Evaluation of New Morphometric Parameters of Neoangiogenesis in Human Colorectal Cancer Using Confocal Laser Endomicroscopy (CLE) and Targeted Panendothelial Markers

The tumor microcirculation is characterized by an abnormal vascular network with dilated, tortuous and saccular vessels. Therefore, imaging the tumor vasculature and determining its morphometric characteristics represent a critical goal for optimizing the cancer treatment that targets the blood vessels (i.e. antiangiogenesis therapy). The aim of this study was to evaluate new vascular morphometric parameters in colorectal cancer, difficult to achieve through conventional immunohistochemistry, by using the confocal laser endomicroscopy method. Fresh biopsies from tumor and normal tissue were collected during colonoscopy from five patients with T3 colorectal carcinoma without metastasis and were marked with fluorescently labeled anti-CD31 antibodies. A series of optical slices spanning 250 mm inside the tissue were immediately collected for each sample using a confocal laser endomicroscope. All measurements were expressed as the mean +/- standard error. The mean diameter of tumor vessels was significantly larger than the normal vessels (9.46 +/- 0.4 mu m vs. 7.60 +/- 0.3 mu m, p = 0.0166). The vessel density was also significantly higher in the cancer vs. normal tissue samples (5541.05 +/- 262.81 vs. 3755.79 +/- 194.96 vessels/mm(3), p = 0.0006). These results were confirmed by immunohistochemistry. In addition, the tortuosity index and vessel lengths were not significantly different (1.05 +/- 0.016 and 28.30 +/- 3.27 mu m in normal tissue, vs. 1.07 +/- 0.008 and 26.49 +/- 3.18 mu m in tumor tissue respectively, p = 0.5357 and p = 0.7033). The daughter/mother ratio (ratio of the sum of the squares of daughter vessel radii over the square of the mother vessel radius) was 1.15 +/- 0.09 in normal tissue, and 1.21 +/- 0.08 in tumor tissue (p = 0.6531). The confocal laser endomicroscopy is feasible for measuring more vascular parameters from fresh tumor biopsies than conventional immunohistochemistry alone. Provided new contrast agents will be clinically available, future in vivo use of CLE could lead to identification of novel biomarkers based on the morphometric characteristics of tumor vasculature.