The human monocytic THP-1 cell line was obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan) and cultured in RPMI-1640 medium (Life Technologies, Co., Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Nichirei Biosciences, Inc., Tokyo, Japan), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Co.). THP-1 cells were treated with 320 nM phorbol-12-myristate-13-acetate (PMA; EMD Millipore, Billerica, MA, USA) for 24 h to induce macrophages and washed three times with phosphate-buffered saline (PBS) to remove PMA. Monocytes were isolated from a healthy human donor, who provided written informed consent, in accordance with the protocols approved by the Ethics Committees at Kanazawa University Hospital (study no. 1463). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Peripheral blood was collected from the donor into conical tubes containing 1–2 ml heparin and mixed with an equal volume of saline. PBMCs were isolated using LymphoPrep tubes (Axis-Shield, Dundee, Scotland), according to the manufacturer's protocol, and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, penicillin and streptomycin. After 24 h, the non-adherent cells were removed by gentle aspiration, and the adherent cells were regarded as monocytes. To induce M2 macrophages, these monocytes were cultured with monocyte colony stimulating factor (M-CSF; 100 ng/ml; Wako, Tokyo, Japan) for up to 5 days, followed by treatment with interleukin (IL)-4 (20 ng/ml; PeproTech, Rocky Hill, NJ, USA), IL-10 (20 ng/ml; MACS, Cambridge, MA, USA) and IL-13 (20 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) for 24 h (34). The human gastric cancer cell lines, TMK-1 (poorly differentiated adenocarcinoma) and MKN45 (poorly differentiated adenocarcinoma), were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured as described for THP-1 cells. After being grown to confluence, the cells were harvested by treatment with 0.25% trypsin/EDTA (Life Technologies, Co.). The cells were seeded in gelatin-coated 75-cm² flasks (BD BioCoat, San Jose, CA, USA) and cultured in 10 ml of medium at 37°C in a humidified atmosphere of 5% CO₂ in air.

PTX was purchased from Bristol-Myers Squibb Co. (Tokyo, Japan), dissolved in distilled water at appropriate concentrations and stored at −20°C until used.

The viability of THP-1 macrophages and PBMC-derived M2 macrophages treated with PTX was determined by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 1×10⁴/well in 96-well plates and incubated overnight at 37°C. The supernatants were discarded and replaced with fresh serum-free medium. PTX dissolved in PBS was added to the cell culture medium at concentrations of 0–3,000 nM. After incubation for 24 and 48 h, the supernatants were discarded, MTT solution was added to each well (final concentration, 500 µg/ml) and the cells were incubated at 37°C for 3 h. The supernatants were removed, 150 µl of dimethyl sulfoxide (Wako) was added to each well, and the absorbance of each at 535 nM was determined using a microplate reader (Bio-Rad 550; Bio-Rad, Tokyo, Japan). The percentage of inhibition was calculated by comparing the density of the drug-treated to the untreated control cells. All experiments were repeated a minimum of three times.

An in vitro co-culture system was used to assess the indirect effects of cancer cells on THP-1 macrophages. Briefly, 5×10⁵ THP-1 macrophages were seeded into the lower chamber of each well of a 6-well Transwell dish and incubated in the absence (control) or presence of the same number of MKN45 or TMK-1 cells using 1-µm pore Boyden chambers (BD Falcon, San Jose, CA, USA). To investigate whether PTX modulates THP-1 macrophages, the cells were exposed to 1 and 5 nM PTX at the start of the co-culture.

M2 macrophages derived from PBMC preparations were incubated in the absence or presence of 1, 5 or 10 nM PTX for 48 h, followed by western blot analysis.

Cells were lysed in RIPA buffer [50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l sodium chloride, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 1.0% (w/v) NP-40 substitute (Wako)] containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor (both from Sigma-Aldrich, Inc., St. Louis, MO, USA). The protein concentration of each lysate was measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Whole-cell lysates were boiled in SDS sample buffer and subjected to SDS polyacrylamide gel electrophoresis (Bio-Rad, Philadelphia, PA, USA). Proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad) and blocked with commercial gradient buffer (EzBlock; ATTO, Corp., Tokyo, Japan) at room temperature for 30 min. The membranes were incubated with the following primary antibodies: mouse monoclonal anti-TLR4 IgG, diluted 1:200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); mouse monoclonal anti-CD204 IgG, diluted 1:200 (TransGenic, Inc., Kobe, Japan); mouse monoclonal anti-NOS2 IgG, diluted 1:200 (Santa Cruz Biotechnology, Inc.); rabbit monoclonal anti-pSTAT1 IgG, diluted 1:1,000; rabbit monoclonal anti-pSTAT3 IgG, diluted 1:1,000 (both from Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-NF-κB p65 IgG, diluted 1:200 (Santa Cruz Biotechnology, Inc.); rabbit monoclonal anti-histone H3 IgG, diluted 1:1,000 (Cell Signaling Technology); and mouse monoclonal anti-β-actin IgG, diluted 1:10,000 (Sigma-Aldrich, Inc.). Following incubation with secondary antibodies, the antibody-antigen complexes were detected using an ECL western blotting detection kit (GE Healthcare, Ltd., Tokyo, Japan) and the Light-Capture system (ATTO).

Conditioned media of MKN45 cells were prepared by plating 5×10⁶ tumor cells in 10 ml complete RPMI-1640 supplemented with FBS in 75-cm² flasks for 48 h, and harvesting and centrifuging the supernatants. THP-1 macrophages (3×10⁵ cells/slide) were seeded onto Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY, USA) for 24 h and exposed to 50% conditioned medium of MKN45 cells for 2 days in the absence or presence of 1 or 5 nM PTX. The cells were fixed in a mixture of methanol and acetone (1:1) for 15 min, incubated with normal goat serum (5% in PBS) to block non-specific binding and incubated overnight at 4°C with mouse monoclonal anti-CD204 antibody, diluted 1:200 (TransGenic, Inc.). The slides were washed in PBS and incubated with goat anti-mouse IgG antibody conjugated to Alexa Fluor 488 (Life Technologies, Co.), diluted 1:400 for 1 h at room temperature. The slides were counterstained with 4′,6-diamidino-2-phenylindole (Life Technologies, Co.), diluted 1:2000, to visualize the nuclei and examined under a fluorescence microscope (Olympus BX51; Olympus, Tokyo, Japan).

NF-κB activation was determined by measuring the increased expression of its p65 subunit by western blotting. Briefly, THP-1 macrophages (5×10⁵ cells/well) were seeded into 6-well plates and cultured in RPMI-1640 medium containing 1% FBS for 24 h. The cells were stimulated with LPS (1,000 ng/ml; Wako), PTX (1 or 5 nM), 50% conditioned medium of MKN45 cells, or 50% conditioned medium of MKN45 cells plus 5 nM PTX for 1 h. The cells were harvested with trypsin-EDTA and centrifuged at 500 × g for 5 min. Nuclear extract was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol.

Total RNA was extracted from THP-1 or M2 macrophages using RNeasy Mini kits and treated with RNase-free DNase (both from Qiagen, Germantown, MD, USA). The integrity of isolated RNA was verified by analytical agarose gel electrophoresis. First stand cDNA was prepared from 2-µg aliquots of DNase-treated RNA using cDNA synthesis kits. Primers for VEGF-A (forward, 5′-CCTTGCTGCTCTACCTCCAC-3′ and reverse, 5′-ATGATTCTGCCCTCCTCCTT-3′); VEGF-C (forward, 5′-CACGAGCTACCTCAGCAAGA-3′ and reverse, 5′-GCTGCCTGACACTGTGGTA-3′); IL-10 (forward, 5′-GTCATCGATTTCTTCCCTGTG-3′ and reverse, 5′-ACTCATGGCTTTGTAGATGCCT-3′); arginase (Arg)-1 (forward, 5′-TTCTCAAAGGGACAGCCACG-3′ and reverse, 5′-TCAAGCAGACCAGCCTTTCT-3′); CD163 (forward, 5′-AGGATGCTGGAGTGATTTGC-3′ and reverse, 5′-CCAGCCGTCATCACATATTG-3′); CD204 (forward, 5′-GGGAACATTCTCAGACCTTG-3′ and reverse, 5′-AATCCTCGTGGACCACTTTC-3′); PD-L1 (forward, 5′-GAACTACCTCTGGCACATCCT-3′ and reverse, 5′-CACATCCATCATTCTCCCTTT-3′); PD-L2 (forward, 5′-GTCTTGGGAGCCAGGGTGAC-3′ and reverse, 5′-TGAAAAGTGCAAATGGCAAGC-3′); and TATA box binding protein (TBP; forward, 5′-TGCACAGGAGCCAAGAGTGAA-3′ and reverse, 5′-CACATCACAGCTCCCCACCA-3′) were designed using Primer Express software. The expression of each of the above genes was normalized relative to that of 18S rRNA in the same sample. Each PCR mixture for VEGF-A, VEGF-C, IL-10, Arg-1 and TBP contained 2X SYBR-Green Master Mix (PE Biosystems, Foster City, CA, USA), cDNA template, and optimized primer concentrations, diluted to a final volume of 25 µl with nuclease-free water. All PCR reactions were performed using an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA).

M2 macrophages derived from PBMC were suspended in 10% FBS/RPMI-1640. Cells were plated at density of 5×10⁵ cells/wells in 6-well culture plates. After removing the cell culture supernatants, M2 macrophages were stimulated with PTX (5 nM) in the presence or absence of TAK-242 (20 nM; ChemScene, Monmouth Junction, NJ, USA), a small-molecule-specific inhibitor of TLR4 signaling for 24 h, followed by RT-PCR as mentioned above.

Differences were analyzed by one-way analysis of variance or two-sided Student's t-tests using the computer software package SPSS 10.0 (SPSS, Inc., Chicago, IL, USA). All in vitro data represent the results of three independent experiments. A P-value <0.05 indicated statistical significance.

PTX has been shown to be a ligand of TLR4, a member of the Toll-like receptor family, a class of pattern recognition molecules in innate and acquired immune responses (33). Assays of TLR4 expression in THP-1 macrophages and PBMC-derived M2 macrophages by western blotting showed that both cell types were positive for TLR4 expression (Fig. 1A).

MTT assays of THP-1 macrophages and PBMC-derived M2 macrophages were performed to determine the minimum concentrations of PTX that were cytotoxic towards these cells. The viability of THP-1 macrophages and PBMC-derived M2 macrophages at 48 h was significantly inhibited by 1,000 and 2,000 nM PTX, respectively (P<0.01 each; Fig. 1B). Since the concentrations of PTX in plasma and ascites are ~10 nM up to 72 h after intravenous administration of 80 mg/m² to patients with gastric cancer (35), subsequent experiments used PTX concentrations of 1, 5 and 10 nM.

The interactions among macrophages, gastric cancer cells, and PTX were analyzed using an in vitro co-culture system. Western blot analysis showed that the expression of the M2 marker CD204 was increased when THP-1 macrophages were co-cultured with MKN45 or TMK-1 cells, and that this expression was suppressed by treatment with 1 or 5 nM PTX for 48 h (Fig. 2A). Densitometric analyses showed the same results (Fig. 2B). In contrast, expression of the M1 marker NOS2 was not altered when THP-1 macrophages were co-cultured with MKN45 or TMK-1 cells, but was increased when THP-1 macrophages were treated with 5 nM PTX and co-cultured with MKN45 cells, and when THP-1 macrophages were treated with 1 and 5 nM PTX and co-cultured with TMK-1 cells (Fig. 2A and C).

Although THP-1 macrophages cultured in the absence of conditioned medium of MKN45 cells did not express CD204 (Fig. 3A), cells cultured in the presence of conditioned medium showed induction of CD204 expression (Fig. 3B). Moreover, treatment with 1 and 5 nM PTX suppressed the expression of CD204 (Fig. 3C and D).

The signaling transducer and activator of transcription (STAT) pathways play pivotal roles in the transcriptional profile of macrophages. STAT1 has been shown essential for M1 tumoricidal activity and to trigger the expression of pro-inflammatory cytokines (36). In contrast, cells with M2 phenotype harboring activated STAT3 are not tumoricidal; rather, they facilitate tumor development (37). We, therefore, investigated whether STAT1 and STAT3 in the THP-1 macrophages are phosphorylated after co-culture with the gastric cancer cell lines for 12 h, and found that STAT3 was significantly phosphorylated (Fig. 3E). However, treatment with 1 and 5 nM PTX significantly suppressed the phosphorylation of STAT3 while enhancing the phosphorylation of STAT1.

PBMC-derived M2 macrophages exhibited a spindle pattern morphology in the absence of PTX (Fig. 4A), but had a round shape in the presence of 1, 5 and 10 nM PTX (Fig. 4B-D), suggesting that treatment with PTX may have altered their morphology into M1-like macrophages. Western blot analysis showed that CD204 expression in the PBMC-derived M2 macrophages was suppressed by treatment with 1, 5 and 10 nM PTX (Fig. 4E). Moreover, STAT3 phosphorylation was significantly suppressed by treatment with PTX.

As NF-κB signaling is one of the major pathways involved in LPS and PTX-TLR4-mediated inflammation in macrophages (38), the interactions of LPS and PTX with NF-κB signaling were examined in the THP-1 macrophages. Treatment of THP-1 macrophages with LPS or PTX for 1 h induced marked intranuclear translocation of NF-κB p65 (Fig. 5A). Whereas conditioned medium of MKN45 cells did not induce intranuclear translocation of NF-κB p65, conditioned medium plus 5 nM PTX resulted in significant intranuclear translocation of this protein. This result showed that TLR4-NF-κB p65 signaling is stimulated by co-culture with gastric cancer cells.

Expression of CD204 mRNA in the M2 macrophages treated with PTX was significantly reduced, when compared to M2 macrophages without any treatments as determined by RT-PCR. However, treatment with TAK-242, a TLR4 antagonist, significantly cancelled the lower CD204 mRNA level (P=0.01; Fig. 5B). These data indicate that low-dose PTX can change M2 polarization to M1 polarization in human macrophages via TLR4.

M2 TAMs upregulate the expression of VEGF-A, VEGF-C, IL-10, Arg-1 and CD163 (39), making them target molecules in cancer treatment. To investigate the mechanism of action of PTX in macrophages, the levels of expression of these cytokine mRNAs were assayed in macrophages co-cultured with gastric cancer cells in the presence or absence of 1 nM PTX. The levels of VEGF-A and VEGF-C mRNAs in macrophages were upregulated by co-culture with cancer cells, and downregulated by incubation with 1 nM PTX (P=0.02; Fig. 5C). IL-10, Arg-1 and CD163 mRNAs showed the same trends. These results suggest that PTX can alter macrophage phenotype and functions. We further analyzed PD-L1 and PD-L2 expression. As a result, we did not find any differences in PD-L1 expression among the groups. In contrast, the expression of PD-L2 mRNA was higher in the macrophages co-cultured with cancer cells than that in the macrophages cultured alone, while PTX treatment could not significantly inhibit the upregulated expression.