Maria Elena Jockovich

Targeting Hypoxia, a Novel Treatment for Advanced Retinoblastoma

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.

Retinal tumor imaging and volume quantification in mouse model using spectral-domain optical coherence tomography

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.

Blood vessel maturation in retinoblastoma tumors: Spatial distribution of neovessels and mature vessels and its impact on ocular treatment

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.

Heterogeneous tumor vasculature in retinoblastoma: Implications for vessel targeting therapy

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 α smooth muscle actin (α-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.

Blood vessel maturation in human uveal melanoma: spatial distribution of neovessels and mature vasculature.

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.

Impact of Tumor-Associated Macrophages in LHBETATAG Mice on Retinal Tumor Progression: Relation to Macrophage Subtype

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.

Posterior juxtascleral injection of anecortave acetate: Magnetic resonance and echographic imaging and localization in rabbit eyes

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.

Lack of Effect of SU1498, an Inhibitor of Vascular Endothelial Growth Factor Receptor-2, in a Transgenic Murine Model of Retinoblastoma

SU1498, a tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2), has activity against retinal neovascular diseases. To determine if this drug might have clinical utility against retinoblastoma, we evaluated the effects of SU1498, as well as the expression of VEGFR-2, in a transgenic animal model of retinoblastoma. Optical coherence tomography (OCT) was evaluated as a technology to measure retinal tumors in vivo, in response to treatment. Immunofluorescence analysis was performed to evaluate the distribution and expression of VEGFR-2 in enucleated eyes from LHβTag transgenic mice and controls at 4, 8, 12, and 16 weeks of age. VEGFR-2 and phosphorylated (p)VEGFR-2 levels were quantitated by Western blot. OCT was used to pair 10-week-old animals based on tumor volume (n=10), and these animals were treated with 6 periocular injections of SU1498 (50mg/kg, given twice weekly) or vehicle for 3 weeks. Tumor burden was determined by histology and in vivo imaging by OCT. VEGFR-2 and pVEGFR-2 expression levels were upregulated during tumorigenesis. However, SU1498 did not significantly reduce tumor burden compared to vehicle (p=0.29). OCT imaging of one matched pair demonstrated equivalent, linear tumor growth despite treatment with SU1498. Retinal tumors can be followed non-invasively and quantitatively measured with OCT. VEGFR-2 is strongly upregulated during tumorigenesis in transgenic retinoblastoma; however, SU1498 does not decrease tumor volume in transgenic murine RB at the studied dose and route of administration.

Quantitative evaluation of retinal tumor volume in mouse model of retinoblastoma by using ultra high-resolution optical coherence tomography

An ultra high resolution spectral-domain optical coherence tomography (SD-OCT) together with an advanced animal restraint and positioning system was built for noninvasive non-contact in vivo three-dimensional imaging of rodent models of ocular diseases. The animal positioning system allowed the operator to rapidly locate and switch the areas of interest on the retina. This function together with the capability of precise spatial registration provided by the generated OCT fundus image allows the system to locate and compare the same lesion (retinal tumor in the current study) at different time point throughout the entire course of the disease progression. An algorithm for fully automatic segmentation of the tumor boundaries and calculation of tumor volume was developed. The system and algorithm were successfully applied to monitoring retinal tumor growth quantitatively over time in the LHBETATAG mouse model of retinoblastoma.

Basic fibroblast growth factor impact on retinoblastoma progression and survival.

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.