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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

Microglia ablation in brain tumors using the CD11b-HSVTK/GCV system.(A) Immunocytochemical detection of microglia in tumors developed in C57BL/6 and CD11b-HSVTK mice brains after local GCV administration. The tumors were generated by intracranial implantation of GL261 glioma cells. GCV was delivered to the tumor area through mini-osmotic pumps. Image shows the significant reduction of microglia in tumors developed in CD11b-HSVTK compared to C57BL/6 mice. Anti-Iba 1 antibody was used to detect microglial cells (green) and DAPI was used to detect all cell nuclei (blue). (B) Western blot detection of Iba-1 in tumors extracted from C57BL/6 and CD11b-HSVTK mice brains after the treatment with GCV. (C) The graph shows corresponding levels of Iba-1 protein expression in C57BL/6 and CD11b-HSVTK mice brain tumors after GCV administration determined by western blot. Mean ± S.E and significant difference from control (*) are shown (p < 0.05).
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pone.0131059.g004: Microglia ablation in brain tumors using the CD11b-HSVTK/GCV system.(A) Immunocytochemical detection of microglia in tumors developed in C57BL/6 and CD11b-HSVTK mice brains after local GCV administration. The tumors were generated by intracranial implantation of GL261 glioma cells. GCV was delivered to the tumor area through mini-osmotic pumps. Image shows the significant reduction of microglia in tumors developed in CD11b-HSVTK compared to C57BL/6 mice. Anti-Iba 1 antibody was used to detect microglial cells (green) and DAPI was used to detect all cell nuclei (blue). (B) Western blot detection of Iba-1 in tumors extracted from C57BL/6 and CD11b-HSVTK mice brains after the treatment with GCV. (C) The graph shows corresponding levels of Iba-1 protein expression in C57BL/6 and CD11b-HSVTK mice brain tumors after GCV administration determined by western blot. Mean ± S.E and significant difference from control (*) are shown (p < 0.05).

Mentions: To further evaluate the participation of microglia in activation of the Pyk2 pathway in glioma cells we used a murine model of local microglia ablation. Our preliminary investigation with use of western blot and immunohistochemical staining did not reveal any difference in microglial infiltration of implanted tumors in CD11b-HSVTK and C57BL/6mice without administration of GCV. In order to assess the effectiveness of microglia ablation in tumor site, animals were sacrificed after 7 days of GCV administration and frozen brain sections were prepared followed by immunocytochemical detection of Iba-1. The images of tumors presented in Fig 4A demonstrate that microglial cells were significantly reduced in CD11b-HSVTK mice compared to C57BL/6 animals where microglial cells were still present. For further evaluation of the efficacy of microglia ablation in CD11b-HSVTK mice, quantitative analysis of Iba-1 by western blot was performed in C57BL/6 and CD11b-HSVTK mice brain tumor samples (Fig 4B and 4C). Results showed a significant reduction in the expression of Iba-1 in CD11b-HSVTK mice, confirming the substantial reduction of brain microglia in CD11b-HSVTK as compared to wild type animals.


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)

Microglia ablation in brain tumors using the CD11b-HSVTK/GCV system.(A) Immunocytochemical detection of microglia in tumors developed in C57BL/6 and CD11b-HSVTK mice brains after local GCV administration. The tumors were generated by intracranial implantation of GL261 glioma cells. GCV was delivered to the tumor area through mini-osmotic pumps. Image shows the significant reduction of microglia in tumors developed in CD11b-HSVTK compared to C57BL/6 mice. Anti-Iba 1 antibody was used to detect microglial cells (green) and DAPI was used to detect all cell nuclei (blue). (B) Western blot detection of Iba-1 in tumors extracted from C57BL/6 and CD11b-HSVTK mice brains after the treatment with GCV. (C) The graph shows corresponding levels of Iba-1 protein expression in C57BL/6 and CD11b-HSVTK mice brain tumors after GCV administration determined by western blot. Mean ± S.E and significant difference from control (*) are shown (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476590&req=5

pone.0131059.g004: Microglia ablation in brain tumors using the CD11b-HSVTK/GCV system.(A) Immunocytochemical detection of microglia in tumors developed in C57BL/6 and CD11b-HSVTK mice brains after local GCV administration. The tumors were generated by intracranial implantation of GL261 glioma cells. GCV was delivered to the tumor area through mini-osmotic pumps. Image shows the significant reduction of microglia in tumors developed in CD11b-HSVTK compared to C57BL/6 mice. Anti-Iba 1 antibody was used to detect microglial cells (green) and DAPI was used to detect all cell nuclei (blue). (B) Western blot detection of Iba-1 in tumors extracted from C57BL/6 and CD11b-HSVTK mice brains after the treatment with GCV. (C) The graph shows corresponding levels of Iba-1 protein expression in C57BL/6 and CD11b-HSVTK mice brain tumors after GCV administration determined by western blot. Mean ± S.E and significant difference from control (*) are shown (p < 0.05).
Mentions: To further evaluate the participation of microglia in activation of the Pyk2 pathway in glioma cells we used a murine model of local microglia ablation. Our preliminary investigation with use of western blot and immunohistochemical staining did not reveal any difference in microglial infiltration of implanted tumors in CD11b-HSVTK and C57BL/6mice without administration of GCV. In order to assess the effectiveness of microglia ablation in tumor site, animals were sacrificed after 7 days of GCV administration and frozen brain sections were prepared followed by immunocytochemical detection of Iba-1. The images of tumors presented in Fig 4A demonstrate that microglial cells were significantly reduced in CD11b-HSVTK mice compared to C57BL/6 animals where microglial cells were still present. For further evaluation of the efficacy of microglia ablation in CD11b-HSVTK mice, quantitative analysis of Iba-1 by western blot was performed in C57BL/6 and CD11b-HSVTK mice brain tumor samples (Fig 4B and 4C). Results showed a significant reduction in the expression of Iba-1 in CD11b-HSVTK mice, confirming the substantial reduction of brain microglia in CD11b-HSVTK as compared to wild type animals.

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