doubts about its suitability as an in vivo tracer. However, a number of groups have recently developed several NIR fluorescent proteins that enable real-time imaging free of autofluorescence interference thus making possible to take a deep view into the tissues. With all of this in mind, we devised a novel fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1α, which appears upregulated in growing tumors. At the same time, by fusing a fluorescent to a bioluminescent protein we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor. We also tested the inducibility and performance of this hypoxia sensor by imaging artificially activated cells both in culture (in vitro) and inside an animal model (in vivo). Finally, the breast cancer cell line MDA-MB 231, which is known by its invasiveness and ability to metastasize, was modified to carry the hypoxia sensor and subsequently injected intravenously in live mice. Metastases were detected in lung and brain by fluorescence and/or bioluminescence. In conclusion, we have described the designing and development of a functional dual hypoxia-sensing system able to display BRET activity between the firefly luciferase and the NIR fluorochrome. In addition a metastatic tumoral cell line was genetically modified to demonstrate that the biosensor is able to detect hypoxic conditions in metastasis in vivo, yielding valuable functional information about the tumor besides size and spatial localization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 469.