GlioM&M: Web-based tool for studying circulating and infiltrating monocytes and macrophages in glioma

Abstract

Monocytes, macrophages and microglia make up a large part of the glioma environment and have an important role in maintaining and propagating glioma progression. Targeting these cells to inhibit their tumor-promoting effect and reprogramming them into an anti-tumor phenotype is a potential therapeutic approach for glioma. In this study we analyzed the transcriptomes of eight different monocyte subgroups derived from the brain and the blood of glioma-bearing mice. We compared the expression profile of blood-derived monocytes versus tumor-infiltrating monocytes and found increased expression of both pro- and anti-inflammatory pathways in tumor infiltrating monocytes. To help disseminate these datasets, we created a user-friendly web-based tool accessible at www.glioma-monocytes.com. This tool can be used for validation purposes and to elucidate gene expression profiles of tumor-interacting monocytes and macrophages as well as blood-derived circulating monocytes. This tool can also be used to identify new markers and targets for therapy in these different cell populations.

Figure 1

Isolation of blood and brain infiltrated monocytes and macrophages from glioma mouse model. (a) Schematic overview of experiment shows timeline and methods used. (b) Glioma cell line GL261.BpalmGFP was injected intracranially into syngeneic mouse to establish brain tumor model. Scale bar 50 µm (c) Glioma monocytes and macrophages were separated from microglia by FACS based on the expression level of CD45 and CD11b. Monocytes were isolated based on the presence of F4/80 and high expression of Ly6C, macrophages were further divided into CCR2 high and low. (d) Blood from tumor-bearing mice was harvested using cardiac puncture. Inflammatory monocytes (CD45^high, CD115^high, CD11c^low and Ly6c^high) and patrolling monocytes (CD45^high, CD115^high, CD11c^low, Ly6c^low) were isolated from whole blood by FACS.

Figure 2

Global analyses of gene expression. (a) Top 250 most differentially expressed genes among 8 cell subtypes isolated from blood and brain. (b) PCA plot illustrates difference in principle components between brain and blood samples. (c) MA plot reveals the number of genes with a significant change in expression in comparison between GFP^pos and GFP^neg brain monocytes. (d) Significantly differentiated gene expression shown in MA plot between blood-derived classical infiltrating monocytes (Ly6C^high) and patrolling monocytes (Ly6C^low). (e) Significantly differentiated gene expression shown in MA plot between blood-derived monocytes and glioma monocytes.

Figure 3

Different cell populations show expression of specific markers. (a) Gene expression of markers specific to inflammatory Ly6C^high (Cx3cr1^mid Ccr2⁺ Cd62l⁺ Cd43^low Treml4⁺) and patrolling Ly6c^low (Cx3cr1^high Ccr2^- Cd62l^- Cd43^high Treml4^-) monocytes (b) Normalized read counts of cell markers used to identify and differentiate monocytes from macrophages (CCR2^high and CCR2^low), including Ly6C, Ccr2, Cx3cr1, Fcgr1, Mertk, Itgax, Ptprc, Adgre1, Mrc1, Ccr7 and Nr4a1. (c) Expression of activation markers Cd74, H2-Aa, H2-Eb1, IL1b (d) Arg1, H2-DMb1, Il4ra, Tnf (E) Mrc1, Cd80, Cd86 and Nos2 shows that after infiltration into the glioma monocytes and macrophages are in an activated state. Data represents 3 independent experiments and are presented as the mean with SEM (error bars). Differential expression analysis with fold₂change, SEM and adjusted p-values are listed in Supplementary Table S1.

Figure 4

Analysis of various cytokine pathways in glioma monocytes and macrophages indicated upregulation of both pro- and anti-inflammatory pathways. (a) Relative expression of IFNγ related genes in the pro-inflammatory pathway, showing an overall upregulation in glioma-infiltrating cells of the IFNγ pathway. (b) IL10 pathway, which is anti-inflammatory, is upregulated as shown by the high relative expression in glioma-infiltrating cell groups. (c,d) Gene set enrichment analysis (GSEA) for the ranked genes based on the differential expression of genes comparing GFP^neg glioma monocytes to Ly6C^high blood monocytes, identified significant upregulation (FDR p-value < 0.05) of the IFNγ and IL10 pathways.

Figure 5

Web-based tool for studying circulating and glioma monocytes and macrophages in glioma model. (a) On the homepage of website (www.glioma-monocytes.com) the experimental setup is summarized as a schematic and in text. Expression of individual genes in various cell populations can be consulted using the search bar. (b) Example of output of inquiry focusing on Ccr2, showing normalized read count and differential expression between all cell groups.