Effect of miR-142-3p on the M2 Macrophage and Therapeutic Efficacy Against Murine Glioblastoma

Isolation of Human Glioblastoma-Infiltrating Macrophages and CD14⁺ Monocytes

This study was approved by the institutional review board of the University of Texas M. D. Anderson Cancer Center and conducted under protocol LAB03-0687. Informed consent was obtained from each subject. Tumors were confirmed as glioblastoma based on the 2007 World Health Organization classification of tumors of the central nervous system (20) by a board-certified neuropathologist. Further details are provided in the Supplementary Methods (available online).

Real-Time Polymerase Chain Reaction to Confirm Relative miR-142 Expression Levels

Total RNA extracted was used as the template for reverse transcription using the TaqMan real-time polymerase chain reaction (PCR) kit (Applied Biosystems, Carlsbad, CA) in the 7500 real-time PCR system (Applied Biosystems). Primers for reverse transcription were purchased for human miR-142-3p (PN4427975, assay ID: 000464; Life Technologies, Grand Island, NY) and U18 (PN4427975, assay ID: 001204; Life Technologies). Further details are provided in the Supplementary Methods (available online).

In Vivo Experiments

The miR-142-3p duplex that mimics pre-miR-142-3p (sense: 5′-UGUAGUGUUUCCUACUUUAUGGAU-3′, antisense: 5′-CC AUAAAGUAGGAAACACUACAAA-3′) and the scramble control miRNA duplex (sense: 5′-AGUACUGCUUACGAUACGGTT-3′, antisense: 5′-CCGUAUCGUAAGCAGUACUTT-3′) were synthesized (Avetra Bioscience, San Carlos, CA). The sequence of murine miR-142-3p is identical to human miR-142-3p on the basis of National Center for Biotechnology Information blast data (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The treatment regimens consisted of 2 µL of miR-142-3p or scramble control (10 µg/µL) + 48 µL of phosphate-buffered saline (PBS) mixed with the vehicle (40 µL PBS + 10 µL lipofectamine 2000; Life Technologies) or the vehicle control (90 µL PBS + 10 µL lipofectamine 2000). Preliminary pharmacokinetic data indicate that the elimination half-life of miRNA administered in this manner is 6 hours in serum and 12 hours within the peripheral blood immune cells. Mice were maintained in the M. D. Anderson Isolation Facility in accordance with Laboratory Animal Resources Commission standards and handled according to the approved protocol 08-06-11831.

Syngeneic Subcutaneous Model

The murine glioma GL261 cell line was obtained from the National Cancer Institute–Frederick Cancer Research Tumor Repository and cultured in a humidified atmosphere of 5% carbon dioxide and 95% air at 37°C in Dulbecco’s modified Eagle medium (Life Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% penicillin/streptomycin/L-glutamine (Mediatech, Manassas, VA). The cells were kept in −80°C and used within 15 passages after the thawing. To induce subcutaneous tumors, logarithmically growing GL261 cells were injected into the right hind flanks of 6-week-old C57BL/6J female mice or nude mice at a dose of 1×10⁶ cells suspended in 100 µL of matrigel basement membrane matrix (BD Biosciences, San Jose, CA). When palpable tumors formed that were approximately 0.5cm in diameter, the mice were treated by local tumor injection in blinding and randomization (n = 5 per group). Tumors were measured twice per week. Tumor volume was calculated with slide calipers using the following formula: V = (L x W x H)/2, where V is volume (mm³), L is the long diameter, W is the short diameter, and H is the height.

Syngeneic Intracranial Clonotypic Glioma Model

To induce intracerebral tumors in C57BL/6J mice, GL261 cells were collected in logarithmic growth phase and mixed with an equal volume of 10% methyl cellulose in improved modified Eagle medium (zinc option; Life Technologies), and loaded into a 250-μL syringe (Hamilton, Reno, NV) with an attached 25-gauge needle. The mice were anesthetized with ketamine (100mg/kg) and xylazine (10mg/kg), and the needle was positioned 2mm to the right of bregma and 4mm below the surface of the skull at the coronal suture using a stereotactic frame (Kopf Instruments, Tujunga, CA), as previously described (21). The intracerebral tumorigenic dose for GL261 cells was 5×10⁴ in a total volume of 5 μL. Mice were randomly assigned to scramble control or the miR-142-3p treatment group (n = 10 per group) and observed and weighed three times per week. When they showed signs of neurological symptoms, they were compassionately killed by prolonged carbon dioxide inhalation. Their brains were removed, placed in 4% paraformaldehyde, and embedded in paraffin.

Pharmacokinetic Study

Non-tumor-bearing C57BL/6J mice (n = 3 per time point) were administered miRNA + lipofectamine 2000 intravenously once and subsequently killed at 0, 15 minutes, 1, 4, 8 and 24 hours. The liver, peripheral blood mononuclear cells, and serum were subsequently analyzed for miRNA expression by quantitative PCR. Further details are provided in the Supplementary Methods (available online).

Genetically Engineered Murine Model of Glioma

The use of immune-competent Ntv-a mice with RCAS-platelet-derived growth factor B + RCAS-B-cell lymphoma 2 induced high-grade gliomas for testing immune therapeutics has been previously described (22). To transfer genes by RCAS vectors, DF-1 producer cells transfected with a particular RCAS vector (1×10⁵ DF-1 cells in 1–2 µL of PBS) were injected on postnatal day 1 or 2 into the frontal lobes of Ntv-a mice at the coronal suture. Twenty-one days after introducing the glioma-inducing transgenes, the mice were randomly assigned to the treatment or control group (n = 9 per group). Mice were treated intravenously on Monday, Wednesday, and Friday for 3 weeks. The mice were killed 90 days after injection or sooner if they demonstrated morbidity related to tumor burden.

Statistical Analysis

All quantified data represent a mean of triplicate samples ± standard deviation (SD) or as indicated. Statistical significance was determined by Student t test or as indicated. P values less than .05 were considered statistically significant. Outliers were eliminated by the Grubbs test. All of the statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA), except for the survival comparison with log-rank test performed by SAS version 9.3 (SAS Institute, Cary, NC) and TIBCO Spotfire S+ version 8.2 (Somerville, MA). For the subcutaneous tumor measurement, linear mixed models and F test were fit to assess tumor growth after adjusting for treatment effect and taking into account the associations among repeated measures within each subject. All statistical tests were two-sided.

miR-142-3p Expression in Monocyte-Derived Glioblastoma-Associated Macrophages

Using the Human miRNA OneArray Microarray v2 to assess miRNA expression profiling, the miRNA expression profile in glioblastoma-infiltrating macrophages was matched to that of monocytes from the peripheral blood. With a mean 4.95-fold decrease relative to the level in matched peripheral monocytes, miR-142-3p emerged as a leading candidate (Figure 1A; Table 1). Meanwhile, miR-142-3p downregulation was not detected in glioblastoma tissue relative to normal brain tissue by total tissue miRNA microarray (Supplementary Table 1, available online). Subsequent real-time PCR analysis revealed that, although miR-142-3p expression was detected in total glioblastoma tumor tissues, glioblastoma cell lines or glioma cancer stem cells seldom express this miRNA. Rather it is the monocytes that are the main source of miR-142-3p. Intriguingly, miR-142-3p is downregulated within the glioblastoma-infiltrating macrophages relative to peripheral blood monocytes (mean relative expression of infiltrating CD11b+ macrophages = 2.03, SD = 1.17; mean relative expression of peripheral CD14+ monocytes = 9.45, SD =0.69; P < .001) (Figure 1B). There was no statistically significant difference of miR-142-3p expression in peripheral monocytes from patients with glioblastoma compared with healthy donors.

miR-142-3p Expression in M1 vs M2 Macrophages

Based on the observation of less miR-142-3p expression in the glioblastoma-infiltrating macrophages relative to its monocyte precursor, we further clarified the miR-142-3p expression in the proinflammatory M1 subset and immunosuppressive M2 subset. These subsets were induced ex vivo and characterized based on their morphological, phenotypic, and phagocytic characteristics. Specifically, the M1 macrophages appeared more round, whereas the M2 macrophages assumed a more elongated phenotype, as previously described (Figure 2A) (23). Moreover, the M2 macrophages expressed lower levels of CD11b, CD86, and major histocompatibility complex II than the M1 macrophages but higher levels of CD163 (Figure 2B) and exhibited more robust phagocytosis (mean fluorescence intensity of M1 macrophages: 1.01, SD = 0.05; mean fluorescence intensity of M2 macrophages = 2.25, SD = 0.50; P < .001; n = 6) (Figure 2C), consistent with prior reports (24,25). Quantitative reverse-transcription PCR confirmed the downregulation of miR-142-3p during macrophage differentiation; however, immune-suppressive M2 macrophages were found to have lower miR-142-3p expression. Specifically, the mean expression ± standard deviation of miR-142-3p in M1 macrophages relative to matched M2 macrophages was 1.38±0.36 vs 1.00±0.08 (P = .03; n = 6) (Figure 2D).

miR-142-3p Interacts With the TGF-β Pathway

Using multiple bioinformatics tools (TargetScan 6.2, PicTar, miRanda, and miRDB), potential target genes of miR-142-3p were identified. A cluster of TGF-β pathway genes with conserved target sites in their 3’-untranslated regions (UTRs) were identified, including transforming growth factor beta receptor 1 (TGFBR1) and transforming growth factor beta 2 (TGFB2). The TGF-β pathway is well known for its potent role in immune suppression and tumorigenesis, including polarization of tumor-promoting M2 macrophages (26). Thus, we investigated the effect of miR-142-3p overexpression on the TGF-β pathway in the monocyte-derived M1 and M2 subsets (Supplementary Figure 1A, available online). In the miR-142-3p overexpressing cells, the TGFBR1 mRNA levels remained unchanged (Supplementary Figure 1B, available online), whereas the TGFBR1 protein levels were repressed in the M2 macrophages (Figure 3, A and B), which indicates that miR-142-3p mediates its effect on TGFBR1 by post-transcriptional regulation rather than by targeting mRNA degradation. TGF-β2 cytokine secretion was not inhibited by miR-142-3p in either the M1 or M2 macrophages (Supplementary Figure 1C, available online). Anti-miR-142-3p treatment resulted in increased TGFBR1 protein levels only in the M2 macrophages (Figure 3A), signifying that TGF-β pathway regulation by miR-142-3p is contextual and cell specific. Other predicted targets of miR-142-3p such as integrin beta 8 (ITGB8), integrin alpha V (ITGAV), SMAD4, NF-κB, and TGFB2 were not found to be inhibited in either the M1 or M2 macrophage populations (Figure 3A). Furthermore, we detected the activation of the TGFBR1 downstream protein, SMAD2, after stimulation of M1 and M2 cells with TGF-β1. As expected, p-SMAD2 was inhibited by miR-142-3p only in M2 macrophages but not in the M1 macrophages (Figure 3B). TGFBR1 SMAD-independent targets such as p-AKT and RhoA were not appreciably altered by miR-142-3p. p-TAK1 was not expressed in either M1 or M2 populations (data not shown).

To further prove that miR-142-3p binds to TGFBR1 mRNA, a luciferase assay was conducted by mutating the predicted miR-142-3p TGFBR1 3’-UTR-luciferase reporter binding site (Figure 3C). In cotransfected HeLa cells, TGFBR1 luciferase activity was substantially inhibited by miR-142-3p compared with a parental luciferase vector control, whereas mutational alteration of the miR-142-3p TGFBR1 3’-UTR binding site resulted in full abolishment of the inhibition (Figure 3D).

Overexpression of miR-142-3p Does Not Alter Macrophage Phenotype

We next assessed whether miR-142-3p could shift macrophage differentiation or phenotype based on the expression of the general macrophage marker CD68 and the M2 specific marker CD163 (27–29). After transfection with either miR-142-3p or an miR-142-3p inhibitor, CD68 expression levels in the M1 and M2 macrophages were unchanged (mean CD68 expression percentage of scramble control group = 87.8%, SD = 14.4%; mean CD68 expression percentage of miR-142-3p = 87.8%, SD = 14.9%; P = .98; n = 8) (Supplementary Figure 2, A and C, available online). CD163 expression was occasionally downregulated by over-expression of miR-142-3p (mean CD163 expression percentage of scramble control group = 74.6%, SD = 7.95%; mean CD163 expression percentage of miR-142-3p = 63.9%, SD = 16.9%; P = .02; n = 8) (Supplementary Figure 2, B and C, available online), but this was inconsistently observed.

miR-142-3p Overexpression Induces M2 Macrophage Apoptosis

During ex vivo culture of miR-142-3p overexpressing macrophages, we observed a significant decrease in the number of M2 macrophages. Further evaluation revealed that miR-142-3p transfection induced more early (Annexin V⁺ 7-AAD^-) and late apoptosis/cellular death (Annexin V⁺ 7-AAD⁺) in M2-committed macrophages (mean fold relative to control after excluding one outlier = 2.12; SD = 1.01; n = 8) compared with M1-committed macrophages (mean fold relative to control after excluding one outlier = 1.17; SD = 0.34; n = 8) at 48 hours (P = .01 after excluding the outliers by Grubbs test) (Figure 4A). Because TGF-β has been shown to have a role in autocrine growth of cells (30), we next investigated whether TGFBR1 blockade in the M2 cells, which may have autocrine dependency on this pathway, would induce selective apoptosis. As expected, more apoptosis was observed within the M2-committed macrophages treated with either SB431542 (mean fold relative to control of M2-comitted macrophages = 2.38, SD = 1.04; mean fold relative to control of M1-comitted macrophages = 1.23, SD = 0.26; P = .04; n = 6) or LY364947 (mean fold relative to control of M2-comitted macrophages = 2.29, SD = 0.35; mean fold relative to control of M1-comitted macrophages = 1.28, SD = 0.37; P = .001; n = 6) compared with the M1-committed macrophages at 48 hours (Figure 4B). Identical results were obtained with specific TGFBR1 small interfering RNA (Supplementary Figure 3, A and B, available online), and this appeared to be cell-specific because miR-142-3p overexpression did not induce apoptosis in glioma cancer stem cells (Supplementary Figure 3, C and D, available online).

miR-142-3p Inhibits Glioma Growth

We next investigated whether miR-142-3p could exert an antiglioma effect in vivo (Figure 5A). Aggressive subcutaneous GL261 glioma growth was observed in the control group of syngeneic C57BL/6J mice, whereas glioma growth was markedly inhibited during the 3-week course of miR-142-3p local treatment (P = .03; n = 5 per group) (Figure 5B).To determine whether miR-142-3p treatment can exert a similar effect against intracerebral glioma, syngeneic C57BL/6J mice harboring GL261 were treated with miR-142-3p intravenously for 3 weeks (Figure 5C). The median survival duration for the scramble control group was 23.5 days, which was extended to 31 days for mice treated with miR-142-3p (P = .03; n = 10 per group) (Figure 5D). During the treatment course, no behavioral or neurological abnormalities were observed in the mice. Necropsies of the brains of miR-142-3p–treated mice demonstrated no evidence of demyelination, macrophage infiltration, or lymphocytic infiltration in the non-tumor-bearing areas that would indicate the induction of autoimmunity. Furthermore, administration of miR-142-3p did not induce a peripheral lymphocytosis or monocytosis (Supplementary Figure 4, available online).

miR-142-3p Inhibits Glioma-Infiltrating Macrophages

In a genetically engineered Ntv-a murine model system of high-grade glioma, miR-142-3p–treated mice also had extended survival (median survival: 32 days vs 24 days; P = .03; n = 9) (Figure 6, A and B). This model system has robust glioma-infiltrating macrophages (22), and treatment with miR-142-3p resulted in lower F4/80+ macrophage infiltration compared with the control group (F4/80 positive percentage in control mice = 19.1%, SD = 10.2%; F4/80 positive percentage in miR-142-3p–treated mice = 5.35%, SD = 2.23%; P = .009; n = 8) (Figure 6C). A negative linear correlation was established between the infiltrating F4/80+ macrophages and survival in the miR-142-3p–treated mice (R ² = .303) (Figure 6D), indicating that miR-142-3p is exerting a therapeutic effect by modulating glioma-infiltrating macrophages. miR-142-3p suppressed CD11b+ macrophages elaborating interleukin 6 (IL-6) consistent with M2 skewed cells by 29% (mean percentage of CD11b+ IL-6+ M2 macrophages in controls = 71.8%, SD = 6.5%; mean percentage of CD11b+ IL-6+ M2 macrophages in miR-142-3p = 51.1%, SD = 5.2%; P = .01; n = 3) and enhanced CD11b+ macrophages elaborating interferon gamma (IFN-γ) consistent with M1 skewed cells by 83% (mean percentage of CD11b+ IFN-γ+ M1 macrophages in controls = 43.4%, SD = 11.9%; mean percentage of CD11b+ IFN-γ+ M1 macrophages in miR-142-3p = 79.6%, SD = 16.9%; P = .04; n = 3) within the spleen. In contrast, miR142-3p did not affect the functional M1 and M2 composition in blood and lymph nodes (Supplementary Figure 5A, available online). However, there was no direct effect of miR-142-3p on dendritic cell expansion, CD8+ effector responses as reflected by the production of effector cytokines such as IFN-γ, tumor necrosis factor alpha, and interleukin 2, or T-cell–mediated tumor cytotoxicity (Supplementary Figure 5, B–D, available online).