Russell B. Foxall

Kinetics of miRNA 142-5p expression in different macrophage populations

Target Scan online network search for miRNAs broadly conserved across vertebrates and cross referenced with known M2-like markers.

Association among macrophage phenotype, miR-142-5p expression, and survival outcomes in patients with DLBCL. A, Kaplan–Meier curves showing (PFS in patients with high (miRNA score of 3–4) versus low (miRNA score of 0–2) miR-142-5p expression (red and blue lines, respectively). B, Kaplan–Meier curves showing PFS in patients with high (>30 positive cells) versus low (<30 positive cells) CD206 expression (red and blue lines, respectively). C, Kaplan–Meier curves showing PFS in patients with high miR-142-5p and CD206 expression (n = 9) compared with those patients with low CD206 and/or miR-142-5p expression; n = 38 (median PFS, 12 months vs. not reached; P = 0.0096), generated using GraphPad Prism and analyzed using a Gehan–Breslow–Wilcoxon test.

Baseline characteristics of patients with high (miRNA score of 3-4) versus low (miRNA score of 0-2) miRNA 142-5p expression

Baseline characteristics of patients with high CD206 expression (>30 positive cells) versus low CD206 expression (<30 positive)

Baseline characteristics of patients with high miRNA 142-5p and CD206 expression compared to those patients with low CD206 and/or miR-142-5p expression

Clinical and pathological features of treatment naïve DLBCL FFPE patient samples with low miRNA/CD206 expression.

Clinical and pathological details of treatment naïve DLBCL FFPE patient samples with high miRNA 142-5p plus CD206 expression

CD163+ tumor-associated macrophages (TAM) are associated with poor response to immunochemotherapy in patients with diffuse large B-cell lymphoma and comprise a significant proportion of the tumor microenvironment (TME). Targeting these TAMs to reverse immunosuppression is an attractive approach for improving treatment response rates. A change in microRNA (miRNA) expression has the potential to modify hundreds of genes by interacting with multiple messenger RNA (mRNA) targets, thereby altering the wider cellular transcriptional profile. The manipulation of miRNAs that control macrophage behavior may switch the global transcriptomic profiles of immunosuppressive macrophages to more activated, antitumor states, resulting in the reversal of immune resistance and enhancement of monoclonal antibody (mAb) therapy. Our studies show a significant upregulation of miR-142-5p in immunosuppressive macrophages stimulated with IL4/IL13 compared with lipopolysaccharide (LPS)/IFNγ-stimulated macrophages and nonpolarized controls. This upregulation of miR-142-5p in macrophages stimulated with immunoinhibitory cytokines IL10 and TGFβ was also demonstrated. Inhibition of miR-142-5p expression in IL4/IL13-stimulated macrophages led to a significant increase in FcγRIIIA, CD40, and CD47 expression with enhanced phagocytosis of chronic lymphocytic leukemia (CLL) cells opsonized with rituximab when compared with nontransfected controls. Expression of miR-142-5p in diagnostic samples of patients with diffuse large B-cell lymphoma (DLBCL) was assessed using miRScope assays. These were combined with immunohistochemistry (IHC) staining of CD206 to identify immunosuppressive macrophages. Combination of high miR-142-5p and CD206 expression correlated with a reduction in progression-free survival (PFS) following standard rituximab containing chemotherapy regimens in 38 patients. Together, these studies indicate the potential of targeting miRNA to repolarize macrophages and improve treatment success. Significance:The manipulation of macrophage polarization within the TME has the potential to enhance immunotherapies used across many different cancer types. The use of miRNA inhibitors to target the immune system in a cell-specific way using antibody oligonucleotide conjugates has the potential to reduce toxicity historically associated with miRNA therapeutics and change the phenotype of cells at the transcriptomic level.

Bioinformatics analysis of miRNA expression in DLBCL and immunosuppressive macrophages within TCGA, miRBase, and TargetScan. A, Heatmap showing correlation of modules with each other, in which blue represents positive correlation and red represents negative correlation; 13,799 expressed genes summarized by 60 modules; the size and color of each circle indicates the strength of correlation between two modules using the Spearman correlation method. B, Network topology of the macrophage-enriched (cyan) co-expression module. Each point represents one gene within the cyan module. The x-axis shows scaled intramodular connectivity (normalized sum of adjacencies to other module genes; higher values denote hub genes). The y-axis shows maximum adjacency ratio (MAR; the fraction of a gene’s total connectivity contributed by its strongest single connection; higher MAR indicates dependence on a single partner). Points are colored according to the clustering coefficient (local neighborhood interconnectedness). The observed relationship between connectivity and MAR indicates that highly connected genes participate through distributed co-expression rather than single dominant edges, consistent with a robust macrophage transcriptional program.