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  • Author or Editor: Frederick F. Lang x
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Claudio E. Tatsui, Frederick F. Lang, Joy Gumin, Dima Suki, Naoki Shinojima and Laurence D. Rhines

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.

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Daniel K. Fahim, Claudio E. Tatsui, Dima Suki, Joy Gumin, Frederick F. Lang and Laurence D. Rhines

Object

There is currently no reproducible animal model of human primary malignant bone tumors in the spine to permit laboratory investigation of the human disease. Therefore, the authors sought to adapt their previously developed orthotopic model of spinal metastasis to a model for primary malignant bone tumors of the spine.

Methods

A transperitoneal surgical approach was used to implant osteosarcoma (Krib-1) into the L-3 vertebral body of nude mice via a drill hole. Motor function was evaluated daily using the previously validated qualitative key milestones of tail dragging, dorsal stepping, hindlimb sweeping, and paralysis. A subset of these animals was euthanized upon reaching the various milestones, and the spines were removed, sectioned, and stained. The degree of spinal cord compression was correlated with the occurrence of milestones and assessed by a ratio between the neural elements divided by the area of the spinal canal. Another subset of animals received stably transfected Krib-1 cells with the luciferase gene, and bioluminescence was measured at 10, 20, and 30 days postimplantation.

Results

Osteosarcoma xenografts grew in all animals according to a reliable and reproducible time course; the mean time for development of behavioral milestones was noted in relation to the day of implantation (Day 1). Tail dragging (Milestone 1) occurred on Day 19.06 (95% CI 16.11–22.01), dorsal stepping (Milestone 2) occurred on Day 28.78 (95% CI 26.79–30.77), hindlimb sweeping (Milestone 3) occurred on Day 35.61 (95% CI 32.9–38.32), and paralysis of the hindlimb (Milestone 4) occurred on Day 41.78 (95% CI 39.31–44.25). These clinically observed milestones correlated with increasing compression of the spinal cord on histological sections. The authors observed a progressive increase in the local bioluminescence (in photons/cm2/sec) of the implanted level over time with a mean of 2.17 (range 0.0–8.61) at Day 10, mean 4.68 (range 1.17–8.52) at Day 20, and mean 5.54 (range 1.22–9.99) at Day 30.

Conclusions

The authors have developed the first orthotopic murine model of a primary malignant bone tumor in the spine, in which neurological decline reproducibly correlates with tumor progression as evidenced by pathological confirmation and noninvasive bioluminescence measurements. Although developed for osteosarcoma, this model can be expanded to study other types of primary malignant bone tumors in the spine. This model will potentially allow animal testing of targeted therapies against specific primary malignant tumor types.