Journal: bioRxiv

Article Title: Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution

doi: 10.1101/2023.01.26.522174

Figure Lengend Snippet: (A) Schematic illustration of the in vitro scRadiotracing workflow in human glioma samples (n=20), leading to calculation of radioactivity per single tumor cell and TAM. Pseudocolor plots derived from flow cytometry show the applied gating strategy. The single cell suspension (left) was separated into TAM-enriched (CD11b(+), second from left) and tumor enriched (third from left) fractions. Tumor cells were defined via GFAP or 5-ALA after confirmation of 5-ALA-positivity during surgery or after confirmation of GFAP-positivity during neuropathological workup of the same samples. 5-ALA positive cells co-localized with GFAP-positive cells in samples where both markers allowed tumor cell identification (right). (B) Relative distribution of tumor cells and TAMs in the single cell suspension of human glioma samples revealed similar heterogeneity in human high-grade glioma (HGG, n=10) compared to low-grade glioma (LGG, n=2). The initial two patients did not receive an analysis of the single cell suspension. (C) Regression model indicated strong contribution of TAMs (CD11b(+) cells) but not of CD11b(−)/non-tumor cells to the measured activity in the investigated TAM-enriched samples of patients that underwent biopsy (n=10). Linear regression, β = standardized regression coefficient, error bands represent 95% confidence interval. (D) Comparison of single cell tracer uptake (scTSPO) of tumor cells and TAMs in samples of human HGG (n=11) and LGG (n=9). Tumor cells of HGG indicated higher scTSPO than tumor cells of LGG, whereas there was no significant difference for TAMs (unpaired t-test). Tumor cells of HGG showed higher scTSPO than TAMs of HGG (paired t-test). Mean±SEM. (E) Correlation of TSPO-PET signals with scTSPO elucidated a strong correlation between PET signals and tumor cell TSPO enrichment and no correlation between PET signals and TAM TSPO enrichment. N=13, R = Pearson’s coefficient of correlation. Error bands represent 95% confidence interval. (F) Example of three patients with HGG investigated via in vitro scRadiotracing. All three patients showed similar signals in amino acid PET (FET-PET) and only little contrast enhancement in MRI. The patient with high tumoral TSPO-PET signal (upper row, glioblastoma, WHO grade IV, high-affinity binding status (HAB)) showed distinctly more TSPO tracer uptake in both tumor cells and TAMs when compared to the patients with only faint (middle row, astrocytoma, WHO grade III, high-affinity binding status) or low (bottom row, glioblastoma, WHO grade IV, low-affinity binding status (LAB)) tumoral signal in TSPO-PET. The patient with glioblastoma and low-affinity binding status (bottom row) showed nearly absent single cell tracer uptake of TAMs but notable TSPO tracer uptake of tumor cells. T1w = T1-weighted, FLAIR = Fluid-attenuated inversion recovery.

Article Snippet: Cell pellets were resuspended in 100 μl cold D-PBS and stained with fluorochrome-conjugated antibodies recognizing human CD11b and GFAP (Miltenyi Biotec, 130-113-810 and 130-123-846) as described above.

Techniques: In Vitro, Radioactivity, Derivative Assay, Flow Cytometry, Suspension, Activity Assay, Comparison, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: The EP2 receptor induces expression of inflammatory enzymes and cytokines in mouse peritoneal macrophages. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, values are mean ± SEM. A, EP2 immunostaining is detected in wild-type but not EP2−/− C57BL/6 primary peritoneal macrophages costained for Cd11b. Scale bar, 100 μm. B, Peritoneal macrophages were stimulated with LPS (10 ng/ml) for 1 and 6 h. qPCR demonstrates a rapid upregulation of EP2 receptor mRNA by 1 h and subsequent downregulation by 6 h following LPS stimulation (n = 4–6 per group; two-way ANOVA for effect of time ##p < 0.01, and effect of interaction p = 0.02; Bonferroni's multiple-comparison tests comparing mean of 1 h vehicle (veh) and 1 h LPS *p < 0.05). C, Peritoneal macrophages were stimulated with LPS (10 ng/ml) +/− the EP2 agonist butaprost (1 μm) or vehicle. qPCR demonstrates an induction of proinflammatory mediators COX-2, iNOS, and gp91phox with LPS stimulation that is further enhanced by costimulation with butaprost (time points 1 h for COX-2 and 6 h for iNOS and gp91phox; n = 4–6 per group; two-way ANOVA for effect of LPS #p < 0.05, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost *p < 0.05, **p < 0.01). D, LPS-induced macrophage NO release is increased by EP2 receptor activation with butaprost (1 μm), whereas EP2−/− macrophages show reduced NO levels compared with EP2+/+ macrophages (n = 4 per group; Student's tests #p < 0.05, ##p < 0.01 comparing EP2+/+ to EP2−/−). E, qPCR demonstrates induction of IL-6 mRNA at 1 h and IL1β mRNA at 6 h, and a trend of decreased TNFα mRNA at 6 h in LPS-stimulated macrophages with addition of butaprost (n = 4–6 per group; two-way ANOVA for effect of LPS ##p < 0.01, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost **p < 0.01 for IL-6 at 1 h and IL1β at 6 h).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Expressing, Immunostaining, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Conditional deletion of the EP2 receptor in macrophages suppresses oxidative enzyme and cytokine gene expression. A, Genomic DNA PCR is shown for EP2+/+, EP2lox/+, and EP2lox/lox C57BL/6 mice. B, Quantitative genomic PCR was assayed for EP2+/+ wild-type, EP2+/−, EP2−/−, Cd11bCre;EP2lox/lox, Cd11bCre;EP2lox/+, and Cd11bCre;EP2+/+; values are relative to wild-type EP2+/+ control DNA. C, Peritoneal macrophages were isolated from adult Cd11bCre;EP2lox/lox and Cd11bCre;EP2+/+ mice and sorted using Cd11b antibody-conjugated magnetic beads before qPCR analysis. Basal levels of EP2 mRNA, assayed by qPCR, are reduced by 91% in Cd11bCre;EP2lox/lox compared with control macrophages (left; Cd11bCre;EP2lox/lox vs Cd11bCre;EP2+/+) and are reduced by 56% in LPS-stimulated Cd11bCre;EP2lox/lox macrophages (right; **p < 0.01; n = 5–6 per group). D, Conditional deletion of EP2 in macrophages reduces LPS-mediated increases in proinflammatory gene expression (two-way ANOVA for effect of LPS treatment is represented by ###p < 0. 001; Bonferroni's multiple-comparisons tests comparing mean of Cd11bCre;EP2+/+/LPS and Cd11bCre;EP2lox/lox/LPS were ***p < 0.001; n = 5–6 per group).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Gene Expression, Control, Isolation, Magnetic Beads

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Examination of levels of monocytic populations in Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice. Splenocytes and peripheral blood immune cells were isolated from 3-month-old mice. A, Representative plots for CD11b+ cells gated on CD115 and Ly6C yielding four populations of cells, including CD115−/Ly6C− macrophages, CD115−/Ly6Cint-hi neutrophils, CD115int/Ly6Cint resident monocytes, and CD115hi-int/Ly6Chi inflammatory monocytes in vehicle and LPS-treated mice. B, Quantification of levels of monocytic populations, including macrophages, resident monocytes, and inflammatory monocytes does not show differences between genotypes in vehicle or LPS-treated mice. Levels of neutrophils are decreased in peripheral blood with LPS, but not vehicle stimulation (*p < 0.05; n = 4 mice per group). C, Quantification of CD11b-positive microglia derived from brains of Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice does not show differences in number (n = 5–7 mice per genotype). D, Comparison of copy number of EP2/copy number of 18S is shown for adult microglia and peritoneal macrophages from Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice (n = 4–6 per group; p < 0.05 unpaired t test). Macrophage expression of EP2 in Cd11bCre;EP2+/+ mice was 28-fold higher; however, the percentage reduction of expression with conditional deletion of EP2 was similar in both microglia and macrophages, and was 62.2 and 62.1%, respectively.

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Isolation, Derivative Assay, Comparison, Expressing