More circulating anti-51 integrin antibody accumulated in small tumors than in normal islets of wild-type mice (Figure 5; A to F)

More circulating anti-51 integrin antibody accumulated in small tumors than in normal islets of wild-type mice (Figure 5; A to F). normally be isolated by the endothelial barrier. However, not all tumor vessels are equally leaky, leakiness is heterogeneous along individual tumor Rabbit Polyclonal to CDK1/CDC2 (phospho-Thr14) vessels, and SIS-17 the amount of leakage is limited by the high interstitial fluid pressure in tumors.19,21,22 The effectiveness of molecular targets in tumors for macromolecular therapeutics in the bloodstream is thus determined by their amount of expression, cellular distribution, and accessibility. Integrin 51 and its ligand fibronectin have been found to be overexpressed in blood vessels of human and mouse tumors.13 The oncofetal isoform of fibronectin with an extra domain B (ED-B) is up-regulated in tumor vessels and is being explored as a diagnostic and therapeutic target.23 Selective antagonists targeted to 51 integrin reverse tumor growth in preclinical models13,14,24 and have entered clinical trials.25 Selective inhibitors of SIS-17 51 integrin are thought to lead to endothelial cell apoptosis by anoikis and other mechanisms.13,14 The present study sought to obtain a better understanding of the distribution of 51 integrin expression and to identify which sites are accessible to antibodies in the bloodstream. Specifically, we 1) compared the cellular distribution of 51 integrin in tumors and normal organs, 2) examined the accessibility of 51 integrin on tumor blood vessels to antibodies in the bloodstream, and 3) determined the extent and uniformity of expression of 51 integrin on blood vessels in different mouse tumor models and during tumor progression. Our approach was first to use conventional immunohistochemistry to determine the distribution of 51 integrin in mice. Next, we tested the accessibility of the integrin by injecting the same anti-51 integrin antibody intravenously and examining sites of binding 10 minutes to 24 hours later. Using these two approaches, we compared the distribution and accessibility of 51 integrin in pancreatic islet tumors in RIP-Tag2 transgenic mice,26 intestinal adenomas in adenomatous polyposis coli (mice (C57BL/6J-or tumors, or MCa-IV tumors). CCD camera gain was standardized as follows. First, after the brightest section (reference specimen) of a data set was identified, a digital image was acquired with the CCD camera. Next, the distribution of pixel fluorescence intensities of the image was displayed using the histogram function of Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Camera gain was then adjusted, another image acquired, and the process repeated until the intensity histogram for the reference specimen image was well distributed, as judged by pixels throughout the entire intensity range with few or no pixels at maximal intensity of 255. When the camera gain for the reference specimen was optimized, the entire data set of images was acquired using this gain. Pancreatic islet tumors in SIS-17 RIP-Tag2 mice were classified by their diameters in tissue sections as small ( 500 m), medium (500 1000 m), or large ( 1000 m), which approximately correspond to stage of tumor progression.26 Digital images of three to four tumors of each size in one or more sections of pancreas from each mouse were acquired. Regions of interest (ROI), defined by tissue boundaries identified by their vascular pattern in specimens stained for 51 integrin and CD31 immunoreactivities, were outlined with the freehand tool of ImageJ (Figures 1 and 2). The ROI of normal islets and small and medium size tumors was completely included in a single image. Large tumors, which exceeded the size of a single image, were sampled by acquiring three images at 4, 8, and 12 oclock around the tumor, each image representing one ROI. Fluorescence intensities of the pixels within the ROI were then recorded with ImageJ. The extent of co-localization of anti-51 integrin and anti-CD31 antibodies in RIP-Tag2 tumors was calculated using the co-localization function of ImageJ (Figure 2). Open in a separate window Figure 1 Rapidly accessible 51 integrin on blood vessels of RIP-Tag2 tumors. Confocal microscopic images showing 51 integrin immunoreactivity (red) and CD31 immunoreactivity (green) in pancreas of RIP-Tag2 mice. A and B: Blood vessels (arrows) in tumors (defined) and pancreatic ducts (arrowheads) both have strong 51 integrin immunoreactivity after staining by standard immunohistochemistry. A: However, co-localization of CD31 (yellow-green) immunoreactivity co-localizes with 51 integrin on tumor vessels but not on ducts (reddish) or normal blood vessels (green) of the acinar pancreas. C: At 10 minutes after intravenous injection of anti-51 integrin.