An orthotopic murine model of human spinal metastasis: histological and functional correlations

Laboratory investigation

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Object

There is currently no reproducible animal model of human spinal metastasis that allows for laboratory study of the human disease. Consequently, the authors sought to develop an orthotopic model of spinal metastasis by using a human lung cancer cell line, and to correlate neurological decline with tumor growth.

Methods

To establish a model of spinal metastasis, the authors used a transperitoneal surgical approach to implant PC-14 lung tumors into the L-3 vertebral body of nude mice via a drill hole. In 24 animals, motor function was scored daily by using the validated semiquantitative Basso-Beattie-Bresnahan (BBB) scale. A second group of 26 animals (6 or 7 per time point) were sacrificed at specific times, and the spines were removed, sectioned, and stained. Canal compromise was analyzed quantitatively by determining the ratio of the area of the neural elements to the area of the spinal canal on histological sections (neural/canal ratio). Correlations between BBB score and histological evaluation of tumor growth were assessed.

Results

Lung cancer xenografts grew in all animals undergoing functional evaluation (24 mice) according to a reliable and reproducible time course, with paraplegia occurring at a median interval of 30 days following tumor implantation (95% CI 28.1–31.9 days). Importantly, the analysis defined 4 key milestones based on components of the BBB score; these were observed in all animals, were consistent, and correlated with histological progression of tumor. From Days 1 to 14, the mean BBB score declined from 21 to 19. The animals progressed from normal walking with the tail up to walking with the tail constantly touching the ground (milestone 1). The median time to tail dragging was 12 days (95% CI 10.8–13.2). Histological studies on Day 14 demonstrated that tumor had progressed from partial to complete VB infiltration, with initial compression of the neural elements and epidural tumor extension to adjacent levels (mean neural/canal ratio 0.32 ± 0.05, 7 mice). From Days 15 to 20/21 (left/right leg), the mean BBB score declined from 19 to 14. Animals showed gait deterioration, with the development of dorsal stepping (milestone 2). The median time to dorsal stepping was 21 days (95% CI 19.4–22.6) in the left hindlimb and 23 days (95% CI 20.6–25.4) in the right hindlimb. Histological studies on Day 21 demonstrated an increase in the severity of the neural element compression, with tumor extending to adjacent epidural and osseous levels (mean neural/canal ratio 0.19 ± 0.05, 6 mice). From Days 22 to 26/27 (left/right leg), the mean BBB score declined from 14 to 8. Animals had progressive difficulty ambulating, to the point where they showed only sweeping movements of the hindlimb (milestone 3). The median time to hindlimb sweeping was 26 days (95% CI 23.6–28.4) and 28 days (95% CI 27.1–28.9) in the left and right hindlimbs, respectively. Histological studies on Day 28 revealed progressive obliteration of the spinal canal (mean neural/canal ratio 0.09 ± 0.01, 7 mice). From Days 29 to 36, the animals progressed to paralysis (milestone 4). The median time to paralysis was 29 days (95% CI 27.6–30.4) and 30 days (95% CI 28.1–31.9) in the left and right hindlimbs, respectively.

Conclusions

The authors have developed an orthotopic murine model of human spinal metastasis in which neurological decline reproducibly correlates with severity of tumor progression. Although developed for lung cancer, this model can be expanded to study other types of metastatic or primary spinal tumors. Ultimately, this will allow testing of targeted therapies against specific tumor types.

Abbreviations used in this paper: BBB = Basso-Beattie-Bresnahan; BMS = Basso Mouse Scale; IVP = internal venous plexus; PMMA = polymethylmethacrylate; VB = vertebral body.

Article Information

Address correspondence to: Frederick F. Lang, M.D., Department of Neurosurgery, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 442, Houston, Texas. 77030. email: flang@mdanderson.org.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Intraoperative photograph showing landmarks for exposure of the L-3 VB. The retroperitoneal space was opened between the left kidney and the vascular bundle, exposing the left psoas muscle. The L3–4 intervertebral disc is located in a plane caudal to the inferior pole of the left kidney (white dashed line).

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    Graph depicting the mean BBB scores of the mice over time. The mean BBB score was calculated each day and plotted against time to show the trend in neurological impairment. These daily averages are distinct from the median times to each of the 4 milestones shown in Table 2, which were determined using the Kaplan-Meier method.

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    Photomicrographs of histological specimens depicting the invasion of PC-14 implants into the spinal canal. A: Sagittal section of a normal vertebral level demonstrating the muscle layer anterior to the VB and a normal venous foramen with a patent IVP. B: Higher magnification of the inset in panel A, demonstrating red blood cells within a normal venous foramen and IVP. C: Section showing PC-14 cells infiltrating the VB 7 days after implantation. Tumor cells are seen within the venous foramen and occluding the IVP. D: Higher magnification of the inset in panel C, demonstrating tumor cells occluding the IVP. E: Sagittal section demonstrating a normal level (upper part of the panel) containing healthy bone marrow, and a level completely colonized by PC-14 cells (lower part of the panel). Note the extension of tumor cells through the venous foramen and IVP, forming an early epidural mass. F: Tumor cells initially replace the bone marrow, and only gradually alter the architecture of the trabecular bone. H & E, original magnification × 50 (A, C, and E); × 200 (B and D); × 100 (F). Tu = tumor; VF = venous foramen.

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    Photomicrographs of histological specimens depicting the method used to calculate the neural/canal ratio. A: Axial section of a specimen harvested 14 days after tumor implantation, with the area of the spinal canal outlined. B: Specimen from panel A, with the area of the spinal cord and nerve roots outlined. Note that the tumor is deforming the round contour of the neural elements. C: Axial section of a specimen harvested 28 days after tumor implantation, with the area of the spinal canal outlined. D: Specimen from panel C, with the area of the spinal cord and nerve roots outlined. Note that the epidural tumor is severely compressing the neural elements. H & E, original magnification × 50. AR = area (in square millimeters); N = neural elements; T = tumor.

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    Graph depicting the mean neural/canal ratio, with the SEM represented by error bars, at 7, 14, 21, and 28 days after tumor implantation. The ratio decreases from Day 7 to Day 28, corresponding to progressive tumor infiltration into the spinal canal and compression of the neural elements.

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    Photomicrographs showing histological specimens obtained at Day 7 after tumor implantation. A: In this specimen, tumor is infiltrating the epidural space, surrounding the nerve roots, but causing no mass effect on the spinal cord. B: Note that the venous foramen is invaded by tumor cells, enabling access to the epidural space. C: In this axial cut, the tumor is less advanced. There is partial infiltration of the VB. D: Higher magnification of the inset in panel C, showing a small venous foramen crossing the posterior cortex of the vertebra. These channels provide an access route for tumor cells in the marrow to reach the epidural space. H & E, original magnification × 100 (A and B); × 50 (C); × 200 (D). CE = cauda equina; CM = conus medullaris; NR = nerve root.

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    Photomicrographs showing histological specimens obtained at Day 14 after tumor implantation, and photograph of a mouse at milestone 1. A: Tumor cells are infiltrating the VB, posterior elements, and epidural space, with mild circumferential spinal cord compression. B: Sagittal section cut at the level caudal to the tumor implantation, demonstrating the extensive epidural disease. H & E, original magnification × 50 (A and B). C: Photograph of mouse obtained at neurological examination, which at this time typically reveals tail dragging (arrow). SC = spinal cord; SP = spinous process.

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    Photomicrographs showing histological specimens obtained at 21 days after tumor implantation, and photograph of a mouse at milestone 2. A: Tumor is infiltrating the VB, posterior elements, and epidural space with severe circumferential spinal cord compression. B: Sagittal section cut at a level caudal to the site of tumor implantation, showing infiltration into the adjacent VBs. H & E, original magnification × 50 (A and B). C: Photograph of mouse obtained at neurological examination; at this stage, functional assessment of the animals is expected to reveal dorsal stepping (arrow). Disc = intervertebral disc.

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    Photomicrograph showing histological specimens obtained at 28 days after tumor implantation, and photographs of mice at milestones 3 and 4. A: Tumor has filled the spinal canal and obliterated the spinal cord. H & E, original magnification × 50. B: Photographs of mice obtained at neurological examination; the predicted neurological findings at this stage are hindlimb sweeping (arrowhead) progressing rapidly to paralysis (arrow).

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