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Purpose: To assess the safety and efficacy of a customized ablation treatment (InnovEyes) to correct myopia and myopic astigmatism with femtosecond laser-assisted in situ keratomileusis (Femto LASIK). Patients and Methods: In this prospective, nonrandomized, multicenter study, 113 patients (225 eyes) with preoperative myopia less than -9.0 diopters (D) and astigmatism 0 to -4.0 D (based on InnovEyes refraction) underwent wavefront, tomography, and biometry assessment using a single diagnostic device (InnovEyes sightmap). These data were imported and used unmodified by the InnovEyes algorithm to automatically calculate and optimize correction of lower- and higherorder aberrations (HOAs) treated by the EX500 ablation profile. Visual acuity, refractive error, HOAs, and patient satisfaction were evaluated over 3 months. Results: A total of 106 patients (212 eyes) completed the study and were included in the analysis. Mean preoperative manifest refraction spherical equivalent (MRSE) was -3.38±1.76 D. At Month 3, uncorrected distance visual acuity was 20/20 or better in 208/212 (98.1%) eyes, and it was the same as, or better than, the preoperative best-corrected distance visual acuity (CDVA) in 162/212 (76.4%) eyes; 76/212 (35.8%) eyes gained ≥1 line of CDVA. MRSE was within ±0.5 D in 195/212 (92.0%) eyes. Additionally, 201/209 (96.2%) eyes had no change (defined as a change between -0.1 µm and 0.1 µm, inclusive) in HOAs, and 105/106 (99.1%) patients reported to be satisfied with the results. Conclusion: Customizing ray-tracing Femto LASIK with this platform appeared safe and effective in correcting myopic astigmatism and also achieved a significant percentage of eyes gaining lines of vision, potentially by addressing HOAs, along with a consistently high level of patient satisfaction.
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PURPOSE: To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model. METHODS: The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate. RESULTS: The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056). CONCLUSIONS: This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.
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Macrófagos/fisiología , Neoplasias de la Retina/patología , Retinoblastoma/patología , Animales , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Recuento de Células , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Microglía/citología , Neovascularización Patológica/patología , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguíneaRESUMEN
PURPOSE: Gelatinases, matrix metalloproteinase (MMP)-2, and MMP-9 are known for their importance in angiogenesis and tumor biology. The purpose of this study was to test the hypothesis that anecortave acetate (AA) decreases transgenic retinoblastoma (RB) tumor burden by modulating gelatinase activity. METHODS: To assess the possible gelatinase modulation after AA treatment, a single subconjunctival injection of AA (300 microg) was delivered to the right eyes of 10-week-old LH(BETA)T(AG) mice. Eyes were evaluated for gelatinase expression and activity by gel and in situ zymography at 24 hours, 48 hours, and 1 week after treatment. RESULTS: Gel zymography of whole eye extracts and in situ zymography of retinal tumors showed strong gelatinase expression and activity within transgenic RB tumors. AA treatment in RB transgenic mice resulted in a significant decrease of gelatinase activity 1 week after AA treatment. Surprisingly, there was an initial transient upregulation of MMP-9 activity in whole eye extracts at 24 and 48 hours after AA treatment in both LH(BETA)T(AG) transgenic and wild-type mice. This increase was not observed in the tumors. CONCLUSIONS: As suggested by our data, inhibition of gelatinase activity appears to be a mechanism of action of AA. AA treatment results in a decrease in gelatinase activity that correlates with the significant decrease in tumor burden shown by the authors' previous studies. However, the significance of the initial, transient upregulation of gelatinase by AA injection is unknown, and further studies are warranted. Combining antiangiogenic agents with multiple mechanisms of action has the potential to enhance RB tumor control.
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Inhibidores de la Angiogénesis/uso terapéutico , Modelos Animales de Enfermedad , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Pregnadienodioles/uso terapéutico , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Animales , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Transgénicos , Neoplasias de la Retina/enzimología , Neoplasias de la Retina/patología , Retinoblastoma/enzimología , Retinoblastoma/patología , Regulación hacia ArribaRESUMEN
PURPOSE: The purpose of this study was to evaluate the effects of vessel targeting and chemotherapy agents on inducing hypoxic regions in LH(BETA)T(AG) murine retinal tumors. METHODS. Twelve- and 16-week-old LH(BETA)T(AG) transgenic retinoblastoma mice were treated with periocular injections to the right eye only of saline (n = 42), anecortave acetate (a single injection; 300 microg/20 microL; n = 42), or carboplatin (two injections per week for 3 weeks; 62.5 microg/20 microL; n = 42). Eyes were enucleated 1 day, 1 week, and 1 month after injection. To assess hypoxia, mice received 60 mg/kg pimonidazole via intraperitoneal injection. Eyes were enucleated, and tumor sections were analyzed. RESULTS: Levels of hypoxia significantly increase in 16-week-old animals 1 day and 1 week after treatment with anecortave acetate, a known angiostatic agent. Eyes treated with anecortave acetate showed a 28% (P < 0.001) increase in hypoxic regions in comparison with the saline-treated control group 1 day after injection and a 17% (P < 0.001) increase 1 week after injection. In early tumors of 12-week-old animals, levels of hypoxia increased by 2.0% (P = 0.011) 1 day after anecortave acetate injection compared to controls. Levels of hypoxia significantly decrease in 16-week-old animals 1 week and 1 month after treatment with carboplatin, a chemotherapeutic agent. Eyes treated with carboplatin showed a 21.7% (P = 0.017) decrease in hypoxic regions in comparison with the saline-treated control group 1 week after injection and a 4.51% (P < 0.001) decrease 1 month after injection. In early tumors of 12-week-old animals, levels of hypoxia decreased by 0.0429% (P < 0.001) 1 month after carboplatin injection compared with controls. CONCLUSIONS: Treatment with a vessel-targeting agent results in changes in the tumor microenvironment as early as 1 day after treatment. By increasing hypoxia in tumors, vessel-targeting agents can be combined with glycolytic inhibitors which have been shown previously to target hypoxic regions in this transgenic model. This approach may have benefits for children with this disease and should be further investigated.
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Inhibidores de la Angiogénesis/uso terapéutico , Modelos Animales de Enfermedad , Hipoxia/etiología , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguínea , Animales , Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Hipoxia/diagnóstico , Ratones , Ratones Transgénicos , Microscopía Confocal , Nitroimidazoles/farmacología , Pregnadienodioles/uso terapéutico , Neoplasias de la Retina/patología , Retinoblastoma/patologíaRESUMEN
PURPOSE: The aims of this study are (1) to evaluate the spatial distribution of neovessels and mature vessels in human uveal melanoma tumors and (2) to determine whether vessel maturation is associated with the major indicators for poor prognosis. METHODS: Immunohistochemical analyses were performed on human tissue specimens from enucleated eyes (n = 14) to assess total vessels, neovessels, mature vessels, and cell proliferation. Tumor morphology was analyzed by hematoxylin and eosin and modified periodic acid-Schiff (PAS) staining.The spatial distribution of neovessels and mature vessels was analyzed by immunohistochemistry, and correlated with major indicators of poor prognosis (i.e., aggressive PAS patterns, epithelioid cytology, mitotic figures, extraocular extension, anterior tumor location, ciliary body involvement, large tumor size, cell proliferation, and angiogenic activity). RESULTS: Neovesseldensity was greater than mature vessel density in apical (p = 0.17), central (p = 0.036), and peripheral (p = 0.31) regions of the tumors, while mature vessel density was greater than neovessel density in basal areas of the tumor (p = 0.47). This pattern indicated that vessel maturation begins at the base of the tumor and later extends to the peripheral and apical regions. The difference between mature and neovessel densities for the apical (-0.8 +/- 1.9) and central areas (-0.8 +/- 1.3) of the tumor was significantly higher than the difference obtained for the basal area (0.3 +/- 1.6; p = 0.014 and p = 0.012, respectively), indicating a higher density of mature vessels compared to neovessels at the base. Statistical correlations were found between mature vessel density and tumor size (r = 0.48, p = 0.084), cell proliferation (r = 0.62, p = 0.042), and mitotic figures (r = 0.76, p = 0.001). CONCLUSIONS: Significant differences exist in the spatial distribution of mature versus neovessels in human uveal melanoma. Vessel maturation is associated with known clinical and pathologic indicators of poor prognosis (e.g., cell proliferation). Antiangiogenic therapy should be considered for the treatment of ocular malignancies; however, the results of this study indicate that blood vessel maturation heterogeneity may limit the efficacy of vessel targeting agents.
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Vasos Sanguíneos/patología , Melanoma/irrigación sanguínea , Neovascularización Patológica/patología , Neoplasias de la Úvea/irrigación sanguínea , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Vasos Sanguíneos/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Enucleación del Ojo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Melanoma/patología , Melanoma/cirugía , Microscopía Fluorescente , Persona de Mediana Edad , Neovascularización Patológica/metabolismo , Pericitos/metabolismo , Pericitos/patología , Pronóstico , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/cirugíaRESUMEN
We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.
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Neoplasias de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Algoritmos , Animales , Cinamatos/farmacología , Modelos Animales de Enfermedad , Procesamiento de Imagen Asistido por Computador , Hormona Luteinizante de Subunidad beta/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/etiología , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/etiología , Retinoblastoma/patología , Tomografía de Coherencia Óptica/estadística & datos numéricos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
PURPOSE: The purposes of this study were to evaluate the spatial distribution of neovessels versus mature vessels in both human retinoblastoma (RB) and LH(BETA)T(AG) tumors, assess similarities and differences between the animal model and the human RB specimens, and determine whether vessel maturation is associated with risk factors for metastasis. METHODS: Immunohistochemical analyses were performed on human (n = 10) and LH(BETA)T(AG) (n = 11) enucleation specimens to evaluate the spatial distribution of neovessels and mature vessels. In human RB, vessel maturation was correlated with treatment history and metastatic risk factors. RESULTS: In human RB, the percentage of neovessels was higher in the periphery of the tumor than in the center (P = 0.021). This finding was mostly attributed to the distribution of large-caliber vessels (i.e., neovessels were higher in the periphery for large [P = 0.050]- and medium [P = 0.032]-caliber vessels; and mature vessels were higher in the center for large-caliber vessels [P = 0.032]). In this small series, vessel maturation did not correlate with risk for metastasis. Similar results were observed in LH(BETA)T(AG) tumors. The percentage of large-caliber neovessels was higher in the periphery than in the center (P = 0.038). CONCLUSIONS: There is a spatially distributed, heterogeneous vessel population containing neovessels and mature vessels in advanced RB disease. There is a significantly higher concentration of mature, large-caliber vessels in the center of tumors that is similar in human RB and LH(BETA)T(AG) retinal tumors. From these data the authors hypothesize that tumor vessel maturation in RB initiates in central regions of the tumor and radiates toward the periphery.
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Modelos Animales de Enfermedad , Neovascularización Patológica/patología , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguínea , Actinas/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Vasos Sanguíneos/patología , Preescolar , Colágeno Tipo IV/metabolismo , Endoglina , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lactante , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Receptores de Superficie Celular/metabolismo , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patologíaRESUMEN
PURPOSE: Chemotherapy resistance is a problem in the treatment of advanced retinoblastoma (RB). Since basic fibroblast growth factor (bFGF) is a survival factor for neural precursor cells, bFGF was evaluated as a growth and chemoresistance factor in RB. METHODS: bFGF expression was analyzed in the LH-betaTag transgenic mouse model of RB and human RB cell lines by immunofluorescence, RT-PCR, and Western blot. Proliferation and apoptosis (TUNEL) assays were performed. RESULTS: bFGF levels significantly increased during tumorigenesis in transgenic RB, as a function of tumor status (P = 0.005). PCR and confocal microscopy confirmed that the human cell lines and primary tumors expressed bFGF. bFGF was localized to vascular and tumor cells and rarely to glial cells. Exogenous 18-kDa bFGF induced proliferation in two RB cell lines (WERI and Y79). Western blot analysis demonstrated 34-, 22-, and 18-kDa isoforms in transgenic RB and both cell lines. In TUNEL assays, chemoresistance to carboplatin-induced apoptosis was observed in the Y79 line, which expressed a higher ratio of high (34 kDa)- to low-molecular-weight bFGF isoforms, compared with the WERI line. Similar to other bFGF tumor studies, exogenous low-molecular-weight (18 kDa) bFGF (1 ng) significantly enhanced carboplatin-induced apoptosis in the more chemosensitive WERI, but not the chemoresistant Y79 line. CONCLUSIONS: RB tumors produce significant amounts of bFGF, and the differential production and response to isoforms of bFGF may have implications for invasive tumor growth and chemoresistance.
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Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Carboplatino/farmacología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Transgénicos , Microscopía Confocal , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/mortalidad , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/mortalidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Células Tumorales Cultivadas/metabolismoRESUMEN
PURPOSE: The aim of this study is to correlate tumor size of retinoblastoma tumor samples with blood vessel maturation to assess how these factors may affect vessel targeting therapy. METHODS: Analysis was performed on retinoblastoma tumor specimens (n = 5) enucleated as primary treatment from May 2005 to February 2006. Tumor size was measured as the largest cross sectional area of the tumor, measured during pathologic assessment. Vessel density and heterogeneity was measured by immunohistochemical analysis. Total microvessel density was detected by staining endothelial cells using a lectin from Bandeira simplicifolia; novel vasculature was detected with the endothelial cell marker endoglin (CD105). Blood vessel basement membrane was detected with an antibody against type IV collagen. Vessel maturation was assessed by pericyte recruitment, detected with alpha smooth muscle actin (alpha-sma). RESULTS: A statistically significant correlation was detected between tumor burden and age at enucleation (P = 0.008). All retinoblastoma tumor samples harbored a high degree of blood vessel heterogeneity containing both immature neovessels as well as pericyte-committed mature vasculature. There was a statistically significant correlation between type IV collagen and age at enucleation (P = 0.045). CONCLUSIONS: This study provides a framework for a better understanding of tumor and vessel development in retinoblastoma. Results of this study provide insight into the relationship between age and tumor burden in these tumors. Knowledge of the degree of heterogeneity detected in these tumors will aid in the selection of novel blood vessel targeting strategies for children with this disease and other diseases with pathologic neovascularization.
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Neovascularización Patológica/patología , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguínea , Actinas/metabolismo , Adolescente , Adulto , Antígenos CD/metabolismo , Niño , Colágeno Tipo IV/metabolismo , Endoglina , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Enucleación del Ojo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Neovascularización Patológica/tratamiento farmacológico , Receptores de Superficie Celular/metabolismo , Neoplasias de la Retina/patología , Neoplasias de la Retina/cirugía , Retinoblastoma/patología , Retinoblastoma/cirugíaRESUMEN
PURPOSE: The purpose of this study was to evaluate the presence and extent of hypoxia in murine retinoblastoma tumors and the feasibility of targeting hypoxic cells as a novel therapeutic strategy. METHODS: Hypoxic and vascular areas in LH(BETA)T(AG) mouse retinal tumors were measured using immunohistochemistry. The glycolytic inhibitor 2-deoxy-d-glucose (2-DG) was used to test the efficacy of targeting hypoxic cells in retinoblastoma. Sixteen-week-old LH(BETA)T(AG) mice received injections of saline, carboplatin (31.25 microg/20 microL), 2-DG (500 mg/kg), and carboplatin (31.25 microg/20 microL) + 2-DG (500 mg/kg). Carboplatin was administered through biweekly subconjunctival injections to right eyes only for 3 weeks. 2-DG was administered through intraperitoneal injection three times a week for 5 weeks. Saline was administered using both methods. Eyes were enucleated at 21 weeks of age and examined for residual tumor. RESULTS: Hypoxic regions were observed in tumors larger than 3.28 mm(2). When 2-DG was combined with carboplatin, a marked decrease in tumor burden was observed that was significantly more pronounced than when either agent was given alone. The hypoxic tumor cell population as measured by pimonidazole was markedly reduced by carboplatin + 2-DG (P < 0.01) and by 2-DG alone (P < 0.01), but not by carboplatin alone, indicating that 2-DG effectively killed hypoxic retinoblastoma cells in vivo. CONCLUSIONS: Treatment with glycolytic inhibitors as adjuvants to chemotherapy has the potential to increase the efficacy of chemotherapy in advanced retinoblastoma. This approach may have benefits for children with this disease and should be further investigated.
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Antimetabolitos/uso terapéutico , Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Desoxiglucosa/uso terapéutico , Hipoxia/tratamiento farmacológico , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Animales , Quimioterapia Adyuvante , Progresión de la Enfermedad , Combinación de Medicamentos , Sinergismo Farmacológico , Estudios de Factibilidad , Glucólisis/efectos de los fármacos , Hipoxia/etiología , Hipoxia/patología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Neoplasias de la Retina/complicaciones , Neoplasias de la Retina/patología , Vasos Retinianos/patología , Retinoblastoma/complicaciones , Retinoblastoma/patologíaRESUMEN
PURPOSE: To evaluate the mechanism and timing of retinal tumor cell death in the LH(BETA)T(AG) mouse model of retinoblastoma after treatment with vascular targeting therapies and conventional therapies (focal chemotherapy and radiation). METHODS: For vascular targeting therapy, 12- or 16-week-old mice were treated with a single subconjunctival injection of either anecortave acetate (300 microg) or combretastatin A4 (1.5 mg). Eyes were analyzed at 1 day and 1 week after treatment. Tumor cell death was evaluated using TUNEL assays or immunofluorescence analysis of activated caspase 3 to detect apoptosis. Histopathologic analysis was performed to identify areas of necrosis. For conventional therapy, LH(BETA)T(AG) mice were treated with six serial subconjunctival injections of focally delivered carboplatin chemotherapy (100 microg/delivery) or hyperfractionated external beam radiotherapy (EBRT; 15 Gy total dose). Cell death was analyzed by TUNEL assay. RESULTS: The highest levels of apoptotic cell death were seen 1 day after treatment in all treatment groups compared with vehicle controls. At 1 week after treatment, apoptotic cell death remained significantly elevated in the EBRT and carboplatin groups, but not after vessel targeting therapy. No significant necrosis was detected by histology in tumors of treated or of control eyes. CONCLUSIONS: Conventional therapies (focal carboplatin chemotherapy and EBRT) and vascular targeting agents significantly increase cell death through apoptosis, while not having a significant effect on necrosis in this murine model of retinoblastoma. These studies will aid in the optimization of delivery schemes of combined treatment modalities.
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Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Modelos Animales de Enfermedad , Neoplasias de la Retina/patología , Retinoblastoma/patología , Animales , Carboplatino/uso terapéutico , Caspasa 3/metabolismo , Endotelio Vascular/patología , Etiquetado Corte-Fin in Situ , Ratones , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Pregnadienodioles/uso terapéutico , Radioterapia Conformacional/métodos , Neoplasias de la Retina/irrigación sanguínea , Neoplasias de la Retina/terapia , Retinoblastoma/irrigación sanguínea , Retinoblastoma/terapia , Estilbenos/uso terapéutico , Factores de TiempoRESUMEN
PURPOSE: The purpose of this study was to investigate tumor control efficacy of paclitaxel in the treatment of retinal tumors harbored by the LH beta-Tag murine transgenic model of retinoblastoma. METHODS: LH beta-Tag-positive mice (n = 5) 10 weeks of age received two 20-microL subconjunctival injections delivered with a 72-hour interval to right eyes only. Paclitaxel was dissolved in 100% dimethyl sulfoxide (DMSO) and delivered at doses of 0.5 mg, 0.25 mg, 0.125 mg, 0.0625 mg, 0.0313 mg, and 0.0152 mg in a 20-microL volume. Control mice received two subconjunctival injections of DMSO or of saline solution to right eyes only or they remained untreated. Eyes were analyzed at 16 weeks of age for residual tumor burden, which was measured by gauging the cross-sectional area of largest tumor focus. RESULTS: Linear regression analysis of tumor burden demonstrates a statistically significant (P < 0.001) dose-response relationship between paclitaxel concentration and tumor size. Transient ocular toxicities were observed after treatment, but most had subsided at the time of analysis, 6 weeks after injection. After histologic assessment, the 0.25-mg paclitaxel dose had effectively reduced tumor burden and was associated with minimal toxicity, including mild conjunctival chemosis and fibrosis, corneal epitheliopathy, and corneal edema. CONCLUSIONS: Subconjunctival delivery of paclitaxel effectively inhibits intraocular tumor burden in the LH beta-Tag model of retinoblastoma. This treatment modality may represent a novel strategy for the clinical management of retinoblastoma.
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Antineoplásicos Fitogénicos/farmacología , Paclitaxel/farmacología , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/toxicidad , Conjuntiva , Modelos Animales de Enfermedad , Inyecciones Intralesiones , Modelos Lineales , Hormona Luteinizante de Subunidad beta/genética , Ratones , Ratones Transgénicos , Paclitaxel/toxicidad , Neoplasias de la Retina/patología , Retinoblastoma/patologíaRESUMEN
PURPOSE: The aim of this study was to quantify tumor cell proliferation and growth, analyze tumor blood vessel development, and determine the efficacy of antiangiogenic and angiostatic therapy in targeting mature vessels in retinal tumors of the LH(BETA)T(AG) mouse model for retinoblastoma. METHODS: LH(BETA)T(AG) mouse retinas were analyzed at 4, 8, 12, and 16 weeks of age. Tumor burden was analyzed by histology; cell proliferation, vessel density, angiogenesis, and vessel maturation were detected by immunofluorescence. To assess the efficacy of mature vessel targeting, 16-week-old mice were treated with single subconjunctival injections of the selective vascular-targeting drug combretastatin A4 prodrug (CA4P) or anecortave acetate, and eyes were analyzed 1 day and 1 week after injection to determine microvessel density and the number of angiogenic and mature vessels. RESULTS: Increased cell proliferation and angiogenesis were detected in the retinal inner nuclear layer (INL) before morphologic neoplastic changes were evident. As tumor size increased, angiogenesis diminished concomitantly with the appearance of mature vessels. Treatment with CA4P and anecortave acetate resulted in significant reductions in total vessel density. However, neither drug reduced the amount of alpha-smooth muscle actin (SMA)-positive, mature vessels. CONCLUSIONS: Results of this study provide new insight into the relationship between tumor growth and blood vessel development in the LH(BETA)T(AG) mouse and establish the framework for defining the selective action of two vessel-targeting drugs against new blood vessels compared with mature blood vessels. These findings suggest a high potential value in targeting the process of angiogenesis in the treatment of children with retinoblastoma.
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Modelos Animales de Enfermedad , Neovascularización Patológica/patología , Neoplasias de la Retina/irrigación sanguínea , Retinoblastoma/irrigación sanguínea , Actinas/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Bibencilos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Endoglina , Endotelio Vascular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Pericitos/metabolismo , Pregnadienodioles/uso terapéutico , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/metabolismo , Retinoblastoma/patología , Estilbenos/uso terapéuticoRESUMEN
PURPOSE: To confirm juxtascleral delivery of anecortave acetate in rabbit eyes by ocular imaging techniques and to determine drug localization and distribution as a function of time after injection. METHODS: Four female New Zealand white rabbits (weight, 2.5-3.0 kg) received a single juxtascleral posterior sub-Tenon capsule injection of 0.5 mL or 1 mL of 30 mg/mL anecortave acetate. Rabbit eyes were imaged with ultrasonography and magnetic resonance imaging (MRI) before injection, immediately after injection, and at 2 hours, 1 week, and 4 weeks after injection. Rabbit eyes were also imaged with b-mode ultrasonography during the juxtascleral injections. RESULTS: Ultrasonography and MRI demonstrated that juxtascleral posterior sub-Tenon capsule injection of anecortave acetate effectively delivered the drug in direct apposition to the posterior pole of the rabbit eye. The drug remained in the juxtascleral site for at least 5 weeks. The drug was visualized clearly by MRI immediately after injection, decreasing in intensity thereafter. Cannula insertion and the drug delivery process were clearly visualized by real-time ultrasound analysis. Immediately after drug injection, ultrasonography indirectly localized anecortave acetate localization as an echolucent zone posterior to the scleral surface. At the later time points, however, the juxtascleral location of the drug was verified with ultrasonography as a relatively echogenic focus in the same location. CONCLUSIONS: Juxtascleral administration of anecortave acetate via a posterior sub-Tenon capsule approach effectively delivered the drug to the desired position in direct apposition to the globe posteriorly. MRI and ultrasonography both demonstrated that anecortave acetate remained localized to this location for at least 5 weeks after initial injection.
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Inhibidores de la Angiogénesis/administración & dosificación , Tejido Conectivo/diagnóstico por imagen , Sistemas de Liberación de Medicamentos , Pregnadienodioles/administración & dosificación , Esclerótica/efectos de los fármacos , Esclerótica/diagnóstico por imagen , Inhibidores de la Angiogénesis/farmacocinética , Animales , Tejido Conectivo/efectos de los fármacos , Femenino , Inyecciones , Imagen por Resonancia Magnética , Órbita/diagnóstico por imagen , Pregnadienodioles/farmacocinética , Conejos , Retina/diagnóstico por imagen , Distribución Tisular , UltrasonografíaRESUMEN
Retinoblastoma is a pediatric retinal tumor caused by mutational inactivation of the tumor suppressor pRb. Additional genetic changes, as yet unidentified, are believed to be required for tumor initiation. Mutations in the Wnt signaling pathway have been implicated in the pathogenesis of many cancers. Multiple Wnt pathway genes are expressed in the retina and the pRb and Wnt pathways interact biochemically, raising the possibility that alterations in the Wnt pathway contribute to retinoblastoma. Our studies showed that Wnt signaling activation significantly decreased the viability of retinoblastoma cell lines by inducing cell cycle arrest, which was associated with upregulated p53. Furthermore, immunolocalization of the Wnt signaling mediator beta-catenin in human and mouse retinoblastoma tissue indicated that canonical Wnt signaling is suppressed in tumors in vivo. These studies are consistent with the Wnt pathway acting as a tumor suppressor in retinoblastoma and suggest that loss of Wnt signaling is tumorigenic in the retina.
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Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Retinoblastoma/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Ciclo Celular , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Mutación , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , beta Catenina/metabolismoRESUMEN
PURPOSE: To evaluate the efficacy and dose-response of transcorneoscleral Coulomb controlled iontophoresis (CCI) of carboplatin in the treatment of retinal tumors of a murine model of retinoblastoma. METHODS: Thirty 6-week-old LHBETATAG mice underwent a total of six, serial iontophoretic treatments administered two times per week using a current density of 2.57 mA/cm2 for 5 minutes. Fourteen animals received carboplatin treatments at concentrations of 1.4, 7.0, 10.0, or 14.0 mg/mL without current. Ten control mice underwent treatment with balanced saline solution. RESULTS: A dose-dependent inhibition of intraocular tumor was observed after repetitive iontophoretic treatment. At carboplatin concentrations of 7 mg/mL, 50% of the treated eyes (4/8) exhibited tumor control. No corneal toxicity was observed in eyes treated at carboplatin concentrations under 10 mg/mL. CONCLUSIONS: CCI delivery of carboplatin safely and effectively controls intraocular tumors in a dose-dependent manner in this murine model of retinoblastoma. CCI is a noninvasive, painless option for the focal delivery of carboplatin. However, further clinical and laboratory research is needed before this method of drug delivery is available for children with retinoblastoma.
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Antineoplásicos/administración & dosificación , Carboplatino/administración & dosificación , Sistemas de Liberación de Medicamentos , Iontoforesis/métodos , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ratones , Ratones Transgénicos , Neoplasias de la Retina/patología , Retinoblastoma/patología , Resultado del TratamientoRESUMEN
PURPOSE: To evaluate the tumor control efficacy of the antiangiogenic agent anecortave acetate as single and combined therapy, in retinal tumor reduction using the LH(BETA)T(AG) mouse model of retinoblastoma. METHODS: Group A: Ten-week-old, LH(BETA)T(AG) mice received a single subconjunctival injection of anecortave acetate (1200, 600, 300, and 150 microg) delivered to right eyes only. Group B: Ten-week-old, LH(BETA)T(AG) mice received a single subconjunctival injection of anecortave acetate (600, 300, and 150 microg) delivered to right eyes only, either during a cycle of carboplatin (six subconjunctival deliveries) or after the completed cycle. Carboplatin was delivered at the subtherapeutic concentration of 62.5 microg. All animals were euthanatized at 16 weeks of age, and the eyes were examined histopathologically. RESULTS: A statistically significant reduction in tumor burden was detected after a single periocular injection of anecortave acetate. The reduction of tumor burden followed a U-shaped dose-response curve. Tumor burden was significantly decreased when anecortave acetate and carboplatin were combined. However, varying doses and delivery schedule of these agents had significant impact on the effectiveness of the combined treatment. The most effective scheme was delivering a low dose (150-300 microg) of anecortave acetate after a complete cycle of carboplatin. Histopathological evaluation showed no signs of retinal toxicity to anecortave acetate delivery alone or in combination with carboplatin. CONCLUSIONS: Anecortave acetate, as monotherapy or as adjuvant therapy, significantly controlled tumor burden in a murine model of retinoblastoma. Moreover, adjuvant therapy enabled the use of typically subtherapeutic carboplatin doses without decreasing efficacy of the therapy.
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Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Modelos Animales de Enfermedad , Pregnadienodioles/uso terapéutico , Neoplasias de la Retina/tratamiento farmacológico , Retinoblastoma/tratamiento farmacológico , Animales , Quimioterapia Adyuvante , Relación Dosis-Respuesta a Droga , Inyecciones , Ratones , Ratones TransgénicosRESUMEN
Employment of animal models in basic research has significantly advanced the understanding of fundamental processes underlying tumorigenesis in retinoblastoma, including elucidating the complex role of pRb and other related protein products in cell cycle regulation, apoptosis, DNA damage responses, and terminal differentiation. The evolution of therapy for retinoblastoma has also been facilitated through translational research using in vivo models, including xenograft and genetically engineered systems. In retinoblastoma, these models provide a valuable preclinical context for testing the efficacy and safety of conventional chemotherapy, radiotherapy, or novel agents on tumor growth, tissue invasion, and metastasis.
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Modelos Animales de Enfermedad , Neoplasias de la Retina/patología , Retinoblastoma/patología , Animales , Humanos , Neoplasias de la Retina/genética , Neoplasias de la Retina/terapia , Retinoblastoma/genética , Retinoblastoma/terapiaRESUMEN
PURPOSE: To evaluate the effect of subconjunctival injections of combretastatin A-4 phosphate (CA-4P) prodrug treatment on tumor vasculature and growth in an animal model of hereditary retinoblastoma. METHODS: Twenty-four, 12-week-old simian virus-40 T-antigen-positive mice received six subconjunctival CA-4P injections at doses of 0.5, 1.0, 1.5, and 2.0 mg delivered at 72-hour intervals to the right eye only. Six control animals received placebo treatment. All animals underwent serial ophthalmic evaluations and were euthanatized at 16 weeks of age, and eyes were obtained for histopathologic examination. Eyes were graded for presence or absence of tumor, delay of tumor growth, and intratumoral vascularity. RESULTS: The use of subconjunctivally injected CA-4P prodrug induced an extensive, dose-dependent decrease in microvessel density and led to significant tumor reduction in treated eyes compared with the placebo control (P <0.001). No evidence of corneal, lenticular, choroidal, or retinal toxicity was observed by histopathologic evaluation. CONCLUSIONS: Subconjunctival delivery of CA-4P is associated with extensive dose-dependent reduction in blood vessel count in this murine model of retinoblastoma. A combination treatment of retinoblastoma incorporating CA-4P may allow enhanced tumor reduction enabling a decrease in standard treatment doses of both chemotherapy and external beam radiotherapy.