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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

IL1β treatment enhances a stem cell phenotype in vitro, which correlates with Cx3Cr1 loss in vivoA) SP analysis of freshly dissociated tumor cells cultured in 10% FBS medium, in NSC medium as spheres, and cultured in NSC medium like monolayer. Cells were treated with or without 100pM IL1β for 24h. The right graph shows relative SP from three independent glioma lines in three different conditions. B) qPCR data for relative mRNA expression of Nanog, Oct4, Sox2, CD44 and Musashi in GBM cells cultured in NSC medium with geltrex as a monolayer. Data represent average value of three independent cell lines. Error bars represent SD. *p < 0.05 by unpaired t-test. C) SP analysis of GBM samples generated in B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. Inserts show FTC-treated samples. The right graph represents relative SP from for 4 independent tumors per genotype.
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Figure 6: IL1β treatment enhances a stem cell phenotype in vitro, which correlates with Cx3Cr1 loss in vivoA) SP analysis of freshly dissociated tumor cells cultured in 10% FBS medium, in NSC medium as spheres, and cultured in NSC medium like monolayer. Cells were treated with or without 100pM IL1β for 24h. The right graph shows relative SP from three independent glioma lines in three different conditions. B) qPCR data for relative mRNA expression of Nanog, Oct4, Sox2, CD44 and Musashi in GBM cells cultured in NSC medium with geltrex as a monolayer. Data represent average value of three independent cell lines. Error bars represent SD. *p < 0.05 by unpaired t-test. C) SP analysis of GBM samples generated in B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. Inserts show FTC-treated samples. The right graph represents relative SP from for 4 independent tumors per genotype.

Mentions: A) Representative images of brain tumor sections from B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice were stained with anti-Iba1 (green), anti-BrdU (red), and counterstained with nuclear DAPI (blue). B) Quantified bar graphs from A showing that there was no statistically significant difference in the number of BrdU+Iba1+ cells in tumors from the three different genotypes. C) Representative images of brain tumor sections from B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice were stained with anti-Iba1 (green), anti-Cleaved Caspase-3 (red), and counterstained with nuclear DAPI (blue). D) Quantified bar graphs from C showing that there was no statistically significant difference in the number of BrdU+ Cleaved Caspase-3+ cells in tumors from the three different genotypes. For B and D, one-way ANOVAs with Tukey's multiple comparisons tests were performed and demonstrated that there were no statistically significant differences observed in the number of BrdU+Iba1+ and BrdU+ Cleaved Caspase-3+ cells in the three genotypes. E) Dot plots representing the number of CD11b+Ly-6Chi Ly-6G− inflammatory monocytes were analyzed from the blood of the same mice before and 20 days after tumor cell transplantation. There was a significant decrease in Ly6Chi inflammatory monocytes in Cx3cr1GFP/GFP mice, suggesting increased infiltration to GBM. F) Dot plots represent the number of CD11b+Ly-6Chi Ly-6G− inflammatory monocytes when mice were sacrificed at the end-point of survival. There was a significant difference in CD11b+Ly-6Chi Ly-6G− inflammatory monocytes when the three genotypes were compared and they all showed a reduction compared to naïve mice before the transplant, see E. A paired t-test was used in Figure 6E and one-way ANOVA with Tukey's multiple comparisons test was performed for F. Scale bars for A and C represent 50 μm.


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)

IL1β treatment enhances a stem cell phenotype in vitro, which correlates with Cx3Cr1 loss in vivoA) SP analysis of freshly dissociated tumor cells cultured in 10% FBS medium, in NSC medium as spheres, and cultured in NSC medium like monolayer. Cells were treated with or without 100pM IL1β for 24h. The right graph shows relative SP from three independent glioma lines in three different conditions. B) qPCR data for relative mRNA expression of Nanog, Oct4, Sox2, CD44 and Musashi in GBM cells cultured in NSC medium with geltrex as a monolayer. Data represent average value of three independent cell lines. Error bars represent SD. *p < 0.05 by unpaired t-test. C) SP analysis of GBM samples generated in B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. Inserts show FTC-treated samples. The right graph represents relative SP from for 4 independent tumors per genotype.
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Figure 6: IL1β treatment enhances a stem cell phenotype in vitro, which correlates with Cx3Cr1 loss in vivoA) SP analysis of freshly dissociated tumor cells cultured in 10% FBS medium, in NSC medium as spheres, and cultured in NSC medium like monolayer. Cells were treated with or without 100pM IL1β for 24h. The right graph shows relative SP from three independent glioma lines in three different conditions. B) qPCR data for relative mRNA expression of Nanog, Oct4, Sox2, CD44 and Musashi in GBM cells cultured in NSC medium with geltrex as a monolayer. Data represent average value of three independent cell lines. Error bars represent SD. *p < 0.05 by unpaired t-test. C) SP analysis of GBM samples generated in B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice. Inserts show FTC-treated samples. The right graph represents relative SP from for 4 independent tumors per genotype.
Mentions: A) Representative images of brain tumor sections from B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice were stained with anti-Iba1 (green), anti-BrdU (red), and counterstained with nuclear DAPI (blue). B) Quantified bar graphs from A showing that there was no statistically significant difference in the number of BrdU+Iba1+ cells in tumors from the three different genotypes. C) Representative images of brain tumor sections from B6, Cx3cr1GFP+ and Cx3cr1GFP/GFP mice were stained with anti-Iba1 (green), anti-Cleaved Caspase-3 (red), and counterstained with nuclear DAPI (blue). D) Quantified bar graphs from C showing that there was no statistically significant difference in the number of BrdU+ Cleaved Caspase-3+ cells in tumors from the three different genotypes. For B and D, one-way ANOVAs with Tukey's multiple comparisons tests were performed and demonstrated that there were no statistically significant differences observed in the number of BrdU+Iba1+ and BrdU+ Cleaved Caspase-3+ cells in the three genotypes. E) Dot plots representing the number of CD11b+Ly-6Chi Ly-6G− inflammatory monocytes were analyzed from the blood of the same mice before and 20 days after tumor cell transplantation. There was a significant decrease in Ly6Chi inflammatory monocytes in Cx3cr1GFP/GFP mice, suggesting increased infiltration to GBM. F) Dot plots represent the number of CD11b+Ly-6Chi Ly-6G− inflammatory monocytes when mice were sacrificed at the end-point of survival. There was a significant difference in CD11b+Ly-6Chi Ly-6G− inflammatory monocytes when the three genotypes were compared and they all showed a reduction compared to naïve mice before the transplant, see E. A paired t-test was used in Figure 6E and one-way ANOVA with Tukey's multiple comparisons test was performed for F. Scale bars for A and C represent 50 μm.

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