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In vivo bioluminescence imaging for leptomeningeal dissemination of medulloblastoma in mouse models

View Article: PubMed Central - PubMed

ABSTRACT

Background: The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images.

Methods: Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding.

Results: The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 106 UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients.

Conclusion: The results and described method are valuable resources for further translational research to overcome MB seeding.

No MeSH data available.


Related in: MedlinePlus

The intracisternal injection and intracerebellar injection methods. (A) BLI of mice with cell injection into the cisterna magna show that the signals are observed at day 0, expand at day 6, and begin to spread to the spinal cord at day 9. (B) The signals were detected first at day 6 and gradually migrated to the spinal cord from day 12 in mice with cells injected into the cerebellum. (C) BLI quantification of tumor-occupied areas during the study. (D) The median survival was 22 and 34 days in the intracisternal injection model and intracerebellar injection model, respectively. (E) Histopathology of xenograft MB seeding. Inlet figures denote the subfrontal area (a), cerebellum (b), upper thoracic spinal cord (c), lower thoracic spinal cord (d), and conus medullaris (e). The intracisternal injection model displays strong similarity to the histopathological character and widespread dissemination pattern of MB seeding. (F) Representative immunofluorescence images (DAPI: blue, Human Nuclei: green, Ki-67: red, Merge: yellow) show that both the intracisternal- and intracerebellar-injected UW426-effLuc cells are highly proliferative in vivo. Scale bars represent 50 μm
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Fig4: The intracisternal injection and intracerebellar injection methods. (A) BLI of mice with cell injection into the cisterna magna show that the signals are observed at day 0, expand at day 6, and begin to spread to the spinal cord at day 9. (B) The signals were detected first at day 6 and gradually migrated to the spinal cord from day 12 in mice with cells injected into the cerebellum. (C) BLI quantification of tumor-occupied areas during the study. (D) The median survival was 22 and 34 days in the intracisternal injection model and intracerebellar injection model, respectively. (E) Histopathology of xenograft MB seeding. Inlet figures denote the subfrontal area (a), cerebellum (b), upper thoracic spinal cord (c), lower thoracic spinal cord (d), and conus medullaris (e). The intracisternal injection model displays strong similarity to the histopathological character and widespread dissemination pattern of MB seeding. (F) Representative immunofluorescence images (DAPI: blue, Human Nuclei: green, Ki-67: red, Merge: yellow) show that both the intracisternal- and intracerebellar-injected UW426-effLuc cells are highly proliferative in vivo. Scale bars represent 50 μm

Mentions: Intracisternal (1.2 × 106 cells, N = 7) and intracerebellar injections (1.2 × 105 cells, N = 5) using UW426-effLuc cells were performed. In mice that had injections into the cisterna magna, BLI signals were observed at day 0 and gradually spread along the spinal axis over time [day 0: (0.91 ± 0.49) × 106 p/s/cm2/sr; day 16, (4.43 ± 0.76) × 106 p/s/cm2/sr; Fig. 4a and c]. However, in mice that had injections into the cerebellar hemisphere, the signals were detected on day 9 [(0.16 ± 0.19) × 106 p/s/cm2/sr], suddenly increased and migrated to the spinal cord from day 16 [(1.11 ± 0.21) × 106 p/s/cm2/sr; Fig. 4b and c]. The median survival was 22 days for the intracisternal injection model and 34 days for the intracerebellar injection model (Fig. 4d). To analyze the histopathology, mice that had injections into the cisterna magna (N = 5) were sacrificed at 22 days and mice that had injections into the cerebellar hemisphere (N = 5) were sacrificed at 35 days. In the intracisternal injection model, tumor cells spread in the entire neuraxis from the subfrontal area and cerebellar surface to the distal spinal cord. In the intracerebellar injection model, tumor cells aggregated in the cerebellum and did not disseminate into the subfrontal area or distal spinal cord in some animals examined (Fig. 4e). Table 2 summarizes the dissemination pattern of each cell injection method. Immunofluorescence staining revealed that abundant human nuclei and Ki67 double positive cells were observed in mice that had injections into the cisterna magna or cerebellar hemisphere (Fig. 4f).Fig. 4


In vivo bioluminescence imaging for leptomeningeal dissemination of medulloblastoma in mouse models
The intracisternal injection and intracerebellar injection methods. (A) BLI of mice with cell injection into the cisterna magna show that the signals are observed at day 0, expand at day 6, and begin to spread to the spinal cord at day 9. (B) The signals were detected first at day 6 and gradually migrated to the spinal cord from day 12 in mice with cells injected into the cerebellum. (C) BLI quantification of tumor-occupied areas during the study. (D) The median survival was 22 and 34 days in the intracisternal injection model and intracerebellar injection model, respectively. (E) Histopathology of xenograft MB seeding. Inlet figures denote the subfrontal area (a), cerebellum (b), upper thoracic spinal cord (c), lower thoracic spinal cord (d), and conus medullaris (e). The intracisternal injection model displays strong similarity to the histopathological character and widespread dissemination pattern of MB seeding. (F) Representative immunofluorescence images (DAPI: blue, Human Nuclei: green, Ki-67: red, Merge: yellow) show that both the intracisternal- and intracerebellar-injected UW426-effLuc cells are highly proliferative in vivo. Scale bars represent 50 μm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016924&req=5

Fig4: The intracisternal injection and intracerebellar injection methods. (A) BLI of mice with cell injection into the cisterna magna show that the signals are observed at day 0, expand at day 6, and begin to spread to the spinal cord at day 9. (B) The signals were detected first at day 6 and gradually migrated to the spinal cord from day 12 in mice with cells injected into the cerebellum. (C) BLI quantification of tumor-occupied areas during the study. (D) The median survival was 22 and 34 days in the intracisternal injection model and intracerebellar injection model, respectively. (E) Histopathology of xenograft MB seeding. Inlet figures denote the subfrontal area (a), cerebellum (b), upper thoracic spinal cord (c), lower thoracic spinal cord (d), and conus medullaris (e). The intracisternal injection model displays strong similarity to the histopathological character and widespread dissemination pattern of MB seeding. (F) Representative immunofluorescence images (DAPI: blue, Human Nuclei: green, Ki-67: red, Merge: yellow) show that both the intracisternal- and intracerebellar-injected UW426-effLuc cells are highly proliferative in vivo. Scale bars represent 50 μm
Mentions: Intracisternal (1.2 × 106 cells, N = 7) and intracerebellar injections (1.2 × 105 cells, N = 5) using UW426-effLuc cells were performed. In mice that had injections into the cisterna magna, BLI signals were observed at day 0 and gradually spread along the spinal axis over time [day 0: (0.91 ± 0.49) × 106 p/s/cm2/sr; day 16, (4.43 ± 0.76) × 106 p/s/cm2/sr; Fig. 4a and c]. However, in mice that had injections into the cerebellar hemisphere, the signals were detected on day 9 [(0.16 ± 0.19) × 106 p/s/cm2/sr], suddenly increased and migrated to the spinal cord from day 16 [(1.11 ± 0.21) × 106 p/s/cm2/sr; Fig. 4b and c]. The median survival was 22 days for the intracisternal injection model and 34 days for the intracerebellar injection model (Fig. 4d). To analyze the histopathology, mice that had injections into the cisterna magna (N = 5) were sacrificed at 22 days and mice that had injections into the cerebellar hemisphere (N = 5) were sacrificed at 35 days. In the intracisternal injection model, tumor cells spread in the entire neuraxis from the subfrontal area and cerebellar surface to the distal spinal cord. In the intracerebellar injection model, tumor cells aggregated in the cerebellum and did not disseminate into the subfrontal area or distal spinal cord in some animals examined (Fig. 4e). Table 2 summarizes the dissemination pattern of each cell injection method. Immunofluorescence staining revealed that abundant human nuclei and Ki67 double positive cells were observed in mice that had injections into the cisterna magna or cerebellar hemisphere (Fig. 4f).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images.

Methods: Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding.

Results: The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 106 UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients.

Conclusion: The results and described method are valuable resources for further translational research to overcome MB seeding.

No MeSH data available.


Related in: MedlinePlus