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Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis.

Feng X, Szulzewsky F, Yerevanian A, Chen Z, Heinzmann D, Rasmussen RD, Alvarez-Garcia V, Kim Y, Wang B, Tamagno I, Zhou H, Li X, Kettenmann H, Ransohoff RM, Hambardzumyan D - Oncotarget (2015)

Bottom Line: Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice.The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β.Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences at Cleveland Clinic, Cleveland, Ohio, USA.

ABSTRACT
The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G-F4/80-/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.

No MeSH data available.


Related in: MedlinePlus

Cx3cr1 deficiency has no impact on microglial accumulation at the close edge of GBM and has no impact on tumor growth in organotypic slice culturesA) Representative images of Iba1 staining in tumors from the three genotypes. The white broken-lines in the images are drawn to show the close and distant edge of the tumors (170 μm each) and DAPI-positive nuclear density was used as guide to separate the tumor from the peri-tumoral area. B) Quantified bar graphs for the entire peri-tumoral areas that were constructed from a series of 20x images that cover the entire peri-tumoral area (n=7, 6, 7 for B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP, correspondingly). C) Quantification of tumor areas in organotypic brain slice cultures from the three genotypes at 6 days post-tumor cell inoculation are presented as dot plots. D) Representative images of GFP-positive cells from slices generated from Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. The white lines in the images are drawn to show the inner and outer layers of the tumors (80 μm each). The quantified numbers of GFP-positive cells in the inside, inner and outer layer of tumors are presented as dot blots. A one-way ANOVA with Tukey's multiple comparisons test was performed and demonstrated that there were no statistically significant differences observed in tumor volumes in the three genotypes (C and D). Scale bar represents 50 μm for A and 150 μm for D.
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Figure 7: Cx3cr1 deficiency has no impact on microglial accumulation at the close edge of GBM and has no impact on tumor growth in organotypic slice culturesA) Representative images of Iba1 staining in tumors from the three genotypes. The white broken-lines in the images are drawn to show the close and distant edge of the tumors (170 μm each) and DAPI-positive nuclear density was used as guide to separate the tumor from the peri-tumoral area. B) Quantified bar graphs for the entire peri-tumoral areas that were constructed from a series of 20x images that cover the entire peri-tumoral area (n=7, 6, 7 for B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP, correspondingly). C) Quantification of tumor areas in organotypic brain slice cultures from the three genotypes at 6 days post-tumor cell inoculation are presented as dot plots. D) Representative images of GFP-positive cells from slices generated from Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. The white lines in the images are drawn to show the inner and outer layers of the tumors (80 μm each). The quantified numbers of GFP-positive cells in the inside, inner and outer layer of tumors are presented as dot blots. A one-way ANOVA with Tukey's multiple comparisons test was performed and demonstrated that there were no statistically significant differences observed in tumor volumes in the three genotypes (C and D). Scale bar represents 50 μm for A and 150 μm for D.

Mentions: Loss of one copy of the Cx3cr1 gene has been shown to reduce accumulation of microglia (by 50%) and delay optic nerve glioma development [33]. Thus we decided to evaluate microglia/macrophage accumulation in peri-tumoral areas in tumors from the three genotypes by quantifying the total number of Iba1+ cells in two concentric peri-tumoral layers of tissue (Fig. 7A and 7B). There were no significant differences in the numbers of Iba1+ cells in the first and second peri-tumoral layers in GBM tissue from B6 and Cx3cr1GFP/+ mice compared to the tumors in Cx3cr1GFP/GFP mice. These data suggest that loss of Cx3cr1 does not affect accumulation of microglia in peri-tumoral areas. In order to assess the functional consequences of CX3CR1 deficiency on microglial migration and the effect it has on tumor growth, we generated gliomas ex vivo in naïve organotypic slice cultures generated from B6, Cx3cr1GFP/+ and Cx3cr1GFP/GFP mice as described in Supplemental Figure 9. This method allowed us to investigate the role of microglial CX3CR1 on tumor growth. There were no significant differences in the size of the tumors at 6 days post-tumor cell inoculation. Next, we fixed the organotypic slices, sectioned them into 8μm sections, and quantified GFP+ microglial density in tumors generated ex vivo in slices from the three genotypes. Quantification of GFP+ microglia inside of the tumor and inner and outer peri-tumoral areas showed no significant differences in microglia density whether one or two copies of Cx3cr1 were deleted. These data suggest that the effect of Cx3cr1 loss on tumor growth and increased stem cell-like cell phenotype in vivo is driven by increased infiltration of inflammatory monocytes and their increased expression of IL1β.


Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis.

Feng X, Szulzewsky F, Yerevanian A, Chen Z, Heinzmann D, Rasmussen RD, Alvarez-Garcia V, Kim Y, Wang B, Tamagno I, Zhou H, Li X, Kettenmann H, Ransohoff RM, Hambardzumyan D - Oncotarget (2015)

Cx3cr1 deficiency has no impact on microglial accumulation at the close edge of GBM and has no impact on tumor growth in organotypic slice culturesA) Representative images of Iba1 staining in tumors from the three genotypes. The white broken-lines in the images are drawn to show the close and distant edge of the tumors (170 μm each) and DAPI-positive nuclear density was used as guide to separate the tumor from the peri-tumoral area. B) Quantified bar graphs for the entire peri-tumoral areas that were constructed from a series of 20x images that cover the entire peri-tumoral area (n=7, 6, 7 for B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP, correspondingly). C) Quantification of tumor areas in organotypic brain slice cultures from the three genotypes at 6 days post-tumor cell inoculation are presented as dot plots. D) Representative images of GFP-positive cells from slices generated from Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. The white lines in the images are drawn to show the inner and outer layers of the tumors (80 μm each). The quantified numbers of GFP-positive cells in the inside, inner and outer layer of tumors are presented as dot blots. A one-way ANOVA with Tukey's multiple comparisons test was performed and demonstrated that there were no statistically significant differences observed in tumor volumes in the three genotypes (C and D). Scale bar represents 50 μm for A and 150 μm for D.
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Figure 7: Cx3cr1 deficiency has no impact on microglial accumulation at the close edge of GBM and has no impact on tumor growth in organotypic slice culturesA) Representative images of Iba1 staining in tumors from the three genotypes. The white broken-lines in the images are drawn to show the close and distant edge of the tumors (170 μm each) and DAPI-positive nuclear density was used as guide to separate the tumor from the peri-tumoral area. B) Quantified bar graphs for the entire peri-tumoral areas that were constructed from a series of 20x images that cover the entire peri-tumoral area (n=7, 6, 7 for B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP, correspondingly). C) Quantification of tumor areas in organotypic brain slice cultures from the three genotypes at 6 days post-tumor cell inoculation are presented as dot plots. D) Representative images of GFP-positive cells from slices generated from Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. The white lines in the images are drawn to show the inner and outer layers of the tumors (80 μm each). The quantified numbers of GFP-positive cells in the inside, inner and outer layer of tumors are presented as dot blots. A one-way ANOVA with Tukey's multiple comparisons test was performed and demonstrated that there were no statistically significant differences observed in tumor volumes in the three genotypes (C and D). Scale bar represents 50 μm for A and 150 μm for D.
Mentions: Loss of one copy of the Cx3cr1 gene has been shown to reduce accumulation of microglia (by 50%) and delay optic nerve glioma development [33]. Thus we decided to evaluate microglia/macrophage accumulation in peri-tumoral areas in tumors from the three genotypes by quantifying the total number of Iba1+ cells in two concentric peri-tumoral layers of tissue (Fig. 7A and 7B). There were no significant differences in the numbers of Iba1+ cells in the first and second peri-tumoral layers in GBM tissue from B6 and Cx3cr1GFP/+ mice compared to the tumors in Cx3cr1GFP/GFP mice. These data suggest that loss of Cx3cr1 does not affect accumulation of microglia in peri-tumoral areas. In order to assess the functional consequences of CX3CR1 deficiency on microglial migration and the effect it has on tumor growth, we generated gliomas ex vivo in naïve organotypic slice cultures generated from B6, Cx3cr1GFP/+ and Cx3cr1GFP/GFP mice as described in Supplemental Figure 9. This method allowed us to investigate the role of microglial CX3CR1 on tumor growth. There were no significant differences in the size of the tumors at 6 days post-tumor cell inoculation. Next, we fixed the organotypic slices, sectioned them into 8μm sections, and quantified GFP+ microglial density in tumors generated ex vivo in slices from the three genotypes. Quantification of GFP+ microglia inside of the tumor and inner and outer peri-tumoral areas showed no significant differences in microglia density whether one or two copies of Cx3cr1 were deleted. These data suggest that the effect of Cx3cr1 loss on tumor growth and increased stem cell-like cell phenotype in vivo is driven by increased infiltration of inflammatory monocytes and their increased expression of IL1β.

Bottom Line: Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice.The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β.Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences at Cleveland Clinic, Cleveland, Ohio, USA.

ABSTRACT
The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b+CD45hiCX3CR1lowLy-6ChiLy-6G-F4/80-/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.

No MeSH data available.


Related in: MedlinePlus