No abstract

Our group's work on late radiation effects has been governed by the hypothesis that the effects observed in normal tissues are a consequence of multicellular interactions through a network of mediators. Further, we believe that inflammation is a necessary component of this process. We therefore investigated whether the recruitment of mononuclear cells, observed during the pneumonitic period in the irradiated normal lung, is dependent on the expression of chemokines, notably Mcp1. Since statins have been shown to reduce chemokine expression and inflammatory cell recruitment, we specifically examined whether statins could be used to reduce monocyte recruitment. Mice received 15 Gy whole-lung irradiation; treated groups were administered lovastatin three times weekly starting either immediately or 8 weeks postirradiation. At subsequent intervals, animals were killed humanely, and cellular, mRNA and protein analyses were undertaken. Statin-treated animals demonstrated a statistically significant reduction in both macrophage and lymphocyte populations in the lung compared to radiation alone as well as improved rates of survival and decreased collagen content. In addition, ELISA measurements showed that radiation-induced increases in Mcp1 protein were reduced by statin treatment. Additional experiments are needed to assess whether statins offer a potential treatment for the amelioration of late effects in breast and lung cancer patients undergoing radiation therapy.

Tumour oxygenation has been shown to play an important role in radiotherapeutic response both in experimental tumours and in the clinic. To eliminate microregions of hypoxia, i.e. low oxygenation, numerous strategies have been used, generally aiming either to increase oxygen delivery to the tumour (Horsman et al, 1994) or to directly target the hypoxic cells using radiosensitizers or hypoxic cell cytotoxic agents (Brown and Giaccia, 1994). Several recent reports have also suggested the use of angiogenesis inhibitors (Klauber et al, 1997;OÕReilly et al, 1997) or thrombocytic agents (Huang et al, 1997) to directly target the tumour vasculature. A major shortcoming in evaluating the use of any of these approaches, however, is the inability to adequately quantify and understand the accompanying changes in tumour physiology.The current study presents a method for combining several sophisticated techniques to obtain a comprehensive two-dimensional mapping of the relationships among tumour vascular configuration, oxygen transport and hypoxic development. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO 2 ) cryospectrophotometrically in a frozen tumour block. Second, hypoxic development, in relation to the intravascular oxygen availability, is quantified in an adjacent histological section, using immunohistochemical detection of a recently developed nitroheterocyclic hypoxia marker (EF5). Third, a combination of fluorescent and immunohistological stains is used to define the distribution of distances from the viable tumour cells to the nearest anatomical or perfused blood vessel. MATERIALS AND METHODS Mice and tumour modelsThe KHT tumour, a sarcoma maintained in vivo, was passaged approximately every 2 weeks by i.m. inoculation of single-cell suspensions prepared by a mechanical dissociation procedure (Thomson and Rauth, 1974). Using 6-to 8-week-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME, USA), 2×10 5 KHT cells were inoculated i.m. into the hind limbs. Tumours were selected for analysis when they reached volumes of between 320 and 1100 mm 3 (as measured by callipers (volume = πqdiam-eter 3 /6). Guidelines for the humane treatment of animals were followed as approved by the University Committee on Animal Resources. Injection of fluorescent stains and EF5 hypoxic markerLocalized areas of tumour hypoxia were assessed in frozen tissue sections by immunohistochemical identification of sites of 2-nitroimidazole metabolism. A pentafluorinated derivative (EF5) of etanidazole (synthesized by Dr R Vishnuvajjala of the National Cancer Institute) was injected i.v., 1 h before tumour freezing (0.2 ml of 10 mM EF5). Protein conjugates of EF5 have been used previously to immunize mice from which monoclonal antibodies were developed (Lord et al, 1993). These antibodies are extremely specific for the EF5 drug adducts that form when the drug is incorporated by Summary Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the under...

Although antiangiogenic strategies have proven highly promising in preclinical studies and some recent clinical trials, generally only combinations with cytotoxic therapies have shown clinical effectiveness. An ongoing question has been whether conventional therapies are enhanced or compromised by antiangiogenic agents. The present studies were designed to determine the pathophysiologic consequences of both single and combined treatments using fractionated radiotherapy plus AG-013736, a receptor tyrosine kinase inhibitor that preferentially inhibits vascular endothelial growth factor receptors. DU145 human prostate xenograft tumors were treated with (a) vehicle alone, (b) AG-013736, (c) 5 Â 2 Gy/wk radiotherapy fractions, or (d) the combination. Automated image processing of immunohistochemical images was used to determine total and perfused blood vessel spacing, overall hypoxia, pericyte/collagen coverage, proliferation, and apoptosis. Combination therapy produced an increased tumor response compared with either monotherapy alone. Vascular density progressively declined in concert with slightly increased A-smooth muscle actin-positive pericyte coverage and increased overall tumor hypoxia (compared with controls). Although functional vessel endothelial apoptosis was selectively increased, reductions in total and perfused vessels were generally proportionate, suggesting that functional vasculature was not specifically targeted by combination therapy. These results argue against either an AG-013736-or a combination treatment-induced functional normalization of the tumor vasculature. Vascular ablation was mirrored by the increased appearance of dissociated pericytes and empty type IV collagen sleeves. Despite the progressive decrease in tumor oxygenation over 3 weeks of treatment, combination therapy remained effective and tumor progression was minimal. [Cancer Res 2007;67(20):9921-8]

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP þ vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-g). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.