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¶¶
From the Biomedical Graduate Program,* the Departments of Medicine and Pathology and Laboratory Medicine, and the Cancer Center, University of Pennsylvania School of Medicine, Philadelphia; and the Veterans Affairs Medical Center,¶ Philadelphia, Pennsylvania
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Abstract |
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As originally conceived, anti-angiogenesis therapy would inhibit the formation of new blood vessels without affecting existing vessels.4 However, the dramatic tumor regression observed with some angiogenesis inhibitors6 implies that they may also affect existing tumor vessels and, indeed, studies have demonstrated that anti-angiogenic therapy induces tumor EC apoptosis, even when tumor growth is slowed but not stopped.7 Our own studies of murine tumors treated with the angiogenesis inhibitor recombinant murine interleukin-12 (rmIL-12) showed that control of tumor growth is associated with induction of tumor ischemia and ischemic cell death.3 The reduction in tumor vascularity observed during treatment resulted predominantly from loss of small-caliber vessels,8 which suggested that the pattern of vessel loss was not essentially stochastic. Rather, it seemed likely to be related to the process by which tumor vessels develop and mature.
The developmental process underlying new blood vessel formation and maturation has been well-studied, although it remains incompletely understood.9 Neovascularization in adult life is characterized by angiogenesis, a process in which new vessels are formed by cells derived from nearby pre-existing vessels. Early studies of angiogenesis describe an initial phase of EC mitosis associated with expansion of the existing vascular network and formation of new capillary sprouts.10,11 Subsequently, newly-formed vessels become invested with pericytes (also known as periendothelial cells or Rouget cells), which coincides with cessation of the angiogenic response.12 The developmental progression of angiogenesis has been visualized best in the postnatal rodent retina.13,14 Here, sprouting angiogenesis produces an expanding endothelial plexus during the first 2 weeks of postnatal development. A programmed lag in pericyte recruitment allows the nascent vasculature to undergo extensive remodeling, with pericyte investment serving later to preserve the vasculature in its final configuration and provide resistance to regression. The importance of this endothelial-pericyte interaction and vessel maturation process is shown by transgenic mice lacking platelet-derived growth factor B, in which embryonic lethality is associated with pericyte deficiency and capillary rupture.15
We postulated that vascular development in tumors would essentially parallel the process in normal tissues and give rise to a diversity of vessels with respect to age and maturation. Understanding this process in the murine tumors commonly used in preclinical studies would lead to a better appreciation of the primary therapeutic target of anti-vascular agents that, in turn, might reveal their therapeutic potential and limitations. Accordingly, in this study we characterized the development and maturation of blood vessels in transplanted murine tumors and in these tumors after they were subjected to anti-vascular therapy. Our results indicate that vessels develop in transplanted murine tumors in an ordered process with features that can be identified by immunostaining of conventional histological sections. The level of vessel maturation is strongly correlated with their response to anti-vascular therapy, which leads us to predict that the maturation profile of vessels within tumors will influence or determine vascular and tumor response to anti-vascular agents.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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C57Bl/6 and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME), and C3H/HeN mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All mice were 6- to 8-week-old females maintained in microisolator cages under sterile conditions. The K173516 and B16F1017 murine melanoma cell lines (syngeneic with C3H/HeN and C57Bl/6 strains, respectively), along with the RENCA renal cell carcinoma line18 were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and penicillin/streptomycin. All three cell lines were tested and demonstrated to be mycoplasma-free.
Tumor Studies
For tumor growth studies, 106 tumor cells were injected subcutaneously into the lower left flank of syngeneic mice. Injected cells were derived from low-passage frozen stocks that had been established in culture less than 1 week before injection. When established tumors reached a diameter of 1 to 2 mm, rmIL-12 or phosphate-buffered saline (PBS) vehicle was administered intraperitoneally at or under the maximum tolerated dose for each strain on a five dose per week schedule (five daily injections followed by 2 days of rest) for up to 3 weeks. C3H/HeN mice received their maximum tolerated dose of 125 ng per injection, and C57Bl/6 and BALB/c mice received 500 ng per injection. Tumors were measured bidirectionally by calipers at regular intervals, and tumor volume was calculated using the formula for approximating the volume of a spheroid [0.52 x (width)2 x (length)]. Mice were euthanized according to guidelines established by the Institutional Animal Care and Use Committee.
Immunohistochemistry
Four-µm sections from formalin-fixed, paraffin-embedded tumors were stained for ECs and pericytes using anti-von Willebrand factor (vWF; DAKO, Carpinteria, CA) and anti-
-smooth muscle actin (SMA; DAKO) antibodies, respectively. Deparaffinized sections underwent antigen retrieval by incubation in 0.08% pronase (Roche, Indianapolis, IN) followed by incubation in PBS and 10% goat serum. Anti-vWF antibody diluted 1:800 was added for 2 hours followed by ALEXA488-conjugated goat anti-rabbit Ig antibody (Molecular Probes, Eugene, OR) diluted 1:200 for 1 hour. Blocking of endogenous mouse tissue immunoglobulin was achieved using the Mouse-On-Mouse kit (Vector Laboratories, Burlingame, CA). Anti-SMA antibody was applied at 1:60 dilution for 60 minutes, followed by Texas Red-conjugated goat anti-mouse Ig antibody (Molecular Probes) for 1 hour. Proliferating cells were detected by staining with a peroxidase-conjugated antibody against proliferating cell nuclear antigen (PCNA, DAKO) along with a biotinyl tyramide amplification kit (New England Nuclear, Boston, MA) and 3-amino-9-ethylcarbonazole (AEC) substrate (Vector Laboratories). Apoptotic cells were detected using the Apoptag indirect fluorescein detection kit (Intergen, Purchase, NY). Three-color staining for ECs, pericyte, and proliferating/apoptotic nuclei was performed on the same sections. For Hoechst dye perfusion studies, 200 µl of 10 mmol/L Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) in PBS was administered intravenously 5 minutes before tumor excision. Excised tumors were then embedded in OCT compound (Sakura Finetek, Torrance, CA), frozen in liquid nitrogen, and cut with a cryostat. Ten-µm sections were fixed in 4% paraformaldehyde in PBS (pH 7.4) and stained for ECs as described below. All histological specimens were viewed under a Microphot-FX light microscope (Nikon, Melville, NY) equipped with a Photometrics Coolsnap digital camera and image acquisition software (Roper Scientific, Trenton, NJ).
Confocal Microscopy of Tumor Vasculature
Tumor-bearing mice were injected with 150 µl of 2 mg/ml fluorescein isothiocyanate-conjugated tomato (Lycopersicon esculentum) lectin (Vector Laboratories) in PBS intravenously into the tail vein. Ten minutes later, the mice were anesthetized and then perfused with 4% paraformaldehyde in PBS (pH 7.4) intracardially. For confocal imaging of the vasculature alone, tumors were excised and sectioned essentially as described previously.8 For histological staining of thick tumor sections, tumors were embedded in OCT compound, frozen, and cut with a cryostat. Thick (60 µm) tumor sections were stained for the endothelial marker CD31 (PECAM) and SMA according to a method adopted from Hashizume and colleagues.19 Sections were blocked with Mouse-on-Mouse reagent and incubated overnight at room temperature in anti-SMA antibody diluted 1:200 in PBS and 0.3% Triton X-100, washed, and then incubated in Cy5-conjugated goat anti-mouse Ig (Amersham Pharmacia, Piscataway, NJ). Subsequently, sections were incubated overnight in rat anti-mouse PECAM antibody (Pharmingen, San Diego, CA) diluted 1:500, washed, and incubated in Texas Red anti-rat Ig (Molecular Probes) diluted 1:200 for 3 hours.
All thick sections were imaged using an upright Nikon (Augusta, GA) E-600 Eclipse microscope equipped with a Bio-Rad (Hercules, CA) 1024-ES confocal system. Fluorescein isothiocyanate, Texas Red, and Cy5 fluorescence were detected by a three-line, 15 mW argon-krypton laser system (American Laser, Fraser, MI). For each image set acquired, individual fluorescence channel data were saved along with composite data sets indicating regions of fluorescence overlap. Image stacks were integrated to create a composite maximum intensity projection of tumor vasculature imaged in three dimensions.
Image Analysis
For microvessel density measurements, slides were scanned at low power (x40) to identify areas of highest vascularity. Ten to 20 high-powered (x200) fields were then selected randomly within these areas, and microvessel densities were calculated based on the number of vWF-positive structures. In addition, vessel lumen cross-sectional areas were determined for all counted vessels automatically (ImageTool; University of Texas, San Antonio, TX) based on spatial calibration parameters established with a slide micrometer. Microvessel counting was performed by multiple blinded observers in conjunction with a pathologist. A pericyte-positive vessel was defined as a vWF-positive vessel surrounded by at least one cell staining positive for -SMA. Proliferating and apoptotic vessels were defined as vessels in which at least one vWF-positive cell contained a nucleus that stained positive for PCNA and terminal dUTP nick-end labeling (TUNEL), respectively. Two to three sections were examined per tumor, and at least five tumors were examined per treatment group.
Statistical Analysis
Assessment of statistical significance was performed either by Student’s t-test (for normally distributed data sets) or Mann-Whitney U-test (for nonnormally distributed data sets). Correlation coefficients were derived from Pearson’s correlation method. All statistical analysis was performed using Instat software for the Macintosh version 2.0 (Graphpad Software, Philadelphia, PA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We began by examining blood vessels in untreated K1735 tumors. Examination of thin (4 µm) tumor sections stained with anti-vWF antibody (Figure 1A)
revealed that tumor vessels varied greatly in size, with small-caliber (<15 µm diameter) vessels predominating (Figure 1B)
. At the smallest end of the spectrum were individual vWF-positive cells lacking a visible lumen. These stained for other EC markers (CD31 and Tie2) but not for the melanocyte-specific antigen S100 expressed by K1735 cells (data not shown) and accounted for 18% of total K1735 tumor vascular structures. To assess whether these single endothelial cells (SECs) were functional vessels, tumor-bearing mice were injected with Hoechst 33342 intravenously to label the nuclei of cells exposed to blood flow.20,21
It failed to stain SECs (Figure 1C
, arrowheads) or the cells around them (Figure 1C
, top row) but did stain the vast majority (>90%) of larger vessels (Figure 1C
, bottom row), indicating that SECs were not perfused. This result also showed that SECs were discrete endothelial structures rather than tangentially cut sections of larger vessels. Dual staining for vWF and PCNA revealed that a much greater percentage of SECs (54 ± 15%) were proliferating compared to ECs in other vessels (18 ± 6%; P < 0.01, Mann-Whitney U-test). To test the idea that SECs were related to tumor angiogenesis, K1735 cells were engineered to overexpress the proangiogenic factor, vascular endothelial growth factor (VEGF164). These K1735.VEGF cells, which produce 20-fold more VEGF in 21% O2 cultures and induce 62% more angiogenesis in in vivo Matrigel neovascularization assays than control cells (data not shown), gave rise to tumors with a microvessel density (MVD) of 42.6 ± 15.5 vessels/high-power field compared to 14.5 ± 4.3 vessels/high-power field for control tumors. SECs were four to five times more abundant in these tumors, constituting 26.6 ± 5.3% of all vessels in K1735.VEGF tumors compared to 17.9 ± 2.4% of vessels in K1735 tumors (Figure 1D)
. To place SECs in the context of overall tumor vasculature, thick (60 µm) sections from tumors whose perfused vessels had been previously labeled in situ with a fluorochrome-conjugated tomato lectin,8
were stained for CD31. Laser-scanning confocal imaging of these sections revealed numerous thin tendrils of unperfused (lectin unstained) ECs sprouting from larger perfused vessels (Figure 1E
, arrows). These structures were undoubtedly angiogenic sprouts, which suggested that the corresponding SECs seen in thin tumor sections also reflected tumor angiogenic activity.
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K1735 tumor vessels were also characterized with respect to pericyte coverage by staining tumor sections with anti-vWF and anti--SMA antibodies; pericyte-positive vessels were defined as vWF-positive structures associated with at least one SMA-positive cell. Analysis of 10 untreated K1735 tumors ranging in size from 4 to 13 mm in diameter revealed that overall, 38 ± 5% of vessels were pericyte covered. Because of the possibility that pericytes surrounding some vessels might be missed because of the thinness of the histological sections, we traced 100 apparently pericyte-negative vessels through five sequential 4-µm sections (20 µm of tumor depth) to determine how many were pericyte covered on subsequent sections. Pericytes were only detected in 6 of these 100 vessels, indicating that the absence of pericyte coverage could be determined on the basis of one tumor section with 94% accuracy. Pericyte-negative vessels tended to be smaller (mean vessel cross-sectional area, 110 ± 75 µm2) than their pericyte-invested counterparts (mean area, 430 ± 215 µm2) (Figure 2A)
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, which appeared at higher magnification as thin, lectin-perfused EC tubes (Figure 2, C and D)
. Angiogenic sprouts frequently (Figure 2E)
emanated from perfused vessels (Figure 2F)
, with the vast majority (>95%) sprouting from distal pericyte-negative vessels rather than proximal pericyte-covered vessels (Figure 2, G and H)
. This is consistent with our observation on thin tumor sections that SECs were rarely associated with pericytes. When EC proliferative status was examined as a function of pericyte coverage by sequential staining for SMA/vWF and PCNA (Figure 2I)
, 34.8 ± 6.5% of pericyte-negative tumor vessels contained one or more PCNA-positive ECs, compared with 14.7 ± 3.9% of pericyte-positive vessels (P < 0.01, Mann-Whitney U-test). Thus, from the standpoints of vessel size, endothelial sprouting, and proliferation status, pericyte coverage marks more established tumor vessels, just as pericyte coverage of normal vessels denotes maturation.13
To investigate whether the spectrum of vessels in tumors changed as a function of tumor growth, we analyzed the vasculature of 27 K1735 tumors ranging from 3 to 14 mm in diameter (Figure 3)
. Compared to large tumors (5 to 14 mm), small tumors (<5 mm) had a higher mean MVD (13.4 ± 1.9 versus 11.3 ± 1.4, P < 0.01), a larger fraction of vessels that were SECs (26.0 ± 3.6 versus 18.7 ± 4.4, P < 0.01), and a lower fraction of pericyte-covered vessels (29.5 ± 5.7 versus 34.5 ± 4.5, P < 0.05). Thus, the vasculature of small K1735 tumors exhibited a more angiogenic and less mature phenotype. However, differences were not large, overlap was considerable, and vascular characteristics could not be predicted based on individual tumor size (Figure 3)
. Furthermore, significant differences in vascular parameters were not found among the larger tumors.
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We next characterized the pattern of K1735 vascular change with anti-angiogenesis therapy. Mice bearing either untreated or rmIL-12-treated tumors were injected with fluorescein isothiocyanate-tomato lectin, and tumor vasculature was imaged in thick sections (Figure 4, A and B)
. In untreated tumors, the vasculature consisted of a fine, reticulated network of small diameter vessels (mean diameter, 11 µm). Therapy led to a decrease in vessel density, with the remaining vessels being predominantly of larger size (mean diameter, 24 µm). When thin sections from size-matched untreated and rmIL-12-treated tumors were stained for vWF and SMA (Figure 4, C and D)
, treatment was seen to reduce overall MVD (Figure 4E
; 11.5 ± 2.8 versus 6.8 ± 1.8 vessels/high-power field; P < 0.05, t-test) but not the density of pericyte-positive vessels (Figure 4E
, shaded bars; 4.41 ± 0.92 versus 4.35 ± 0.57 vessels/high-power field, respectively). Rather, a decrease in the density of pericyte-negative tumor vessels during treatment (from 7.1 to 2.5 vessels/high-power field; P < 0.05 t-test) accounted for the decrease in overall MVD. The numbers of endothelial sprouts (Figure 4, F and G)
and SECs (5.41 ± 0.55% versus 17.9 ± 2.41% of total vessels, P < 0.01 Student’s t-test; Figure 1D
) were also markedly reduced, indicating that rmIL-12 therapy inhibited angiogenesis. Together, these changes resulted in a marked increase in the proportion of pericyte-covered vessels in treated tumors (62% in treated versus 38% in untreated tumors, P < 0.01).
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. Apoptotic vessels were defined as those in which at least one vWF-positive cell contained a TUNEL-positive nucleus (Figure 5, B and C)
. Few, if any, vessels were apoptotic in untreated K1735 tumors (Figure 5G)
. rmIL-12 treatment increased apoptosis among pericyte-negative vessels (1.05 ± 0.25 vessels/high-power field or 26% of vessels treated versus 0.27 ± 0.15 or 3.2% of vessels untreated; P < 0.05 Mann-Whitney U-test) but not among pericyte-positive vessels (0.15 ± 0.16 vessels/high-power field treated versus 0.14 ± 0.10 vessels/high-power field untreated). Additionally, we did not observe pericyte apoptosis in treated tumors. The apoptosis in pericyte-negative vessels accompanying their selective decrease with treatment indicated that rmIL-12 reduced K1735 tumor vascularity by inducing vessel regression as well as by inhibiting new vessel formation.
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and vascularity8
of these other two tumor types were consistently suppressed by rmIL-12 treatment. Untreated RENCA tumors had an overall MVD of 9.56 ± 1.23 vessels/high-power field, of which 27% were pericyte-covered (Table 2)
. After rmIL-12 treatment, MVD was reduced by 33% while the density of pericyte-covered vessels was not significantly altered. Untreated B16F10 tumors had a similar MVD and level of pericyte coverage as untreated RENCA tumors, and rmIL-12 therapy led to a 58% reduction in overall MVD. There was a 33% reduction in the density of pericyte-covered vessels but a more marked 68% reduction in the density of pericyte-negative vessels (P < 0.005). A feature of pericyte-covered vessels in treated B16F10 tumors was the occasional finding of TUNEL staining in both pericytes and ECs, notably in vessels located in regions of massive tumor cell death (data not shown). This indicates that pericyte-positive tumor vessels are not invulnerable and will regress under certain conditions, as may exist when the vessel microenvironment is disrupted. Angiogenesis was decreased in both RENCA and B16F10 tumors after rmIL-12 treatment, with SECs decreasing from 11.2% of total vessels to 4.6% in RENCA (P < 0.05) and from 19.5% to 5.5% in B16F10 (P < 0.005). As in K1735 tumors, the preferential loss of pericyte-negative vessels in RENCA and B16F10 tumors with rmIL-12 treatment resulted in a higher percentage of pericyte-covered vessels after therapy (48% and 47%, respectively).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. Larger tumors tend to have a lower fraction of nascent vessels and a higher fraction of mature vessels, suggesting that the spectrum of tumor vessels shifts as tumors enlarge. A recent study using anti-desmin and anti-SMA antibodies to stain pericytes and confocal imaging of thick tumor sections suggested that pericytes are associated with the vast majority of blood vessels in tumors and even cover endothelial sprouts.22
We cannot explain why we saw a more restricted pattern of pericyte coverage both in thin and thick sections, but the tumors studied were different. That pericytes invest only a subset of tumor vessels has been described by others in mouse7,23
and human24,25
tumors, and the association between coverage and vessel maturation recalls the process of retinal neovascularization in the neonatal rodent eye.13
The limited distribution that we observe correlates with vessel development and patterns of vessel therapeutic response and should be useful for discriminating between different categories of tumor vessels. Given the heterogeneity of tumor vessels based on stage of development or maturation, we examined the latter for determinants of vessel response. In multiple tumor models, the decrease in vessel density with therapy is attributable primarily to loss of pericyte-negative vessels, resulting in a higher proportion of pericyte-positive vessels after therapy. Loss of pericyte-negative vessels is because of both angiogenesis inhibition and regression of existing vessels. Although pericyte-negative vessels regress, some may also be lost through acquisition of pericytes during therapy. Maturation would reduce the number of these vessels and help maintain the density of pericyte-positive vessels during therapy, when there is continued, albeit slow, tumor growth. Preferential survival of pericyte-covered vessels may be because of pericyte production of EC survival factors26 or cell contacts formed with ECs providing survival signals. It is also possible that pericyte coverage is an indicator of protected vessels and that factors other than pericytes but associated with pericyte coverage actually provide protection. Pericyte coverage marks vessels protected from anti-vascular therapies in addition to rmIL-12, including VEGF withdrawal24 and anti-vascular thalidomide analogs (MS Gee, unpublished observations).
A tumor’s vascular response to anti-vascular therapy will be the primary determinant of its clinical response, although factors such as tumor cell dependence on vascular supply21 and response to ischemic stress27 may influence outcome. Because of this, pericyte coverage and protection of tumor vessels may strongly influence tumor therapeutic response. Transplanted mouse tumors we studied have a relatively low fraction of pericyte-covered vessels, indicating that most of their vessels are newly formed, and angiogenesis is vigorous. Many of their vessels are susceptible to regression, and angiogenesis inhibition would slow or stop the rapid addition of new vessels, so effective anti-vascular agents produce readily detectable effects. Tumors with a high fraction of pericyte-covered vessels have a greater proportion of mature vessels, fewer newly formed vessels, and may be less angiogenic. Active agents may have less effect on these tumors because fewer vessels are susceptible. This may help explain why autochthonous MMTV-induced mouse mammary tumors with a substantial fraction of pericyte-covered vessels do not respond to rmIL-12 therapy, whereas transplanted Mm5MT mammary tumors, derived from similar cells and with a much lower fraction of pericyte-covered vessels, respond well.28 A correlation between tumor vessel pericyte coverage and response is strengthened by the fact that modulation of pericyte coverage in K1735 tumors alters their response to rmIL-12 therapy in the expected direction (NM Yeilding, unpublished observations). This has implications for human cancers in which some of the most common types, eg, breast, colon, lung, and prostate, have vessels with moderate to extensive pericyte coverage.25 Although the response of these cancers to anti-vascular agents has not been described in detail, there is an impression of disparity between the striking results seen in mouse studies and the effects obtained in human trials.29
Clinical application of pericyte information will require reliable identification of pericytes in human tumor specimens. Although pericyte phenotype may be variable,22 reproducible discrimination between pericyte-positive and pericyte-negative vessels in human tumors has been reported.25 Quantitation of SECs may be useful because it provides an indicator of tumor angiogenic activity distinct from, but related to, pericyte coverage. The adverse influence of pericyte coverage on anti-vascular efficacy may apply only to certain agents. Efficacy of agents targeting ECs in more mature vessels or targeting other vascular components may not be limited by pericyte coverage.20,30 For agents that do selectively target angiogenic ECs, pericyte and SEC information may provide additional evidence of therapeutic anti-vascular effect. This requires tumor samples from before and after treatment, but an increase in pericyte coverage and a decrease in SECs would confirm that an effect was achieved. A better understanding of vessel development and maturation in human cancers and of the factors affecting pericyte coverage of their vessels may advance anti-vascular therapy of human cancers.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grants R01 CA 77851 and CA 83042 to W. M. F. L. and an Medical Scientist Training Program (MSTP) grant to M. S. G.), the American Cancer Society (CSM-98759 to N. M. Y.), and the United States Department of Defense (BC 990509 to N. M. Y.).
Accepted for publication October 1, 2002.
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 A. M. Munchhof, F. Li, H. A. White, L. E. Mead, T. R. Krier, A. Fenoglio, X. Li, J. Yuan, F.-C. Yang, and D. A. Ingram Neurofibroma-associated growth factors activate a distinct signaling network to alter the function of neurofibromin-deficient endothelial cells Hum. Mol. Genet., June 1, 2006; 15(11): 1858 - 1869. [Abstract] [Full Text] [PDF]
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 P. Martinive, J. De Wever, C. Bouzin, C. Baudelet, P. Sonveaux, V. Gregoire, B. Gallez, and O. Feron Reversal of temporal and spatial heterogeneities in tumor perfusion identifies the tumor vascular tone as a tunable variable to improve drug delivery. Mol. Cancer Ther., June 1, 2006; 5(6): 1620 - 1627. [Abstract] [Full Text] [PDF]
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N. Jo, C. Mailhos, M. Ju, E. Cheung, J. Bradley, K. Nishijima, G. S. Robinson, A. P. Adamis, and D. T. Shima Inhibition of Platelet-Derived Growth Factor B Signaling Enhances the Efficacy of Anti-Vascular Endothelial Growth Factor Therapy in Multiple Models of Ocular Neovascularization Am. J. Pathol., June 1, 2006; 168(6): 2036 - 2053. [Abstract] [Full Text] [PDF]
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 P. Atluri, G. P. Liao, C. M. Panlilio, V. M. Hsu, M. J. Leskowitz, K. J. Morine, J. E. Cohen, M. F. Berry, E. E. Suarez, D. A. Murphy, et al. Neovasculogenic therapy to augment perfusion and preserve viability in ischemic cardiomyopathy. Ann. Thorac. Surg., May 1, 2006; 81(5): 1728 - 1736. [Abstract] [Full Text] [PDF]
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 Z. Kan, S. Phongkitkarun, S. Kobayashi, Y. Tang, L. M. Ellis, T. Y. Lee, and C. Charnsangavej Functional CT for Quantifying Tumor Perfusion in Antiangiogenic Therapy in a Rat Model Radiology, October 1, 2005; 237(1): 151 - 158. [Abstract] [Full Text] [PDF]
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 E. W. B. Jeffes, J. G. Zhang, N. Hoa, A. Petkar, C. Delgado, S. Chong, A. Obenaus, R. Sanchez, S. Khalaghizadeh, T. Khomenko, et al. Antiangiogenic Drugs Synergize with a Membrane Macrophage Colony-Stimulating Factor-Based Tumor Vaccine to Therapeutically Treat Rats with an Established Malignant Intracranial Glioma J. Immunol., March 1, 2005; 174(5): 2533 - 2543. [Abstract] [Full Text] [PDF]
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M. F. McCarty, J. Wey, O. Stoeltzing, W. Liu, F. Fan, C. Bucana, P. F. Mansfield, A. J. Ryan, and L. M. Ellis ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor tyrosine kinase, inhibits orthotopic growth and angiogenesis of gastric cancer Mol. Cancer Ther., September 1, 2004; 3(9): 1041 - 1048. [Abstract] [Full Text] [PDF]
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B. M. Fenton, S. F. Paoni, and I. Ding Pathophysiological Effects of Vascular Endothelial Growth Factor Receptor-2-Blocking Antibody plus Fractionated Radiotherapy on Murine Mammary Tumors Cancer Res., August 15, 2004; 64(16): 5712 - 5719. [Abstract] [Full Text] [PDF]
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M. Furuhashi, T. Sjoblom, A. Abramsson, J. Ellingsen, P. Micke, H. Li, E. Bergsten-Folestad, U. Eriksson, R. Heuchel, C. Betsholtz, et al. Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate Cancer Res., April 15, 2004; 64(8): 2725 - 2733. [Abstract] [Full Text] [PDF]
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 R. C. Cattley and B. R. Radinsky Cancer Therapeutics: Understanding the Mechanism of Action Toxicol Pathol, January 1, 2004; 32(1_suppl): 116 - 121. [Abstract] [PDF]
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M. S. Gee, S. Makonnen, K. al-Kofahi, B. Roysam, F. Payvandi, H.-W. Man, G. W. Muller, and W. M. F. Lee Selective Cytokine Inhibitory Drugs with Enhanced Antiangiogenic Activity Control Tumor Growth through Vascular Inhibition Cancer Res., December 1, 2003; 63(23): 8073 - 8078. [Abstract] [Full Text] [PDF]
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P. Baluk, S. Morikawa, A. Haskell, M. Mancuso, and D. M. McDonald Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors Am. J. Pathol., November 1, 2003; 163(5): 1801 - 1815. [Abstract] [Full Text] [PDF]
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S. J. McAlhany, S. J. Ressler, M. Larsen, J. A. Tuxhorn, F. Yang, T. D. Dang, and D. R. Rowley Promotion of Angiogenesis by ps20 in the Differential Reactive Stroma Prostate Cancer Xenograft Model Cancer Res., September 15, 2003; 63(18): 5859 - 5865. [Abstract] [Full Text] [PDF]
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