Regulated Polarization of Tumor-Associated Macrophages by miR-145 via Colorectal Cancer–Derived Extracellular Vesicles

Haruka Shinohara, Yuki Kuranaga, Minami Kumazaki, Nobuhiko Sugito, Yuki Yoshikawa, Tomoaki Takai, Kohei Taniguchi, Yuko Ito and Yukihiro Akao
J Immunol August 15, 2017, 199 (4) 1505-1515; DOI: https://doi.org/10.4049/jimmunol.1700167

Abstract

Macrophages are polarized into functional classically activated and alternatively activated (M2) phenotypes depending on their microenvironment, and these cells play an important role in the immune system. M2-like polarization of tumor-associated macrophages (TAMs) is activated by various secretions from cancer cells; however, the interaction between cancer cells and TAMs is not well understood. Recent studies showed that cancer cell–derived extracellular vesicles (EVs) contribute to tumor development and modulation of the tumor microenvironment. In the current study, we investigated colorectal cancer–derived EVs containing miR-145 with respect to the polarization of TAMs. Colorectal cancer cells positively secreted miR-145 via EVs, which were taken up by macrophage-like cells. Interestingly, colorectal cancer–derived EVs polarized macrophage-like cells into the M2-like phenotype through the downregulation of histone deacetylase 11. An in vivo study showed that EV-treated macrophages caused significant enlargement of the tumor volumes. These findings suggest that colorectal cancer cells use miR-145 within EVs to efficiently modulate M2-like macrophage polarization and tumor progression.

This article is featured in In This Issue, p.1211

Introduction

Macrophages play an important role in the immune system. Depending on their microenvironment, macrophages can become polarized into distinct functional phenotypes: classically activated (M1) macrophages and alternatively activated (M2) macrophages (1–3). M1 macrophages appear in response to LPS and IFN-γ. In contrast, M2 macrophages are activated by IL-4 and IL-13 (1–3). Macrophages can be polarized into the “M2-like” state in response to IL-10, TGF-β, immune complexes, or glucocorticoid (3, 4). Several groups described the expression profiles of microRNAs (miRNAs) in polarized macrophages and microglia, showing that miR-145 is strongly associated with polarization of the M2-like phenotype (5, 6).

Although tumor-associated macrophages (TAMs) have been reported to contain various phenotypes, TAMs generally exhibit the M2-like state and perform some functions that allow cancer growth, invasion, metastasis, and immune evasion (2, 3). Recent studies revealed that secretions from cancer cells promote M2-like polarization of TAMs (7); however, the signals coming from cancer cells to elicit functional polarization of TAMs are not fully understood.

Cancer cells secrete large amounts of extracellular vesicles (EVs), which carry genetic information, including proteins, mRNA, and miRNA (8). As initially described in the 1980s, EVs were considered garbage bags for discarding unnecessary molecules in cells (9). In a previous study, we found that colorectal cancer cells positively secrete antioncogenic miR-145 via EVs, which contributes to the maintenance of low levels of intracellular miR-145 in colorectal cancer cells (10). However, the role of extracellular miR-145 within EVs has not been clarified. EVs are now considered to have important roles in cell-to-cell communication and in the modulation of microenvironments (11, 12). Earlier, we reported that colorectal cancer cell–derived EVs promote angiogenesis and induce phenotypic alteration of regulatory T-like cells, suggesting that EVs contribute to the establishment of a positive tumor microenvironment (13–15).

In this study, we investigated the role of extracellular miR-145 within colorectal cancer–derived EVs in the functional polarization of TAMs. We found that miR-145 secreted from colorectal cancer cells via EVs was taken up by macrophage-like cells and promoted polarization of the M2-like phenotype through the downregulation of histone deacetylase 11 (HDAC11). We also confirmed that EV-treated macrophages promoted substantial tumor growth in vivo. These findings suggest that colorectal cancer cells secrete miR-145 via EVs as one of the communication tools between cancer cells and TAMs to efficiently modulate the tumor microenvironment.

Materials and Methods

Cell culture and treatment

Human monocytic leukemia THP-1 and NOMO-1 cells and human colon cancer DLD-1 cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). All cell lines were tested for mycoplasma contamination using a MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza, Rockland, ME). Cells were cultured under a 95% air/5% CO2 atmosphere at 37°C in RPMI-1640 (code number 189-02025; Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, St. Louis, MO). THP-1 and NOMO-1 cells were used as macrophage-like cells by treating them with 12-O-tetradecanoylphorbol 13-acetate (50 ng/ml) for 24 h. The phenotypic alteration from monocytic leukemia cells to macrophage-like cells was confirmed by flow cytometry (Supplemental Fig. 1A). The macrophage-like cells were cocultured with DLD-1 cells by placing an insert with 0.4-μm pores (catalog number 140660; Thermo Fisher Scientific, Waltham, MA) above the macrophage-like cell layer.

Real-time RT-PCR

Total RNA was isolated from cells using a NucleoSpin miRNA kit (TaKaRa, Otsu, Japan), according to the manufacturer’s protocol. Total RNAs of normal human tissues (Human Total RNA Master Panel II) were purchased from Clontech (Mountain View, CA). The expression levels of miRNAs and mRNAs were determined as described previously (16, 17). The following primer sequences were used: IL-12p40 sense, 5′-CTCTGGCAAAACCCTGACC-3′; IL-12p40 antisense, 5′-GCTTAGAACCTCGCCTCCTT-3′; IL-10 sense, 5′-AAGACCCAGACATCAAGGCG-3′; IL-10 antisense, 5′-AGGCATTCTTCACCTGCTCC-3′; TNF sense, 5′-AGGCGGTGCTTGTTCCTCAG-3′; TNF antisense, 5′-GTTATCTCTCAGCTCCACGC-3′; VEGFA sense, 5′-CAGAATCATCACGAAGTGGT-3′; VEGFA antisense, 5′-AGGAACATTTACACGTCTGC-3′; GAPDH sense, 5′-CAACCCATGGCAAATTCCATGGCA-3′; and GAPDH antisense, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′.

Isolation of cancer cell–derived EVs

For the collection of cancer cell–derived EVs, RPMI-1640 medium supplemented with EV-deprived FBS was made as follows: FBS was centrifuged at 3000 rpm for 5 min, and its supernatant was filtered through a Millex-HV Filter Unit (0.45-μm pores; Merck Millipore, Darmstadt, Germany). The flow-through fraction was ultracentrifuged at 100,000 rpm for 4 h. Without disturbing the EV pellet, the supernatant was carefully removed and used as EV-deprived FBS, which was added to the RPMI-1640 medium (EV-deprived RPMI-1640). DLD-1 cells were cultured for 48 h at 37°C in dishes containing 20 ml of EV-deprived RPMI-1640. The culture medium was collected and centrifuged at 2000 rpm for 5 min. The resulting supernatant was filtered through a 0.45-μm pore filter to remove cellular debris and then through a 0.22-μm pore filter to isolate EVs and/or apoptotic bodies. The flow-through fraction was ultracentrifuged at 90,000 rpm for 3 h. The collected EV pellet was suspended in 1 ml of PBS and incubated with macrophage-like cells. Where indicated, EVs were purified by iodixanol density gradient centrifugation, as described by Tauro et al. (18), with some modifications. For preparation of the discontinuous iodixanol gradient, 40% (w/v) iodixanol working solution was prepared by combining a buffer working solution (0.75 M sucrose/30 mM Tris-HCl) and OptiPrep (Alere Technologies, Oslo, Norway). Solutions of 5, 10, and 20% iodixanol were made by mixing appropriate amounts of a homogenization buffer (0.25 M sucrose/10 mM Tris-HCl) and the iodixanol working solution. EVs were overlaid on top of an iodixanol gradient, which was formed by layering 40, 20, 10, and 5% solution, and the gradient was ultracentrifuged at 90,000 rpm for 16 h. The gradient was aliquoted into 16 fractions, which were diluted in PBS and centrifuged for 3 h at 90,000 rpm. The density of each fraction was estimated by the absorbance value at 340 nm. For analysis of miRNAs in EVs, RNA lysis buffer was added to an EV pellet, and RNA samples were prepared using a NucleoSpin miRNA kit (TaKaRa). To analyze protein expression of EVs, we added protein extraction buffer to a separate EV pellet.

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) is a method used to detect particles ranging in size from ∼30 to 1000 nm in a liquid sample. NTA permits determination of the size distribution and the relative concentration of EVs. NTA measurements were performed with a NanoSight LM10 (NanoSight, Wiltshire, U.K.). A collected EV pellet was suspended in 1 ml of PBS and then diluted at 1:100 in PBS before the analysis. NTA Version 2.3 (NanoSight) was used to capture and analyze data.

Transmission electron microscopy

EVs were incubated with magnetic beads coated with anti-CD63 Ab. After incubation, magnetic beads were washed and fixed in 2% paraformaldehyde in 0.2 M phosphate buffer (PB; pH 7.4). The magnetic beads were washed with PB and postfixed in 2% osmium tetroxide for 2 h. After washing in PB, the beads were progressively dehydrated in a graded ethanol series. Thereafter, they were embedded in EPON 812 resin (TAAB Laboratories Equipment, Reading, U.K.), and thin sections were prepared, stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy.

Western blotting

Protein extraction and Western blotting experiments were performed as described previously (17). Abs against CD9 (sc-13118) and CD81 (sc-166029) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD63 Ab (CBL553) was obtained from Merck Millipore, and anti–β-actin Ab was purchased from Sigma-Aldrich. Anti-HDAC11 (ab47036) and anti–histone H3 (acetyl K9+K14+K18+K23+K27; ab47915) Abs were purchased from Abcam (Cambridge, MA). The quantity loaded was verified using anti–α-tubulin (code number PM054; MBL, Nagoya, Japan).

Flow cytometry

To measure the expression of cell surface markers, cells were harvested, suspended in staining buffer (PBS containing 1% FBS), and incubated with FITC-conjugated anti-human CD11b (catalog number 301329), allophycocyanin-conjugated anti-human CD206 (catalog number 321109), and PE-conjugated anti-human CD68 (catalog number 333807; all from BioLegend, San Diego, CA). The cells were acquired and analyzed with an EC800 Cell Analyzer (Sony Biotechnology, Tokyo, Japan).

ELISA

The concentration of IL-12p40 and IL-10 in culture supernatants was measured using a Quantikine ELISA Kit (R&D Systems), according to the manufacturer’s protocol.

Immunocytochemistry

12-O-tetradecanoylphorbol-13-acetate was added to THP-1 and NOMO-1 cells, which were then seeded into the wells of a Lab-Tek II Chamber Slide System (Thermo Fisher Scientific) the day before incubation with DLD-1 cell–derived EVs. EVs were prestained with Vybrant DiO Cell-Labeling Solution (Molecular Probes, Eugene, OR), as described previously (13). Labeled EVs were added to cultures of THP-1 macrophage-like cells. The cells were stained with Hoechst33342 (5 μg/ml) and anti-phalloidin Ab (Cytoskeleton, Denver, CO), according to the immunofluorescence protocol of Cell Signaling Technology. Labeled cells were observed with a BIOREVO fluorescence microscope (Keyence, Osaka, Japan).

Tracking of nascent miR-145 derived from DLD-1 cells to macrophage-like cells via EVs

Nascent miR-145 was traced using a Click-iT RNA capture Kit (Invitrogen), as described previously (13). Briefly, donor DLD-1 cells were incubated overnight with 0.2 mM 5-ethynyl uridine (EU), which is naturally incorporated into nascent RNA. After the incubation, total RNA was extracted from the donor cells. EVs derived from DLD-1 cells were isolated from DLD-1 culture medium and added to the medium of the recipient macrophage-like cells. After overnight incubation, total RNA was extracted from the recipient cells. EU-labeled RNA was purified and subjected to quantitative RT-PCR.

Luciferase reporter assay

A predictive binding site in the 3′ untranslated region (UTR) of human HDAC11 for miR-145 was inserted into a pMIR-REPORT Luciferase miRNA Expression Reporter Vector (Applied Biosystems), according to the manufacturer’s protocol. The vector with a mutation in the binding site contained a seed region mutated using a PrimeSTAR Mutagenesis Basal Kit (TaKaRa). Mutation of the vector was confirmed by sequence analysis. The vectors (0.1 μg/ml) and miR-145 (20 nM) or nonspecific control miRNA (Dharmacon) were used to cotransfect THP-1 macrophage-like cells. Luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Assay for HDAC11 overexpression

The HDAC11 expression vector was generated by inserting the open reading frame of HDAC11 cDNA into the Sgfl and Pmel sites of the pF5A-CMV neo vector (Promega). Macrophage-like cells were transfected with this vector (0.2 μg/ml) using Lipofectamine 2000 (Invitrogen).

Human tumor xenograft model

The Committee for Animal Research and Welfare of Gifu University approved all animal experimental protocols. Animal experiments were conducted in accordance with the guidelines for Animal Experiments of Gifu International Institute of Biotechnology. DLD-1 cells (1 × 106 cells in 100 μl of PBS) were coinjected s.c. with EV-treated THP-1 macrophage-like cells (1 × 106 cells in 100 μl of PBS) into two sites on the back of five female athymic nude mice. DLD-1 cells and EV/antagomiR-145–treated THP-1 macrophage-like cells were coinjected into two sites on right flanks of the mice. DLD-1 cells and nontreated THP-1 macrophage-like cells were inoculated into two sites on their left flank. Tumor size was monitored by measuring the length (L) and width (W), and the volume (V) was estimated according to the following formula: V = (L × W2) × 0.5.

Results

miR-145 was secreted via EVs by colorectal cancer cells

As shown previously (19), miR-145 was confirmed to be highly expressed in normal colon tissue (Fig. 1A). The expression level of miR-145 is frequently decreased in colorectal adenomas and cancers compared with adjacent nontumor mucosal tissue in the same patient (20–22). Such a decrease is also observed in human colorectal cancer cell lines (20, 21). Earlier, we reported that the constant secretion of miR-145 via EVs contributes to the downregulation of intracellular miR-145 in colorectal cancer cells (10). The levels of extracellular miR-145 within EVs were significantly greater than the intracellular ones (Fig. 1B), suggesting that miR-145 was secreted from the various colorectal cancer cell lines tested. DLD-1 cells secreted a relatively higher level of miR-145 compared with other cell lines. To further examine the purity of EVs derived from DLD-1 cells, we examined the protein-expression profiles of various tetraspanins (CD9, CD63, and CD81). As shown in Fig. 1C, these proteins were dominantly expressed in EVs compared with their cellular expression. Cytosolic protein Tsg101 was also observed in the EVs. In contrast, calnexin (endoplasmic reticulum marker), histone H3 (nuclear protein), and β-actin (cytoskeletal marker) were barely detected in EVs. Use of the nanoparticle tracking system (NTA) revealed that DLD-1 cells secreted EVs that were heterogeneous in size (average size: 115.3 ± 1.5 nm, Fig. 1D). The morphology of EVs was assessed using electron microscopy (Fig. 1E). These data indicated that our isolation method yielded EVs as a mixture of exosomes and shed microvesicles. Furthermore, we performed iodixanol density gradient centrifugation and confirmed the presence of miR-145 in EVs. The expression of miR-145 was concentrated in the EV fraction (Supplemental Fig. 1B, 1C). Considering these data, we concluded that our isolation method efficiently yielded EVs.

FIGURE 1.
FIGURE 1.

miR-145 was secreted via EVs by colorectal cancer cells. (A) Relative expression (2−ΔΔCt) of miR-145 in various normal human tissues. (B) Relative expression levels (2−ΔΔCt) of intracellular (Cell) and extracellular (EV) miR-145 per total RNA in colorectal cancer cell lines. EVs were collected from the medium of each cell culture 48 h after establishment of the culture. (A and B) Data are expressed as the mean ± SD of three experiments. ***p < 0.001 versus intracellular expression, Student t test. (C) Protein-expression profiles of EVs from DLD-1 cells. (D) Characterization of DLD-1 cell–derived EVs by NTA. Average size of EVs is shown as the mean ± SD. (E) Electron microscopic image of EVs isolated from the supernatant of a DLD-1 cell culture. EVs (exosomes and shed microvesicles) were captured by poly-l-lysine coated beads.

Macrophage-like cells exhibited the M2-like phenotype after being cocultured with DLD-1 cells

To validate the phenotype of TAMs around DLD-1 cells, THP-1 or NOMO-1 macrophage-like cells were cocultured with DLD-1 cells. The expression levels of IL-12p40 and TNF-α mRNAs were decreased in these macrophage-like cells, whereas those of IL-10 and VEGFA mRNAs were increased (Fig. 2A). The increased production of IL-12p40 and IL-10 protein also was detected by ELISA (Fig. 2B). Flow cytometry analysis showed that the population of CD11b+ CD68+ CD206+ cells was dramatically increased after coculture with DLD-1 cells (Fig. 2C). These results suggested that TAMs around DLD-1 cells primarily exhibited the M2-like phenotype. Moreover, the expression level of miR-145 was increased significantly in the macrophage-like cells (Fig. 2D).

FIGURE 2.
FIGURE 2.

Macrophage-like cells cocultured with DLD-1 cells exhibited an M2-like phenotype. THP-1 and NOMO-1 macrophage-like cells were cocultured with DLD-1 cells for 48 h. Control cells were cultured in the absence of DLD-1 cells. (A) Expression levels of IL-12p40, TNF, IL-10, and VEGFA mRNA in macrophage-like cells. IL-12p40 and TNF were used as M1 macrophage markers, and IL-10 and VEGFA were used as M2 macrophage markers. (B) IL-12p40 and IL-10 levels in supernatants of macrophage-like cells, as measured by ELISA. (C) Characterization of phenotypes of macrophage-like cells by their cell surface markers. Macrophage-like cells were stained for CD11b, CD68, and CD206. Cells were gated for CD11b+ and CD68/CD206. A representative analysis is shown. (D) Expression levels of miR-145 in macrophage-like cells. (A, B, and D) Data are expressed as the mean ± SD of three experiments. **p < 0.01, ***p < 0.001 versus control, Student t test.

miR-145 secreted via EVs promoted polarization of the M2-like phenotype

The microarray data from several groups indicated that miR-145 is strongly associated with polarization of the M2-like phenotype (5, 6). Therefore, we examined whether miR-145 regulated macrophage polarization. Ectopic expression of miR-145 significantly increased the intracellular level of miR-145 (Fig. 3A) and led to downregulation of IL-12p40 and TNF-α mRNAs and upregulation of IL-10 and VEGFA mRNAs (Fig. 3B). IL-12p40 and IL-10 production and the population of CD11b+ CD68+ CD206+ cells were also increased after transfection with miR-145 (Fig. 3C, 3D). These results suggested that miR-145 promoted polarization to the M2-like phenotype.

FIGURE 3.
FIGURE 3.

miR-145 promoted polarization of the M2-like phenotype. THP-1 and NOMO-1 macrophage-like cells were transfected with miR-145 (10 or 20 nM) or nonspecific RNA (Control; C) for 72 h. (A) Expression levels of miR-145 in macrophage-like cells after transfection with miR-145 (10 nM). (B) Expression levels of IL-12p40, TNF, IL-10, and VEGFA mRNA in macrophage-like cells. (C) IL-12p40 and IL-10 levels in supernatants of macrophage-like cells, as measured by ELISA. (D) Characterization of phenotypes of macrophage-like cells by cell surface markers. Macrophage-like cells were stained for CD11b, CD68, and CD206. Cells were gated for CD11b+ and CD68/CD206. A representative analysis is shown. (A–C) Data are expressed as the mean ± SD of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control, Student t test.

Cancer cell–derived EVs are suggested to be an important tool for the establishment of a conducive tumor microenvironment (12). To investigate the interactions between tumor and macrophages via EVs, we incubated macrophage-like cells with DLD-1 cell–derived EVs and confirmed the uptake of EVs by these macrophage-like cells using fluorescence-labeled EVs. As shown in Fig. 4A, the labeled EVs, shown as green dot-like shapes, were definitely contained within the macrophage-like cells. Incubation with EVs significantly increased the expression level of miR-145 in the macrophage-like cells (Fig. 4B), decreased IL-12p40 and TNF-α mRNA levels, and increased IL-10 and VEGFA mRNA levels (Fig. 4C). Decreased production of IL-12p40 and increased production of IL-10 protein were also observed by ELISA (Fig. 4D). CD11b+ CD68+ CD206+ cells were markedly increased in number after incubation with EVs (Fig. 4E). Moreover, we traced the EV-containing nascent miR-145 from donor DLD-1 cells to recipient macrophage-like cells. EU-labeled nascent miR-145 was detected in DLD-1 cells and in their EVs. EU-labeled miR-145 was also detected within the macrophage-like cells (Fig. 4F). These results suggested that miR-145 was carried by EVs from DLD-1 cells to macrophage-like cells, thus promoting M2-like polarization.

FIGURE 4.
FIGURE 4.

miR-145 within DLD-1 cell–derived EVs promoted polarization of the M2-like phenotype. THP-1 and NOMO-1 macrophage-like cells were coincubated or not with DLD-1 cell–derived EVs (∼2 × 109 particles) for 48 h. (A) Immunocytochemistry of macrophage-like cells after incubation with labeled EVs. EVs (green), F-actin (red), and nuclei (blue) are shown. Scale bar, 30 μm. Expression levels of miR-145 (B) and IL-12p40, TNF, IL-10, and VEGFA mRNA (C) in macrophage-like cells. (D) IL-12p40 and IL-10 levels in supernatants of macrophage-like cells, as measured by ELISA. (E) Characterization of phenotypes of macrophage-like cells by cell surface markers. Macrophage-like cells were stained for CD11b, CD68, and CD206. Cells were gated for CD11b+ and CD68/CD206. A representative analysis is shown. (F) EU-labeled nascent miR-145 levels in donor DLD-1 cells, DLD-1 cell–derived EVs, and recipient macrophage-like cells. (B–D) Data are expressed as the mean ± SD of three experiments. **p < 0.01, ***p < 0.001 versus EV (−), Student t test.

miR-145 promoted IL-10 production by targeting HDAC11

To elucidate which signaling pathway was involved in the polarization of M2-like macrophages by miR-145, we examined the effect of miR-145 on potential target genes. Previous reports showed that histone deacetylase, HDAC11, which is one of the miR-145 target genes, regulates IL-10 expression (23, 24). Ectopic expression of miR-145 suppressed HDAC11 expression in the macrophage-like cells (Fig. 5A). The increased acetylation level of histone H3 was negatively correlated with HDAC11 expression (Fig. 5A). In contrast, treatment with antagomiR-145 reversed the expression level of HDAC11 (Fig. 5B). To further confirm HDAC11 as a direct target gene of miR-145, we cloned the predicted binding site for miR-145 in the 3′UTR of human HDAC11 mRNA (from 1452 to 1717) into the pMIR-REPORT vector (Supplemental Fig. 2A). The luciferase activity of wild-type pMIR was decreased significantly when the cells were cotransfected with miR-145, whereas mutation of the seed sequence abolished the ability of miR-145 to regulate luciferase activity (Supplemental Fig. 2A). These results indicate that miR-145 directly targeted HDAC11 expression in macrophage-like cells. The silencing of HDAC11 also led to upregulation of histone H3 acetylation (Supplemental Fig. 2B). As with transfection with miR-145 (Fig. 3B), expression of IL-12p40 was decreased and that of IL-10 was increased after transfection with siR-HDAC11 (Supplemental Fig. 2C). Furthermore, overexpression of HDAC11 diminished histone H3 acetylation and attenuated the effects on IL-10 expression (Supplemental Fig. 2D, 2E). These results indicate that miR-145 directly silenced HDAC11 expression and promoted IL-10 production in the macrophage-like cells.

FIGURE 5.
FIGURE 5.

miR-145 promoted IL-10 production by targeting HDAC11. (A) Expression levels of HDAC11, acetyl-histone H3, and histone H3 in macrophage-like cells after transfection with miR-145 (10 or 20 nM) or nonspecific RNA (Control; C) for 72 h. (B) Effects of antagomiR-145 on the expression of HDAC11. Cells were transfected with nonspecific RNA (Control; C), miR-145 (10 nM), or antagomiR-145 (10 or 20 nM) for 72 h. The numbers below HDAC11 indicate the density of each band relative to that of control (taken as 100). Expression levels of HDAC11, acetyl-histone H3, and histone H3 (C) and miR-145 (D) in macrophage-like cells after coincubation with DLD-1 cell–derived EVs (∼2 × 109 or 4 × 109 particles) for 48 h. (E) Expression levels of HDAC11, acetyl-histone H3, and histone H3 in macrophage-like cells after coculture with DLD-1 cells for 48 h. (F and G) Macrophage-like cells were transfected with pCMV–HDAC11–3′UTR (WT) or pCMV–HDAC11–3′UTR mutant (Mut) vectors for 24 h and then coincubated with EVs (∼4 × 109 particles) for 48 h. The vector contained the inserted sequence region 186–1717 containing the putative binding sequence of miR-145, as described in Supplemental Fig. 2A. Mutant-type vector (Mut) contained the inserted mutated seed sequence (from CUG to GAC) for miR-145. (F) The expression level of HDAC11 was confirmed by Western blotting. (G) Characterization of phenotypes of macrophage-like cells by cell surface markers. Macrophage-like cells were stained for CD11b, CD68, and CD206. Cells were gated for CD11b+ and CD68/CD206. A representative analysis is shown.

Next, we investigated whether miR-145 within EVs regulated HDAC11 expression. Incubation with EVs decreased HDAC11 expression, which was negatively correlated with the upregulation of miR-145 expression (Fig. 5C, 5D). Downregulation of HDAC11 was also observed in the macrophage-like cells cocultured with DLD-1 cells (Fig. 5E). To further investigate the association between HDAC11 and M2-like polarization via EVs, we constructed HDAC11 expression vectors containing the open reading frame and 3′UTR. As shown in Fig. 5F and 5G, the vector that contained the wild-type 3′UTR barely affected M2-like polarization by EVs; however, the vector having a mutated 3′UTR binding site attenuated the effects of EVs on M2-like polarization, suggesting that miR-145 within EVs silenced HDAC11 and promoted M2-like macrophage polarization.

Based on the TargetScan database (http://www.targetscan.org/), we speculated that TLR4 is also a key regulator of M2-like polarization. We confirmed that miR-145 directly targeted TLR4 in macrophage-like cells (data not shown). The knockdown of TLR4 decreased the expression of IL-12p40; however, the effects on downstream signaling and the expression of IL-10 were not significant (data not shown). Considering these results, we concluded that perturbation of TLR4 signaling by miR-145 might have partly caused suppression of the M1 phenotype polarization; however, HDAC11 contributed to M2-like polarization more so than did TLR4.

Secreted miR-145 was one of the molecules most closely associated with M2-like macrophage polarization

Our data suggested that miR-145 was strongly associated with M2-like macrophage polarization; however, other miRNAs contained in EVs might be involved in the phenotypic regulation of macrophages. The TargetScan database showed that miR-124 also predictively targets HDAC11 and TLR4. The extracellular level of miR-124 was higher than its intracellular one (Supplemental Fig. 3A); however, the expression level of miR-124 was not significantly changed in the macrophage-like cells after coculture with DLD-1 cells (Supplemental Fig. 3B). Moreover, ectopic expression of miR-124 increased IL-12p40 mRNA expression and decreased IL-10 mRNA expression (Supplemental Fig. 3C). These results suggested that miR-145 was more likely to be involved in M2-like macrophage polarization by EVs compared with other miRNAs targeting HDAC11 and TLR4. To further confirm the association between miR-145 within EVs and M2-like macrophage polarization, we transfected DLD-1 cells with miR-145 or antagomiR-145, isolated their EVs from the medium of each cell culture, and incubated them with THP-1 and NOMO-1 macrophage-like cells. As shown in Fig. 6A, the expression level of miR-145 was markedly increased in miR-145–transfected DLD-1 cell–derived EVs (EV/miR-145). In contrast, transfection with antagomiR-145 slightly, but significantly, decreased the level of miR-145 within EVs (EV/antagomiR-145). EV/miR-145 enhanced the suppression of HDAC11 expression in macrophage-like cells compared with EV/control (Fig. 6B). EV/miR-145 also enhanced the effects on IL-12p40 and IL-10 mRNA expression (Fig. 6C). Furthermore, EV/antagomiR-145 increased HDAC11 protein expression (Fig. 6B). The decrease in IL-12p40 and increase in IL-10 mRNA expression were partially canceled by EV/antagomiR-145 compared with EV/control (Fig. 6C). The production of IL-12p40 was slightly decreased in EV/antagomiR-145–treated cells; however, there was no difference in IL-10 production (Fig. 6D). The population of CD11b+ CD68+ CD206+ cells was changed minimally after incubation with EV/antagomiR-145 (Fig. 6E) compared with EVs (Fig. 4E). Based on these data, we considered miR-145 to be one of the molecules that made an important contribution to M2-like macrophage polarization by DLD-1 cell–derived EVs by silencing HDAC11.

FIGURE 6.
FIGURE 6.

Secreted miR-145 was one of the molecules most closely associated with M2-like polarization. EVs were collected from the supernatant of each DLD-1 cell culture after transfection for 72 h (EV/Control: EVs from nonspecific control RNA–transfected DLD-1 cells; EV/miR-145: EVs from miR-145 mimic–transfected DLD-1 cells; EV/antagomiR-145: EVs from antagomiR-145–transfected DLD-1 cells). (A) Expression level of miR-145 in each type of EV. Data are expressed as the mean ± SD of three experiments. *p < 0.05, ***p < 0.001 versus EV/Control, Student t test. (B) Expression level of HDAC11 in macrophage-like cells after coincubation with each type of EV for 48 h. The numbers below HDAC11 indicate the density of each band relative to that of EV (−). (C) Effects of each type of EV on the expression of IL-12p40 and IL-10 mRNA. Data are expressed as the mean ± SD of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus EV (−), Student t test. (D) IL-12p40 and IL-10 levels in supernatants of macrophage-like cells, as measured by ELISA. *p < 0.05 versus EV (−), Student t test. (E) Characterization of phenotypes of macrophage-like cells by cell surface markers. Macrophage-like cells were stained for CD11b, CD68, and CD206. Cells were gated for CD11b+ and CD68/CD206. A representative analysis is shown.

DLD-1 cell–derived EVs regulated functions of the M2-like phenotype

Previous reports showed that M2-like TAMs perform some functions that allow tumor growth (3, 7). To determine the functions of EV-treated macrophage-like cells, we compared the tumor volumes of DLD-1 cell–xenografted mice coinjected with nontreated macrophage-like cells, EV-treated macrophage-like cells, or EV/antagomiR-145–treated macrophage-like cells. Coinjection with EV-treated macrophage-like cells resulted in significantly larger tumors (Fig. 7A, 7B). In these enlarged DLD-1 tumors, connective tissues with capillary vessels were clearly observed (Fig. 7C, upper panels). The expression level of CD206 in macrophage-like cells was further elevated in tumors coinjected with EV-treated macrophages compared with nontreated macrophages (Fig. 7C, lower panels). In contrast, coinjection with EV/antagomiR-145–treated macrophage-like cells partially abrogated the enlargement of tumor volumes compared with coinjection with EV-treated macrophage-like cells (Fig. 7A, 7B). These results suggested that macrophages polarized via DLD-1 cell–derived EVs containing miR-145 contributed to tumor progression.

FIGURE 7.
FIGURE 7.

Colorectal cancer–derived EVs regulated the M2-like phenotype. (A and B) Tumor volumes of DLD-1 cell–xenografted mice coinjected with nontreated macrophages, EV-treated macrophages, or EV/antagomiR-145–treated macrophages. Representative images are shown. (C) Immunohistochemical staining of DLD-1 tumors from mice coinjected with nontreated macrophages or EV-treated macrophages. Representative photomicrographs of H&E-stained tumors (upper panels) and those stained with anti-CD206 Ab (lower panels). Scale bars, 50 μm. (D) Schematic diagram of the regulation of macrophage polarization by miR-145 via colorectal cancer–derived EVs. miR-145 within colorectal cancer–derived EVs is taken up by macrophages, increasing their intracellular miR-145 level. miR-145 directly targets HDAC11, leading to IL-10 production by inhibition of histone deacetylation. The M2-like macrophage polarized by EVs promotes tumor growth. miR-145 also targets TLR4, which may partially cause diminished M1 phenotype polarization. *p < 0.05, ***p < 0.001 versus nontreated macrophages. CA, capillary vessel; TU, tumor.

Discussion

This study revealed that miR-145 secreted from colorectal cancer cells via EVs were taken up by macrophages, promoting M2-like phenotype polarization by targeting HDAC11 and TLR4 (Fig. 7D). TAMs polarized by miR-145 within EVs may contribute to modulation of the tumor microenvironment and tumor progression.

Much evidence has revealed that miR-145 acts as a tumor suppressor to promote apoptosis and modulate the cell cycle and that ectopic expression of miR-145 results in apoptotic cell death (20, 25). The induction of apoptosis barely occurred in THP-1– and NOMO-1–derived macrophage-like cells after their transfection with miR-145 (data not shown). In various cancer cells, expression levels of miR-145 are extremely downregulated to avoid antioncogenic functions (21, 26–28). Generally, antioncogenes are downregulated by genomic abnormality, epigenetic changes, aberrant transcription, or impaired processing. In the case of miR-145, deletion of transcriptional factor p53 was reported (25). In addition, disposition via EVs contributes, in part, to the low levels of intracellular miR-145 (10). In this study, we demonstrated that nascent miR-145 in colorectal cancer cells was definitely sorted into their EVs. A recent study revealed that specific sequence motifs in miRNAs control their localization into EVs (29). The sequence of miR-145 contains some exosome-dominant miRNA motifs, such as CCCU. Thus, these motifs might cause the sorting of miR-145 into EVs.

Some exosomal miRNAs were reported to be increased in the serum or plasma from cancer patients (30). We examined the expression level of miR-145 within EVs in the plasma of DLD-1 cell–xenografted mice; however, the difference compared with the control was not statistically significant (p = 0.109, Supplemental Fig. 4). Previous reports showed that the miR-145 level is decreased in the plasma from colorectal cancer patients (31, 32). Based on these findings, we speculate that miR-145 does not circulate throughout the total body system but instead adjusts to the topical tumor microenvironment. Further studies will be needed to discover the fate of secreted miR-145.

This study demonstrated that HDAC11, as a target gene of miR-145, was associated with M2-like macrophage polarization. First, we speculated that TLR4 is a key regulator of M2-like polarization. TLR4 signaling is transduced through downstream adaptors and activates cytokine production (33). One of these adaptors, TIRAP (also known as Mal), which was also reported to be a target gene of miR-145, has a crucial role in the production of proinflammatory cytokines (34, 35). We speculate that perturbation of TLR4 signal transduction by miR-145 may inhibit, in part, the production of proinflammatory cytokines, such as IL-12 or TNF, to diminish M1 phenotype polarization. LPS-stimulated TAMs produce higher levels of anti-inflammatory cytokines compared with nonstimulated TAMs (36), suggesting that TLR4 might also be needed for the production of anti-inflammatory cytokines in TAMs. Lin et al. (24) also showed that the overexpression of miR-145 has little effect on the TLR4 signaling pathway, and they identified HDAC11 as a critical target of miR-145. HDAC11 was identified in 2002 as a member of HDAC class IV (37). Although the function of HDAC11 remains poorly understood, recent studies showed that it negatively regulates IL-10 expression. The disruption of HDAC11 leads to acetylation of histone H3 and H4 and subsequent recruitment of transcription factors Sp1 and STAT3, followed by upregulation of IL-10 expression (23). Recent studies also revealed the role of HDAC11 as an epigenetic regulator of myeloid-derived suppressor cell expansion (38). Our data suggested that HDAC11 was one of the essential targets of miR-145 for M2-like macrophage polarization; however, overexpression of HDAC11 did not completely cancel the effects of EVs. Thus, we could not exclude the possibility that some other target molecule(s) might be involved in the regulated macrophage polarization by miR-145; further investigation is needed.

In this study, we showed that miR-145 within EVs is one of the molecules most closely involved in M2-like macrophage polarization. To validate the involvement of miR-145 in macrophage polarization, we performed experiments using EV/antagomiR-145 (Fig. 6). EV/antagomiR-145 attenuated M2-like polarization; however, the effects on the suppression of M1 phenotype polarization were not entirely canceled. We could not completely exclude the possibility that other molecules might be involved, because EVs contain various proteins, mRNAs, and miRNAs. We recommend that more investigation and discussion are needed. There are some limitations to our investigation. We showed the modulation of tumor progression by DLD-1 cell–derived EVs in a xenografted nude mice model, which rules out the involvement of adaptive immunity. Macrophages promote angiogenesis and subsequent tumor growth by VEGF (39). EV-treated macrophage-like cells highly expressed VEGF (Fig. 4C), which might be one of the causes of promoted tumor growth (Fig. 7A, 7B).

We demonstrated the functions of EV-treated macrophage-like cells. Our data from in vitro and in vivo systems indicate that macrophages polarized by EVs could contribute to tumor progression. Taken together, our findings suggest that, to maintain themselves, cancer cells use miR-145 within EVs to modulate the functions of surrounding macrophages.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by a grant-in-aid for a research fellow from the Japan Society for the Promotion of Science (16J08131 to H.S.) and a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (24659157 to Y.A.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    EU
    5-ethynyl uridine
    EV
    extracellular vesicle
    HDAC11
    histone deacetylase 11
    M1
    classically activated
    M2
    alternatively activated
    miRNA
    microRNA
    NTA
    nanoparticle tracking analysis
    PB
    phosphate buffer
    TAM
    tumor-associated macrophage
    UTR
    untranslated region.

  • Received February 1, 2017.
  • Accepted June 21, 2017.

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