Mapping chemotherapeutic drug distribution in cancer cell spheroids using 2D-TOF-SIMS and LESA-TIMS-MS.

Three-dimensional (3D) cancer cell cultures grown in the form of spheroids are effective models for the study of in vivo-like processes simulating cancer tumor pharmacological dynamics and morphology. In this study, we show the advantages of Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) combined with in situ Liquid Extraction Surface Analysis coupled to trapped Ion Mobility Spectrometry Mass Spectrometry (LESA-TIMS-TOF MS) for high spatial resolution mapping and quantitation of ABT-737, a chemotherapeutic drug, at the level of single human colon carcinoma cell spheroids (HCT 116 MCS). 2D-TOF-SIMS studies of consecutive sections (∼16 µm thick slices) showed that ABT-737 is homogenously distributed in the outer layers of the HCT 116 MCS. Complementary in situ LESA-TIMS-TOF MS/MS measurements confirmed the presence of the ABT-737 drug in the MCS slides by the observation of the molecular ion [M + H]+m/z and mobility, and the charateristic fragmentation pattern. LESA-TIMS-TOF MS allowed a quantitative assessment of the ABT-737 drug of the control MCS slice spiked with ABT-737 standard over the 0.4-4.1 ng range and MCS treated starting at 10 µM for 24 h. These experiments showcase an effective protocol for unambigous characterization and 3D mapping of chemotherapeutic drug distribution at the single MCS level.

Feasibility of functional precision medicine for guiding treatment of relapsed or refractory pediatric cancers.

Children with rare, relapsed or refractory cancers often face limited treatment options, and few predictive biomarkers are available that can enable personalized treatment recommendations. The implementation of functional precision medicine (FPM), which combines genomic profiling with drug sensitivity testing (DST) of patient-derived tumor cells, has potential to identify treatment options when standard-of-care is exhausted. The goal of this prospective observational study was to generate FPM data for pediatric patients with relapsed or refractory cancer. The primary objective was to determine the feasibility of returning FPM-based treatment recommendations in real time to the FPM tumor board (FPMTB) within a clinically actionable timeframe (<4 weeks). The secondary objective was to assess clinical outcomes from patients enrolled in the study. Twenty-five patients with relapsed or refractory solid and hematological cancers were enrolled; 21 patients underwent DST and 20 also completed genomic profiling. Median turnaround times for DST and genomics were within 10 days and 27 days, respectively. Treatment recommendations were made for 19 patients (76%), of whom 14 received therapeutic interventions. Six patients received subsequent FPM-guided treatments. Among these patients, five (83%) experienced a greater than 1.3-fold improvement in progression-free survival associated with their FPM-guided therapy relative to their previous therapy, and demonstrated a significant increase in progression-free survival and objective response rate compared to those of eight non-guided patients. The findings from our proof-of-principle study illustrate the potential for FPM to positively impact clinical care for pediatric and adolescent patients with relapsed or refractory cancers and warrant further validation in large prospective studies. ClinicalTrials.gov registration: NCT03860376 .

Imaging neuroinflammation with TSPO: A new perspective on the cellular sources and subcellular localization.

Translocator Protein 18 kDa (TSPO), previously named Peripheral Benzodiazepine Receptor, is a well-validated and widely used biomarker of neuroinflammation to assess diverse central nervous system (CNS) pathologies in preclinical and clinical studies. Many studies have shown that in animal models of human neurological and neurodegenerative disease and in the human condition, TSPO levels increase in the brain neuropil, and this increase is driven by infiltration of peripheral inflammatory cells and activation of glial cells. Therefore, a clear understanding of the dynamics of the cellular sources of the TSPO response is critically important in the interpretation of Positron Emission Tomography (PET) studies and for understanding the pathophysiology of CNS diseases. Within the normal brain compartment, there are tissues and cells such as the choroid plexus, ependymal cells of the lining of the ventricles, and vascular endothelial cells that also express TSPO at even higher levels than in glial cells. However, there is a paucity of knowledge if these cell types respond and increase TSPO in the diseased brain. These cells do provide a background signal that needs to be accounted for in TSPO-PET imaging studies. More recently, there are reports that TSPO may be expressed in neurons of the adult brain and TSPO expression may be increased by neuronal activity. Therefore, it is essential to study this topic with a great deal of detail, methodological rigor, and rule out alternative interpretations and imaging artifacts. High levels of TSPO are present in the outer mitochondrial membrane. Recent studies have provided evidence of its localization in other cellular compartments including the plasma membrane and perinuclear regions which may define functions that are different from that in mitochondria. A greater understanding of the TSPO subcellular localization in glial cells and infiltrating peripheral immune cells and associated function(s) may provide an additional layer of information to the understanding of TSPO neurobiology. This review is an effort to outline recent advances in understanding the cellular sources and subcellular localization of TSPO in brain cells and to examine remaining questions that require rigorous investigation.

The Translocator Protein (TSPO) Genetic Polymorphism A147T Is Associated with Worse Survival in Male Glioblastoma Patients.

Glioblastoma (GBM) is the most common primary brain tumor in adults, with few available therapies and a five-year survival rate of 7.2%. Hence, strategies for improving GBM prognosis are urgently needed. The translocator protein 18kDa (TSPO) plays crucial roles in essential mitochondria-based physiological processes and is a validated biomarker of neuroinflammation, which is implicated in GBM progression. The TSPO gene has a germline single nucleotide polymorphism, rs6971, which is the most common SNP in the Caucasian population. High TSPO gene expression is associated with reduced survival in GBM patients; however, the relation between the most frequent TSPO genetic variant and GBM pathogenesis is not known. The present study retrospectively analyzed the correlation of the TSPO polymorphic variant rs6971 with overall and progression-free survival in GBM patients using three independent cohorts. TSPO rs6971 polymorphism was significantly associated with shorter overall survival and progression-free survival in male GBM patients but not in females in one large cohort of 441 patients. We observed similar trends in two other independent cohorts. These observations suggest that the TSPO rs6971 polymorphism could be a significant predictor of poor prognosis in GBM, with a potential for use as a prognosis biomarker in GBM patients. These results reveal for the first time a biological sex-specific relation between rs6971 TSPO polymorphism and GBM.

Tyrosyl-DNA Phosphodiesterase 1 and Topoisomerase I Activities as Predictive Indicators for Glioblastoma Susceptibility to Genotoxic Agents.

Glioblastoma (GBM) patients have an estimated survival of ~15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for the selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc), inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan. We treated GBM cell lines with increasing concentrations of irinotecan and compared the IC50 values. We found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.972, based on the average from three sets of experiments) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC50, while the expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. The TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for the selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies.

Potentiation of morphine-induced antinociception and locomotion by citalopram is accompanied by anxiolytic-like effects.

Morphine and related opioids are the mainstay of analgesic treatment, especially in patients suffering chronic pain. Besides their antinociceptive effects they may also exhibit anxiolytic-like properties that could contribute to pain relief. The pharmacological manipulation of the serotonergic system may not only modulate pain transmission and processing but also other behavioral effects of opioids. The present study aimed to analyze the effect of the concurrent treatment with citalopram, a selective serotonin reuptake inhibitor, on the antinociceptive, locomotor and anxiety-related effects induced by acute and subchronic administration of morphine in mice. Citalopram (15mg/kg) enhanced the acute antinociceptive effects of morphine when concurrently administered as evidenced by a two-fold increase in the ED50 for the antinociceptive effect of morphine in the hot-plate test. Chronic studies also revealed that concurrent citalopram treatment (15mg/kg) delayed the development of tolerance to the thermal antinociceptive effects of morphine. Additionally, morphine-induced hyperlocomotion was potentiated by citalopram as assessed in the open-field test and in the spontaneous activity recording in the home cage, a behavioral outcome to which tolerance or desensitization was not developed. Interestingly, chronic administration of both drugs promoted an anxiolytic effect as evidenced by the increased central activity in the open field test. Future investigations on this pharmacological interaction, such as the possible translational research in clinics, might have consequences in future strategies for the therapeutic management of pain.

Analysis of SOX2-Regulated Transcriptome in Glioma Stem Cells.

INTRODUCTION: Glioblastoma is the most malignant brain tumor in adults and is associated with poor survival despite multimodal treatments. Glioma stem-like cells (GSCs) are cells functionally defined by their self-renewal potential and the ability to reconstitute the original tumor upon orthotopic implantation. They have been postulated to be the culprit of glioma chemo- and radio-resistance ultimately leading to relapse. Understanding the molecular circuits governing the GSC compartment is essential. SOX2, a critical transcription regulator of embryonic and neural stem cell function, is deregulated in GSCs however; the precise molecular pathways regulated by this gene in GSCs remain poorly understood. RESULTS: We performed a genome-wide analysis of SOX2-regulated transcripts in GSCs, using a microarray. We identified a total of 2048 differentially expressed coding transcripts and 261 non-coding transcripts. Cell adhesion and cell-cell signaling are among the most enriched terms using Gene Ontology (GO) classification. The pathways altered after SOX2 down-modulation includes multiple cellular processes such as amino-acid metabolism and intercellular signaling cascades. We also defined and classified the set of non-coding transcripts differentially expressed regulated by SOX2 in GSCs, and validated two of them. CONCLUSIONS: We present a comprehensive analysis of the transcriptome controlled by SOX2 in GSCs, gaining insights in the understanding of the potential roles of SOX2 in glioblastoma.

Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01.

Despite the recent advances in the development of antitumor therapies, the prognosis for patients with malignant gliomas remains dismal. Therapy with tumor-selective viruses is emerging as a treatment option for this devastating disease. In this study we characterize the anti-glioma effect of VCN-01, an improved hyaluronidase-armed pRB-pathway-selective oncolytic adenovirus that has proven safe and effective in the treatment of several solid tumors. VCN-01 displayed a significant cytotoxic effect on glioma cells in vitro. In vivo, in two different orthotopic glioma models, a single intra-tumoral administration of VCN-01 increased overall survival significantly and led to long-term survivors free of disease.

The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma.

PURPOSE: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy, more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are a feasible option for osteosarcoma treatment. VCN-01 is a replication-competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma. EXPERIMENTAL DESIGN: We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in orthotopic and metastatic osteosarcoma murine animal models. RESULTS: This study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent antisarcoma effect in vitro and in vivo in mouse models of intratibial and lung metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile. CONCLUSIONS: These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease. Clin Cancer Res; 22(9); 2217-25. ©2015 AACR.

Correction: Characterization of the Antiglioma Effect of the Oncolytic Adenovirus VCN-01.

[This corrects the article DOI: 10.1371/journal.pone.0147211.].

Caracterización molecular del gen GBA en pacientes cubanos con enfermedad de Gaucher / Molecular characterization of the GBA gene in Cuban patients with Gaucher disease

La enfermedad de Gaucher (EG) es el trastorno relacionado con el depósito lisosomal de mayor prevalencia a nivel mundial. Presenta un patrón de herencia autosómico recesivo y se origina fundamentalmente por mutaciones en el gen GBA, localizado en la región cromosómica 1q21, que codifica la enzima lisosomal ácido β-glucosidasa. En el presente trabajo se describen los resultados obtenidos por el laboratorio de Biología Molecular del Centro Nacional de Genética Médica, La Habana, Cuba, en el análisis del gen GBA en pacientes cubanos con sospecha clínica de la EG. Fueron estudiados siete pacientes no emparentados, caracterizados clínicamente y con diagnóstico bioquímico y/o histológico. Se realizó el estudio de cuatro mutaciones mediante amplificación de secuencias del gen GBA y digestión con enzimas de restricción. En los casos en que ninguna de las mutaciones estudiadas por análisis directo estuvo presente, las muestras se sometieron a cribado de mutaciones y secuenciación de ADN. El análisis molecular permitió identificar la mutación presente en el 100(percent) de los cromosomas analizados. Fueron detectadas las mutaciones L444P, N370S e I403T. El presente estudio estableció una metodología para el diagnóstico por biología molecular de la enfermedad de Gaucher en la población cubana que permite ofrecer un servicio diagnóstico efectivo y rápido en pacientes con sospecha de la enfermedad y en posibles portadores…(AU)

Aspectos bioquímicos, genéticos y comorbilidades de la enfermedad de Gaucher, diagnóstico molecular en Cuba / Co-morbidities, biochemical and genetic aspects in Gauchers disease: molecular diagnosis in Cuba

La enfermedad de Gaucher es la esfingolipidosis más común, causada por la deficiencia de la glucocerebrosidasa, una enzima lisosomal que cataliza la hidrólisis de la glucosilceramida en glucosa y ceramida. Se han descrito más de 250 mutaciones que se expanden a lo largo del gen que codifica esta enzima. Las manifestaciones clínicas son multisistémicas con un amplio espectro y en ocasiones no se correlacionan bien con el genotipo específico. Este trabajo presenta una actualización sobre distintos aspectos de la enfermedad de Gaucher y su diagnóstico molecular en Cuba...(AU)

Haplotipo del gen del factor VIII en el diagnóstico molecular de la hemofilia A: estudio de una familia / Factor VIII gene haplotype in the haemophilia a molecular dagnosis: a family study

La hemofilia A se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudio indirecto, por ser causada por mutaciones heterogéneas en el gen del factor VIII. Se estudió una familia afectada, para la detección de portadora de la gestante y posteriormente se realizó el diagnóstico prenatal. La extracción de ADN se hizo por el método de precipitación salina Salting Out a tres muestras de sangre y una de líquido amniótico. Se efectuó el análisis de los polimorfismos St14, Bcl I y Hind III. Para la determinación de sexo fetal, se estudió el gen AMXY. La técnica empleada fue la reacción en cadena de la polimerasa. El análisis del marcador Bcl I arrojó que la gestante era portadora de hemofilia A, pero al ser homocigótica no era informativa; el polimorfismo St14 por sí solo no brindaba la información de la condición de la gestante, pero al ser heterocigótica para el mismo y conociendo de antemano la información de ser portadora, se pudo realizar diagnóstico prenatal, gracias al análisis conjunto de los marcadores. El polimorfismo Hind III no fue informativo. El feto resultó ser varón sano(AU)

Hemophilia A is a coagulation disorder congenital disease which consist in a recessive disorder linked to X chromosome. HA is cause by heterogeneous mutations in factor VIII gen, that´s why, the study was carry out by indirect studies. We studied one family afected; we were determinated of carrier status of pregnancy woman and later we relizated of prenatal diagnosis. The DNA extraction from the three blood samples and one amniotic fluid was obtained by the saline precipitation procedure (Salting Out). We studied the polymorphisms St14, Hind III and Bcl1. The determination of fetal sex was studied of AMXY gen. The technique used was Polymerase Chain Reaction. The Bcl I marker analysis showed that the mother was a carrier of haemophilia A but being homozygous was not informative, ST14 polymorphism alone did not provide information on the condition of the mother but to be heterozygous for it and knowing beforehand information of being a carrier prenatal diagnosis was possible thanks to joint analysis of the markers. Hind III polymorphism was not informative. The male fetus was found to be healthy(AU)

Haplotipo del gen del factor VIII en el diagnóstico molecular de la hemofilia A: estudio de una familia / Factor VIII gene haplotype in the haemophilia a molecular dagnosis: a family study

La hemofilia A se caracteriza por ser una enfermedad congénita del trastorno de la coagulación y constituye un desorden recesivo ligado al cromosoma X. El estudio molecular se realiza por estudio indirecto, por ser causada por mutaciones heterogéneas en el gen del factor VIII. Se estudió una familia afectada, para la detección de portadora de la gestante y posteriormente se realizó el diagnóstico prenatal. La extracción de ADN se hizo por el método de precipitación salina Salting Out a tres muestras de sangre y una de líquido amniótico. Se efectuó el análisis de los polimorfismos St14, Bcl I y Hind III. Para la determinación de sexo fetal, se estudió el gen AMXY. La técnica empleada fue la reacción en cadena de la polimerasa. El análisis del marcador Bcl I arrojó que la gestante era portadora de hemofilia A, pero al ser homocigótica no era informativa; el polimorfismo St14 por sí solo no brindaba la información de la condición de la gestante, pero al ser heterocigótica para el mismo y conociendo de antemano la información de ser portadora, se pudo realizar diagnóstico prenatal, gracias al análisis conjunto de los marcadores. El polimorfismo Hind III no fue informativo. El feto resultó ser varón sano (AU)

Hemophilia A is a coagulation disorder congenital disease which consist in a recessive disorder linked to X chromosome. HA is cause by heterogeneous mutations in factor VIII gen, that's why, the study was carry out by indirect studies. We studied one family afected; we were determinated of carrier status of pregnancy woman and later we relizated of prenatal diagnosis. The DNA extraction from the three blood samples and one amniotic fluid was obtained by the saline precipitation procedure (Salting Out). We studied the polymorphisms St14, Hind III and Bcl1. The determination of fetal sex was studied of AMXY gen. The technique used was Polymerase Chain Reaction. The Bcl I marker analysis showed that the mother was a carrier of haemophilia A but being homozygous was not informative, ST14 polymorphism alone did not provide information on the condition of the mother but to be heterozygous for it and knowing beforehand information of being a carrier prenatal diagnosis was possible thanks to joint analysis of the markers. Hind III polymorphism was not informative. The male fetus was found to be healthy (AU)