Prior to sacrifice, four animals from each treatment group were randomly selected for collection of tumors for molecular assays. Five tumors were collected from each animal with exception of one rat treated with combination therapy that had no tumors (instillation likely missed the trachea). After weighing the remaining lungs, they were inflated with 10% neutral-buffered formalin at a constant hydrostatic pressure of 25 cm for 6 hours. Lung volume (VL) was determined by fluid displacement. Lung lobes were trimmed at 3-4 mm intervals in a direction perpendicular to the axial airways with the first slice placed randomly near the cranial and/or hilar end. Paraffin embedded lungs were sectioned at 5 μm thickness and stained with hematoxylin and eosin. Approximately 30 histology sections were generated per set of lung lobes. For measurement of tumor size, a mylar morphometry grid with points spaced one mm apart was overlaid upon each of the microscope slides. The total area of the lung sections and the area occupied by tumor were estimated by counting points overlying tissue (15 (link)). An estimate of total tumor volume was determined by the percentage of tissue points overlying tumor multiplied by the VL.
Gross pathology images from a PowerShot S3IS digital camera (Canon USA, Inc.) were processed with Adobe Photoshop CS3 (v. 10.0.1; Adobe Systems, Inc.). Contrast was adjusted to +50; images were cropped, rotated, and resized. Measurement bars of 4 mm length were added with the line tool of Photoshop. Photomicrographs were made using Olympus equipment and software (BX41 with UPlanFL objectives, DP25 camera; Olympus America, Inc.). Automatic exposure settings were used, contrast was adjusted to +50, images were resized, and image magnification was indicated using measurement bars of known length.