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. 2013 Oct 11;8(10):e77571.
doi: 10.1371/journal.pone.0077571. eCollection 2013.

F11R Is a Novel Monocyte Prognostic Biomarker for Malignant Glioma

Free PMC article

F11R Is a Novel Monocyte Prognostic Biomarker for Malignant Glioma

Winnie W Pong et al. PLoS One. .
Free PMC article


Objective: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.

Methods: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.

Results: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.

Significance: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following potential conflicts; there are no direct conflicts of interest. Dr. Pong reports grants from the W. M. Keck Foundation during the conduct of the study. Dr. Feng reports personal fees from National Cancer Institute and Cleveland Clinic, and non-financial support from University of Michigan outside the submitted work. Dr. Mardis reports personal fees from Pacific Biosciences Inc and from Illumina Inc, and other from Life Technologies, outside the submitted work. Dr. Gutmann reports grants from National Cancer Institute and from National Institutes of Health during the conduct of the study; personal fees from Biomarin, outside the submitted work; In addition, Dr. Gutmann has Neurofibromatosis type 1 patents. None of these organizations were involved in the study design; collection, analysis and interpretation of data; writing of the paper; and/or decision to submit for publication. This does not alter their adherence to all the PLOS ONE policies on sharing data and materials.


Figure 1
Figure 1. Differentially-expressed brainstem microglia (BSM) and bone marrow monocyte (BMDM) transcripts.
(A) A heat map was generated from unsupervised hierarchical clustering analysis that included all genes expressed (FPKM >1 in ≥1/6 samples). (B) Candidate genes selected for further validation for comparison to CX3CR1 and CCR2.
Figure 2
Figure 2. Transcript and protein validation of genes differentially expressed between BMDM and BSM.
(A) BMDM-enriched transcripts, including Sell (p=0.0019), Met (p=0.0074), Cd93 (p=0.0104), Kit (p=0.0377), and Clec12a (p=0.0049), were more highly expressed in independently-generated BMDM (n=5) relative to BSM (n=6) using TaqMan and SYBR Green qPCR. BSM-enriched transcripts, including Mertk (p=0.0001), F11r (p<0.0001), P2ry13 (p=0.0002), Cadm1 (p=0.0002), and Cd81 (p=0.0001), were more highly expressed in BSM. (B) Flow cytometry analysis of BMDM (CD11b+ and CD115+ cells; R1, left panel) and BSM (CD11b+ CD45low cells; R1, right panel) verified that SELL (C) and CLEC12A (D) were detected on BMDM, and not on BSM, while F11R (E) and CD81 (F) were detected on BSM and not BMDM. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 3
Figure 3. High-grade murine gliomas contain F11r+ microglia and macrophages.
(A) Ntv-a Ink4a-Arf-/-;Gli-luc mice develop tumors following intracranial RCAS-PDGFB injection (n=4). Sell and F11r expression was examined by flow cytometry in lymphocytes (R1, CD11b- CD45+ cells), microglia (R2, CD11b+ CD45low cells), and macrophages (R3, CD11b+ CD45high cells) within the tumor and in control naïve brains (n=4) in four separate experiments. (B) More F11r+ microglia and macrophages were identified in the gliomas relative to the control brains, most notably in the expansion of the R3 population (34% of positively labeled cells), which is typically a very small percentage in control brains (<2%). The majority of the labeled macrophages in the glioma are positive for F11r only (Q1; 94%), with few cells infiltrating the glioma positive for Sell only (Q3; 5%) or double positive for both F11r and Sell (Q2; 1.4%). (C) Bar graphs illustrate the mean (SEM) percentage and SEM for each immune cell population as well as their corresponding F11r and Sell surface expressions. White bars = control, black bars = glioma.
Figure 4
Figure 4. BMDM acquire F11r expression following brain infiltration.
(A) GVHD was induced in recipient BALB/cJ mice following total body irradiation (TBI) and bone marrow transplantation (BMT) from T cell depleted (TCD) B6.SJL-Ptprc a Pepcb/BoyJ donors. Following C57BL/6J mouse donor lymphocyte infusions (DLI), immune cell infiltration was assessed by flow cytometry at 1 week, 2 weeks, and 3 weeks post-DLI (n=6 GVHD and n=6 BMT-only control per time point). (B) Control BMT-only mice do not exhibit GVHD and lack substantial macrophage (R3) or lymphocyte (R1, and R4 in Figure S4) infiltration. There is a very small CD11b+, CD45.1high monocyte population that expresses F11r in the control brain (<2%; R3, right panel). (C) Chimeric mice with GVHD have donor monocyte infiltration (CD11b+, CD45.1high cells; R3) with negligible CD11b+, CD45.1low cells (R2). F11r and Sell expression (right column) of positively-labeled infiltrating donor monocytes (R3) over the course of 3 weeks of GVHD demonstrates a shift from F11r+ Sell+ cells to F11r+ only cells. (D) Bar graphs illustrate the mean and SEM for each population of immune system cells over the course of the three weeks following DLI.
Figure 5
Figure 5. F11R expression correlates with GBM malignancy grade and survival.
(A) Immunohistochemistry demonstrates that human bone marrow sections (n=3) have few F11R+ cells (arrows), while neurologically-normal post-mortem human frontal cortex brain sections contain numerous F11R+ mononuclear cells (n=3) (p= 0.0016). Endothelial cell labeling in the upper left quadrant of the brain section represent a positive control for staining. Scale bar = 50µm. Insets depict representative positively-labeled mononuclear cells. (B) A representative high-grade glioma, GBM, contains many F11R+ cells. Increased percentages of F11R+ cells (Kruskal-Wallis test/Dunn's Multiple Comparison Test, p<0.0001) are observed in high-grade glioma (AA, anaplastic astrocytoma, n=23; GBM, n=52) relative to low-grade tumors (PA, pilocytic astrocytoma, n=73) or normal brain (NB, n=23). (C) Kaplan-Meier curves and log rank test demonstrate that increased F11R expression negatively correlated with patient survival (GEO database: GSE16011, n=159, p=0.0037). (D) F11R was highly expressed in GSE16011 GBM samples assigned to Classical and Mesenchymal TCGA subtypes relative to the Proneural subtype (p=3.94E-12).

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