Christina Decatur

Intravitreal and Subconjunctival Melphalan for Retinoblastoma in Transgenic Mice

Purpose. To measure the chemotherapeutic effects of focal melphalan (intravitreal and subconjunctival) on tumor burden, hypoxia, and vasculature in LHBETATAG murine retinoblastoma model. Methods. LHBETATAG transgenic mice were treated with a single 1 mcg intravitreal injection of melphalan, 100 mcg subconjunctival injection, or semiweekly 10 mcg subconjunctival injections for 3 weeks. At 1 or 3 weeks, eyes were enucleated, serially sectioned, and processed with haematoxylin and eosin (H&E) for tumor burden measurements and probed with immunofluorescence to analyze tumor hypoxia and vasculature. Results. Focal melphalan significantly reduced retinal tumor size (P < 0.02) when given intravitreally or subconjunctivally. Eyes treated with a one-time intravitreal injection of 1 mcg melphalan had significantly smaller tumors at both 1 week (P = 0.017) and at 3 weeks after injection (P = 0.005). Intratumoral hypoxia showed a significant decline in hypoxia at 1 week following intravitreal injection and after maximum dosage of subconjunctival melphalan. Total vasculature was not significantly affected following intravitreal administration. Conclusion. Focal delivery of melphalan via intravitreal or subconjunctival injection has a significant effect on reducing tumor burden, hypoxia, and vasculature, in the treatment of murine retinoblastoma tumors.

Retinoblastoma Treatment: Utilization of the Glycolytic Inhibitor, 2-deoxy-2-fluoro-D-glucose (2-FG), to Target the Chemoresistant Hypoxic Regions in LH BETA T AG Retinal Tumors

PURPOSE To analyze the effect of the glycolytic inhibitor 2-deoxy-2-fluoro-d-glucose (2-FG) on tumor burden, hypoxia, and blood vessels in LH(BETA)T(AG) retinal tumors. METHODS Seventeen-week-old LH(BETA)T(AG) retinal tumor eyes (n = 36) were treated with 2-FG and analyzed at 1 day and 1 week post a single treatment, and 1 day post a biweekly treatment for 3 weeks. Tumor sections were analyzed for hypoxia, tumor burden, and vasculature. To assess tumor burden, sections were processed for standard hematoxylin-eosin (H&E) staining. Immunofluorescent techniques were used to stain for new and mature blood vessels. RESULTS Hypoxia and tumor burden reduction are significantly different between the treatment schedules used (P < 0.001). Eyes treated with 2-FG for 3 weeks showed a significant decrease in hypoxia (P = 0.001) and tumor burden (P = 0.009); whereas those treated with one injection and evaluated at 1 day and 1 week postinjection did not show a decrease in either hypoxia (P = 0.373 and P = 0.782, respectively) or tumor burden (P = 0.203 and P = 0.836, respectively). When evaluating the spatial distribution of hypoxic regions in the different areas of the tumor, 2-FG showed a differential effect on hypoxia depending on the area. Hypoxia was most decreased in the base of the treated eyes with a 95% reduction (P < 0.001). CONCLUSIONS This is the first study to elucidate that 2-FG treatment in retinoblastoma produces an impact on hypoxia and a concomitant decrease on tumor burden. In this study, the authors validate their previous studies by revealing that glycolytic inhibitors effectively target hypoxia in retinoblastoma tumors. The future application of 2-FG as an adjuvant treatment to standard chemotherapy may enhance the treatment of retinoblastoma.

Abstract 4642: Combined glycolytic inhibition and anti-vascular treatment in retinoblastoma: Novel strategy for tumor control avoids chemotherapy

Purpose: The purpose of this study was to evaluate the combination treatment of glycolytic inhibitors and anti-angiogenic agents on tumor burden and hypoxia in advanced retinoblastoma using the LHbetaTag murine transgenic retinoblastoma model. Methods: Under IACUC protocol, thirty advanced LHbetaTag mice (16 weeks of age) were divided into 5 groups (n=6 per group) and treated with periocular injections of (a) saline, (b) 2-deoxy-glucose (2-DG), (c) anecortave acetate (AA) or (d) 2-DG plus AA (one day post AA treatment) or (e) 2-DG plus AA (one week post AA treatment). Eyes were enucleated at 21 weeks and tumor sections were analyzed for tumor burden and intra-tumoral hypoxia. Results: Combined treatment with 2-DG and AA (both 2-DG one day and one week post AA treatment) showed significant reduction in tumor burden compared to saline control (61% and 56% respectively, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4642. doi:1538-7445.AM2012-4642

Abstract 2359: Using the glycolytic inhibitor 2-fluorodeoxy-D-glucose, a novel glycolytic inhibitor approach to target the chemoresistant, hypoxic cell population in advanced LHBETATAG retinal tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: The aim of the current study is to assess the impact of 2-fluorodeoxy-D-glucose on tumor burden and hypoxia in the LHBETATAG retinal tumors. Methods: The study protocol was approved by the University of Miami Institutional Animal Care and Use Review Board Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 17-week-old (n=54) LHBETATAG transgenic mice were treated with 2-fluorodeoxy-D-glucose or saline control. These animals received three different treatments. They were treated: (1) with one injection and sacrificed at one day post-treatment, (2) with one injection and sacrificed at one week post-treatment, or (3) twice a week for three weeks and sacrificed at one day post-last injection. At the time of enucleation, all eye samples were snap frozen and analyzed for tumor burden and hypoxia using immunohistochemical techniques. Average densities of the different groups were statistically analyzed using ANOVA. Results were considered significant if p≤ 0.05. Results: There was no apparent toxicity associated with 2-fluorodeoxy-D-glucose treatment. There was a significant reduction in tumor burden following treatment with 2-fluorodeoxy-D-glucose at 1 day (86%) and 3 weeks (63%) post-treatment (p≤0.05). There was no reduction of tumor burden observed when mice were treated with 1 injection and eyes harvested at 1 week post-treament (2%, p=0.0640). There was a significant reduction of hypoxia areas following treatment with 2-fluorodeoxy-D-glucose at 1 day (100%) and 3 weeks (75%) post-treatment (p≤0.05). There was an increase in hypoxia of 12% following treatment at 1 week post-injection, but this increase was not statistically significant. Conclusions: 2-FDG significantly reduces tumor burden and tumor hypoxia following a single injection, with continued efficacy following repeated injections for 3 weeks. 2-FDG treatment is efficacious in murine retinoblastoma tumors and may enhance tumor control when combined with other therapies. 2-FDG appears to target hypoxic cells, a population that has been resistant to chemotherapy and radiation. Additionally, 2-FDG is commonly used in medical imaging and does not pose significant toxicities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2359. doi:1538-7445.AM2012-2359

Abstract 2332: The glucose analog 2-DG selectively inhibits AKT and ERK in endothelial cells via interference of N-linked glycosylation and induces endothelial cell apoptosis in vitro and in vivo.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BACKGROUND: As every step in tumor angiogenesis is an energy consuming process, targeting endothelial (EC) glucose metabolism is a rational strategy for angiogenesis inhibition. We recently showed that interference with endothelial glucose metabolism by the glucose analog 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis in vitro and in vivo, at concentrations lower than those required to induce tumor cell cytotoxicity. The objectives of the current study are to further understand the basis for EC sensitivity to 2-DG in vitro and in vivo. METHODS: Time dependent effects of 2-DG were studied on EC tube formation at 4, 8 and 18 hrs. Apoptosis was assessed by the TUNEL assay at 24, 48 and 72 hours. The effects of low antiangiogenic concentrations (0.6 mM) of 2-DG on ERK, AKT and mTOR pathways were assessed in endothelial (HUVEC) and cancer (MDA-MB-231, HT-29, 786-0) cells by western blot. The in vivo effects of intraocular 2-DG administration on newly forming (CD-105) and total (lectin) tumor vessels were evaluated in the LHBETATAG retinoblastoma model. In vivo EC apoptosis after 2-DG treatment was assessed by TUNEL/CD31 staining of treated tumors. RESULTS: Inhibition of HUVEC capillary formation by 2-DG occurred as early as 8 hours after treatment, and the effects were clear at 18 hours compared to controls. EC apoptosis was non-significantly increased at 24 hours, and was prominent at 48 and 72 hours. Antiangiogenic 2-DG concentrations (0.6 mM) significantly inhibited phosphorylation of Akt473, S6 and Erk at 24 hours. On the other hand, 2-DG did not affect these pathways in MDA-MB-231 or HT-29 cells, while they occurred at a lower level in 786-0 cells. Mannose successfully reversed AKT, S6 and ERK inhibition only in HUVECs, but not in 786-0 cells, suggesting that the EC selective inhibition of ERK and AKT are mediated by inhibition of N-linked glycosylation, while other mechanisms mediate the effects in cancer cells. Importantly, these effects were 2-DG specific, as other glucose analogs (FDG) and the glycolytic inhibitor oxamate did not significantly affect ERK or AKT. Intraocular 2-DG administration decreased tumor microvasculature, and importantly, induced EC apoptosis, as demonstrated by TUNEL+/CD 31 co-localization. CONCLUSIONS: Our data indicate that in angiogenic ECs, 2-DGs inhibition of N-linked glycosylation targets critical proliferation and survival pathways. 2-DG mediated Inhibition of AKT and ERK pathways may explain the significant endothelial sensitivity cytotoxic and pro-apoptotic effects of 2-DG in vitro and in vivo, and further support targeting EC glucose metabolism as a viable strategy for angiogenesis inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2332. doi:1538-7445.AM2012-2332

Abstract 5352: Novel treatment approaches in retinoblastoma: Impact of combination therapy on tumor burden

Purpose: The purpose of the current study is to examine vessel targeting, chemotherapy, and mammalian target of rapamycin (mTOR) inhibitor agents in LHBETATAG retinal tumors and their impact on tumor burden. Methods: Group A: Ten-week-old, LHBETATAG mice (n=30) received a single subconjunctival injection of anecortave acetate (AA; 1200, 600, 300, and 150 µg) delivered to right eyes only. Group B: Ten-week-old, LHBETATAG mice (n=30) received a single subconjunctival injection of AA (600, 300, and 150 µg) 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 µg. All animals were euthanatized at 16 weeks of age, and the eyes were examined histopathologically. Group C: Eighteen-week-old, LHBETATAG mice received (n=30) subconjunctival injections of rapamycin once weekly for two consecutive weeks (0.00333, 0.167, 3.33, and 6.67 mg/kg). Tumor sections were analyzed for tumor burden with immunohistochemistry techniques. Results: A statistically significant reduction in tumor burden was detected after a single periocular injection of AA. The reduction of tumor burden followed a U-shaped dose-response curve. Tumor burden was significantly decreased when AA 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 µg) of AA after a complete cycle of carboplatin. Reduction in tumor burden were significantly different between rapamycin doses and control (p Conclusions: AA, 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. Inhibition of mTOR reduced tumor burden during late disease in the LHBETATAG retinoblastoma tumor model. Rapamycin may have a role in combination with chemotherapy or other adjuvant therapies to enhance retinoblastoma tumor control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2011-5352

Abstract 1517: Retinoblastoma tumor development: Role of tumor-associated macrophages and their sub-type in LHBETATAG retinal tumor progression

Purpose: The purpose of the current study is to establish the distribution of tumor associated macrophages (TAMs) during tumor development in a transgenic retinoblastoma mouse model, study the contribution of bone-marrow derived macrophages in these tumors, and assess the supportive role of TAMs in retinoblastoma tumor growth. Methods: Macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. To establish the origin of TAMs in transgenic retinoblastoma tumors, 107 bone marrow cells from green fluorescent protein (GFP) positive 16-week-old mice were transplanted into age-matched, irradiated LHBETATAG mice via tail vein injections. Macrophage depletion was performed by subconjuctival (SC) delivery of liposomal clodronate. Results: TAMs’ density increased from 4- to 12-weeks of age in mice with small to medium-sized tumors (p=0.037), and remained stable in the later stages of the disease (i.e., 16-week-old with large tumors; p=0.20). 38% of TAMs (2.5 ± 3.2 cells per 400X hpf) in 16-week old mice were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was correlated with a significant decrease in the expression levels of MMP-9 (p=0.014) and mature vessels (p Conclusions: The current study expands our understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1517. doi:10.1158/1538-7445.AM2011-1517

Abstract 3865: Regional and temporal variations in gene expression and vasculature during retinoblastoma tumorigenesis and its impact on ocular treatment

Purpose: The aims of this study were to evaluate the hypothesis that LHBETATAG retinoblastoma tumors exhibit regional and temporal variations in gene expression and vascular development in retinoblastoma. Methods: To evaluate intratumoral gene expression, LHBETATAG mice aged 12, 16, and 20 weeks were euthanized (n=9). Specimens were taken from 5 tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized with an Affymetrix GeneChip Mouse Gene ST 1.0 arrays. To examine the spatial distribution of new and mature blood vessels, immunohistochemical analyses were conducted on enucleated human (n=10) and LHBETATAG retinal tumors from 16-week-old mice (n=11). Results: There were significant temporal differences in gene expression for LHBETATAG retinoblastoma tumors (p 2.5 there were significant changes in gene expression for 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes were analyzed with significant involvement in angiogenesis (p Conclusions: There are significant temporal and regional variations in the gene expression and vasculature of retinoblastoma. Differentially expressed genes overlap with angiogenesis, hypoxia, and cellular metabolism. There is a heterogeneous vessel population in advanced retinoblastoma disease. These findings provide further understanding of the mechanisms involved in tumor progression, and emphasize that tumor biopsies cannot be used for prognostication secondary to tumor regionalization. Finally, optimally-timed adjuvant therapies that target critical components of identified pathways may potentially enhance retinoblastoma tumor control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3865. doi:10.1158/1538-7445.AM2011-3865

Abstract 2050: 2-Deoxy-D-Glucose (2-DG) as a glycolytic inhibitor in the treatment of retinoblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: The aim of the current study is to assess the impact of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on tumor burden and hypoxia in the LHBETATAG retinal tumors. Methods: Group A: 17-week-old LHBETATAG transgenic mice (n=30) received periocular injections of saline and 2-DG (62.5, 125, 250, and 500mg/kg). Injections were given 2 times a week for 3 weeks. Group B: 4-week-old mice received oral delivery of 2-DG in custom made food pellets and were treated for either 8 or 18 weeks. 17-week-old mice received oral 2-DG for 8 weeks. At the time of enucleation, all eye samples were snap frozen and analyzed for tumor burden and hypoxic regions using immunohistochemistry. Results: Following injections of 2-DG, tumor control was not different between the control and the lowest two doses (62.5 and 125mg/kg). However, the difference in tumor burden was significant from 250mg/kg dose (p<0.015) and 500mg/kg dose (p<0.001) with 9.7 and 23% tumor burden decrease, respectively. At all doses of periocular 2-DG, the percent hypoxia following drug treatment were lower relative to controls (p<0.001), with a hypoxia decrease in the lowest dose to 2.4% compared to 21% in controls. Following oral delivery of 2-DG, there was a difference between the groups treated with 2-DG and the control groups (p<0.010). A 20% reduction in tumor burden was found in the 8 weeks early treated, a 50% reduction in the 18 weeks early treated, and a 35% reduction in the 8 weeks late treated group. The percent hypoxia was lower relative to controls in the 8 weeks and 18 weeks early treated groups (p<0.001) with a 40% and 50% reduction, respectively. The percent hypoxia did not change following treatment in the treated late group. Conclusions: This study exhibits the efficacy of focal 2-DG as potential therapy to decrease both intratumoral hypoxia and tumor burden. The current study also shows that the non-invasive oral delivery 2-DG treatment has similar or better effects on the tumor depending on the treatment schedule used. In advanced disease of LHBETATAG retinal tumors, hypoxia is increasingly present. The use of glycolytic inhibitors as a therapeutic strategy has the potential to enhance current retinoblastoma treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2011-2050