The intrabronchial implantation of Lewis lung carcinoma (LLC) cells in mice is a well established model for lung tumors. The aim of this study was firstly a three dimensional presentation of intrapulmonary tumors and secondly the quantification of their vascularization by micro-computed tomography (micro-CT).
METHOD AND MATERIALS
Six mice (C57BL/6N) were inoculated with LLC cells via intratracheal injection of 10 million cells. For investigation in micro-CT blood vessels were filled with blood-pool contrast agent (Microfil, Flow Tech, Massachusetts, US), forming a vascular cast. Two different methods of perfusion of the pulmonary vascular tree were applied in 3 mice each. (1) A complete filling of the pulmonary vascular tree was achieved by intravenous in vivo application of contrast medium (CM) via the jugular vein. (2) Successful entire filling of the pulmonary arteries down to the capillary level was enabled by direct injection of CM into the main pulmonary artery ex vivo. After consolidation of the contrast medium the lungs were harvested and investigated with micro-CT (Skyscan1072, Skyscan, Belgium).
Both preparation procedures kept the lungs fully inflated after extirpation. It was possible to detect the multiple lung tumors and to quantify their volume by micro-CT. The results were in accordance with the histological measurements. Depending on the mode of application the intrapulmonary vascular tree was completely filled by contrast medium or respectively only the pulmonary arterial branches were filled. This revealed the pulmonary arterial origin of tumor vascularization and indicates, that the bronchial arteries are of only minor importance in the neovascularization. In the entirely filled lungs it was possible to quantify the vascular content of the tumors by micro-CT and therefore to calculate the vascular fraction.
Micro-CT is a reliable method to quantify the volume of intrapulmonary tumors in the mouse model. Compared to histomorphometric procedures the micro-CT provides a possibility to quantify the vascular parameters entirely and completely which makes it a highly valuable tool in the investigation of angiogenesis.