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Intraocular retinoblastoma treatment has changed radically over the last decade, leading to a notable improvement in ocular survival. However, eyes that relapse remain difficult to treat, as few alternative active drugs are available. More challenging is the scenario of central nervous system (CNS) metastasis, in which almost no advancements have been made. Both clinical scenarios represent an urgent need for new drugs. Using an integrated multidisciplinary approach, we developed a decision process for prioritizing drug selection for local (intravitreal [IVi], intrathecal/intraventricular [IT/IVt]), systemic, or intra-arterial chemotherapy (IAC) treatment by means of high-throughput pharmacological screening of primary cells from two patients with intraocular tumor and CNS metastasis and a thorough database search to identify clinical and biopharmaceutical data. This process identified 169 compounds to be cytotoxic; only 8 are FDA-approved, lack serious toxicities and available for IVi administration. Four of these agents could also be delivered by IT/IVt. Twelve FDA-approved drugs were identified for systemic delivery as they are able to cross the blood-brain barrier and lack serious adverse events; four drugs are of oral usage and six compounds that lack vesicant or neurotoxicity could be delivered by IAC. We also identified promising compounds in preliminary phases of drug development including inhibitors of survivin, antiapoptotic Bcl-2 family proteins, methyltransferase, and kinesin proteins. This systematic approach may be applied more broadly to prioritize drugs to be repurposed or to identify novel hits for use in retinoblastoma treatment.
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Descubrimiento de Drogas/organización & administración , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Infusiones Intraventriculares , Inyecciones Espinales , Inyecciones Intravítreas , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Neoplasias de la Retina/patología , Retinoblastoma/patologíaRESUMEN
In this retrospective study of patients with overt orbital retinoblastoma, we evaluated minimally disseminated disease (MDD) in bone marrow and cerebrospinal fluid (CSF) using CRX and/or GD2 synthase as markers. Ten patients were evaluated-five (50%) at diagnosis and five upon relapse. MDD was detected in four cases (one in the bone marrow, two in the CSF, and in one case in both sites). All patients received chemotherapy and four received orbital radiotherapy. Seven patients relapsed or progressed and all of them died. Three patients remain in complete remission. There was no apparent correlation between MDD and the outcome.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/metabolismo , Proteínas de Homeodominio/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Neoplasia Residual/mortalidad , Neoplasias Orbitales/mortalidad , Retinoblastoma/mortalidad , Transactivadores/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/líquido cefalorraquídeo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Médula Ósea/patología , Femenino , Estudios de Seguimiento , Proteínas de Homeodominio/líquido cefalorraquídeo , Proteínas de Homeodominio/genética , Humanos , Masculino , N-Acetilgalactosaminiltransferasas/líquido cefalorraquídeo , N-Acetilgalactosaminiltransferasas/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Neoplasia Residual/metabolismo , Neoplasia Residual/patología , Neoplasia Residual/terapia , Neoplasias Orbitales/metabolismo , Neoplasias Orbitales/patología , Neoplasias Orbitales/terapia , Pronóstico , Estudios Prospectivos , Dosificación Radioterapéutica , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/mortalidad , Neoplasias de la Retina/patología , Neoplasias de la Retina/terapia , Retinoblastoma/metabolismo , Retinoblastoma/patología , Retinoblastoma/terapia , Estudios Retrospectivos , Tasa de Supervivencia , Transactivadores/líquido cefalorraquídeo , Transactivadores/genética , Adulto JovenRESUMEN
A preclinical model could aid in understanding retinoblastoma vitreous seeds behavior, drug penetration, and response to chemotherapy to optimize patient treatment. Our aim was to develop a tridimensional in vitro model of retinoblastoma vitreous seeds to assess chemotherapy penetration by means of live-cell imaging. Cell cultures from patients with retinoblastoma who underwent upfront enucleation were established and thoroughly characterized for authentication of human tumor origin. The correlation of the in vitro tridimensional structures resembling human spheres and dusts vitreous seeds was established. Confocal microscopy was used to quantify real-time fluorescence of topotecan as a measure of its penetration into different sizes of spheres. Cell viability was determined after chemotherapy penetration. The in vitro spheres and dusts models were able to recapitulate the morphology, phenotype, and genotype of patient vitreous seeds. The larger the size of the spheres, the longer the time required for the drug to fully penetrate into the core (p < 0.05). Importantly, topotecan penetration correlated with its cytotoxic activity. Therefore, the studied tridimensional cell model recapitulated several characteristics of vitreous seeds observed in patients with retinoblastoma and were successfully used to assess live-cell imaging of chemotherapy penetration for drug distribution studies.
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Antineoplásicos/farmacología , Organoides/efectos de los fármacos , Retinoblastoma/tratamiento farmacológico , Topotecan/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Imagenología Tridimensional/métodos , Organoides/diagnóstico por imagen , Cultivo Primario de Células/métodos , Topotecan/uso terapéuticoAsunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Idarrubicina/farmacología , Neoplasias de la Retina/patología , Retinoblastoma/patología , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
MicroRNAs (miRNAs) are short (about 18-24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, including cancers. Due to their short lengths, the specificity often becomes an issue in conventional amplification-based methods. Next-generation sequencing techniques could be an alternative, but the long analysis time and expensive costs make them less suitable for routine clinical diagnosis. Therefore, it is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. In this work, we report a CRISPR/Cas13a-based miRNA biosensing using point-of-care dark-field (DF) imaging. We utilized magnetic-gold nanoparticle (MGNPs) complexes as signal probes, which consist of 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) linked by DNA hybridization. Once the CRISPR/Cas13a system recognized the target miRNAs (miR-21-5p), the activated Cas13a cleaved the bridge linker containing RNA sequences, releasing 60 nm-AuNPs detected and quantified by a portable DF imaging system. The combination of CRISPR/Cas13a, MGNPs, and DF imaging demonstrated amplification-free detection of miR-21-5p within 30 min at a detection limit of 500 attomoles (25 pM) and with single-base specificity. The CRISPR/Cas13a-assisted MGNP-DF assay achieved rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis.
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ANALYSIS: of rare circulating extracellular vesicles (EV) from early cancers or different types of host cells requires extremely sensitive EV sensing technologies. Nanoplasmonic EV sensing technologies have demonstrated good analytical performances, but their sensitivity is often limited by EVs' diffusion to the active sensor surface for specific target EV capture. Here, we developed an advanced plasmonic EV platform with electrokinetically enhanced yields (KeyPLEX). The KeyPLEX system effectively overcomes diffusion-limited reactions with applied electroosmosis and dielectrophoresis forces. These forces bring EVs toward the sensor surface and concentrate them in specific areas. Using the keyPLEX, we showed significant improvements in detection sensitivity by â¼100-fold, leading to the sensitive detection of rare cancer EVs from human plasma samples in 10 min. The keyPLEX system could become a valuable tool for point-of-care rapid EV analysis.
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Técnicas Biosensibles , Vesículas Extracelulares , Neoplasias , Humanos , Neoplasias/diagnóstico , ElectroósmosisRESUMEN
Molecular classification of medulloblastoma is critical for the treatment of this brain tumor. Array-based DNA methylation profiling has emerged as a powerful approach for brain tumor classification. However, this technology is currently not widely available. We present a machine-learning decision support system (DSS) that enables the classification of the principal molecular groups-WNT, SHH, and non-WNT/non-SHH-directly from quantitative PCR (qPCR) data. We propose a framework where the developed DSS appears as a user-friendly web-application-EpiGe-App-that enables automated interpretation of qPCR methylation data and subsequent molecular group prediction. The basis of our classification strategy is a previously validated six-cytosine signature with subgroup-specific methylation profiles. This reduced set of markers enabled us to develop a methyl-genotyping assay capable of determining the methylation status of cytosines using qPCR instruments. This study provides a comprehensive approach for rapid classification of clinically relevant medulloblastoma groups, using readily accessible equipment and an easy-to-use web-application.t.
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Purpose: Although there have been improvements in the management of metastatic retinoblastoma, most patients do not survive, and all patients suffer from multiple short- and long-term treatment toxicities. Reliable and informative models to assist clinicians are needed. Thus we developed and comprehensively characterized a novel preclinical platform of primary cell cultures and xenograft models of metastatic retinoblastoma to provide insights into the molecular biology underlying metastases and to perform drug screening for the identification of hit candidates with the highest potential for clinical translation. Methods: Orbital tumor, bone marrow, cerebrospinal fluid, and lymph node tumor infiltration specimens were obtained from seven patients with metastatic retinoblastoma at diagnosis, disease progression, or relapse. Tumor specimens were engrafted in immunodeficient animals, and primary cell lines were established. Genomic, immunohistochemical/immunocytochemical, and pharmacological analysis were performed. Results: We successfully established five primary cell lines: two derived from leptomeningeal, two from orbital, and one from lymph node tumor dissemination. After the intravitreal or intraventricular inoculation of these cells, we established cell-derived xenograft models. Both primary cell lines and xenografts accurately retained the histological and genomic features of the tumors from which they were derived and faithfully recapitulated the dissemination patterns and pharmacological sensitivity observed in the matched patients. Conclusions: Ours is an innovative and thoroughly characterized preclinical platform of metastatic retinoblastoma developed for the understanding of tumor biology of this highly aggressive tumor and has the potential to identify drug candidates to treat patients who currently lack effective treatment options.
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Neoplasias de la Retina , Retinoblastoma , Animales , Humanos , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Recurrencia Local de Neoplasia , Línea Celular , Modelos Animales de Enfermedad , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/genéticaRESUMEN
Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management.
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Importance: Comprehensive understanding of the genomic and gene-expression differences between retinoblastoma tumors from patients with bilateral disease may help to characterize risk and optimize treatment according to individual tumor characteristics. Objective: To compare the genomic features between each eye and a specimen from an orbital relapse in patients with bilateral retinoblastoma. Design, Setting, and Participants: In this case, 2 patients with retinoblastoma underwent upfront bilateral enucleation. Tumor samples were subjected to genomic and gene-expression analysis. Primary cell cultures were established from both of the tumors of 1 patient and were used for gene-expression studies. Main Outcomes and Measures: Whole-exome sequencing was performed on an Illumina platform for fresh tumor samples and DNA arrays (CytoScan or OncoScan) were used for paraffin-embedded samples and cell lines. Gene-expression analysis was performed using Agilent microarrays. Germinal and somatic alterations, copy number alterations, and differential gene expression were assessed. Results: After initial bilateral enucleation, patient 1 showed massive choroidal and laminar optic nerve infiltration, while patient 2 showed choroidal and laminar optic nerve invasion. Patient 1 developed left-eye orbital recurrence and bone marrow metastasis less than 1 year after enucleation. Both ocular tumors showed gains on 1q and 6p but presented other distinct genomic alterations, including an additional gain in 2p harboring the N-myc proto-oncogene (MYCN) in the left tumor and orbital recurrence. Similar copy number alterations between the orbital recurrence and the left eye supported the origin of the relapse, with an additional 11q loss only detected in the orbital relapse. Specimens from patient 2 showed common copy number gains and losses, but further evolution rendered a 2p gain spanning MYCN in the left tumor. For this patient, microarray expression analysis showed differential expression of the MYCN and the forkhead box protein G1 (FOXG1) gene pathways between the left and right tumors. Conclusions and Relevance: Differential genomic and gene expression features were observed between tumors in 2 patients with bilateral disease, confirming intereye heterogeneity that might be considered if targeted therapies are used in such patients. Chromosomal alteration profile supported the origin of the orbital recurrence from the homolateral eye in 1 patient. Loss in chromosome 11q may have been associated with extraocular relapse in this patient.
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Regulación Neoplásica de la Expresión Génica/genética , Heterogeneidad Genética , Genómica , Neoplasias de la Retina/genética , Retinoblastoma/genética , Transcriptoma , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , Enucleación del Ojo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pérdida de Heterocigocidad , Polimorfismo de Nucleótido Simple , Proto-Oncogenes Mas , Neoplasias de la Retina/patología , Retinoblastoma/patología , Secuenciación del ExomaRESUMEN
An uncommon subgroup of unilateral retinoblastomas with highly aggressive histological features, lacking aberrations in RB1 gene with high-level amplification of MYCN (MCYNamplRB1+/+) has only been described as intra-ocular cases treated with initial enucleation. Here, we present a comprehensive clinical, genomic, and pharmacological analysis of two cases of MCYNamplRB1+/+ with orbital and cervical lymph node involvement, but no central nervous system spread, rapidly progressing to fatal disease due to chemoresistance. Both patients showed in common MYCN high amplification and chromosome 16q and 17p loss. A somatic mutation in TP53, in homozygosis by LOH, and high chromosomal instability leading to aneuploidy was identified in the primary ocular tumor and sites of dissemination of one patient. High-throughput pharmacological screening was performed in a primary cell line derived from the lymph node dissemination of one case. This cell line showed resistance to broad spectrum chemotherapy consistent with the patient's poor response but sensitivity to the synergistic effects of panobinostat-bortezomib and carboplatin-panobinostat associations. From these cells we established a cell line derived xenograft model that closely recapitulated the tumor dissemination pattern of the patient and served to evaluate whether triple chemotherapy significantly prolonged survival of the animals. We report novel genomic alterations in two cases of metastatic MCYNamplRB1+/+ that may be associated with chemotherapy resistance and in vitro/in vivo models that serve as basis for tailoring therapy in these cases.
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BACKGROUND: Intravitreal injection of chemotherapy in retinoblastoma eyes with vitreous seeds may lead to a risk of extraocular tumour dissemination that has not been assessed so far. AIMS: To develop a sensitive and clinically feasible technique to assess for potential retinoblastoma cell reflux after intravitreal injection of melphalan. METHODS: Filter papers were cut in 6 mm diameter circles and sterilised before use. Eyes with retinoblastoma vitreous seeds (group D, International Classification) received weekly intravitreal melphalan injections (20 µg or 30 µg/dose) followed by cryotherapy as part of local treatment. Immediately after finishing the injection and cryotherapy, filter papers were placed on the injection site and on the cryoprobe tip to assess for the expression of the cone-rod homeobox gene (CRX) by real-time qPCR as a surrogate of retinoblastoma RNA. The assay was developed and validated to determine sensitivity, linearity, recovery, repeatability and reproducibility. RESULTS: The assay for quantitation of CRX expression was linear in the range of 1 to 1000 cells. The lowest limit of detection was one retinoblastoma cell and allowed to recover 100% of the cell load in external supplementation. A total of 14 eyes received 22 cycles of intravitreal melphalan and were evaluated for potential extraocular tumour cell dissemination using the developed technique. None of the cycles were positive for CRX in samples from the scar or from the cryoprobe tip. CONCLUSIONS: A sensitive and simple method of tumour cell assessment has been developed that can be used in the clinics to assess for potential extraocular dissemination after intravitreal injections to assure its performance.
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Antineoplásicos Alquilantes/administración & dosificación , Proteínas de Homeodominio/genética , Melfalán/administración & dosificación , Siembra Neoplásica , ARN Neoplásico/análisis , Neoplasias de la Retina/genética , Retinoblastoma/genética , Transactivadores/genética , Biomarcadores de Tumor , Crioterapia , Humanos , Inyecciones Intravítreas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Estudios Retrospectivos , Células Tumorales CultivadasRESUMEN
OBJECTIVES: We characterized and compared the in-vivo absorption of topotecan into the aqueous humor after instillation of aqueous and ointment formulations. METHODS: A lanolin/petrolatum ointment was used. New Zealand rabbits were instilled with topotecan solution (6 µg, group A), a single 10 µg dose of topotecan ointment (group B) or with five 10 µg doses of topotecan ointment (group C). Aqueous humor samples were collected at different times. Corneal samples were collected only for group A. Topotecan was quantified using HPLC, and pharmacokinetic parameters were calculated. Acute corneal epithelial toxicity was assessed after multiple instillations of topotecan ointment. KEY FINDINGS: Total topotecan maximum aqueous humor concentration (Cmax ) was 16.1, 69.9 and 287 ng/ml in group A, B and C, respectively. A single dose of topotecan ointment increased threefold and sevenfold the aqueous humor Cmax , and exposure compared to the aqueous formulation. Aqueous humor concentrations from group C eyes were substantially above the cytotoxic concentration for retinoblastoma cells. No corneal toxicity was evident after ointment instillation. CONCLUSIONS: Topotecan penetrated into the aqueous humor of the rabbit eye after multiple doses of an ointment in concentrations pharmacologically active against retinoblastoma cells without eliciting acute toxicity. Topotecan ointment may translate to the clinical treatment of anterior segment disseminated retinoblastoma.
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Soluciones Oftálmicas/administración & dosificación , Soluciones Oftálmicas/farmacocinética , Retinoblastoma/tratamiento farmacológico , Topotecan/administración & dosificación , Topotecan/farmacocinética , Cuerpo Vítreo/efectos de los fármacos , Administración Tópica , Animales , Humor Acuoso/efectos de los fármacos , Córnea/efectos de los fármacos , Conejos , Distribución TisularRESUMEN
PURPOSE: Although widely used for vitreous seed control in retinoblastoma patients, currently there are no data on melphalan pharmacokinetics after intravitreal injections. Therefore, in this study, we characterized the ocular and systemic disposition of melphalan after intravitreal injection in the rabbit eye. METHODS: New Zealand rabbits received a single intravitreal injection of 15 µg of melphalan. Vitreous, aqueous, retina, and blood samples were collected at different times up to 12 h after the injection. Melphalan was quantitated in the biological samples using a validated high-performance liquid-chromatography technique and pharmacokinetic parameters were calculated by means of compartmental models. RESULTS: Model-predicted melphalan maximum vitreous, aqueous, and retina concentrations were 7.8 µg/mL, 0.024 µg/mL, and 9.8 µg/g tissue, respectively, attained immediately and at 0.8 and 0.25 h after intravitreal injection. Melphalan vitreous concentrations were higher than 0.3 µg/mL for 5 h after dosing. The elimination half-life from the vitreous, aqueous humor, and retina was 1.0, 0.2, and 1.2 h, respectively. Aqueous exposure [area under the curve (AUC)] was only 0.7% of that of the vitreous AUC. Melphalan concentrations in the retina were still detectable 12 h after dosing, while plasma exposure was under the limit of quantitation. CONCLUSION: Intravitreal administration of 15 µg melphalan leads to pharmacological vitreous levels with low aqueous exposure. Melphalan concentrations in the retina were measurable up to 12 h after dosing, but we report nondetectable systemic exposure in the rabbit. The results correlate with the clinical features of retinoblastoma patients that show control of vitreous seeds without systemic toxicity using intravitreal melphalan.
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Melfalán/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Inyecciones Intravítreas , Melfalán/administración & dosificación , Melfalán/análisis , Conejos , Retina/química , Retina/metabolismo , Distribución TisularRESUMEN
Current treatment of retinoblastoma involves using the maximum dose of chemotherapy that induces tumor control and is tolerated by patients. The impact of dose and schedule on the cytotoxicity of chemotherapy has not been studied. Our aim was to gain insight into the cytotoxic and antiangiogenic effect of the treatment scheme of chemotherapy used in retinoblastoma by means of different in vitro models and to evaluate potential effects on multi-drug resistance proteins. Two commercial and two patient-derived retinoblastoma cell types and two human vascular endothelial cell types were exposed to increasing concentrations of melphalan or topotecan in a conventional (single exposure) or metronomic (7-day continuous exposure) treatment scheme. The concentration of chemotherapy causing a 50% decrease in cell proliferation (IC50) was determined by MTT and induction of apoptosis was evaluated by flow cytometry. Expression of ABCB1, ABCG2 and ABCC1 after conventional or metronomic treatments was assessed by RT-qPCR. We also evaluated the in vivo response to conventional (0.6 mg/kg once a week for 2 weeks) and metronomic (5 days a week for 2 weeks) topotecan in a retinoblastoma xenograft model. Melphalan and topotecan were cytotoxic to both retinoblastoma and endothelial cells after conventional and metronomic treatments. A significant decrease in the IC50 (median, 13-fold; range: 3-23) was observed following metronomic chemotherapy treatment in retinoblastoma and endothelial cell types compared to conventional treatment (p<0.05). Metronomic topotecan or melphalan significantly inhibited in vitro tube formation in HUVEC and EPC compared to vehicle-treated cells (p<0.05). Both treatment schemes induced apoptosis and/or necrosis in all cell models. No significant difference was observed in the expression of ABCB1, ABCC1 or ABCG2 when comparing cells treated with melphalan or topotecan between treatment schedules at the IC50 or with control cells (p>0.05). In mice, continuous topotecan lead to significantly lower tumor volumes compared to conventional treatment after 14 days of treatment (p<0.05). Continuous exposure to melphalan or topotecan increased the chemosensitivity of retinoblastoma and endothelial cells to both chemotherapy agents with lower IC50 values compared to short-term treatment. These findings were validated in an in vivo model. None of the dosing modalities induced multidrug resistance mechanisms while apoptosis was the mechanism of cell death after both treatment schedules. Metronomic chemotherapy may be a valid option for retinoblastoma treatment allowing reductions of the daily dose.
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Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Retinoblastoma/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Desnudos , Retinoblastoma/patologíaRESUMEN
Treatment of retinoblastoma, the most common primary ocular malignancy in children, has greatly improved over the last decade. Still, new devices for chemotherapy are needed to achieve better tumor control. The aim of this project was to develop an ocular drug delivery system for topotecan (TPT) loaded in biocompatible hydrogels of poly(ε-caprolactone)-poly(ethyleneglycol)-poly(ε-caprolactone) block copolymers (PCL-PEG-PCL) for sustained TPT release in the vitreous humor. Hydrogels were prepared from TPT and synthesized PCL-PEG-PCL copolymers. Rheological properties and in vitro and in vivo TPT release were studied. Hydrogel cytotoxicity was evaluated in retinoblastoma cells as a surrogate for efficacy and TPT vitreous pharmacokinetics and systemic as well as ocular toxicity were evaluated in rabbits. The pseudoplastic behavior of the hydrogels makes them suitable for intraocular administration. In vitro release profiles showed a sustained release of TPT from PCL-PEG-PCL up to 7days and drug loading did not affect the release pattern. Blank hydrogels did not affect retinoblastoma cell viability but 0.4% (w/w) TPT-loaded hydrogel was highly cytotoxic for at least 7days. After intravitreal injection, TPT vitreous concentrations were sustained above the pharmacologically active concentration. One month after injection, animals with blank or TPT-loaded hydrogels showed no systemic toxicity or retinal impairment on fundus examination, electroretinographic, and histopathological assessments. These novel TPT-hydrogels can deliver sustained concentrations of active drug into the vitreous with excellent biocompatibility in vivo and pronounced cytotoxic activity in retinoblastoma cells and may become an additional strategy for intraocular retinoblastoma treatment.
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Hidrogeles/química , Topotecan/administración & dosificación , Topotecan/química , Animales , Línea Celular Tumoral , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos/métodos , Humanos , Poliésteres/química , Polietilenglicoles/química , Conejos , Retina/metabolismo , Retinoblastoma/tratamiento farmacológico , Topotecan/uso terapéuticoRESUMEN
Translational research in retinoblastoma - a pediatric tumor that originates during the development of the retina - would be improved by the creation of new patient-derived models. Using tumor samples from enucleated eyes we established a new battery of preclinical models that grow in vitro in serum-free medium and in vivo in immunodeficient mice. To examine whether the new xenografts recapitulate human disease and disseminate from the retina to the central nervous system, we evaluated their histology and the presence of molecular markers of dissemination that are used in the clinical setting to detect extraocular metastases. We evaluated GD2 synthase and CRX as such markers and generated a Taqman real-time quantitative PCR method to measure CRX mRNA for rapid, sensitive and specific quantification of local and metastatic tumor burden. This approach was able to detect 1 human retinoblastoma cell in 100.000 mouse brain cells. Our research adds novel preclinical tools for the discovery of new retinoblastoma treatments for clinical translation.
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Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/enzimología , Movimiento Celular , Proteínas de Homeodominio/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Neoplasias Experimentales/enzimología , Neoplasias de la Retina/enzimología , Retinoblastoma/enzimología , Transactivadores/metabolismo , Animales , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Preescolar , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Proteínas de Homeodominio/genética , Humanos , Lactante , Ratones Desnudos , N-Acetilgalactosaminiltransferasas/genética , Micrometástasis de Neoplasia , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/secundario , Transducción de Señal , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
PURPOSE: To assess in vitro cytotoxic activity and antiangiogenic effect, ocular and systemic disposition, and toxicity of digoxin in rabbits after intravitreal injection as a potential candidate for retinoblastoma treatment. METHODS: A panel of two retinoblastoma and three endothelial cell types were exposed to increasing concentrations of digoxin in a conventional (72-hour exposure) and metronomic (daily exposure) treatment scheme. Cytotoxicity was defined as the digoxin concentration that killed 50% of the cells (IC50) and was assessed with a vital dye in all cell types. Induction of apoptosis and cell-cycle status were evaluated by flow cytometry after both treatment schemes. Ocular and systemic disposition after intravitreal injection as well as toxicity was assessed in rabbits. Electroretinograms (ERGs) were recorded before and after digoxin doses and histopathological examinations were performed after enucleation. RESULTS: Digoxin was cytotoxic to retinoblastoma and endothelial cells under conventional and metronomic treatment. IC50 was comparable between both schedules and induced apoptosis in all cell lines. Calculated vitreous digoxin Cmax was 8.5 µg/mL and the levels remained above the IC50 for at least 24 hours after intravitreal injection. Plasma digoxin concentration was below 0.5 ng/ml. Retinal toxicity was evident after the third intravitreal dose with considerable changes in the ERG and histologic damage to the retina. CONCLUSIONS: Digoxin has antitumor activity for retinoblastoma while exerting antiangiogenic activity in vitro at similar concentrations. Metronomic treatment showed no advantage in terms of dose for cytotoxic effect. Four biweekly injections of digoxin led to local toxicity to the retina but no systemic toxicity in rabbits.