To identify transcripts that potentially distinguish infiltrating monocytes from brain microglia, we isolated and examined gene expression of BMDM (CD11b+
cells) and brainstem microglia (BSM) (CD11b+
cells) pooled from 6-week-old C57BL/6 male mice (ten mice/set) by FACS. Despite low RNA yields (0.4-100ng of total RNA), overall good quality RNA was obtained (Agilent R
umber, RIN=7.6-9.0) [24
], and the resulting cDNA yields (4.9-5.8µg) were used to perform parallel Affymetrix Mouse Exon 1.0ST microarray and Illumina RNA-sequencing (Figure S2
, Table S3
Complementary analysis pipelines Cufflinks and ALEXA-Seq for RNA-Seq [18
] and Aroma and Partek for microarray data [26
] were employed to analyze each platform. Fold changes and q-values were calculated for each gene, and a mean fold-change rank was generated by merging the four independent analyses. High-confidence gene lists were subsequently generated that included only differentially-expressed genes identified to have q-values <0.05 by at least three of the four analysis methods (Table S5
). A global unsupervised analysis, excluding genes not expressed or not variably expressed by Cufflinks analysis, demonstrated clear separation of BMDM from BSM samples ().
Differentially-expressed brainstem microglia (BSM) and bone marrow monocyte (BMDM) transcripts.
We next prioritized candidate transcripts based on their expression of protein products potentially amenable to flow cytometry analysis (cell surface markers), and selected ten transcripts of predicted membrane-associated proteins (5 BSM and 5 BMDM; ) for real-time quantitative PCR (qPCR) verification (). We secondarily validated two representative transcripts for BSM (F11R and CD81) and BMDM (SELL and CLEC12A) by flow cytometry (). Since CD81 resides inside the cell membrane of unstimulated microglia, and CLEC12A is expressed at relatively low levels in monocytes, we chose F11R and SELL as representative differentially-expressed flow cytometry markers to study monocyte infiltration in murine glioblastoma.
Genes of predicted membrane-associated proteins of brain microglia across a wide range of fold changes are highlighted relative to Cx3cr1.
Transcript and protein validation of genes differentially expressed between BMDM and BSM.
Genes of predicted membrane-associated proteins of bone marrow monocytes across a wide range of fold changes are highlighted relative to Ccr2.
Since F11r and Sell are expressed in <2% of the total cells isolated from bone marrow and brain, respectively, we first sought to determine whether F11r and Sell would retain their distinctive expression between infiltrative BMDM and resident microglia populations in the setting of induced murine malignant glioma using Ntv-a Ink4a-Arf-/-
;Gli-luc mice injected with RCAS-PDGFB () [11
]. Whereas control naïve mice do not form gliomas nor contain significant numbers of CD11b+
BMDM cells (R3; <2% cells) (Figure S3
, ), murine gliomas exhibit increased numbers of lymphocytes (R1; 27% cells) as well as CD11b+
cells (R2, microglia; 39% cells) and CD11b+
cells (R3, macrophages; 34% cells). While CD11b+
microglia were >99% F11r+
, the population of macrophages was not exclusively Sell+
. Surprisingly, the majority of these glioma-associated CD11b+
macrophages were also F11r+
(94%, Q1) (). Because fewer than 6% of these glioma-associated F11r+
cells were dendritic cells, B cells, or NK cells as determined by flow cytometry using CD3, CD4, CD11c, CD8a, CD19, CD103, and NK1.1 antibodies (data not shown), we hypothesized that Sell+
BMDM acquire F11r expression following brain infiltration in GBM.
High-grade murine gliomas contain F11r+ microglia and macrophages.
To demonstrate that Sell+
monocytes acquire F11r expression as a consequence of brain infiltration, we employed allogeneic BMT to track infiltrating monocytes and resident microglia by virtue of unique CD45 antigen expression (). In this model, chimeric mice with GVHD have CD45.2-expressing resident microglia and CD45.1-expressing donor BMDM and exhibit brain mononuclear infiltration following DLI, whereas control animals (BMT-only) have negligible monocyte infiltration (). Resident CD45.2 microglia from both control and GVHD mice are F11r+
and remain F11r+
throughout the course of the disease (3 weeks) without increasing CD45.2 surface expression (Figure S4
, Table S6
) or expressing CD45.1 (, CD11b+
population below R2). As observed in induced murine GBM, infiltrating CD45.1+
BMDM (, ) become F11r+
as a function of time after entry into the brain: in the first week post-DLI, the majority of the CD45.1+
infiltrating macrophages (R3) express surface Sell (53% F11r+
, 25% F11r-
); however, by the third week post-DLI, 90% of the positively-labeled R3 cells express only F11r. No other population of infiltrating cells changed their F11r and Sell expression (data not shown). Collectively, these data strongly suggest that F11r is a marker of brain macrophages in the context of murine GBM, regardless of tissue origin, prompting us to examine its prognostic value in patients with high-grade glioma.
BMDM acquire F11r expression following brain infiltration.
F11r and Sell surface expression of donor monocyte infiltration in chimeric mice with GVHD.
In human gliomas, 30-50% of the cells are CD68+
tissue macrophages; however, we previously showed that the percentage of CD68+
cells in these human tumors did not correlate with glioma malignancy grade [2
]. To determine whether F11R expression was associated with increasing glioma malignancy grade, we first confirmed the differential protein expression of F11R in histological sections of human bone marrow and brain (p=0.0016) (). Second, we quantified the percent of F11R+
cells (both resident and infiltrating macrophages) in a series of glioma tissue microarrays containing all glioma malignancy grades. In contrast to our prior findings with CD68 and IBA1, we found a positive correlation between the percent of F11R+
cells and high-grade glioma (p<0.0001) ().
F11R expression correlates with GBM malignancy grade and survival.
Because the percent of F11R+
cells was greatest in the high-grade gliomas, we next asked whether F11R expression had prognostic value in predicting patient survival. Using GBM data containing 159 specimens (GSE16011) [23
] dichotomized by median F11R
expression, high F11R
expression was associated with reduced patient survival (HR=1.33, log rank test p=0.0037) (). Previous studies have demonstrated that differences in survival relate to specific GBM molecular subtypes [29
], and that expression of macrophage/microglia-related genes are associated with the Mesenchymal subtype [3
]. In this regard, we also found that IBA1
) and CD68
expression was increased in Mesenchymal subtype tumors and was associated with decreased survival (Table S7
). However, using a multivariate Cox model to account for molecular subtype assignments, survival outcomes using AIF1
were strongly influenced by the molecular subtype (continuous expression analysis: Subtype overall p=0.0065 and p= 0.0188, respectively; dichotomized expression analysis: Subtype overall p=0.0072 and p= 0.0128, respectively; Table S9
). As a result, CD68
was no longer prognostic for patient survival when we account for these robust subtype contributions (continuous expression analysis: HR=1.16 p=0.1222, dichotomous expression analysis: HR=1.36 p=0.0886). AIF1
retained overall prognostic value (continuous expression analysis: HR=1.32 p=0.0155, dichotomous expression analysis: HR=1.42 p=0.0496); however, it is strongly dependent on the survival difference between the Proneural vs. Mesenchymal subtypes. While F11R
was also more highly expressed in the Mesenchymal subtypes relative to the Proneural subtype (p = 5.74E-11) (), F11R
expression had the greatest independent prognostic value regardless of molecular subtype (Dichotomized gene expression HR=1.57, p=0.0189) (). In contrast, the expression levels of two frequently-employed macrophage and microglia markers, CCR2 and CX3CR1, were not predictive of patient survival in GBM (Table S8
). Together, these data establish F11R as a novel monocyte predictor of patient outcome in GBM.
Relative contributions of TCGA subtypes to the survival outcome of GBM patients dichotomized by F11R expression.