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By Helena J. Mauceri, PhD, Rabih M. Salloum, MD, Nora T. Jaskowiak, MD, Mitchell C. Posner, MD, and Ralph R. Weichselbaum, MD
Angiogenesis is a discrete component of tumor phenotype and is required for
tumor progression and metastases formation. Successful vascularization, based on invasion and morphological differentiation of endothelial cells, is controlled by the relative ratios of pro-angiogenic and anti-angiogenic proteins in the tumor microenvironment.1 Unlike proliferating endothelial cells within tumors, normal endothelium is quiescent, and cell division is rare. Treatments designed to inhibit the angiogenesis process are designed to exploit the differences between proliferating endothelial cells in tumor and those of normal tissues. Also, because endothelium is genetically stable, it is unlikely that endothelial cells will develop resistance to cytotoxic agents.2 Approaches to inhibit angiogenesis include the use of neutralizing antibodies to angiogenic proteins, integrin molecules, and growth factor receptors, as well as antibiotic derivatives, such as TNP-470 and minocycline.3-7
Angiostatin, a plasminogen cleavage product, has been shown to induce tumor regression and prolong tumor dormancy in murine model systems.8-10 However, when angiostatin is employed as a single treatment modality, tumor regrowth ensues following cessation of treatment, and cure rate is limited. Because radiotherapy improves local tumor control and cure rates,11-16 we evaluated the interaction between angiostatin and ionizing radiation (IR). We demonstrated that combined treatment with angiostatin and IR enhanced tumor regression in Lewis lung carcinoma (LLC) tumors and three human tumor xenograft model systems.17 We also studied the interaction between IR and endostatin, a c-terminal proteolytic fragment of collagen XVIII, and reported that combined treatment with IR and endostatin produced significant tumor growth inhibition when compared with either treatment alone.18
Another approach to enhance the therapeutic ratio involves blocking a positive regulator of angiogenesis. We conducted experiments in which tumor-bearing mice were treated with an antibody to vascular endothelial growth factor (VEGF). We reported that combined treatment with anti-VEGF antibody and IR produced greater than additive antitumor effects when compared to either treatment alone.19 Collectively, our studies suggest that when combined with IR treatment, angiogenesis inhibition targets both tumor cells and tumor vasculature. Enhancing tumor control without increasing toxicity has important implications for cancer treatment.
The isocoumarin derivative, NM-3 [2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid], has been reported to inhibit the growth of endothelial cells in vitro and exert modest antitumor effects in vivo.20,21 Because NM-3 possesses a low toxicity profile, is orally bioavailable, and is easily produced, we conducted studies to evaluate if treatment with NM-3 and IR enhanced the therapeutic ratio of IR. We report that
NM-3 increases the antitumor effects of IR without a concomitant increase in acute local or systemic toxicity. The bioavailability and non-toxic profile of NM-3 support the testing of this new isocoumarin in combination with radiotherapy in clinical trials.
NM-3 Is Anti-Angiogenic and Selectively Cytotoxic to Endothelial Cells
We evaluated the effects of NM-3 treatment on the three components of angiogenesis (i.e., endothelial cell survival, migration, and tube formation). Cell survival was analyzed using clonogenic assays in which endothelial cells and tumor cells were exposed to NM-3 alone for four hours or exposed to NM-3 and treated with increasing doses of IR. NM-3 was cytotoxic to endothelial cells but not to tumor cells. When endothelial cells were exposed to NM-3 prior to IR exposure, additive killing was observed. No enhancement of IR-induced cytotoxicity was observed in tumor cell cultures.
Endothelial cell migration was evaluated using an inverted, modified Boyden chamber.22 Cells were treated with NM-3 for four hours prior to exposure to IR. NM-3 alone did not affect endothelial cell migration when compared to control. However, IR alone inhibited migration by 34% and the combination of NM-3 and IR produced a 49% inhibition in endothelial cell migration.
To evaluate the effects of NM-3 on tube formation, tumor cells were mixed with Matrigel (synthetic basement matrix) and injected subcutaneously into nude mice. Matrigel plugs from mice receiving daily injections of saline showed a robust angiogenic response, as demonstrated by an extensive network of vessels. In contrast, a dramatic reduction in neovascularization was observed in the Matrigel plugs from mice receiving two daily injections of NM-3.
The Combination of NM-3 and IR Inhibits Primary Tumor Growth
The effects of NM-3 and IR were assessed in one murine tumor and two human tumor xenograft models. In the LLC models, no significant tumor regression was observed following treatment with NM-3 alone. Tumors in the IR alone group regressed initially, but then regrew. However, combined treatment with NM-3 and IR significantly reduced mean tumor volume when compared to IR alone. Equivalent treatment effects were observed in xenografts of human espohageal adenocarcinoma (Seg-1) cells. Treatment with NM-3 alone did not inhibit tumor growth, whereas combined treatment with NM-3 and IR produced a significant reduction in mean tumor volume. The combined treatment group demonstrated a growth delay of 14 days compared to the control group and three days compared to the IR alone group. Human squamous cell carcinoma (SQ-20B) control tumors and tumors treated with NM-3 alone grew steadily for 20 days, at which time animals in these two groups were sacrificed due to tumor burden. Tumors in the IR treatment group initially doubled in volume, regressed, and then regrew to twice original volume (day 0 volume). Animals treated with NM-3 and IR regressed to 77% of original volume but, unlike IR treated tumors, never regrew to original volume. These studies demonstrate that when NM-3 is combined with IR, significant growth inhibition of primary tumors can be achieved.
Primary Tumor Growth Delay Translates to Enhanced Local Cures
We employed the LLC tumor model system to examine the effects of treatment with NM-3 and IR on local cure rate, defined as the absence of measurable tumor at the site of implantation. Mice bearing LLC tumors were treated with a higher dose of NM-3 (50 mg/kg/day) and a higher dose of IR (66 Gy). On day 21 after initiation of treatment, five of 12 mice in the IR alone group were cured locally, whereas nine of 11 animals in the NM-3/IR group were tumor-free.
Combination Treatment with NM-3 and IR Reduces Microvascular Density
To assess the effect of NM-3 and IR on tumor vessels, LLC tumors were excised on days 5 or 11 from mice treated with NM-3, IR, or the combination of NM-3 and IR. Employing rat anti-mouse-CD31 (PECAM-1)
monoclonal antibody, immunohistochemistry was performed and microvascular density was determined using light microscopy. At day 5, tumors in the combined treatment group (NM-3 and IR) had fewer vessels (14.2 ± 1.2) than either the NM-3 alone group (19.2 ± 3.6) or the IR group (18.7 ± 2.7). No further reduction in the total number of vessels was observed in any treatment group at day 11. However, fewer distinct small vessels were present in the IR and combined treatment groups compared to the control and NM-3 alone groups.
NM-3 Alone or in Combination with IR Is Not Toxic to Experimental Animals
We also evaluated the effects of treatment with NM-3 alone and in combination with IR on regional and systemic toxicity. Mice bearing Seg-1 tumor xenografts and mice bearing LLC tumors were employed in these studies. With regard to systemic toxicity, no weight loss or mortality resulted from NM-3 treatment in either experiment. Local toxicity was evaluated in treated hind limbs. When the hind limbs of mice bearing Seg-1 xenografts were scored for superficial injury and scab formation, no differences between treatments were observed. Mice bearing LLC tumors were treated for 11 days with higher doses of both NM-3 and IR. In these animals, hind limbs were scored for superficial injury, scab formation, ulceration, hair loss, and limb shortening. Both the IR alone and the NM-3 and IR groups demonstrated similar local effects of treatment. A greater increase in foot swelling was noted in the combined treatment group at days 17 and 20, but by day 21 no difference between IR alone and NM-3 and IR was evident.
The present studies demonstrate that NM-3, an orally bioavailable isocoumarin, increases the antitumor effects of IR. Combined treatment with NM-3 and IR increased tumor regression and local cure rates when compared to IR alone, without a concomitant increase in toxicity. This enhancement of the therapeutic ratio of IR is attributable to the selective effects of NM-3 on the tumor vasculature, as indicated by the effects of NM-3 on endothelial cell survival, migration, and tube formation. Clinical radiotherapy focuses on targeting the tumor, employing techniques to deliver higher doses of IR to the tumor volume while sparing normal tissues. However dose escalation IR is required to achieve tumor control. The use of chemical modifiers and conventional cytotoxic agents combined with radiotherapy has yielded poor clinical results, mainly due to acute tissue toxicity.
Our studies employing treatment with NM-3 describe a new paradigm in which a non-toxic agent potentiates the effects of IR. We demonstrate selective antitumor effects by employing combined treatment with NM-3 and IR, without local or systemic toxicity. Importantly, the combination of NM-3 and radiotherapy improves both tumor growth delay and radiocurability. The present findings are supported by reports of angiogenesis inhibitors potentiating the effects of IR and strengthen the concept of targeting both tumor cells and the tumor microvasculature to improve the therapeutic ratio.
(Dr. Mauceri is Research Associate and Dr. Weichselbaum is Professor and Chairperson, Department of Radiation and Cellular Oncology; Drs. Salloum and Jaskowiak are Surgical Oncology Fellows and Dr. Posner is Associate Professor, Department of Surgery, University of Chicago.)
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