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Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus

Pyk2 is mostly detected in glioma cells rather than in other cell types in mouse brain.Immunohistochemistry was performed on C57BL/6 mice brain sections containing the tumor area. GL261 glioma cells were implanted into the brains of C57BL/6 mice and grown for 16 days. Photographs show the tumor and surrounding healthy tissue. The dash line outlines the border of tumor. Anti-GFAP antibody was used to detect glioma cells and astrocytes (red, panel A), anti-Iba 1 antibody was used to detect microglial cells (green, panel B) and Pyk2 detection is presented in blue (panel C). The merged images of anti-GFAP and anti-Pyk2, of anti-Iba1 and anti-Pyk2, and of all antibodies together can be seen in merged image boxes D, E, F correspondingly. Insert panels d, e, and f represent enlarged images of astrocytes, microglia, and invading glioma cells. Solid arrows indicate glioma cells, frame arrows indicate astrocytes, and double headed arrows indicate microglia. Scale bar: 40 μm.
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pone.0131059.g002: Pyk2 is mostly detected in glioma cells rather than in other cell types in mouse brain.Immunohistochemistry was performed on C57BL/6 mice brain sections containing the tumor area. GL261 glioma cells were implanted into the brains of C57BL/6 mice and grown for 16 days. Photographs show the tumor and surrounding healthy tissue. The dash line outlines the border of tumor. Anti-GFAP antibody was used to detect glioma cells and astrocytes (red, panel A), anti-Iba 1 antibody was used to detect microglial cells (green, panel B) and Pyk2 detection is presented in blue (panel C). The merged images of anti-GFAP and anti-Pyk2, of anti-Iba1 and anti-Pyk2, and of all antibodies together can be seen in merged image boxes D, E, F correspondingly. Insert panels d, e, and f represent enlarged images of astrocytes, microglia, and invading glioma cells. Solid arrows indicate glioma cells, frame arrows indicate astrocytes, and double headed arrows indicate microglia. Scale bar: 40 μm.

Mentions: To detect the cellular localization of Pyk2 within a brain tumor, we utilized a murine glioma model. Fig 2 shows immunocytochemical analysis of Pyk2, glial fibrillary acidic protein (GFAP, a marker of glioma cells and astrocytes) and Iba1 (a marker of microglial cells) in brain slices obtained from tumor bearing C57BL/6 mice. Brain sections were prepared 16 days after the implantation procedure. Since glioma cells are much larger in size and do not have long branched processes characteristic for astrocytes, glioma cells and astrocytes can be easily distinguished from each other (Fig 2A, 2d and 2f), as we described earlier [35]. The merged image demonstrates that glioma cells express abundant levels of Pyk2 (Fig 2D and 2F) in the tumor core as well as at the invasion area (Fig 2f), whereas astrocytes and microglia do not show a marked amount of Pyk2 (Fig 2D, 2E and 2F). Although there have been previous reports indicating that Pyk2 is present in microglia [39, 40], it is weakly detected in microglia in the present study because the imaging settings were optimized to visualize the large quantities of Pyk2 present in glioma cells.


Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

Pyk2 is mostly detected in glioma cells rather than in other cell types in mouse brain.Immunohistochemistry was performed on C57BL/6 mice brain sections containing the tumor area. GL261 glioma cells were implanted into the brains of C57BL/6 mice and grown for 16 days. Photographs show the tumor and surrounding healthy tissue. The dash line outlines the border of tumor. Anti-GFAP antibody was used to detect glioma cells and astrocytes (red, panel A), anti-Iba 1 antibody was used to detect microglial cells (green, panel B) and Pyk2 detection is presented in blue (panel C). The merged images of anti-GFAP and anti-Pyk2, of anti-Iba1 and anti-Pyk2, and of all antibodies together can be seen in merged image boxes D, E, F correspondingly. Insert panels d, e, and f represent enlarged images of astrocytes, microglia, and invading glioma cells. Solid arrows indicate glioma cells, frame arrows indicate astrocytes, and double headed arrows indicate microglia. Scale bar: 40 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476590&req=5

pone.0131059.g002: Pyk2 is mostly detected in glioma cells rather than in other cell types in mouse brain.Immunohistochemistry was performed on C57BL/6 mice brain sections containing the tumor area. GL261 glioma cells were implanted into the brains of C57BL/6 mice and grown for 16 days. Photographs show the tumor and surrounding healthy tissue. The dash line outlines the border of tumor. Anti-GFAP antibody was used to detect glioma cells and astrocytes (red, panel A), anti-Iba 1 antibody was used to detect microglial cells (green, panel B) and Pyk2 detection is presented in blue (panel C). The merged images of anti-GFAP and anti-Pyk2, of anti-Iba1 and anti-Pyk2, and of all antibodies together can be seen in merged image boxes D, E, F correspondingly. Insert panels d, e, and f represent enlarged images of astrocytes, microglia, and invading glioma cells. Solid arrows indicate glioma cells, frame arrows indicate astrocytes, and double headed arrows indicate microglia. Scale bar: 40 μm.
Mentions: To detect the cellular localization of Pyk2 within a brain tumor, we utilized a murine glioma model. Fig 2 shows immunocytochemical analysis of Pyk2, glial fibrillary acidic protein (GFAP, a marker of glioma cells and astrocytes) and Iba1 (a marker of microglial cells) in brain slices obtained from tumor bearing C57BL/6 mice. Brain sections were prepared 16 days after the implantation procedure. Since glioma cells are much larger in size and do not have long branched processes characteristic for astrocytes, glioma cells and astrocytes can be easily distinguished from each other (Fig 2A, 2d and 2f), as we described earlier [35]. The merged image demonstrates that glioma cells express abundant levels of Pyk2 (Fig 2D and 2F) in the tumor core as well as at the invasion area (Fig 2f), whereas astrocytes and microglia do not show a marked amount of Pyk2 (Fig 2D, 2E and 2F). Although there have been previous reports indicating that Pyk2 is present in microglia [39, 40], it is weakly detected in microglia in the present study because the imaging settings were optimized to visualize the large quantities of Pyk2 present in glioma cells.

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus