An orthotopic lung tumor model for image-guided microirradiation in rats

Abstract

The purpose of this study was to develop a rat orthotopic lung tumor model with a solitary intrapulmonary nodule to study the effects of high-dose radiation. A549-Luc non-small cell lung cancer (NSCLC) cells were implanted into nude rats in the intercostal space between ribs 5 and 6 of the right lung. Bioluminescence and microcomputed tomography (CT) imaging were performed after implantation to confirm the presence of a solitary tumor and to monitor tumor growth. A device using image guidance for localization was developed to facilitate high-precision irradiation in small animals. A pilot irradiation study was performed, and response was assessed by bioluminescence imaging and immunohistochemistry. Radiation response was confirmed through serial bioluminescence imaging, and the strength of the bioluminescence signal was observed to be inversely proportional to dose. Response was also observed by the monoclonal antibody bavituximab, which binds to exposed lipid phosphatidylserine (PS) on tumor vessels. The ability to (1) reproducibly generate solitary tumor nodules in the rat lung, (2) identify and monitor tumor growth by bioluminescence imaging and CT imaging, (3) accurately target these tumors using high doses of radiation, and (4) demonstrate and quantify radiation response using bioluminescence imaging provides significant opportunity to probe the biological mechanisms of high-dose irradiation in preclinical settings.

FIG. 1

The image-guided small animal irradiation device (panel A) with major components indicated, positioned beneath a commercial X-ray source. Panel B: Software for image acquisition and positioning. Panel C: Initial localization image with beam center and tumor indicated. Panel D: Final localization verified by a second image. Panel E: Calibration and centering relative to the beam axis.

FIG. 2

Development of rat orthotopic model for SBRT. Panel A: Dissected specimen showing the desired injection site. Panel B: Rats are anesthetized using rat cocktail and placed on a supporting frame head up inclined at 30° angle. The upper, middle and bottom marks indicate ribs 1, 6 and 12, respectively. Cells mixed with matrigel are implanted using a 28G1/2 needle. A needle guard is used to control the depth. Panels C, E and G: Bioluminescence imaging in three animals 3 weeks after implantation. Panels D, F and H: Corresponding high-resolution digital image of the solitary nodule in the rat lung.

FIG. 3

Panel A: Bioluminescence signal 3 weeks after implantation. Panel B: Micro-CT coronal view, panel C: Micro-CT axial view, and panel D: bioluminescence imaging of the same animal after dissection. Panel E: Bioluminescence imaging of the dissected lung.

FIG. 4

Target validation after Image-guided radiation. Panel A: Normal lung tissue harvested 24 h after irradiation; panel B: tumor tissue harvested 24 h after irradiation; panel C: Unirradiated tumor tissue. In all panels: (i) vascular endothelium detected by mouse anti-rat CD31 antibody followed by Cy3-goat anti-mouse antibody (red), (ii) bavituximab detected using biotinylated goat anti-human IgG followed by Cy2-streptavidin (green), (iii) DNA detected by DAPI (blue), (iv) CD31, bavituximab, and DNA merge image. Panel D: Tumor immunofluorescence staining; merged image of DAPI and phospho-γ-H2AX (0 Gy, upper panel) and irradiated tumors (10 Gy, lower panel). Panel E: DNA-PKcs; panel F: Ki67; panel G, DNA-PKcs and Ki67 merge image; panel H: DNA-PKcs, Ki67 and DAPI merge image.

FIG. 5

Tumor progression after (panel A) no treatment and (panel B) delivery of 60 Gy in three fractions on days 20, 45 and 59. The heat scale is given in signal intensity per unit area. Panel C: Tumor growth curve obtained by integrating the bioluminescence imaging signal over a region of interest.