June 2007
Volume 48, Issue 6
Free
Anatomy and Pathology/Oncology  |   June 2007
Retinoblastoma Tumor Vessel Maturation Impacts Efficacy of Vessel Targeting in the LHBETATAG Mouse Model
Author Affiliations
  • Maria-Elena Jockovich
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • M. Livia Bajenaru
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • Yolanda Piña
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • Fernando Suarez
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • William Feuer
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • M. Elizabeth Fini
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
  • Timothy G. Murray
    From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida.
Investigative Ophthalmology & Visual Science June 2007, Vol.48, 2476-2482. doi:10.1167/iovs.06-1397
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      Maria-Elena Jockovich, M. Livia Bajenaru, Yolanda Piña, Fernando Suarez, William Feuer, M. Elizabeth Fini, Timothy G. Murray; Retinoblastoma Tumor Vessel Maturation Impacts Efficacy of Vessel Targeting in the LHBETATAG Mouse Model. Invest. Ophthalmol. Vis. Sci. 2007;48(6):2476-2482. doi: 10.1167/iovs.06-1397.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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 LHBETATAG mouse model for retinoblastoma.

methods. LHBETATAG 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 α-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 LHBETATAG 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.

Retinoblastoma, at an incidence of 1 in 15,000 live births, is one of the most common malignant tumors of childhood. 1 2 Significant advances in screening and treatment during the past century have led to cure of this primary eye cancer in virtually all children. Recently, clinical advances have focused on increasing tumor control and globe conservation with their attendant preservation of sight. Available treatments include laser therapy, cryotherapy, external beam radiotherapy, charged-particle radiation, and systemic chemotherapy. Nevertheless, serious concerns exist regarding the significant morbidity and the potential mortality associated with these therapies; therefore, new therapeutic modalities are under investigation. 
To survive, growing tumors require the formation of new blood vessels from preexisting ones. 3 This process, called angiogenesis, is complex and highly dynamic and involves a series of events and a multitude of regulatory factors, many of which can be targets for therapy. The formation of new blood vessels may thus be inhibited by the use of antiangiogenic agents. Newly formed vessels may also be targeted by the use of angiostatic agents that functionally disrupt blood flow through immature vasculature. 
Retinoblastoma tumors are highly vascularized and depend on the vascular supply for viability. 4 The capacity of these tumors to promote angiogenesis has been demonstrated. 5 6 Angiogenic factors such as vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and KDR, have been localized to areas of novel vasculature in human retinoblastoma tumors. 7 8 Blood vessel density, as measured by endothelial cell markers, in these tumors correlates with invasive growth and metastasis and is associated with poor prognosis. 9 10 11 Increased blood vessel density and propensity for angiogenesis stimulation in retinoblastoma may make these tumors sensitive to vascular targeting agents. 
Our research group and others 12 13 14 15 have actively studied the efficacy of blood vessel-targeting therapy for retinoblastoma with the LHBETATAG mouse model, which has been used extensively to test therapeutic strategies for retinoblastoma treatment. The response to radiation and cryotherapy treatment in the murine LHBETATAG retinoblastoma model closely correlates with the response observed in human retinoblastoma. 16 17  
Although studies of treatment modalities have been performed using the LHBETATAG mouse model, this model has not been characterized with respect to tumor and blood vessel development. A description of blood vessel development in this model system will provide a point of reference for vessel-targeting therapy and may give insight into possible therapeutic targets for this malignancy. The aim of this study was to characterize and correlate cell proliferation, tumor growth, blood vessel development, and maturation during tumor progression in the LHBETATAG mouse model for retinoblastoma and to determine how these factors affect vessel-targeting therapy. 
Methods
Animals
The study protocol was approved by the University of Miami School of Medicine Animal Care and Use Review Board. All experiments in this study were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
The LHBETATAG transgenic mouse model used in this study has been characterized previously. 18 SV40Tag was detected by PCR analysis of tail biopsy specimens. Littermates negative for SV40Tag served as negative controls. 
Subconjunctival Injections
Each 16-week-old LHBETATAG mouse received a single subconjunctival injection of anecortave acetate (300 μg; Alcon Pharmaceuticals, Forth Worth, TX) or CA4P (2 mg; Oxigene Inc., Cambridge, MA) in the right eye. Injections, 20-μL volume, were delivered with a 33-gauge needle inserted into the superotemporal subconjunctival space. Eyes were analyzed 1 day and 1 week after injection. 
Tumor Size Measurements
Twenty-four LHBETATAG animals (48 eyes) were divided into four groups based on age. Both eyes of each mouse were enucleated and immediately fixed in 10% formalin by immersion. Then they were embedded in paraffin, sectioned serially, and processed for standard hematoxylin-eosin (H&E) staining. Microscopic images of H&E-stained sections (sixty 5-μm sections per eye) were obtained with a digital camera at a magnification of 40×. The section of the eye containing the largest tumor area was chosen, and tumor boundaries were traced (Image Pro Express Software; Media Cybernetics, Silver Spring, MD). Tumor areas for all eyes were averaged, yielding an average area for each time point. 
Immunohistochemistry
Mice were killed with CO2 fumes. Eyes were enucleated, snap frozen in optimum cutting temperature (OCT) compound, and serially sectioned (8 μm). Twenty animals (40 eyes) were divided into four groups based on age. Slides were fixed with 4% paraformaldehyde (room temperature [RT]) or methanol (–20°C). Proliferative cells were labeled with the polyclonal antibody Ki67-proliferation marker (Abcam, Cambridge, MA) and detected by tyramide signal amplification–enhanced immunohistochemistry with the use of a detection kit (TSA-Plus Cyanine 3; Perkin Elmer Life and Analytical Sciences, Waltham, MA), as described previously. 19 Total vascular endothelial cells were detected with the Alexa Fluor 568–conjugated lectin (Bandeira simplicifolia; 1:1000; Invitrogen, Carlsbad, CA). Vascular endothelial cells of newly formed angiogenic vessels were detected by immunofluorescence with a rat monoclonal anti–mouse endoglin (CD105) antibody (1:250; Santa Cruz Biotechnology, Santa Cruz, CA) and a secondary Alexa Fluor 488–conjugated antibody (1:500; Invitrogen). Mature blood vessels associate with pericytes, and α-smooth muscle actin (α-SMA) is a marker for pericytes. Mature blood vessels were identified by α-sma-Cy3 conjugate (1:10,000; Sigma Chemical, St. Louis, MO). Cell nuclei were stained for 5 minutes with 4,6-diamidino-2-phenylindole-2-HCl (DAPI, 1:5000; Invitrogen). 
Serial cross-sections of eyes containing tumors were examined for the presence of the markers described with an upright fluorescence microscope (BX51; Olympus America Inc., Melville, NY). All images were digitally acquired and recompiled (Photoshop CS; Adobe, San Jose, CA). Sections were viewed at 20× magnification. 
Evaluation of Immunostaining and Vessel Quantification
Analyses were performed on digitized fluorescence microscopic images (200×; Photoshop CS; Adobe). Immunostaining intensities were calculated as the average from at least four sections of at least five tumors. Vessel areas, calculated in pixels, were selected and measured as a fraction of tumor area or inner nuclear layer (INL) area. For controls, or images without tumors, vessels inside the INL available section were measured. For tumors smaller than the magnification field, only vessel areas lying within the tumor were measured. For tumors larger than the magnification field, all vessels inside the available field were chosen. In larger tumors, vessel densities were calculated for various random sections and then averaged. 
The Ki67 proliferation index was determined by analysis of digitized fluorescence microscopic images (200×; Photoshop CS; Adobe). Ki67–cyanine 3-labeled nuclei were selected and measured as a fraction of the total DAPI-labeled nuclei in the INL area of the retina. For each age group, the proliferation index was calculated and represented the mean of Ki-67 proliferation index calculated for at least three mice of the same age. 
Statistical Methods
To find the best predictive model of tumor growth in animals between 8 and 16 weeks of age, stepwise multiple regression was performed on data from the right eyes of all animals. Log tumor area was the dependent variable, and age, globe size, and log globe size were independent variables. To assess the variability between both eyes of each animal, a mixed model was fitted with log tumor area as the dependent variable and age as a covariate; variance components for between-animal and between-eye (residual) variation were calculated. The intraclass correlation coefficient was used to summarize variability between eyes as a fraction of total variability. 
Tumor angiogenesis activity at 4 weeks of age was compared between wild-type and LHBETATAG mice with a test of vessel type–animal type interaction. Similarly, among LHBETATAG mice, a test of development week–vessel type interaction was used to check for differences in angiogenesis activity in animals from 8 to 16 weeks of age. Both analyses were performed with mixed-model analysis of variance because some, but not all, animals contributed endoglin and lectin measurements. 
Statistical significance of the increase in density of α-SMA staining over time was assessed by linear regression analysis. Two-sided, two-sample t-tests were used to compare lectin levels and α-SMA–positive cells between controls and animals receiving vascular targeting treatment. 
Results
Tumor Growth
Retinal tumor size in LHBETATAG mice was measured over time. Mean tumor area (in pixels) was 0 (±0) for 4-week-old mice, 8680.4 (±8111.6) for 8 week-old mice, 63,674.3 (±72,067.7) for 12-week-old mice, and 289,814.7 (± 251,329.6) for 16-week-old mice (Fig. 1A) . Because the mean tumor area increased exponentially, log transformation was performed. Box-Cox analysis also suggested this as a suitable transformation for effecting equality of variance. Stepwise multiple regressions of data from the right eyes demonstrated that animal age accounted for 66.4% (r 2) of the variability in log tumor sizes. No further statistical advantage was obtained by adjusting for globe size (P = 0.14) or log globe size (P = 0.12). When data from all eyes were included in a mixed model, age was a significant predictor of log tumor size (P < 0.001), with slope (SE) 0.19/wk (0.033); the estimated 8-week intercept was 3.74. The between-animal variance component was 0.159, and the between-eye (residual) variance component was 0.100, yielding an intraclass correlation coefficient of 0.61. This indicated that 61% of the total variability resulted from differences between animals and that 39% of the variability resulted from differences between eyes of the same animal. 
Tumor Cell Proliferation
Ki67 proliferation index is an important diagnostic standard to assess cell proliferation in tumors. 20 The Ki67 antigen is the prototypic cell cycle–related nuclear protein and is expressed in proliferating cells in all phases of the active cell cycle. The Ki67 antibody is useful in establishing the cell growing fraction in neoplasms. We performed Ki67 immunofluorescence localization analysis in eye sections of LHBETATAG mice 4 to 16 weeks of age and calculated the Ki67 proliferation index (the number of Ki67-positive cells among the number of the resting cells in the tissue area). Ki67-positive proliferative cells were immunodetected in the retinal INL of LHBETATAG-positive mice as early as 4 weeks of age, when no histopathologically identifiable tumors had developed in the INL. These results indicate that cell proliferation precedes any detectable neoplastic morphologic change in the INL cells. Proliferating cells formed characteristic rosettes early in tumor development, at 8 weeks of age. Tumor cell proliferation reached a plateau at 12 weeks of age (Fig. 1B) . Ki67-labeled cells were absent from control retinas of any age group (not shown). The Ki67 proliferation indices of the mouse tumors mirror those observed in human tumors, in which there is a strong correlation between the proliferation index and the rate of tumor growth. 10  
Tumor Angiogenesis Activity
Angiogenesis in developing retinal tumors was assessed by endoglin staining. Endoglin (CD105) is a marker of newly formed blood vessels and is up-regulated in endothelial cells during angiogenesis. 21 To determine total vessel density, we stained with a pan-endothelial cell marker, the lectin derived from B. simplicifolia, which binds specifically to vascular endothelial cells. 22 Endoglin expressing newly formed blood vessels was detected in preneoplastic stages during tumor development in LHBETATAG–positive mice. At 4 weeks of age, LHBETATAG–positive mice had significantly higher numbers of endoglin-stained vessels than wild-type controls (P = 0.029). Total vessel densities in the INL of 4-week-old LHBETATAG mice seemed slightly higher than in wild-type littermates, but they were not significantly different (P = 0.86; Fig. 2A ). In LHBETATAG retinal tumors, the ratio of vessels undergoing angiogenesis to the total number of vessels changed over time (P = 0.005). In larger tumors, hot spots of endoglin-stained, newly formed blood vessels were found primarily in the vicinity of tumor edges. On average, new vessel density decreased in large tumors harbored by 16-week-old mice (Fig. 2B)
Tumor Vessel Maturation
Endothelial cells form the inner lining of the blood vessel wall, whereas perivascular cells, referred as pericytes, vascular smooth muscle cells, and mural cells, envelop the surface of the vascular tube. VEGF signaling initiates the formation of new vessels by recruiting endothelial cells to form tubes. VEGF also triggers a chain of molecular and cellular events that stabilize the blood vessels by recruiting pericytes and by generating an extracellular matrix (ECM), leading to blood vessel maturation. Mature tumor vessels, though stable, are in a constant state of remodeling. 23 24  
We measured tumor vessel maturation by association with pericytes. Pericyte presence was assessed by α-SMA staining. 25 Mature vessels were not detected in the INL of 4-week-old LHBETATAG mice or in small tumors harbored by 8-week-old mice. Pericyte recruitment by tumor vessels was initially detected at 12 weeks of age. Large tumors harbored by 16-week-old mice contained high-caliber vessels with clear lumen and were ensheathed with α-SMA–positive pericytes. Pericyte recruitment by vessels occurred in random hot spots throughout the tumor (Fig. 3) . Linear regression analysis demonstrated a highly significant increase in the density of α-SMA from 8 through 12 to 16 weeks (P < 0.001). No significant difference in total vessel density, as measured by lectin staining, or in angiogenesis, as determined by endoglin immunohistochemistry, was detected in retinas of wild-type controls from different age groups (not shown). 
Efficacy of Vessel-Targeting Agents
To assess the role of vessel maturation in the efficacy of vascular-targeting treatment, 16-week-old LHBETATAG mice were treated with either anecortave acetate or CA4P. Tumor vessels were analyzed 1 day and 1 week after injection. Results revealed a statistically significant decrease in total vessel density after treatment with these agents (Fig. 4A) . Total vessel density significantly decreased (P < 0.001; two-sample t-test) with either drug as early as 1 day after injection. This significant decrease persisted 1 week after injection. A statistically significant decrease in α-SMA cells was not detected after treatment with vessel-targeting agents (0.39; two-sample t-test) (Fig. 4B) . These results suggest that, though the total number of vessels was reduced after treatment with these vascular-targeting agents, the total number of mature vessels was not significantly affected. 
Discussion
The LHBETATAG mouse model has been used to test the efficacy of vascular targeting as treatment for retinoblastoma. Subconjunctival injections of CA4P, a tubulin-binding agent that disrupts blood flow through immature vasculature, effectively reduced retinal tumor burden in these mice. 13 Focal treatment with anecortave acetate, an antiangiogenic agent, reduced tumor burden as monotherapy and effectively controlled tumor burden when used in combination with carboplatin. 15 Antiangiogenic therapy has been proposed as a valuable therapeutic strategy for solid tumors, including pediatric malignancies. 26 27 This type of therapy is a promising option given that retinoblastoma tumors promote angiogenesis and are highly dependent on their vascular supply. However, data from studies using the LHBETATAG mouse model suggest that dose and delivery scheduling of anecortave acetate are essential for optimal tumor response. 15 In the present study, we have characterized cell proliferation, tumor growth, angiogenesis, and blood vessel maturation in the LHBETATAG mouse model. This careful characterization of retinoblastoma tumor growth and vasculature in the LHBETATAG mouse model is essential to achieve an understanding of tumor vascular requirements of this disease in children and to optimize vascular targeting therapeutic schemes. 
The present study suggests that angiogenesis precedes neoplastic transformation in the LHBETATAG retinoblastoma mouse model. In this regard, it is possible that novel vessel formation potentiates neoplastic changes in hyperproliferating Ki67-positive cells. Novel vessel formation is first activated by an “angiogenic switch” during tumor growth. In other murine cancer models, novel vessel formation is detected in preneoplastic stages of tumor development. 28 29 In these models, neither expression of the oncogene nor hyperproliferation of transformed cells appeared to be sufficient to activate the angiogenic switch. This switch seems to be a separate step in the pathway to malignancy and occurs in only a fraction of preneoplastic malignancies. Only malignancies that have switched to an angiogenic phenotype transform into solid tumors. 30 An understanding of the precise timing of the angiogenic switch can lead to better antiangiogenic therapeutic strategies. However, the timing of this molecular trigger may be difficult to assess in pediatric tumors because tissue displaying the different stages of tumor development is not readily available. Enucleation is only performed in children with advanced disease. At this stage, tumors are already highly angiogenic, and the stages of tumor vascular development, including the angiogenic switch, has been missed. The LHBETATAG mouse model offers a window to the development and vascular requirements of these intraocular tumors. 
We have shown that in LHBETATAG mice, tumor vessel maturation does not occur until disease reaches an advanced stage, when mice are 12 to 16 weeks of age. Interestingly, tumor specimens obtained from patients with advanced retinoblastoma (Reese-Ellsworth stage 5b) have a high percentage of α-SMA–positive mature vessels (M-EJ and TGM, unpublished data, 2005). It has been reported that angiogenic potential in retinoblastoma tumors correlates with invasive growth and metastasis and is associated with poor prognosis. 9 10 11 Nevertheless, in these studies, the number of vessels was measured by specifically labeling only the endothelial cells of the blood vessels. Pericytes, associated with mature blood vessels, have gained new attention as functional and critical contributors to tumor angiogenesis and, therefore, as potential new targets for therapy. 23 24 It has been proposed that in addition to endothelial vessel density, the percentage of mature vessels is important in the progression to invasive and metastatic disease. Similarly, the percentage of mature vessels in tumors may also determine the efficacy of a particular vessel-targeting agent. 
Endothelial cells in newly formed vessels require growth factors for survival; in their absence the endothelial cells undergo apoptosis and regression. 31 Mature vessels are stabilized by pericytes and no longer depend on angiogenic stimuli; thus, they may be resistant to antiangiogenic treatment. This suggests that vessel heterogeneity, in particular the number of mature vessels, found in tumors may limit the efficacy of vessel-targeting therapy, as has been reported. 32 33 Our previous studies to test the efficacy of anecortave acetate or combretastatin A4 were performed in younger LHBETATAG mice, 10 to 12 weeks of age. 13 15 At this age, according to the present study, tumors are still undergoing angiogenesis, and vessel maturation has not begun. In this study, we present data suggesting that antiangiogenic therapy has limited efficacy in targeting the vasculature of 16-week-old LHBETATAG mice. At this stage in tumor development, retinal tumors have established vasculatures with associated pericytes. Although a statistically significant reduction in overall vessel density is achieved, levels of mature vasculature are not significantly reduced in 16-week-old LHBETATAG mice. Similar results were observed when CA4P was used in a mouse cutaneous melanoma model. Data from this report suggest that CA4P selectively induced the regression of unstable tumor neovessels but did not target the mature α-SMA–positive vessels. 34 However, this same study showed that chronic treatment with CA4P led to a reduction of α-SMA–positive vasculature, possibly by inducing CA4P neovessel regression during vessel remodeling. It is then possible that multiple injections with CA4P will lead to targeting of mature vessels in LHBETATAG retinal tumors and possibly in children with retinoblastoma. 
In summary, the findings in this report illustrate the usefulness of mouse modeling to study the pathogenesis of retinoblastoma tumors. With an improved understanding of the events leading to tumor vessel formation and tumor development, targeted therapies can be designed. The degree of vessel maturation in retinoblastoma tumors may have profound implications in the selection of the vessel-targeting schemes for the treatment of this disease. If the disease in children follows what is seen in the LHBETATAG mice, then small tumors would require a different set of vessel-targeting agents than more advanced tumors. For example, small tumors could be treated with an antiangiogenic agent such as anecortave acetate; advanced tumors may require adjuvant treatment-targeting pericytes, as documented in other malignancies. 35 36  
 
Figure 1.
 
LHBETATAG retinal tumor development as a function of age. (A) Retinal tumors areas are given in pixels. Tumor areas measured 0 (±0) for 4-week-old mice, 8680.4 (±8111.6) for 8-week-old mice, 63,674.3 (±72,067.7) for 12-week-old mice, and 289,814.7 (±251,329.6) for 16-week-old mice. Inset: linear regression of log-transformed tumor areas onto animal age, accounting for 66.4% (r 2) of the variability in log tumor sizes. (B) Tumors develop from proliferating cells in the INL. Sections were stained with Ki67 (red) to label proliferating cells and DAPI (blue) to stain nuclei.
Figure 1.
 
LHBETATAG retinal tumor development as a function of age. (A) Retinal tumors areas are given in pixels. Tumor areas measured 0 (±0) for 4-week-old mice, 8680.4 (±8111.6) for 8-week-old mice, 63,674.3 (±72,067.7) for 12-week-old mice, and 289,814.7 (±251,329.6) for 16-week-old mice. Inset: linear regression of log-transformed tumor areas onto animal age, accounting for 66.4% (r 2) of the variability in log tumor sizes. (B) Tumors develop from proliferating cells in the INL. Sections were stained with Ki67 (red) to label proliferating cells and DAPI (blue) to stain nuclei.
Figure 2.
 
Angiogenesis in LHBETATAG retinal tumors. (A) At 4 weeks of age, LHBETATAG-positive mice have significantly higher concentrations of endoglin-stained vessels in the INL than wild-type controls (P = 0.042). Total vessel densities in this area are not significantly different between LHBETATAG mice and wild-type littermates (P = 0.86). (B) In LHBETATAG retinal tumors, the total number of vessels and the amount of vessels undergoing angiogenesis changed over time (P = 0.039). Endothelial cells were labeled with lectin (red) and endoglin (CD105, green). Graph represents vessels densities given as fraction of stained vessels to total area.
Figure 2.
 
Angiogenesis in LHBETATAG retinal tumors. (A) At 4 weeks of age, LHBETATAG-positive mice have significantly higher concentrations of endoglin-stained vessels in the INL than wild-type controls (P = 0.042). Total vessel densities in this area are not significantly different between LHBETATAG mice and wild-type littermates (P = 0.86). (B) In LHBETATAG retinal tumors, the total number of vessels and the amount of vessels undergoing angiogenesis changed over time (P = 0.039). Endothelial cells were labeled with lectin (red) and endoglin (CD105, green). Graph represents vessels densities given as fraction of stained vessels to total area.
Figure 3.
 
Vessel maturation in LHBETATAG retinal tumors. Linear regression demonstrated a highly significant increase in the density of SMA from 8 through 12 to 16 weeks (P < 0.001). Sections were stained with α-SMA (red). Graph represents vessels densities given as fraction of stained vessels to total area in the high power field, 200×. Controls from all age groups had the same vessels densities. Representative 16-week-control is shown.
Figure 3.
 
Vessel maturation in LHBETATAG retinal tumors. Linear regression demonstrated a highly significant increase in the density of SMA from 8 through 12 to 16 weeks (P < 0.001). Sections were stained with α-SMA (red). Graph represents vessels densities given as fraction of stained vessels to total area in the high power field, 200×. Controls from all age groups had the same vessels densities. Representative 16-week-control is shown.
Figure 4.
 
Vessel maturation limits vessel-targeting therapy in LHBETATAG retinal tumors. (A) Sixteen-week-old LHBETATAG mice were treated with single subconjunctival injections of anecortave acetate (AA; 300 μg) or combretastatin A4 (CA4P; 2 mg). Tumor vessels were analyzed 1 day and 1 week after injection. Sections were stained with lectin to detect total vessels and α-SMA to detect mature vessels. Graph represents vessel density given as a fraction of stained vessels to total area in the high-power field (200×). (B) No statistical difference was measured between treated groups. Results showed a statistically significant decrease in total vessel density after treatment with these agents and no significant decrease in mature vessels when treated groups were combined and compared with untreated controls.
Figure 4.
 
Vessel maturation limits vessel-targeting therapy in LHBETATAG retinal tumors. (A) Sixteen-week-old LHBETATAG mice were treated with single subconjunctival injections of anecortave acetate (AA; 300 μg) or combretastatin A4 (CA4P; 2 mg). Tumor vessels were analyzed 1 day and 1 week after injection. Sections were stained with lectin to detect total vessels and α-SMA to detect mature vessels. Graph represents vessel density given as a fraction of stained vessels to total area in the high-power field (200×). (B) No statistical difference was measured between treated groups. Results showed a statistically significant decrease in total vessel density after treatment with these agents and no significant decrease in mature vessels when treated groups were combined and compared with untreated controls.
The authors thank Magda Celdran for excellent assistance with histology. 
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Figure 1.
 
LHBETATAG retinal tumor development as a function of age. (A) Retinal tumors areas are given in pixels. Tumor areas measured 0 (±0) for 4-week-old mice, 8680.4 (±8111.6) for 8-week-old mice, 63,674.3 (±72,067.7) for 12-week-old mice, and 289,814.7 (±251,329.6) for 16-week-old mice. Inset: linear regression of log-transformed tumor areas onto animal age, accounting for 66.4% (r 2) of the variability in log tumor sizes. (B) Tumors develop from proliferating cells in the INL. Sections were stained with Ki67 (red) to label proliferating cells and DAPI (blue) to stain nuclei.
Figure 1.
 
LHBETATAG retinal tumor development as a function of age. (A) Retinal tumors areas are given in pixels. Tumor areas measured 0 (±0) for 4-week-old mice, 8680.4 (±8111.6) for 8-week-old mice, 63,674.3 (±72,067.7) for 12-week-old mice, and 289,814.7 (±251,329.6) for 16-week-old mice. Inset: linear regression of log-transformed tumor areas onto animal age, accounting for 66.4% (r 2) of the variability in log tumor sizes. (B) Tumors develop from proliferating cells in the INL. Sections were stained with Ki67 (red) to label proliferating cells and DAPI (blue) to stain nuclei.
Figure 2.
 
Angiogenesis in LHBETATAG retinal tumors. (A) At 4 weeks of age, LHBETATAG-positive mice have significantly higher concentrations of endoglin-stained vessels in the INL than wild-type controls (P = 0.042). Total vessel densities in this area are not significantly different between LHBETATAG mice and wild-type littermates (P = 0.86). (B) In LHBETATAG retinal tumors, the total number of vessels and the amount of vessels undergoing angiogenesis changed over time (P = 0.039). Endothelial cells were labeled with lectin (red) and endoglin (CD105, green). Graph represents vessels densities given as fraction of stained vessels to total area.
Figure 2.
 
Angiogenesis in LHBETATAG retinal tumors. (A) At 4 weeks of age, LHBETATAG-positive mice have significantly higher concentrations of endoglin-stained vessels in the INL than wild-type controls (P = 0.042). Total vessel densities in this area are not significantly different between LHBETATAG mice and wild-type littermates (P = 0.86). (B) In LHBETATAG retinal tumors, the total number of vessels and the amount of vessels undergoing angiogenesis changed over time (P = 0.039). Endothelial cells were labeled with lectin (red) and endoglin (CD105, green). Graph represents vessels densities given as fraction of stained vessels to total area.
Figure 3.
 
Vessel maturation in LHBETATAG retinal tumors. Linear regression demonstrated a highly significant increase in the density of SMA from 8 through 12 to 16 weeks (P < 0.001). Sections were stained with α-SMA (red). Graph represents vessels densities given as fraction of stained vessels to total area in the high power field, 200×. Controls from all age groups had the same vessels densities. Representative 16-week-control is shown.
Figure 3.
 
Vessel maturation in LHBETATAG retinal tumors. Linear regression demonstrated a highly significant increase in the density of SMA from 8 through 12 to 16 weeks (P < 0.001). Sections were stained with α-SMA (red). Graph represents vessels densities given as fraction of stained vessels to total area in the high power field, 200×. Controls from all age groups had the same vessels densities. Representative 16-week-control is shown.
Figure 4.
 
Vessel maturation limits vessel-targeting therapy in LHBETATAG retinal tumors. (A) Sixteen-week-old LHBETATAG mice were treated with single subconjunctival injections of anecortave acetate (AA; 300 μg) or combretastatin A4 (CA4P; 2 mg). Tumor vessels were analyzed 1 day and 1 week after injection. Sections were stained with lectin to detect total vessels and α-SMA to detect mature vessels. Graph represents vessel density given as a fraction of stained vessels to total area in the high-power field (200×). (B) No statistical difference was measured between treated groups. Results showed a statistically significant decrease in total vessel density after treatment with these agents and no significant decrease in mature vessels when treated groups were combined and compared with untreated controls.
Figure 4.
 
Vessel maturation limits vessel-targeting therapy in LHBETATAG retinal tumors. (A) Sixteen-week-old LHBETATAG mice were treated with single subconjunctival injections of anecortave acetate (AA; 300 μg) or combretastatin A4 (CA4P; 2 mg). Tumor vessels were analyzed 1 day and 1 week after injection. Sections were stained with lectin to detect total vessels and α-SMA to detect mature vessels. Graph represents vessel density given as a fraction of stained vessels to total area in the high-power field (200×). (B) No statistical difference was measured between treated groups. Results showed a statistically significant decrease in total vessel density after treatment with these agents and no significant decrease in mature vessels when treated groups were combined and compared with untreated controls.
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