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Alpha-particle radiotherapy has already been shown to be impervious to most resistance mechanisms. However, in established (i.e. large, vascularized) soft-tissue lesions, the diffusion-limited penetration depths of radiolabeled antibodies and/or nanocarriers (up to 50-80µm) combined with the short range of α-particles (4-5 cell diameters) may result in only partial tumor irradiation potentially limiting treatment efficacy. To address this challenge, we combined carriers with complementary intratumoral microdistributions of the delivered α-particles. We use the α-particle generator Actinium-225 (225Ac), and we combine (1) a tumor-responsive liposome that upon tumor uptake releases in the interstitium a highly-diffusing form of its radioactive payload (225Ac-DOTA), which may penetrate the deeper parts of tumors where antibodies do not reach, with (2) a separately administered, less-penetrating radiolabeled-antibody irradiating the tumor perivascular regions from where liposome contents clear too fast. Methods: On a murine model with orthotopic HER2-positive BT474 breast cancer xenografts, the biodistributions of each carrier were evaluated, and the control of tumor growth was monitored after administration of the same total radioactivity of 225Ac delivered (1) by the 225Ac-DOTA-encapsulating liposomes, (2) by the 225Ac-DOTA-SCN-labeled-trastuzumab, and (3) by both carriers at equally split radioactivities. Results: Tumor growth inhibition was significantly more pronounced when the same total injected radioactivity was divided between the two separate carriers, as compared to the growth delay by the same total injected radioactivity when delivered by either of the carriers alone. The combined carriers enabled more uniform intratumoral microdistributions of α-particles, at a tumor delivered dose that was lower than the dose delivered by the antibody alone. Significance: This strategy demonstrates that more uniform microdistributions of the delivered α-particles within established solid tumors improve efficacy even at lower tumor delivered doses. Augmentation of antibody-targeted α-particle therapies with tumor-responsive liposomes may address partial tumor irradiation improving therapeutic effects.

Hypoxia in cancers has evoked significant interest since 1955 when Thomlinson and Gray postulated the presence of hypoxia in human lung cancers, based on the observation of necrosis occurring at the diffusion limit of oxygen from the nearest blood vessel, and identified the implication of these observations for radiation therapy. Coupled with discoveries in 1953 by Gray and others that anoxic cells were resistant to radiation damage, these observations have led to an entire field of research focused on exploiting oxygenation and hypoxia to improve the outcome of radiation therapy. Almost 65 years later, tumor heterogeneity of nearly every parameter measured including tumor oxygenation, and the dynamic landscape of cancers and their microenvironments are clearly evident, providing a strong rationale for cancer personalized medicine. Since hypoxia is a major cause of extracellular acidosis in tumors, here, we have focused on the applications of imaging to understand the effects of hypoxia in tumors and to target hypoxia in theranostic strategies. Molecular and functional imaging have critically important roles to play in personalized medicine through the detection of hypoxia, both spatially and temporally, and by providing new understanding of the role of hypoxia in cancer aggressiveness. With the discovery of the hypoxia-inducible factor (HIF), the intervening years have also seen significant progress in understanding the transcriptional regulation of hypoxia-induced genes. These advances have provided the ability to silence HIF and understand the associated molecular and functional consequences to expand our understanding of hypoxia and its role in cancer aggressiveness. Most recently, the development of hypoxia-based theranostic strategies that combine detection and therapy are further establishing imaging-based treatment strategies for precision medicine of cancer.

Background Harnessing the power of the immune system by using immune checkpoint inhibitors has resulted in some of the most exciting advances in cancer treatment. The full potential of this approach has, however, not been fully realized for treating many cancers such as pancreatic and breast cancer. Cancer metabolism influences many aspects of cancer progression including immune surveillance. An expanded understanding of how cancer metabolism can directly impact immune checkpoints may allow further optimization of immunotherapy. We therefore investigated, for the first time, the relationship between the overexpression of choline kinase-α (Chk-α), an enzyme observed in most cancers, and the expression of the immune checkpoint PD-L1. Methods We used small interfering RNA to downregulate Chk-α, PD-L1, or both in two triple-negative human breast cancer cell lines (MDA-MB-231 and SUM-149) and two human pancreatic ductal adenocarcinoma cell lines (Pa09C and Pa20C). The effects of the downregulation were studied at the genomic, proteomic, and metabolomic levels. The findings were compared with the results obtained by the analysis of public data from The Cancer Genome Atlas Program. Results We identified an inverse dependence between Chk-α and PD-L1 at the genomic, proteomic, and metabolomic levels. We also found that prostaglandin-endoperoxide synthase 2 (COX-2) and transforming growth factor beta (TGF-β) play an important role in this relationship. We independently confirmed this relationship in human cancers by analyzing data from The Cancer Genome Atlas Program. Conclusions Our data identified previously unknown roles of PD-L1 in cancer cell metabolic reprogramming, and revealed the immunosuppressive increased PD-L1 effect of Chk-α downregulation. These data suggest that PD-L1 regulation of metabolism may be mediated through Chk-α, COX-2, and TGF-β. The observations provide new insights that can be applied to the rational design of combinatorial therapies targeting immune checkpoints and cancer metabolism.

Because of the spatial and temporal heterogeneities of cancers, technologies to investigate cancer cells and the consequences of their interactions with abnormal physiological environments, such as hypoxia and acidic extracellular pH, with stromal cells, and with the extracellular matrix, under controlled conditions, are valuable to gain insights into the functioning of cancers. These insights can lead to an understanding of why cancers invade and metastasize, and identify effective treatment strategies. Here we have provided an overview of the applications of MRI/MRS/MRSI to investigate intact perfused cancer cells, their metabolism and invasion, and their interactions with stromal cells and the extracellular matrix.

© 2019 by the authors., Despite important advances in multimodal therapeutic options, glioblastoma (GBM), the most frequent and aggressive form of all astrocytomas, remains with a median overall survival period of 15 months. A direct correlation between GBM hypoxia and higher aggressiveness, poor prognosis and greater resistance to different treatments has been established. However, because of intratumoral and interindividual heterogeneity, it has not been possible to assess accurately the hypoxia degree from physiopathological parameters or neuroimaging methods. This study aims to develop and evaluate a magnetic resonance imaging (MRI) approach to identify more precisely those tumors that could improve the outcome through an oxygen targeted therapy. Methods: To assess the efficacy of radiotherapy in animals irradiated under air and oxygen breathing, we implemented a GBM animal model obtained by intracranial injection of glioma C6 cells in rats. MRI studies, based on the oxygen-induced contrast in blood (BOLD) and tissues (TOLD), were carried out to evaluate the effect of the modulation in oxygen breathing conditions on the tumors in vivo. The efficacy of the oxygen breathing therapies was determined by the relative tumor volume at the end of the experiment, compared to its size on the day before the treatment. Results: Our results categorized the tumors in responding, non-responding and intermediate behaviors. While BOLD analysis did not show any statistical difference between animals, either breathing air or oxygen, TOLD parameters allowed for the identification of the tumors with higher responses to hyperoxygenic radiotherapy. Conclusions: The non-invasive oxygen enhanced MRI acquisitions proposed here show promising potential to identify those tumors that would generally improve their response to a hypoxia targeted treatment., This work was supported by grants from the Ministry of Economy, Industry and Competitiveness (SAF2017-83043-R), and by the Program MULTITARGET&VIEW-CM from the Community of Madrid (S-BIO-0170-2006), involving contributions from FEDER and FSE funds.

Robust neuroimaging markers of neuropsychiatric disorders have proven difficult to obtain. In alcohol use disorders, profound brain structural deficits can be found in severe alcoholic patients, but the heterogeneity of unimodal MRI measurements has so far precluded the identification of selective biomarkers, especially for early diagnosis. In the present work we used a combination of multiple MRI modalities to provide comprehensive and insightful descriptions of brain tissue microstructure. We performed a longitudinal experiment using Marchigian-Sardinian (msP) rats, an established model of chronic excessive alcohol consumption, and acquired multi-modal images before and after 1 month of alcohol consumption (6.8 ± 1.4 g/kg/day, mean ± SD), as well as after 1 week of abstinence with or without concomitant treatment with the antirelapse opioid antagonist naltrexone (2.5 mg/kg/day). We found remarkable sensitivity and selectivity to accurately classify brains affected by alcohol even after the relative short exposure period. One month drinking was enough to imprint a highly specific signature of alcohol consumption. Brain alterations were regionally specific and affected both gray and white matter and persisted into the early abstinence state without any detectable recovery. Interestingly, naltrexone treatment during early abstinence resulted in subtle brain changes that could be distinguished from non-treated abstinent brains, suggesting the existence of an intermediate state associated with brain recovery from alcohol exposure induced by medication. The presented framework is a promising tool for the development of biomarkers for clinical diagnosis of alcohol use disorders, with capacity to further inform about its progression and response to treatment.

[Introduction]: Functional magnetic resonance imaging (fMRI) is one of the most highly regarded techniques in the neuroimaging field. This technique is based on vascular responses to neuronal activation and is extensively used in clinical and animal research studies. In preclinical settings, fMRI is usually applied to anesthetized animals. However, anesthetics cause alterations, e.g. hypothermia, in the physiology of the animals and this has the potential to disrupt fMRI signals. The current temperature control method involves a technician, as well as monitoring the acquisition MRI sequences, also controlling the temperature of the animal; this is inefficient., [Methods]: In order to avoid hypothermia in anesthetized rodents an Open-Source automatic temperature control device is presented. It takes signals from an intrarectal temperature sensor, as well as signals from a thermal bath, which warms up the body of the animal under study, in order to determine the mathematical model of the thermal response of the animal., [Results]: A Proportional-Integral-Derivative (PID) algorithm, which was discretized in an Arduino microcontroller, was developed to automatically keep stable the body temperature of the animal under study. The PID algorithm has been shown to be accurate in preserving the body temperature of the animal., [Conclusion]: This work presents the TherMouseDuino. It is an Open-Source automatic temperature control system and reduces temperature fluctuations, thus providing robust conditions in which to perform fMRI experiments. Furthermore, our device frees up the technician to focus solely on monitoring the MRI sequences., This work was supported in part by the Spanish Ministerio de Economía y Competitividad (MINECO) and FEDER funds under grants BFU2015-64380-C2-2-R (D.M.) and BFU2015-64380-C2-1-R and EU Horizon 2020 Program 668863-SyBil-AA grant (S.C.). S.C. acknowledges financial support from the Spanish State Research Agency, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (ref. SEV-2013-0317). D.R.Q. was supported by grant “Ayudas para la formación de personal investigador (FPI)” from the Vicerrectorado de Investigación, Innovación y Transferencia of the Universitat Politècnica de València.

The development of contrast agents (CAs) for Magnetic Resonance Imaging (MRI) with T1-T2 dual-mode relaxivity requires the accurate assembly of T1 and T2 magnetic centers in a single structure. In this context, we have synthesized a novel hybrid material by monitoring the formation of Prussian Blue analogue Gd(H2O)4[Fe(CN)6] nanoparticles with tailored shape (from nanocrosses to nanorods) and size, and further protection with a thin and homogeneous silica coating through hydrolysis and polymerization of silicate at neutral pH. The resulting Gd(H2O)4[Fe(CN)6]@SiO2 magnetic nanoparticles are very stable in biological fluids. Interestingly, this combination of Gd and Fe magnetic centers closely packed in the crystalline network promotes a magnetic synergistic effect, which results in significant improvement of longitudinal relaxivity with regards to soluble Gd3+ chelates, whilst keeping the high transversal relaxivity inherent to the iron component. As a consequence, this material shows excellent activity as MRI CA, improving positive and negative contrasts in T1- and T2-weighted MR images, both in in vitro (e.g., phantom) and in vivo (e.g., Sprague-Dawley rats) models. In addition, this hybrid shows a high biosafety profile and has strong ability to incorporate organic molecules on the surface with variable functionality, displaying great potential for further clinical application.

Oxygen monitoring is a topic of exhaustive research due to its central role in many biological processes, from energy metabolism to gene regulation. The ability to monitor in vivo the physiological distribution and the dynamics of oxygen from subcellular to macroscopic levels is a prerequisite to better understand the mechanisms associated with both normal and disease states (cancer, neurodegeneration, stroke, etc.). This chapter focuses on magnetic resonance imaging (MRI) based techniques to assess oxygenation in vivo. The first methodology uses injected fluorinated agents to provide quantitative pO

Thyroid hormone deficiency at early postnatal ages affects the cytoarchitecture and function of neocortical and telencephalic limbic areas, leading to impaired associative memory and in a wide spectrum of neurological and mental diseases. Neocortical areas project interhemispheric axons mostly through the corpus callosum and to a lesser extent through the anterior commissure (AC), while limbic areas mostly project through the AC and hippocampal commissures. Functional magnetic resonance data from children with late diagnosed congenital hypothyroidism and abnormal verbal memory processing, suggest altered ipsilateral and contralateral telencephalic connections. Gestational hypothyroidism affects AC development but the possible effect of transient and chronic postnatal hypothyroidism, as occurs in late diagnosed neonates with congenital hypothyroidism and in children growing up in iodine deficient areas, still remains unknown. We studied AC development using in vivo magnetic resonance imaging and electron microscopy in hypothyroid and control male rats. Four groups of methimazole (MMI) treated rats were studied. One group was MMI-treated from postnatal day (P) 0 to P21; some of these rats were also treated with L-thyroxine (T4) from P15 to P21, as a model for early transient hypothyroidism. Other rats were MMI-treated from P0 to P150 and from embryonic day (E) 10 to P170, as a chronic hypothyroidism group. The results were compared with age paired control rats. The normalized T2 signal using magnetic resonance image was higher in MMI-treated rats and correlated with the number and percentage of myelinated axons. Using electron microscopy, we observed decreased myelinated axon number and density in transient and chronic hypothyroid rats at P150, unmyelinated axon number increased slightly in chronic hypothyroid rats. In MMI-treated rats, the myelinated axon g-ratio and conduction velocity was similar to control rats, but with a decrease in conduction delays. These data show that early postnatal transient and chronic hypothyroidism alters AC maturation that may affect the transfer of information through the AC. The alterations cannot be recovered after delayed T4-treatment. Our data support the neurocognitive delay found in late T4-treated children with congenital hypothyroidism., This work was funded by grants of the Fundación Alicia Koplowitz (Spain) and of the Spanish “Ministerio de Economía, Industria y Competitividad” MINECO-SAF2014-58256-R to PB, MINECO-BFU2015-64380-C2-1-R to SC and of the Spanish “Generalitat Valènciana” GV-PROMETEOII/2014/075 to JG-V.

Oxygen monitoring is a topic of exhaustive research due to its central role in many biological processes, from energy metabolism to gene regulation. The ability to monitor in vivo the physiological distribution and the dynamics of oxygen from subcellular to macroscopic levels is a prerequisite to better understand the mechanisms associated with both normal and disease states (cancer, neurodegeneration, stroke, etc.). This chapter focuses on magnetic resonance imaging (MRI) based techniques to assess oxygenation in vivo. The first methodology uses injected fluorinated agents to provide quantitative pO2 measurements with high precision and suitable spatial and temporal resolution for many applications. The second method exploits changes in endogenous contrasts, i.e., deoxyhemoglobin and oxygen molecules through measurements of T2* and T1, in response to an intervention to qualitatively evaluate hypoxia and its potential modulation., Method development and application supported in part by CPRIT RP140399, RP120670-03, P30 CA142543, and P41 EB015908.

The development of contrast agents (CAs) for Magnetic Resonance Imaging (MRI) with T1–T2 dual-mode relaxivity requires the accurate assembly of T1 and T2 magnetic centers in a single structure. In this context, we have synthesized a novel hybrid material by monitoring the formation of Prussian Blue analogue Gd(H2O)4[Fe(CN)6] nanoparticles with tailored shape (from nanocrosses to nanorods) and size, and further protection with a thin and homogeneous silica coating through hydrolysis and polymerization of silicate at neutral pH. The resulting Gd(H2O)4[Fe(CN)6]@SiO2 magnetic nanoparticles are very stable in biological fluids. Interestingly, this combination of Gd and Fe magnetic centers closely packed in the crystalline network promotes a magnetic synergistic effect, which results in significant improvement of longitudinal relaxivity with regards to soluble Gd3+ chelates, whilst keeping the high transversal relaxivity inherent to the iron component. As a consequence, this material shows excellent activity as MRI CA, improving positive and negative contrasts in T1- and T2-weighted MR images, both in in vitro (e.g., phantom) and in vivo (e.g., Sprague-Dawley rats) models. In addition, this hybrid shows a high biosafety profile and has strong ability to incorporate organic molecules on the surface with variable functionality, displaying great potential for further clinical application., Financial support of the Spanish Ministry of Economy and Competitiveness (projects TEC2016-80976-R and SEV-2016-0683) is gratefully acknowledged. Dr E. M. Rivero thanks the Cursol Foundation for a post-doctoral scholarship. A. C. G. also thanks the La Caixa Foundation for a Ph.D. scholarship.

Since its discovery in the early 90s, BOLD signal-based functional Magnetic Resonance Imaging (fMRI) has become a fundamental technique for the study of brain activity in basic and clinical research. Functional MRI signals provide an indirect but robust and quantitative readout of brain activity through the tight coupling between cerebral blood flow and neuronal activation, the so-called neurovascular coupling. Combined with experimental techniques only available in animal models, such as intracerebral micro-stimulation, optogenetics or pharmacogenetics, provides a powerful framework to investigate the impact of specific circuit manipulations on overall brain dynamics. The purpose of this chapter is to provide a comprehensive protocol to measure brain activity using fMRI with intracerebral electric micro-stimulation in murine models. Preclinical research (especially in rodents) opens the door to very sophisticated and informative experiments, but at the same time imposes important constrains (i.e., anesthetics, translatability), some of which will be addressed here.

Since its discovery in the early 1990s, blood oxygen level dependent signal-based functional magnetic resonance imaging (fMRI) has become a fundamental technique for the study of brain activity in basic and clinical research. Functional MRI provides an indirect but robust and quantitative readout of brain activity through the tight coupling between cerebral blood flow and neuronal activation, the so-called neurovascular coupling. Combined with experimental techniques such as electrophysiology, intracerebral microstimulation, optogenetics, or pharmacogenetics, it provides a powerful framework to investigate the impact of specific circuit manipulations on overall brain dynamics. In this chapter we review the contribution of some of these techniques to the understanding of short- and long-term plasticity processes and provide a comprehensive account of the protocols used in our laboratory. Considerations on the interpretation of the results and the limitations of the approach are also discussed.

There is intense interest in developing non-invasive prognostic biomarkers of tumor response to therapy, particularly with regard to hypoxia. It has been suggested that oxygen sensitive MRI, notably blood oxygen level-dependent (BOLD) and tissue oxygen level-dependent (TOLD) contrast, may provide relevant measurements. This study examined the feasibility of interleaved T2*- and T1-weighted oxygen sensitive MRI, as well as R2* and R1 maps, of rat tumors to assess the relative sensitivity to changes in oxygenation. Investigations used cohorts of Dunning prostate R3327-AT1 and R3327-HI tumors, which are reported to exhibit distinct size-dependent levels of hypoxia and response to hyperoxic gas breathing. Proton MRI R1 and R2* maps were obtained for tumors of anesthetized rats (isoflurane/air) at 4.7 T. Then, interleaved gradient echo T2*- and T1-weighted images were acquired during air breathing and a 10 min challenge with carbogen (95% O2 -5% CO2). Signals were stable during air breathing, and each type of tumor showed a distinct signal response to carbogen. T2* (BOLD) response preceded T1 (TOLD) responses, as expected. Smaller HI tumors (reported to be well oxygenated) showed the largest BOLD and TOLD responses. Larger AT1 tumors (reported to be hypoxic and resist modulation by gas breathing) showed the smallest response. There was a strong correlation between BOLD and TOLD signal responses, but ΔR2* and ΔR1 were only correlated for the HI tumors. The magnitude of BOLD and TOLD signal responses to carbogen breathing reflected expected hypoxic fractions and oxygen dynamics, suggesting potential value of this test as a prognostic biomarker of tumor hypoxia.

[EN] The use of functional magnetic resonance imaging (fMRI) to measure spontaneous fluctuations in blood oxygen level dependent (BOLD) signals has become an indispensable tool to investigate how brain regions interact and form longrange networks. Statistical dependency measures between brain regions obtained from BOLD signals can inform about brain functional states in longitudinal studies of neurological and psychiatric disorders. Furthermore, its non-invasive nature allows comparable measurements in clinical and animal studies, providing excellent translational capabilities. In the present study, we apply Network-Based Statistic (NBS) to investigate alterations in the functional connectivity (FC) of the rat brain in a post-dependent (PD) state, an established animal model of clinical relevant features in alcoholism. In contrast to mass-univariate tests, in which comparisons are performed at single link-level, NBS enhances the statistical power by assuming that the connections comprising the effect of interest are interconnected. Brain-wide resting-state fMRI signals were collected in 14 controls and 13 PD rats, and Pearson correlations computed between 47 brain regions of interest (ROIs). The NBS analysis revealed statistically significant differences in a connected network of structures including hippocampus, amygdala, lateral hypothalamus and the raphe nucleus, all regions with known relevance for addictive behaviors. In contrast, no individual connection could be found significant by univariate comparisons with false discovery rate (FDR) correction. Correlations between the structures in the identified subnetwork tend to decrease or become negative (anti-correlated) in the PD state compared to controls. We interpret this result as evidence for a disconnected subnetwork in the PD state., Research supported by the Spanish Ministerio de Economia y Competitividad (MINECO) under grants BFU2015-64380-C2-1-R and BFU2015-64380-C2-2R and European Union's Horizon 2020 research and innovation programme under grant agreement No 668863 (SyBil-AA). Santiago Canals acknowledges financial support from the Spanish State Research Agency, through the "Severo Ochoa" Programme for Centres of Excellence in R&D (ref. SEV-2013-0317). Antonio Diaz-Parra and Ursula Perez-Ramirez are funded by the Spanish Ministerio de Educacion, Cultura y Deporte (MECD) under grant FPU13/01475 and FPU13/03537, respectively.

Current treatments for demyelinating diseases are generally only capable of ameliorating the symptoms, with little to no effect in decreasing myelin loss nor promoting functional recovery. Mesenchymal stem cells (MSCs) have been shown by many researchers to be a potential therapeutic tool in treating various neurodegenerative diseases, including demyelinating disorders. However, in the majority of the cases, the effect was only observed locally, in the area surrounding the graft. Thus, in order to achieve general remyelination in various brain structures simultaneously, bone marrow-derived MSCs were transplanted into the lateral ventricles (LVs) of the cuprizone murine model. In this manner, the cells may secrete soluble factors into the cerebrospinal fluid (CSF) and boost the endogenous oligodendrogenic potential of the subventricular zone (SVZ). As a result, oligodendrocyte progenitor cells (OPCs) were recruited within the corpus callosum (CC) over time, correlating with an increased myelin content. Electrophysiological studies, together with electron microscopy (EM) analysis, indicated that the newly formed myelin correctly enveloped the demyelinated axons and increased signal transduction through the CC. Moreover, increased neural stem progenitor cell (NSPC) proliferation was observed in the SVZ, possibly due to the tropic factors released by the MSCs. In conclusion, the findings of this study revealed that intraventricular injections of MSCs is a feasible method to elicit a paracrine effect in the oligodendrogenic niche of the SVZ, which is prone to respond to the factors secreted into the CSF and therefore promoting oligodendrogenesis and functional remyelination.

This article belongs to the Special Issue Nanomaterials for Cancer Therapies., We describe the synthesis, characterization and application as contrast agents in magnetic resonance imaging of a novel type of magnetic nanoparticle based on Gd-Si oxide, which presents high Gd3+ atom density. For this purpose, we have used a Prussian Blue analogue as the sacrificial template by reacting with soluble silicate, obtaining particles with nanorod morphology and of small size (75 nm). These nanoparticles present good biocompatibility and higher longitudinal and transversal relaxivity values than commercial Gd3+ solutions, which significantly improves the sensitivity of in vivo magnetic resonance images., The authors thank the Spanish MINECO (projects MAT2012-39290-CO2-01 and MAT2012-39290-CO2-02) for financial support. A.C.-G. also thanks “La Caixa” Foundation for a Ph.D. scholarship.

Even though alterations in the microenvironmental properties of tissues underlie the development of the most prevalent and morbid pathologies, they are not directly observable in vivo by Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS) methods. This circumstance has lead to the development of a wide variety of exogenous paramagnetic and diamagnetic MRI and MRS probes able to inform non invasively on microenvironmental variables such as pH, pO(2), ion concentration o even temperature. This review covers the fundamentals of environmental contrast and the current arsenal of endogenous and exogenous MRI and MRS contrast enhancing agents available to visualize it. We begin describing the physicochemical background necessary to understand paramagnetic and diamagnetic contrast enhancement with a special reference to novel magnetization transfer and C-13 hiperpolarization strategies. We describe then the main macrocyclic structures used to support the environmentally sensitive paramagnetic sensors, including CEST and PARACEST pH sensitive probes, temperature probes and enzyme activity or gene expression activatable probes. Finally we address the most commonly used diamagnetic contrast agents including imidazolic derivatives to reveal extracellular pH and tissue pO(2) values by MRS. The potential applications of these agents in mutimodal and molecular imaging approaches are discussed., This work was supported in part by grants: CTQ2006-06505/BQU, CTQ2009-14146-C01/C02, to P.B. and P.L.L., S-BIO/0179/2006 and SAF2008/01381 to SC and EU MEDITRANS Integrated Project NMP4-CT- 2006-02668 to S.C., P.L.L. and P.B. J. P- T, D. C., B.L. and V.N. are predoctoral fellows from the Community of Madrid and CSIC and V.N. and C.U hold contracts from UNED

Introduction: Immune checkpoint inhibition has emerged as an exciting treatment option for several cancers. New insights into the role of immune checkpoints in cellular metabolism can be used to optimize their effectiveness in treatment. Programmed cell death-1 ligand (PD-L1) is an immune checkpoint overexpressed in cancers that has been successfully exploited for immune therapy. Here we investigated the relationship between the aberrant choline metabolism observed in most cancers and PD-L1 expression in triple-negative human MDA-MB-231 breast cancer cells, and characterized the metabolic effects of PD-L1 downregulation. Methods: Experiments were performed with MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against either luciferase (control siRNA), choline kinase-α (Chk-α), or PD-L1, following standard protocols. Total RNA was isolated, complementary cDNA synthesized, and quantitative real-time PCR performed using IQ SYBR Green supermix and gene-specific primers. Cell extracts were obtained using a dual-phase extraction method and analyzed by high resolution 1H magnetic resonance spectroscopy. 3 to 6 independent experiments were performed for each group. Results: Silencing of Chk-α resulted in a significant increase of PD-L1 mRNA expression and silencing of PD-L1 resulted in a significant increase of Chk-α mRNA expression. These increases were not observed when both PD-L1 and Chk-α were silenced. Transfection with control siRNA did not alter Chk-α mRNA, but induced a small increase of PD-L1 mRNA compared to untreated cells. Transfection of cells with Chk-α and PD-L1 siRNA resulted in a significant decreases of Chk-α and PD-L1 mRNA. Consistent with the mRNA results, a significant increase of phosphocholine (PC) was observed in spectra obtained from cells transfected with PD-L1 siRNA compared to cells transfected with control siRNA, confirming that Chk-α increased in these cells. A significant increase of glutamate, glutathione, alanine and lactate was also observed in cells transfected with PD-L1 siRNA. As expected, cells transfected with a combination of Chk-α and PD-L1 siRNA showed a significant decrease of PC due to downregulation of Chk-α. Discussion: Our data have identified, for the first time, the inverse association between PD-L1 and Chk-α expression following downregulation. These data suggest that treatments that decrease Chk-α and PC could result in cancer cells escaping immune surveillance through increased expression of PD-L1. On the other hand, these cells may become more susceptible to checkpoint inhibitors such as anti-PD-L1 or anti-PD-1 antibodies. PD-L1 downregulation also significantly increased glutathione that acts as a major antioxidant. Our ongoing studies are characterizing the effects of anti-PD-L1 antibody treatment on Chk-α expression. Supported by NIH R35CA209960 and R01CA82337, and the Martin Escudero Foundation and Emerson Foundation. Citation Format: Jesus Pacheco-Torres, Marie-France Penet, Yelena Mironchik, Sridhar Nimmagadda, Balaji Krishnamachary, Zaver M. Bhujwalla. The immune checkpoint metabolome and its relationship with choline metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1680.

Liposomal drug delivery vehicles are promising nanomedicine tools for bringing cytotoxic drugs to cancerous tissues selectively. However, the triggered cargo release from liposomes in response to a target-specific stimulus has remained elusive. We report on functionalizing stealth-liposomes with an engineered ion channel and using these liposomes in vivo for releasing an imaging agent into a cerebral glioma rodent model. If the ambient pH drops below a threshold value, the channel generates temporary pores on the liposomes, thus allowing leakage of the intraluminal medicines. By using magnetic resonance spectroscopy and imaging, we show that engineered liposomes can detect the mildly acidic pH of the tumor microenvironment with 0.2 pH unit precision and they release their content into C6 glioma tumors selectively, in vivo. A drug delivery system with this level of sensitivity and selectivity to environmental stimuli may well serve as an optimal tool for environmentally-triggered and image-guided drug release., From the Clinical Editor: Cancer remains a leading cause of mortality worldwide. With advances in science, delivery systems of anti-cancer drugs have also become sophisticated. In this article, the authors designed and characterized functionalized liposomal vehicles, which would release the drug payload in a highly sensitive manner in response to a change in pH environment in an animal glioma model. The novel data would enable better future designs of drug delivery systems, This work was supported in part by grants SAF-2008-01327, SAF2011-23622 and S2010/BMD-2349 to SC, grants CTQ-2010-20960 and CTQ-2013-47669-R to P.B. and P.L.L. and, grants European Research Council-Ideas Program Starting Grant 208814, ERC-Proof of concept Grant (MOCHA), and the Netherlands Organization for Scientific Research Grant (NWO-VIDI) 700.57.427 to A.K, MEDITRANS (NMP4-CT-2006-026668) to A.K., S.C., P.B. and N.M-. J.P-T received a predoctoral contract from CSIC.

We report an easy and efficient synthetic approach to the first series of (nitroimidazolyl)succinic esters and diacids involving the Michael-type addition of the nitroimidazole to the α,β-unsaturated ester. Voltammetric measurements revealed similar redox potentials to those of previous nitroimidazoles used in hypoxia detection. Theoretical calculations were implemented to understand the mechanism of the addition. © Georg Thieme Verlag Stuttgart., This work was supported in part by grants from the Community of Madrid GR/SAL/0328/2004 and SAF2003-01810 to PB and SAF 2004-03197 to SC.

Aims: To develop a low-risk, little-invasive stem cell-based method to treat acute spinal cord injuries. methods: Adult mice were submitted to an incomplete spinal cord injury, and mesenchymal stem cells injected intramuscularly into both hindlimbs. Behavior tests and MRI of the spinal cord were periodically performed for up to 6 months, along with immunohistochemical analysis. Immunohistochemical and PCR analysis of the muscles were used to detect the grafted cells as well as the soluble factors released. Results: The stem cell-treated mice presented significant improvements in their motor skills 5 months after treatment. Spinal cord repair was detected by magnetic resonance and immunohistochemistry. In the hindlimb muscles, the stem cells activated muscle and motor neuron repair mechanisms, due to the secretion of several neurotrophic factors. Conclusion: Bone marrow mesenchymal stem cell injection into hindlimb muscles stimulates spinal cord repair in acute spinal cord lesions.

The main objective of this work is to demonstrate the feasibility of using bone marrow-derived stem cells in treating a neurodegenerative disorder such as Friedreich's ataxia. In this disease, the dorsal root ganglia of the spinal cord are the first to degenerate. Two groups of mice were injected intrathecally with mesenchymal stem cells isolated from either wild-type or Fxntm1Mkn/Tg(FXN)YG8Pook (YG8) mice. As a result, both groups presented improved motor skills compared to nontreated mice. Also, frataxin expression was increased in the dorsal root ganglia of the treated groups, along with lower expression of the apoptotic markers analyzed. Furthermore, the injected stem cells expressed the trophic factors NT3, NT4, and BDNF, which bind to sensory neurons of the dorsal root ganglia and increase their survival. The expression of antioxidant enzymes indicated that the stem cell-treated mice presented higher levels of catalase and GPX-1, which are downregulated in the YG8 mice. There were no significant differences in the use of stem cells isolated from wild-type and YG8 mice. In conclusion, bone marrow mesenchymal stem cell transplantation, both autologous and allogeneic, is a feasible therapeutic option to consider in delaying the neurodegeneration observed in the dorsal root ganglia of Friedreich's ataxia patients., This work was supported by FARA/FARA Ireland (Friedreich's Ataxia Research Alliance), ASOGAF (Friedreich's ataxia association of Granada), Science and Innovation Ministry (MICINN BFU2010-27326), GVA Prometeo grant 2009/028, PROMETEOII GRANT 2014/014, Tercel (RD06/0010/0023 & RD06/0010/24), MEC-CONSOLIDER CSD2007-00023, Cinco P menos Foundation, EUCOMM, Fundacion Diogenes-Elche city government, Walk on Project, and MAPFRE Foundation.

There is intense interest in developing non-invasive prognostic biomarkers of tumor response to therapy, particularly with regard to hypoxia. It has been suggested that oxygen sensitive MRI, notably blood oxygen level-dependent (BOLD) and tissue oxygen level-dependent (TOLD) contrast, may provide relevant measurements. This study examined the feasibility of interleaved T2*- and T1-weighted oxygen sensitive MRI, as well as R2* and R1 maps, of rat tumors to assess the relative sensitivity to changes in oxygenation. Investigations used cohorts of Dunning prostate R3327-AT1 and R3327-HI tumors, which are reported to exhibit distinct size-dependent levels of hypoxia and response to hyperoxic gas breathing. Proton MRI R1 and R2* maps were obtained for tumors of anesthetized rats (isoflurane/air) at 4.7 T. Then, interleaved gradient echo T2*- and T1-weighted images were acquired during air breathing and a 10 min challenge with carbogen (95% O2 -5% CO2). Signals were stable during air breathing, and each type of tumor showed a distinct signal response to carbogen. T2* (BOLD) response preceded T1 (TOLD) responses, as expected. Smaller HI tumors (reported to be well oxygenated) showed the largest BOLD and TOLD responses. Larger AT1 tumors (reported to be hypoxic and resist modulation by gas breathing) showed the smallest response. There was a strong correlation between BOLD and TOLD signal responses, but ΔR2* and ΔR1 were only correlated for the HI tumors. The magnitude of BOLD and TOLD signal responses to carbogen breathing reflected expected hypoxic fractions and oxygen dynamics, suggesting potential value of this test as a prognostic biomarker of tumor hypoxia.

Functional magnetic resonance imaging (fMRI) is a fundamental tool to investigate human brain networks. However, the cellular mechanisms underlying fMRI signals are not fully understood. One hypothetical mechanism is the putative vascular control exerted by cytosolic calcium in perivascular astrocytes. We have performed combined fMRI-electrophysiology experiments in mice lacking the inositol 1,4,5-triphosphate-type-2 receptor, with the primary pathway of cytosolic calcium increase eliminated into astrocytes. Our results show that evoked electrophysiologic activity and fMRI signals acquired during either transient or sustained neuronal activations occur independently of these large calcium signals. This result challenges the suggested intermediary role of astrocytic calcium surges in fMRI-signal generation., Research at SC laboratory is supported by grants from the Spanish MINECO (BFU2012-39958 and PIM2010ERN-00679 as part of the Era-Net NEURONTRANSAL C project). PJ has been supported by the ‘Symbad’ Marie Curie ITN program.

Genetic mouse models of neurodevelopmental disorders are being massively generated, but technologies for their high-throughput phenotyping are missing. The potential of high-resolution magnetic resonance imaging (MRI) for structural phenotyping has been demonstrated before. However, application to the embryonic mouse central nervous system has been limited by the insufficient anatomical detail. Here we present a method that combines staining of live embryos with a contrast agent together with MR microscopy after fixation, to provide unprecedented anatomical detail at relevant embryonic stages. By using this method we have phenotyped the embryonic forebrain of Robo1/2−/− double mutant mice enabling us to identify most of the well-known anatomical defects in these mutants, as well as novel more subtle alterations. We thus demonstrate the potential of this methodology for a fast and reliable screening of subtle structural abnormalities in the developing mouse brain, as those associated to defects in disease-susceptibility genes of neurologic and psychiatric relevance., Supported by grants from the Spanish Ministry of Science and Innovation MICINN to S.C. (CSD2007-00023, BFU2009-09938, BFU2012-39958 and PIM2010ERN-00679 as part of the Era-Net NEURON TRANSALC project) and to V.B. (CSD2007-00023, SAF2009-07367, BFU2012-33473).

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) allows the efficient and complete functionalization of dendrimers with preformed Gd chelates (prelabeling) to give monodisperse macromolecular contrast agents (CAs) for magnetic resonance imaging (MRI). This monodispersity contrasts with the typical distribution of materials obtained by classical routes and facilitates the characterization and quality control demanded for clinical applications. The potential of a new family of PEG-dendritic CA based on a gallic acid-triethylene glycol (GATG) core functionalized with up to 27 Gd complexes has been explored in vitro and in vivo, showing contrast enhancements similar to those of Gadomer-17, which reveals them to be a promising platform for the development of CA for MRI. © 2011 American Chemical Society., We wish to acknowledge the Spanish MICINN (CTQ2009-14146-C02-02 and CTQ2009-10963) and the Xunta de Galicia (10CSA209021PR) for financial support.

Tumor hypoxia results from the negative balance between the oxygen demands of the tissue and the capacity of the neovasculature to deliver sufficient oxygen. The resulting oxygen deficit has important consequences with regard to the aggressiveness and malignancy of tumors, as well as their resistance to therapy, endowing the imaging of hypoxia with vital repercussions in tumor prognosis and therapy design. The molecular and cellular events underlying hypoxia are mediated mainly through hypoxia-inducible factor, a transcription factor with pleiotropic effects over a variety of cellular processes, including oncologic transformation, invasion and metastasis. However, few methodologies have been able to monitor noninvasively the oxygen tensions in vivo. MRI and MRS are often used for this purpose. Most MRI approaches are based on the effects of the local oxygen tension on: (i) the relaxation times of (19)F or (1)H indicators, such as perfluorocarbons or their (1)H analogs; (ii) the hemodynamics and magnetic susceptibility effects of oxy- and deoxyhemoglobin; and (iii) the effects of paramagnetic oxygen on the relaxation times of tissue water. (19)F MRS approaches monitor tumor hypoxia through the selective accumulation of reduced nitroimidazole derivatives in hypoxic zones, whereas electron spin resonance methods determine the oxygen level through its influence on the linewidths of appropriate paramagnetic probes in vivo. Finally, Overhauser-enhanced MRI combines the sensitivity of EPR methodology with the resolution of MRI, providing a window into the future use of hyperpolarized oxygen probes.

Hexamethyldisiloxane (HMDSO) has been identified as a sensitive proton NMR indicator of tissue oxygenation (pO2) based on spectroscopic spin-lattice relaxometry. A rapid MRI approach has now been designed, implemented, and tested. The technique, proton imaging of siloxanes to map tissue oxygenation levels (PISTOL), utilizes frequency-selective excitation of the HMDSO resonance and chemical-shift selective suppression of residual water signal to effectively eliminate water and fat signals and pulse-burst saturation recovery 1H echo planar imaging to map T1 of HMDSO and hence pO2. PISTOL was used here to obtain maps of pO2 in rat thigh muscle and Dunning prostate R3327 MAT-Lu tumor-implanted rats. Measurements were repeated to assess baseline stability and response to breathing of hyperoxic gas. Each pO2 map was obtained in 3 1/2 min, facilitating dynamic measurements of response to oxygen intervention. Altering the inhaled gas to oxygen produced a significant increase in mean pO2 from 55 Torr to 238 Torr in thigh muscle and a smaller, but significant, increase in mean pO2 from 17 Torr to 78 Torr in MAT-Lu tumors. Thus, PISTOL enabled mapping of tissue pO2 at multiple locations and dynamic changes in pO2 in response to intervention. This new method offers a potentially valuable new tool to image pO2 in vivo for any healthy or diseased state by 1H MRI. Copyright © 2008 John Wiley & Sons, Ltd., Funded by: DAMD. Grant Number: W81XWH-06-1-0149; NIH. Grant Number: P41RR02584 and NIH. Grant Number: U24 CA126608.

This is an open access journal., In the title mol-ecule, C9H9N3O6, all bond lengths and angles are normal. The crystal packing shows inter-molecular C - H⋯O hydrogen bonds, with H⋯O distances of 2.299 (16) and 2.374 (15) Å, which link the mol-ecules into ribbons. © International Union of Crystallography 2007., The authors thank the Ministerio de Educación y Ciencia (CTQ2006-06505/BQU) and the Comunidad de Madrid (P-2006BIO-0170) for financial support.

The hypothesis explored in the present work was that the microstructural properties of brain parenchyma, as sensed by multimodal magnetic resonance imaging (MRI), in combination with machine learning classifiers, might provide a strong analytical framework to identify distinct stages in an alcohol use disorder. Furthermore, we hypothesize that …