Orthotopic murine model of a primary malignant bone tumor in the spine: functional, bioluminescence, and histological correlations

Laboratory investigation

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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.

Abbreviation used in this paper:VB = vertebral body.

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.

Abbreviation used in this paper:VB = vertebral body.

The most common oncological condition affecting the spine is metastatic disease. To allow for animal research regarding this relatively common affliction, we previously described an orthotropic murine model of spinal metastases using a human cell line of lung adenocarcinoma.22 In this model, we validated a clinical assessment tool for evaluating neurological decline in mice with progressive spinal cord compression secondary to malignant disease. We identified tail dragging, dorsal stepping, hindlimb sweeping, and paralysis as reproducible and reliable milestones that correlated well with the degree of spinal canal obliteration on pathological evaluation.

Primary malignant tumors of the spine are much less common than spinal metastasis, corresponding to approximately 5% of all primary malignant bone tumors.6,20 Although several animal models of primary malignant bone tumors have been described,3,5,8,9,19,23 there are no animal models of any primary malignant bone tumor in the spine; therefore, none of them addresses the sequence of events between vertebral body (VB) invasion, dissemination to the epidural space, and compression of the spinal cord, which culminate in loss of neurological function.

To accurately evaluate therapeutic treatments, an animal model should mimic the human disease as closely as possible.2 The model should be reproducible, be developed from human cancer cells, and—in the setting of spinal tumors—the neurological decline should correlate with the severity of spinal cord compression. Thus far, no animal models with these characteristics exist for primary malignant tumors of the spine.

Osteosarcoma is the most common primary malignant tumor of bone11 and the third most common primary malignant tumor of the spine.6 Although aggressive surgical treatment of osteosarcoma is associated with improved survival,13,20 there remains a need for further development of preclinical models to develop new therapeutic approaches.16 We report the development of an orthotopic nude mouse model in which the human osteosarcoma cell line, Krib-1, was implanted directly into the VB. We demonstrate that the implanted mice subsequently experience a progressive neurological decline in a reproducible and predictable time frame correlating with increasing severity of spinal canal compression by pathological evaluation. Furthermore, we show the ability to monitor the local bio-luminescence of the implanted level and relate its increase with neurological decline. This provides the first reliable and reproducible orthotopic murine model of a human primary malignant bone tumor with functional and imaging correlates in the spine.

Methods

Animal Subjects

Thirty-five athymic nude mice (20–25 g) were obtained from the Department of Radiation Oncology, MD Anderson Cancer Center. The mice were housed in standard facilities, 5 mice per cage, with free access to water and rodent chow. During invasive procedures animals were anesthetized with a cocktail of ketamine hydrochloride (10 mg/ml) and xylazine (1 mg/ml) via an intraperitoneal injection at a volume of 0.1 ml/10 g of body weight. All animal manipulations were performed in accordance with institutional guidelines under Institutional Animal Care and Use Committee–approved protocols.

Cell Culture

The osteosarcoma cell line (Krib-1) was a gift from Dr. Valerae O. Lewis (MD Anderson Cancer Center) and was maintained in culture with RPMI 1640 Cellgro medium (Mediatech, Inc.) with 10% fetal bovine serum, penicillin (base 80.5 U/ml), and streptomycin (80.5 pg/ml) (all from Gibco BRL) in a humidified incubator with 5% CO2/95% room air at 37°C.

Establishment of Krib-1 Cells Expressing Luciferase

For bioluminescence assays, Krib-1 cells were grown to approximately 80% confluence and were transfected with the plasmid pEF1a-Luc-IRES-N, a gift from Dr. David Spencer (Baylor College of Medicine) using Lipofectamine 2000 (Invitrogen). Drug-resistant clones were selected using 500 μg/ml G418. Luciferase expression was verified using the Luciferase Assay System (Promega) and bioluminescence imaging.

Preparation of Tumor Cells for Implantation

Monolayers of Krib-1 cells were detached by trypsinization, washed, and suspended in phosphate-buffered saline at a concentration of 106 cells per 100 μl. These cells were then mixed with 100 μl of Matrigel, and approximately 5 μl of this solution was implanted within 5 hours of preparation.

Development of the Intervertebral Tumor

The method of implantation was the same as in the previously reported technique.22 The only modification of the technique was the use of Matrigel along with the osteosarcoma cells (see above) for implantation into the VB. The hole was then sealed with a polymethylmethacrylate plug (Stryker) as described by Mantha et al.10 All other aspects of the tumor implantation technique were unchanged.

Functional Assessment

Animals were observed daily for neurological function of their tail and hindlimbs. The interval between tumor implantation and development of each of the 4 milestones (tail dragging, dorsal stepping, hindlimb sweeping, and complete paralysis) was recorded for all of the animals. These key elements are components of the Basso-Beattie-Bresnahan scale1 and have been shown to be reliable neurological milestones in previous work.22

Mice were assessed daily by placing them individually in an open field and observing gait for 5 minutes. We used a stainless steel smooth floor surface, measuring 90 cm in diameter with a 15-cm wall height to obtain reproducible results. The day that each animal reached each milestone was recorded, counting implantation of the tumor into the spine as Day 1. The mean number of days ± SEM for reaching each of the 4 milestones was calculated. Animals were euthanized when paralysis occurred in accordance with institutional guidelines.

Tissue Processing

Three animals were euthanized at each of the following time points for histological analysis: asymptomatic, tail dragging, dorsal stepping, and hindlimb sweeping. In addition, 1 animal in the bioluminescence subgroup was euthanized at 10, 20, and 30 days for histological analysis. All of these animals were excluded from the functional and bioluminescence analysis, as they were used exclusively for confirmation of tumor progression. After being killed by CO2 inhalation according to our animal facility protocol, the spines were harvested, fixed in 10% formalin for 24 hours, and then placed in decalcifying solution for 1–2 days. When the bone was soft, the specimen was dissected with the aid of a surgical microscope and the poly-methylmethacrylate plug was carefully removed, so that the surrounding tissues would not be altered or damaged during cutting. The spines were embedded in paraffin, sectioned at a thickness of 10 μm, and stained with H & E.

Analysis of Neural Compression

Histological sections (H & E) were analyzed at a magnification of ×50. Imaging software (ImagePro, Media Cybernetics Inc.) was used to measure the area of the spinal canal (canal area) and the area of the spinal cord and nerve roots (neural area) at the level of tumor implantation. The ratio of the neural area to the canal area was used as a measure of neural compression. The mean ± SEM at each time point was plotted.

Bioluminescence Imaging and Quantification

Fifteen animals underwent implantation of Krib-1 cells stably transfected with a plasmid containing the firefly luciferase gene (Krib-1-Luc). Mice were imaged using the Xenogen IVIS 200 System (Xenogen Corp.) after administration of 4 mg (or 0.1 ml of 40 mg/ml) luciferin (intraperitoneal injection). Images were collected with 5 minutes of acquisition time. Bioluminescence color images were overlaid on gray-scale photographic images of the animals to allow for localization of the light source using the Living Image software overlay (version 2.11, Xenogen Corp.) and IGOR image analysis software (version 4.02 A, WaveMetrics, Inc.). To equalize comparisons across animals and between groups, the scale was fixed.

Regions of interest were manually selected, and signal intensity was expressed in terms of number of photons per square centimeter per second. Bioluminescence color images were obtained at postimplantation Days 10, 20, and 30. Histological confirmation of tumor growth was obtained at each time point correlating with the increase in bioluminescence in this subset of animals.

Statistical Analysis

The means and their standard errors (expressed as the mean ± SEM) were obtained. The Kaplan-Meier method was used to estimate the means and 95% CIs for all time-to-event variables. The spinal canal ratio and the bioluminescence values at Days 10, 20, and 30 were compared with one another to evaluate for any statistically significant differences between the 3 time points. The repeated measures general linear model and the Mauchly's test of sphericity were used for this task. Pairwise comparisons with Bonferroni correction were performed.

Results

We performed implantation of Krib-1 (osteosarcoma) cells in 20 animals. Of these, a predetermined subset of 12 mice (3 at Day 10, then 3 at each of the first 3 milestones) was euthanized to provide pathological confirmation of progressive disease and quantification of neural compression at each sequential milestone. Additional pathological confirmation was provided from the 8 mice that were euthanized upon reaching the final milestone of paralysis, and their spines were examined.

A second group of 15 mice had implantation of Krib-1 cells transfected with the luciferase gene (Krib-1-Luc). Of those, 3 mice were euthanized at 10, 20, and 30 days postimplantation for histological confirmation of tumor growth (1 mouse at each time point). Two mice died, likely of complications secondary to bladder distention and subsequent infection at Days 23 and 31 postimplantation. These animals were excluded from the analysis, with the exception of the animal that died at Day 31, which was included only in the bioluminescence analysis. Therefore, of these 15 mice, 11 animals underwent bioluminescence assessment at 10, 20, and 30 days, but only 10 animals reached all milestones, warranting inclusion in the functional analysis.

Functional Analysis

Functional analysis was performed in 8 animals implanted with Krib-1 cells and 10 mice implanted with Krib-1-Luc cells. There was no significant difference in the behavioral analysis of these 2 groups; therefore, we combined them to simplify the analysis. A total of 18 animals experienced all of the neurological milestones. The mean time to development of tail dragging was 19.06 days (95% CI 16.11–22.01), followed by dorsal stepping at a mean of 28.78 days (95% CI 26.79–30.77), then hindlimb sweeping at a mean of 35.61 days (95% CI 32.9–38.32), and paralysis at a mean of 41.78 days (95% CI 39.31–44.25); these data are presented in Table 1. All animals developed neurogenic bladder with urinary retention after exhibiting dorsal stepping and required manual expression of the bladder twice a day to drain urine.

TABLE 1:

Time frame for occurrence of key neurological events (milestones) reflecting progressive neurological decline

MilestoneMean Days to Milestone (95% CI)
tail dragging19.06 (16.11–22.01)
dorsal stepping28.78 (26.79–30.77)
hindlimb sweeping35.61 (32.9–38.32)
paralysis41.78 (39.31–44.25)

Histological Analysis: Qualitative Studies

To correlate functional decline with tumor growth, histological analyses were performed in 3 animals at 10 days postimplantation and subsequently at each neurological milestone and finally in all animals at the time of complete paralysis. The specimens were scored according to the degree of tumor infiltration in the VB (partial/complete), compression of the thecal sac (none/mild/moderate/severe), dural invasion (absent/present), and tumor extension (none, adjacent level epidural/adjacent level bone/extraspinal). These data are presented in Table 2.

TABLE 2:

Qualitative analysis of tumor infiltration at each milestone

MilestoneTumoral Infiltration of Vertebral BodyCompression of Thecal SacDural InvasionTumoral Extension
asymptomaticpartialnoneabsentnone
tail draggingpartialmildabsentadjacent-level epidural
dorsal steppingcompletemoderatepresentadjacent-level bone
hindlimb sweepingcompleteseverepresentadjacent-level bone
paralysiscompleteseverepresentextravertebral

Three animals killed 10 days after tumor implantation confirmed tumor engraftment. We observed partial involvement of the VB with normal trabecular bone preserved in most areas. Some early invasion of the epidural space was evident without compression of the nerve roots or thecal sac. At this early stage, the tumor remained localized to the vertebral level of implantation, and all animals were asymptomatic.

Specimens from the animals euthanized at the time of developing tail dragging demonstrated tumor cells destroying most of the trabecular bone of the VB. Infiltration of the posterior elements was present in one specimen, although the integrity of the cortical bone surface and shape of the spinous and transverse processes remained intact. Tumor was extending to the epidural space, and there was encasement of the nerve roots and minimal canal compromise. The epidural tumor was extending to adjacent levels, but no invasion of the adjacent bone was noted. These findings correlated with a subjective rating of “mild” cord compression at this milestone (Fig. 1A).

Fig. 1.
Fig. 1.

Photomicrographs of histological specimens depicting the invasion of Krib-1 implants into the spinal canal and correlative neurological examination. Photographs of mice at these time points are also shown. A: Tumor (Tu) partially infiltrates the VB and the epidural space with mild mass effect on the spinal cord. Photograph of a mouse obtained at neurological examination, which at this time typically reveals tail dragging (arrow). B: Tumor cells are infiltrating the VB, posterior elements, and epidural space, with moderate circumferential spinal cord compression. Photograph of a mouse. Mice at this stage typically present with dorsal stepping (arrowhead). C: Tumor has filled the spinal canal and is causing severe compression of the spinal cord. Photograph of a mouse obtained at neurological examination at this stage demonstrating hindlimb sweeping (double arrowhead) progressing rapidly to paralysis.

Histological analysis of the spines of the animals euthanized at the second milestone, dorsal stepping, showed an even greater amount of bone destruction and neural element compression (Fig. 1B). In these specimens the tumor had spread to both the epidural space and vertebral bodies of adjacent levels. In addition, we noted distortion of the thecal sac and significant destruction of the trabecular bone of the anterior and posterior elements, with early dural invasion and a small number of tumor cells within the subarachnoid space. This correlated with a subjective rating of “moderate” cord compression.

The 3 animals euthanized at the third milestone, hindlimb sweeping, showed complete destruction of the trabecular bone at the implanted VB and early extension of tumor to the paravertebral tissue (Fig. 1C). In addition, there was near-complete compression of the neural elements, including a greater component of focal leptomeningeal disease, correlating with a subjective rating of “severe” cord compression.

Finally, all 18 animals that reached the final milestone (complete paralysis) were euthanized at that time, providing ample histological specimens. All of these spines showed a similar pattern as observed in the third milestone, with significant compression of the spinal cord. However, a common feature in these specimens was extensive extraspinal infiltration with destruction of the paraspinal muscles and nerve roots.

We performed histological examination of the lungs and liver in a subset of animals that reached paralysis. In accordance with the bioluminescence images, no evidence of disseminated disease was found, confirming that the perivertebral and leptomeningeal infiltration represents local invasion of malignant cells.

Histological Analysis: Quantitative Studies

To quantify the degree of neural element compression by tumor, the area of the spinal canal (canal area) and the area of the identified spinal cord and nerve roots within the spinal canal (neural area) were measured in the specimens obtained from the animals in the following phases of neurological decline: asymptomatic, tail dragging, dorsal stepping, and hindlimb sweeping (n = 3 per milestone). The “neural/canal ratio” was calculated by dividing the neural area by the canal area. The mean ± SEM of these measurements was determined. We observed a strong trend for decrease in neural/canal ratio across the consecutive milestones. At the asymptomatic phase (10 days after tumor implantation), the neural/canal ratio was 0.61 ± 0.14, higher than 0.33 ± 0.08 when the first milestone (tail dragging) was reached. Analysis of the spines when the animals reached the second milestone (dorsal stepping) demonstrated a decrease in the neural canal ratio to 0.12 ± 0.03 when compared with the first milestone. When animals reached the third milestone (hindlimb sweeping), the spinal canal was obliterated by tumor, making the spinal cord and nerves difficult to identify. The mean ratio observed at this time (0.07 ± 0.02) was not significantly different from the one observed at the second milestone. These results are illustrated in Fig. 2.

Fig. 2.
Fig. 2.

Graph depicting the mean neural/canal ratio at the different milestones of the neurological examination. The ratio decreases from asymptomatic to hindlimb sweeping, corresponding to progressive tumor infiltration into the spinal canal and compression of the neural elements.

Evaluation of Tumor Growth by Bioluminescence

To determine the tumor engraftment and growth in the lumbar spine in a noninvasive manner, bioluminescence imaging of Krib-1-Luc cells was performed in 11 animals. As shown in Fig. 3, the light signal detected in the lumbar spine of the animals was qualitatively of greater intensity and size as time passed. We observed that at Day 20, most animals had tail dragging and at Day 30 all animals had dorsal stepping, correlating clinical decline with the increase in light emission by the tumor. The mean bioluminescence measured at all 3 time points was as follows: 2.17 photons/cm2/sec (range 0.0–8.61 photons/cm2/sec) at Day 10; 4.68 photons/cm2/sec (range 1.17–8.52 photons/cm2/sec) at Day 20; and 5.54 photons/cm2/sec (range 1.22–9.99 photons/cm2/sec) at Day 30 (Fig. 4). The pairwise comparisons show that there is a statistically significant difference between the measurements of Day 10 and Day 20 (p = 0.043) and those of Day 10 and Day 30 (p = 0.031), but not between Day 20 and Day 30 (p = 0.546).

Fig. 3.
Fig. 3.

Photographs of the 11 animals implanted with Krib-1-Luc, demonstrating an increase in the bioluminescence at the level of the lumbar spines at 10, 20, and 30 days after tumor implantation.

Fig. 4.
Fig. 4.

Graph depicting the mean bioluminescence at 10, 20, and 30 days after tumor implantation. The bioluminescence increases from Day 10 to Day 30, corresponding to progressive tumor growth.

Discussion

We present the first orthotopic murine model of a human primary malignant bone tumor in the spine with functional, histological, and imaging correlates. We demonstrate that neurological decline reproducibly coincides with tumor progression as evidenced by qualitative pathological confirmation, quantitative evaluation of compression of the neural elements within the spinal canal, and noninvasive bioluminescence measurements. This model provides several advantages when compared with our previously described technique.22 First, the implantation of tumor cells mixed with Matrigel into the VB keeps the tumor cells together and avoids the use of a donor animal to harvest a fragment of tumor. Second, we simplified the neurological assessment by utilizing 4 milestones of the neurological examination correlating with decline in function. Third, we introduced the use of bioluminescence for tumor growth tracking, allowing in vivo confirmation of tumor engraftment and progression via a noninvasive technique.

The reliable and reproducible stepwise neurological decline observed in our model closely mimics the sequential neurological compromise that would occur in humans with a primary malignancy of the spine that was permitted to progress without treatment. The 4 milestones occurred at distinct and highly reproducible time points without overlapping confidence intervals (Table 1). This is critical when developing and testing novel treatment strategies, as delays in the development of these milestones can be a measure of therapeutic efficacy.

Our pathological analysis confirmed that the stepwise neurological decline correlated with increasing tumor infiltration, progressing from the tumor contained within the VB to compression of the thecal sac, rostrocaudal extension of disease to adjacent levels, and finally invasion of the actual dura (Fig. 1). Not only did the neurological decline correlate with this qualitative assessment of pathological progression but also with quantitatively verified compromise of the spinal canal and increasing compression of the neural elements (Fig. 2). The correlation of tumor growth, invasion, and development of neurological decline is pertinent to the human condition, since it is known that extent of local invasion has previously been shown to correlate with decreased overall survival in patients with primary malignant spine tumors.12

Animal models that use bioluminescence imaging have allowed for advancements in the field of cancer therapy.4,15,18,21 Researchers have found that when compared with measuring tumor volume, bioluminescence imaging allows for earlier tumor detection and recognition of cell death in response to chemotherapeutic agents.14,16

We believe that addition of the bioluminescence testing capability is a valuable adjunct to our model, since tumor growth can be tracked in vivo without requiring the euthanization of the animal for histopathological analysis.7 Therefore, we propose that our model can be used to test therapies in different stages of disease. Therapeutic interventions aiming at prevention of bone destruction or tumoral spread to adjacent levels could be started after confirmation of tumor engraftment with bioluminescence and before the development of tail dragging. Treatment strategies directed against decompression of the nervous tissue, prevention of neurological decline, or reduction of tumor burden could start after the development of tail dragging. In this case, therapeutic response could be monitored by sequential bioluminescence imaging and careful evaluation of the neurological examination, timing the development of the next milestone. Salvage therapies could be tested at the moment of occurrence of the third milestone (hindlimb sweeping), and again bioluminescence testing can be undertaken to document the intensity and size of the light signal to monitor eventual tumor regression.

Another advantage of our orthotopic model of human osteosarcoma is the possibility to investigate the complex interactions between the tumor cells and the bony microenvironment, including the osteolytic destruction, bone marrow infiltration, and patterns of dissemination. These appear to closely mimic the pattern of disease progression in human patients.

One of the most interesting findings in our model was the occurrence of dural invasion and development of local leptomeningeal disease. It is well known that local leptomeningeal disease can occur with malignant sarcomas, and the dural invasion documented in the early symptomatic phase of our model could be a potential explanation of why spinal osteosarcomas frequently recur, even after en bloc resections.17 It is possible that our model reflects the natural history of human osteosarcomas, since the real incidence of leptomeningeal disease is unknown in patients with advanced disease in terminal stages. A better understanding of the mechanisms of dural invasion must be investigated since it may have an important impact on the development of novel therapeutic approaches and influence the manner in which en bloc resections are performed in such cases.

Although developed for osteosarcoma, this model can easily be expanded to study other types of primary malignant bone tumors in the spine. The possibility of using human cell lines will allow better animal testing of targeted therapies against specific primary malignant tumor types, which will further advance our ability to effectively treat patients with this disease.

Conclusions

We have developed the first orthotopic murine model of a primary malignant bone tumor in the spine. In this model, neurological decline progresses in a reproducible fashion that correlates with tumor progression as confirmed by qualitative histological analysis and quantitatively verified compression of the neural elements. The neurological decline and pathological progressing also correlates with increasing bioluminescence at the implanted level. This new model can potentially be expanded to other primary malignant bone tumors of the spine, allowing for animal testing of targeted therapies tailored to specific tumor types.

Disclosure

Dr. Rhines is a consultant for Stryker.

Author contributions to the study and manuscript preparation include the following. Conception and design: Rhines, Fahim, Tatsui, Lang. Acquisition of data: Fahim, Tatsui, Gumin. Analysis and interpretation of data: Rhines, Fahim, Tatsui, Suki, Lang. Drafting the article: Fahim, Tatsui. Critically revising the article: Rhines, Fahim, Tatsui, Suki, Lang. Reviewed submitted version of manuscript: Rhines, Fahim, Tatsui, Suki, Gumin. Approved the final version of the manuscript on behalf of all authors: Rhines. Statistical analysis: Suki. Administrative/technical/material support: Gumin. Study supervision: Rhines, Lang.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

References

  • 1

    Basso DMBeattie MSBresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1211995

    • Search Google Scholar
    • Export Citation
  • 2

    Céspedes MVCasanova IParreño MMangues R: Mouse models in oncogenesis and cancer therapy. Clin Transl Oncol 8:3183292006

  • 3

    Comstock KEHall CLDaignault SMandlebaum SAYu CKeller ET: A bioluminescent orthotopic mouse model of human osteosarcoma that allows sensitive and rapid evaluation of new therapeutic agents in vivo. In Vivo 23:6616682009

    • Search Google Scholar
    • Export Citation
  • 4

    Cronin MAkin ARCollins SAMeganck JKim JBBaban CK: High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting. PLoS ONE 7:e309402012

    • Search Google Scholar
    • Export Citation
  • 5

    Gomes CMWelling MQue IHenriquez NVvan der Pluijm GRomeo S: Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi. Eur J Nucl Med Mol Imaging 34:179318032007

    • Search Google Scholar
    • Export Citation
  • 6

    Kelley SPAshford RURao ASDickson RA: Primary bone tumours of the spine: a 42-year survey from the Leeds Regional Bone Tumour Registry. Eur Spine J 16:4054092007

    • Search Google Scholar
    • Export Citation
  • 7

    Klerk CPOvermeer RMNiers TMVersteeg HHRichel DJBuckle T: Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals. Biotechniques 43:1 Suppl71330:2007

    • Search Google Scholar
    • Export Citation
  • 8

    Labrinidis AHay SLiapis VPonomarev VFindlay DMEvdokiou A: Zoledronic acid inhibits both the osteolytic and osteoblastic components of osteosarcoma lesions in a mouse model. Clin Cancer Res 15:345134612009

    • Search Google Scholar
    • Export Citation
  • 9

    Lamoureux FPicarda GRousseau JGourden CBattaglia SCharrier C: Therapeutic efficacy of soluble receptor activator of nuclear factor-kappa B-Fc delivered by nonviral gene transfer in a mouse model of osteolytic osteosarcoma. Mol Cancer Ther 7:338933982008

    • Search Google Scholar
    • Export Citation
  • 10

    Mantha ALegnani FGBagley CAGallia GLGaronzik IPradilla G: A novel rat model for the study of intraosseous metastatic spine cancer. J Neurosurg Spine 2:3033072005

    • Search Google Scholar
    • Export Citation
  • 11

    Miretti SRoato ITaulli RPonzetto CCilli MOlivero M: A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by Luciferase imaging. PLoS One 3:e18282008

    • Search Google Scholar
    • Export Citation
  • 12

    Mukherjee DChaichana KLAdogwa OGokaslan ZAaronson OCheng JS: Association of extent of local tumor invasion and survival in patients with malignant primary osseous spinal neoplasms from the surveillance, epidemiology, and end results (SEER) database. World Neurosurg 76:5805852011

    • Search Google Scholar
    • Export Citation
  • 13

    Rao GSuki DChakrabarti IFeiz-Erfan IMody MGMc-Cutcheon IE: Surgical management of primary and metastatic sarcoma of the mobile spine. J Neurosurg Spine 9:1201282008

    • Search Google Scholar
    • Export Citation
  • 14

    Rehemtulla AStegman LDCardozo SJGupta SHall DEContag CH: Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2:4914952000

    • Search Google Scholar
    • Export Citation
  • 15

    Rosol TJTannehill-Gregg SHLeRoy BEMandl SContag CH: Animal models of bone metastasis. Cancer 97:3 Suppl7487572003

  • 16

    Rousseau JEscriou VPerrot PPicarda GCharrier CScherman D: Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in osteosarcoma preclinical models. Cancer Gene Ther 17:3873972010

    • Search Google Scholar
    • Export Citation
  • 17

    Schwab JGasbarrini ABandiera SBoriani LAmendola LPicci P: Osteosarcoma of the mobile spine. Spine (Phila Pa 1976) 37:E381E3862012

    • Search Google Scholar
    • Export Citation
  • 18

    Song HTJordan EKLewis BKLiu WGanjei JKlaunberg B: Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation. J Transl Med 7:882009

    • Search Google Scholar
    • Export Citation
  • 19

    Sottnik JLDuval DLEhrhart EJThamm DH: An orthotopic, postsurgical model of luciferase transfected murine osteosarcoma with spontaneous metastasis. Clin Exp Metastasis 27:1511602010

    • Search Google Scholar
    • Export Citation
  • 20

    Sundaresan NRosen GBoriani S: Primary malignant tumors of the spine. Orthop Clin North Am 40:2136v2009

  • 21

    Szentirmai OBaker CHLin NSzucs STakahashi MKiryu S: Noninvasive bioluminescence imaging of luciferase expressing intracranial U87 xenografts: correlation with magnetic resonance imaging determined tumor volume and longitudinal use in assessing tumor growth and antiangiogenic treatment effect. Neurosurgery 58:3653722006

    • Search Google Scholar
    • Export Citation
  • 22

    Tatsui CELang FFGumin JSuki DShinojima NRhines LD: An orthotopic murine model of human spinal metastasis: histological and functional correlations. Laboratory investigation. J Neurosurg Spine 10:5015122009

    • Search Google Scholar
    • Export Citation
  • 23

    Zhou ZGuan HDuan XKleinerman ES: Zoledronic acid inhibits primary bone tumor growth in Ewing sarcoma. Cancer 104:171317202005

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Article Information

Contributor Notes

Drs. Fahim and Tatsui contributed equally to this work.

Address correspondence to: Laurence D. Rhines, M.D., Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd. Unit 442, Houston, TX 77030. email: lrhines@mdanderson.org.Please include this information when citing this paper: published online June 27, 2014; DOI: 10.3171/2014.5.SPINE13205.

© Copyright 1944-2019 American Association of Neurological Surgeons

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Figures
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    Photomicrographs of histological specimens depicting the invasion of Krib-1 implants into the spinal canal and correlative neurological examination. Photographs of mice at these time points are also shown. A: Tumor (Tu) partially infiltrates the VB and the epidural space with mild mass effect on the spinal cord. Photograph of a mouse obtained at neurological examination, which at this time typically reveals tail dragging (arrow). B: Tumor cells are infiltrating the VB, posterior elements, and epidural space, with moderate circumferential spinal cord compression. Photograph of a mouse. Mice at this stage typically present with dorsal stepping (arrowhead). C: Tumor has filled the spinal canal and is causing severe compression of the spinal cord. Photograph of a mouse obtained at neurological examination at this stage demonstrating hindlimb sweeping (double arrowhead) progressing rapidly to paralysis.

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    Graph depicting the mean neural/canal ratio at the different milestones of the neurological examination. The ratio decreases from asymptomatic to hindlimb sweeping, corresponding to progressive tumor infiltration into the spinal canal and compression of the neural elements.

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    Photographs of the 11 animals implanted with Krib-1-Luc, demonstrating an increase in the bioluminescence at the level of the lumbar spines at 10, 20, and 30 days after tumor implantation.

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    Graph depicting the mean bioluminescence at 10, 20, and 30 days after tumor implantation. The bioluminescence increases from Day 10 to Day 30, corresponding to progressive tumor growth.

References
  • 1

    Basso DMBeattie MSBresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1211995

    • Search Google Scholar
    • Export Citation
  • 2

    Céspedes MVCasanova IParreño MMangues R: Mouse models in oncogenesis and cancer therapy. Clin Transl Oncol 8:3183292006

  • 3

    Comstock KEHall CLDaignault SMandlebaum SAYu CKeller ET: A bioluminescent orthotopic mouse model of human osteosarcoma that allows sensitive and rapid evaluation of new therapeutic agents in vivo. In Vivo 23:6616682009

    • Search Google Scholar
    • Export Citation
  • 4

    Cronin MAkin ARCollins SAMeganck JKim JBBaban CK: High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting. PLoS ONE 7:e309402012

    • Search Google Scholar
    • Export Citation
  • 5

    Gomes CMWelling MQue IHenriquez NVvan der Pluijm GRomeo S: Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi. Eur J Nucl Med Mol Imaging 34:179318032007

    • Search Google Scholar
    • Export Citation
  • 6

    Kelley SPAshford RURao ASDickson RA: Primary bone tumours of the spine: a 42-year survey from the Leeds Regional Bone Tumour Registry. Eur Spine J 16:4054092007

    • Search Google Scholar
    • Export Citation
  • 7

    Klerk CPOvermeer RMNiers TMVersteeg HHRichel DJBuckle T: Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals. Biotechniques 43:1 Suppl71330:2007

    • Search Google Scholar
    • Export Citation
  • 8

    Labrinidis AHay SLiapis VPonomarev VFindlay DMEvdokiou A: Zoledronic acid inhibits both the osteolytic and osteoblastic components of osteosarcoma lesions in a mouse model. Clin Cancer Res 15:345134612009

    • Search Google Scholar
    • Export Citation
  • 9

    Lamoureux FPicarda GRousseau JGourden CBattaglia SCharrier C: Therapeutic efficacy of soluble receptor activator of nuclear factor-kappa B-Fc delivered by nonviral gene transfer in a mouse model of osteolytic osteosarcoma. Mol Cancer Ther 7:338933982008

    • Search Google Scholar
    • Export Citation
  • 10

    Mantha ALegnani FGBagley CAGallia GLGaronzik IPradilla G: A novel rat model for the study of intraosseous metastatic spine cancer. J Neurosurg Spine 2:3033072005

    • Search Google Scholar
    • Export Citation
  • 11

    Miretti SRoato ITaulli RPonzetto CCilli MOlivero M: A mouse model of pulmonary metastasis from spontaneous osteosarcoma monitored in vivo by Luciferase imaging. PLoS One 3:e18282008

    • Search Google Scholar
    • Export Citation
  • 12

    Mukherjee DChaichana KLAdogwa OGokaslan ZAaronson OCheng JS: Association of extent of local tumor invasion and survival in patients with malignant primary osseous spinal neoplasms from the surveillance, epidemiology, and end results (SEER) database. World Neurosurg 76:5805852011

    • Search Google Scholar
    • Export Citation
  • 13

    Rao GSuki DChakrabarti IFeiz-Erfan IMody MGMc-Cutcheon IE: Surgical management of primary and metastatic sarcoma of the mobile spine. J Neurosurg Spine 9:1201282008

    • Search Google Scholar
    • Export Citation
  • 14

    Rehemtulla AStegman LDCardozo SJGupta SHall DEContag CH: Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2:4914952000

    • Search Google Scholar
    • Export Citation
  • 15

    Rosol TJTannehill-Gregg SHLeRoy BEMandl SContag CH: Animal models of bone metastasis. Cancer 97:3 Suppl7487572003

  • 16

    Rousseau JEscriou VPerrot PPicarda GCharrier CScherman D: Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in osteosarcoma preclinical models. Cancer Gene Ther 17:3873972010

    • Search Google Scholar
    • Export Citation
  • 17

    Schwab JGasbarrini ABandiera SBoriani LAmendola LPicci P: Osteosarcoma of the mobile spine. Spine (Phila Pa 1976) 37:E381E3862012

    • Search Google Scholar
    • Export Citation
  • 18

    Song HTJordan EKLewis BKLiu WGanjei JKlaunberg B: Rat model of metastatic breast cancer monitored by MRI at 3 tesla and bioluminescence imaging with histological correlation. J Transl Med 7:882009

    • Search Google Scholar
    • Export Citation
  • 19

    Sottnik JLDuval DLEhrhart EJThamm DH: An orthotopic, postsurgical model of luciferase transfected murine osteosarcoma with spontaneous metastasis. Clin Exp Metastasis 27:1511602010

    • Search Google Scholar
    • Export Citation
  • 20

    Sundaresan NRosen GBoriani S: Primary malignant tumors of the spine. Orthop Clin North Am 40:2136v2009

  • 21

    Szentirmai OBaker CHLin NSzucs STakahashi MKiryu S: Noninvasive bioluminescence imaging of luciferase expressing intracranial U87 xenografts: correlation with magnetic resonance imaging determined tumor volume and longitudinal use in assessing tumor growth and antiangiogenic treatment effect. Neurosurgery 58:3653722006

    • Search Google Scholar
    • Export Citation
  • 22

    Tatsui CELang FFGumin JSuki DShinojima NRhines LD: An orthotopic murine model of human spinal metastasis: histological and functional correlations. Laboratory investigation. J Neurosurg Spine 10:5015122009

    • Search Google Scholar
    • Export Citation
  • 23

    Zhou ZGuan HDuan XKleinerman ES: Zoledronic acid inhibits primary bone tumor growth in Ewing sarcoma. Cancer 104:171317202005

    • Search Google Scholar
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