Interleukin-6 secreted by tumor-associated macrophages promotes proliferation and migration through JAK2/STAT3 signaling pathway in human prostate cancer cells.

1
Introduction
Prostate cancer is one of the most common malignancies worldwide, with over 1.46 million new cases diagnosed annually.1 Early stage disease is typically treated with prostatectomy, radiotherapy, or androgen deprivation therapy. However, many advanced cases eventually develop resistance, resulting in castration-resistant prostate cancer (CRPC).2,3 Traditional therapies have largely targeted androgen receptor signaling. However, recent studies have emphasized the critical role of cytokine-mediated signaling in the tumor microenvironment (TME), offering new therapeutic avenues.4
The TME is composed of a heterogeneous population of cells, including epithelial cells, fibroblasts, immune cells, macrophages, and various cytokines, all of which contribute to tumor progression and metastasis.5, 6, 7 Among these, macrophages, which differentiate from circulating monocytes, are the most abundant stromal cell type. Due to their functional plasticity, macrophages can polarize into either proinflammatory M1 or anti-inflammatory M2 phenotypes depending on environmental stimuli.8 M1 macrophages, activated by lipopolysaccharide (LPS), IFN-γ, tumor necrosis factor (TNF)-α, and GM-CSF, secrete cytokines such as interleukin (IL)-1β, IL-6, IL-12, and TNF-α, thereby enhancing T cell responses and antitumor immunity. In contrast, M2 macrophages, stimulated by IL-4, IL-13, and M-CSF, produce IL-10, TGF-β, and CCL22, which are associated with tissue repair, immune suppression, and tumor progression.9
Tumor-associated macrophages (TAMs), which often exhibit an M2-like phenotype, contribute to immune evasion, angiogenesis, and metastasis. Their abundance in tumors correlates with poor prognosis in various cancer types.10,11 A key mediator in the TME is IL-6, a multifunctional cytokine implicated in tumor progression, chemoresistance, and metastasis.12 Elevated IL-6 levels have been observed in advanced prostate cancer and CRPC, supporting its role in disease aggressiveness.13,14 Furthermore, androgen receptor-negative prostate cancer cell lines such as DU-145 and PC3 exhibit high IL-6 expression, which supports their growth and survival through both autocrine and paracrine mechanisms.15,16
One of the principal pathways activated by IL-6 is the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis. This pathway governs key oncogenic processes, including cell proliferation, survival, invasion, and angiogenesis.17,18 Aberrant STAT3 activation is a hallmark of multiple cancers and is known to drive anti-apoptotic and tumor-promoting signalings.19,20 In prostate cancer specifically, IL-6-induced STAT3 activation has been associated with neuroendocrine differentiation in androgen-sensitive LNCaP cells.21
Taken together, there is extensive evidence that factors secreted by prostate cancer cells promote the differentiation of macrophages within the tumor microenvironment into tumor-associated macrophages (TAMs). Additionally, IL-6 produced by TAMs exerts a paracrine effect on prostate cancer cells by activating the JAK-STAT signaling pathway. Despite the well-established role of TAMs in tumor progression, the direct interaction between prostate cancer cells and TAMs is not well understood. Therefore, this study aims to investigate how PC-3-derived factors induce macrophage differentiation and, further, how paracrine signaling mediated by macrophage-secreted IL-6 promotes PC-3 cell proliferation and motility. To this end, we co-cultured PC-3 cells with undifferentiated macrophages to investigate macrophage differentiation induced by PC-3-derived factors. We also examined the role of IL-6-mediated signaling, secreted by macrophages, in regulating PC-3 proliferation and migration within this co-culture system.

2
Materials and methods
2.1
Cell culture and treatment
PC3 prostate cancer cells (ATCC, Manassas, VA, USA) were cultured in DMEM-LG (Gibco, Waltham, MA, USA) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin (Gibco). THP-1 monocytes (ATCC) were cultured in RPMI 1640 (Gibco, Waltham, MA, USA) with the same supplements. All cells were maintained in a humidified atmosphere of 5 % CO2 at 37°C. To assess IL-6 effects on PC3 cell proliferation, migration, and invasion, recombinant human IL-6 protein (R&D Systems, Minneapolis, MN, USA) was added at 0, 10, and 100 ng/mL. For co-cultures with TAMs, IL-6 activity was inhibited using a 2.5 μg/mL IL-6 neutralizing antibody (Rockland Immunochemicals, Inc., Limerick, PA, USA), and JAK2/STAT3 activity was blocked using 5 μM WP1066 (Merck, Darmstadt, Germany).

2.2
Macrophage differentiation and polarization
THP-1 cells were differentiated into M0 macrophages by treatment with 150 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA) for 24 h, washed with 1X phosphate-buffered saline, and cultured in a PMA-free medium. To polarize the cells into M1 macrophages, the cells were treated with 250 ng/mL LPS (Sigma) and 20 ng/mL IFN-γ (R&D Systems) for 48 h. To polarize the cells to the M2 macrophages, the cells were treated with 20 ng/mL IL-4 (R&D Systems) and 20 ng/mL IL-13 (R&D Systems) for 48 h. To generate TAMs, the M0 macrophages were cultured with PC3 cell-derived conditioned medium (CM) mixed with fresh medium at a ratio of 2:1 (v/v) and incubated for 48 h in a humidified atmosphere of 5 % CO2 at 37°C.

2.3
Collection of CM
PC3 cells and TAMs were grown to 70 % confluence in a humidified atmosphere containing 5 % CO2 at 37°C. After 72 h of incubation, the CM was collected, centrifuged at 800×g for 5 min, and filtered through a 0.2 μm membrane filter. The supernatant was stored at −80°C until further use.

2.4
Indirect co-culture
THP-1 cells were seeded at densities of 2.5 × 105 cells in 6-well plates, 5 × 104 cells in 12-well plates, and 1 × 104 cells in 24-well plates in Transwell inserts (0.4 μm pores; SPL Life Sciences, Pocheon, Korea). Cells were differentiated with PMA, rested for 24 h in fresh RPMI-1640 medium, and then treated with PC3-CM for 48 h to induce TAMs. Inserts containing TAMs were transferred to wells with pre-seeded PC3 cells (5 × 105, 1 × 105, or 2 × 104 cells per well in the corresponding plates). After 48 h of co-culture, the inserts were removed, and PC3 cells were harvested for cell proliferation and migration assays, qRT-PCR, and western blotting. All cells were maintained in a humidified atmosphere containing 5 % CO2 at 37°C.

2.5
RNA extraction and quantitative RT-PCR
Total RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) and then extracted using chloroform, isopropyl alcohol, and 75 % ethanol. The dried RNA pellet was dissolved in RNase-free DEPC-treated water (Invitrogen, Waltham, MA, USA). RNA concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, 1 μg of RNA was mixed with 10 pmol oligo(dT) at 65°C for 5 min, then incubated in 5x RT buffer (Invitrogen) containing 10 mM dNTP (Promega, Madison, WI, USA), 200 U/μL RTase (Invitrogen), and 2 μL of 100 mM Dithiothreitol (DTT) at 37°C for 50 min, followed by enzyme inactivation at 70°C for 15 min. qRT-PCR was performed in 20 μL reactions containing 1 μL of the cDNA template, 10 μL of SYBR Green (Dyne Bio Inc., Seongnam, Korea), 7 μL of RNase-free DEPC-treated water, and 10 pmol of each primer (BIONEER, Daejeon, Korea). The primer sequences for CD38, HLA-DRβ1, TNF-α, CCL2, CXCL12, IL-6, IL-1β, CD163, CD206, IL-10, VEGFA, TGF-β1, TGF-α, mTOR, FOS, CCND1, PXN, and 18S are listed in Table 1. Amplification was performed using a LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) under the following program: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s.

2.6
MTT assay
Cell proliferation was evaluated using the MTT assay. PC3 cells (5 × 103 cells/well) were seeded in 96-well plates and treated with or without recombinant human IL-6 for 24–48 h. To assess TAM effects, THP-1 monocytes were seeded into Transwell inserts (0.4 μm pores), differentiated into M0 macrophages, and exposed to PC3-CM for 48 h to induce TAMs. TAMs were co-cultured with PC3 cells in 24-well plates for 48 h, with or without a 5 μM WP1066 or a 2.5 μg/mL IL-6 antibody. After 24 or 48 h, MTT solution (Duchefa Biochemie) was added and incubated at 37°C for 3 h. Formazan crystals were dissolved in dimethyl sulfoxide, transferred to 96-well plates, and the absorbance was measured at 570 nm using a SpectraMax M2 reader (Molecular Devices, USA).

2.7
Wound-healing assay
A scratch assay was performed to assess PC3 cell motility. For IL-6 treatment, 5 × 105 PC3 cells were seeded into 35 mm dishes, incubated overnight to confluence, and treated with 100 ng/mL IL-6 for 48 h. To evaluate TAM effects, THP-1 monocytes were seeded in Transwell inserts (0.4 μm pores), differentiated into M0 macrophages, and treated with PC3-CM for 48 h to induce TAMs. TAMs were co-cultured with PC3 cells in 24-well plates for 48 h, with or without a 5 μM WP1066 or 2.5 μg/mL IL-6 antibody. Wounds were made with a 1 mL pipette tip, washed with phosphate-buffered saline, and imaged at 0 h and every 3 h for 48 h using a live-cell analyzer (JuLI Br; NanoEnTek, Korea).

2.8
Transwell invasion assay
Transwell invasion was assessed using the CytoSelect 24-Well Cell Invasion Assay Kit (Cell Biolabs, Inc, San Diego, CA, USA). THP-1 monocytes were seeded in 24-well plates and polarized into TAMs for 48 h. The inserts were pre-coated with 200 μg/mL Matrigel for 1 h at 37°C. PC3 cells (7.5 × 104 cells in 150 μL serum-free medium) were seeded into inserts, which were then placed in TAM-containing wells. Co-culture was maintained for 48 h with or without 5 μM WP1066 or 2.5 μg/mL IL-6 antibody. After incubation, the invaded cells were stained with 0.1 % crystal violet and extracted with dimethyl sulfoxide. The extract was transferred to a 96-well plate, and the absorbance was measured at 560 nm using a SpectraMax M2 reader.

2.9
Cytokine array
M0 macrophages, TAMs, PC3 cells, and PC3+TAMs were cultured for 48 h, and the conditioned media were collected for cytokine array analysis. Human cytokine array panel A (R&D Systems) was incubated with blocking buffer and then with the sample-antibody mixture overnight at 4°C. After washing, the membranes were incubated with streptavidin-Horseradish Peroxidase (HRP) for 30 min and developed using a chemiluminescence reagent mix. Signal intensities were quantified using ImageJ software (v1.54k, NIH, Bethesda, MD, USA).

2.10
Western blot
Samples were homogenized in a lysis buffer, and equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Cytiva). Membranes were blocked with 3 % BSA and incubated with primary antibodies: mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-JAK2 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated JAK2(Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), and rabbit anti-phosphorylated STAT3 (Cell Signaling Technology). After washing, the membranes were probed with HRP-conjugated goat anti-mouse or anti-rabbit IgG (Bethyl Laboratories Inc., Montgomery, TX, USA). Protein bands were visualized using ECL Plus detection reagents (Cytiva), and relative expression levels were quantified with ImageJ software (v1.54k, NIH).

2.11
Statistical analysis
Data were presented as mean ± standard error of the mean. Statistical differences between groups were evaluated using Student t test or one-way analysis of variance. A value of P < 0.05 was considered to indicate a statistically significant.

3
Results
3.1
Polarization of THP-1 cells by cytokine or PC3 cell-derived CM
THP-1 cells were differentiated into M0 macrophages using 150 nM PMA for 24 h. M0 macrophages were then polarized into M1 macrophages with 250 ng/mL LPS and 20 ng/mL IFN-γ or into M2 macrophages with 20 ng/mL IL-4 and 20 ng/mL IL-13. TAMs were generated by treating MO macrophages with PC3-CM. After 48 h, M0 macrophages were round and less-elongated, M1 macrophages showed spindle-like shapes with projections, M2 macrophages were elongated but with fewer extensions, and TAMs exhibited heterogeneous M2-like morphologies (Fig. 1). Gene expression analysis showed that M1 macrophages upregulated proinflammatory genes, including HLA-DRβ1, CCL2, CXCL12, IL-6, and TNF-α, whereas M2 macrophages increased genes associated with immune suppression and tissue repair, such as CD206 and IL-10 (Fig. 2A and B). TAMs induced by PC3-CM expressed both pro-inflammatory and immunosuppressive markers, including HLA-DRβ1, IL-6, IL-1β, CD206, VEGFA, and TGF-β1 (Fig. 2A and B).

3.2
Cytokine secretion of macrophages and PC3 cells
Cytokine array analysis was conducted in M0, TAMs, PC3, and TAMs + PC3 groups. M0 and PC3 cells served as controls for TAMs and TAMs + PC3 cells, respectively. TAMs were generated by incubating M0 macrophages with PC3-CM for 48 h. TAMs were generated by incubation with PC3-CM for 48 h, and TAMs + PC3 were generated via indirect co-culture. TAMs showed increased CCL2, ICAM-1, and TNF-α. Both TAMs and TAMs + PC3 upregulated MIP-1α/β, CXCL1, IL-1β, and GM-CSF. Notably, migration inhibitory factor (MIF) was markedly increased only in the TAMs + PC3 group. G-CSF and IL-6 were highly expressed in TAMs, PC3, and TAMs + PC3 cells (Fig. 3).

3.3
Effects of IL-6 on proliferation and migration of PC3 cells
PC3 cells were treated with IL-6 (0, 10, and 100 ng/mL) for 24–48 h, and cell proliferation was measured using the MTT assay. IL-6 induced a significant, dose-dependent increase in cell proliferation (Fig. 4A). To examine cell migration, cells treated with100 ng/mL IL-6 for 48 h were assessed using a wound-healing assay. No significant difference was observed after 24 h, but migration was notably increased at 48 h compared to the control (Fig. 4B).

3.4
Effects of TAMs on proliferation, migration, and invasion of PC3 cells
To assess the influence of TAMs on prostate cancer cells, TAMs were indirectly co-cultured with PC3 cells for 48 h using Transwell inserts. MTT assay showed increased PC3 proliferation over time in the presence of TAMs (Fig. 5A). Wound-healing and Transwell invasion assays revealed enhanced cell migration and invasion (Fig. 5B and C). qRT-PCR analysis of co-cultured PC3 cells showed upregulation of proliferation-related genes, including TGF-β1, FOS, CCND1, and PXN, while TGF-α and mTOR remained unchanged (Fig. 5D).

3.5
Effects of WP1066 and IL-6 neutralization on proliferation, migration, and invasion of PC3 cells co-cultured with TAMs
To determine whether TAM-derived IL-6 mediates effects on PC3 cells, co-cultures were treated with an IL-6 neutralizing antibody and/or WP1066, a STAT3 inhibitor. TAMs significantly increased PC3 proliferation, which was reduced by WP1066, but not by the IL-6 antibody alone. The IL-6 neutralizing antibody alone did not significantly reduce cell proliferation. However, the combined treatment significantly suppressed cell proliferation (Fig. 6A). Wound-healing and invasion assays showed that TAMs promoted migration and invasion, both of which were attenuated by WP1066 or IL-6 antibody (Fig. 6B and C). qRT-PCR revealed that TAMs upregulated TGF-α and FOS in PC3 cells, whereas WP1066 alone or in combination with IL-6 antibody downregulated their expression (Fig. 6D).

3.6
Effects of IL-6 on JAK2/STAT3 phosphorylation in PC3 cells
Western blotting was used to examine JAK2/STAT3 activation in PC3 cells. IL-6 treatment for 48 h increased JAK2/STAT3 phosphorylation in a dose-dependent manner (Fig. 7A and B). Co-culture with TAMs also increased phosphorylation, whereas WP1066 or the IL-6 antibody reduced phosphorylation levels (Fig. 7C and D).

4
Discussion
Prostate cancer is one of the leading causes of cancer-related deaths in men, with resistance to therapy and metastatic progression presenting major clinical challenges. Increasing evidence highlights the critical role of the tumor microenvironment (TME) in tumor growth, metastasis, and therapy resistance.22 Tumor-associated macrophages (TAMs), which are abundant immune cells in the TME, have dual pro-tumor or antitumor functions depending on their activation state.23 Among their soluble mediators, interleukin-6 (IL-6) has received considerable attention for its role in regulating cancer cell proliferation, migration, and immune evasion via the JAK2/STAT3 signaling pathway.24
This study aimed to elucidate the contribution of TAM-derived IL-6 to the proliferation, migration, and invasion of androgen-independent human prostate cancer PC3 cells. Through cytokine profiling, functional assays, and signaling pathway analysis, we demonstrated that TAM-derived IL-6 is a mediator of aggressive prostate cancer behavior via JAK2/STAT3 activation.
THP-1 monocytes were differentiated into M0 macrophages and polarized into M1, M2, or TAM-like phenotypes using cytokines or PC3-derived CM. As expected, M1 macrophages upregulated proinflammatory markers (HLA-DRβ1, CCL2, CXCL12, IL-6, and TNF-α), consistent with their antitumor role.25 Conversely, M2 macrophages expressed immunosuppressive markers (CD206 and IL-10), supporting tumor growth, angiogenesis, and suppression of adaptive immune responses.26 Notably, TAMs generated from PC3-CM exhibited a mixed profile, upregulating both proinflammatory and immunosuppressive markers (HLA-DRβ1, IL-6, IL-1β, CD206, VEGFA, and TGF-β1). This hybrid phenotype is consistent with the reported plasticity of TAMs, where tumor-educated macrophages integrate inflammatory and suppressive properties to create a tumor-supportive niche.27,28
Cytokine array analysis revealed distinct patterns in TAMs and PC3 cells. CCL2, ICAM-1, and TNF-α were increased in TAMs. These factors are known to promote immune cell recruitment, tumor-stroma interactions, and tumor infiltration.29, 30, 31 Additionally, co-culture of TAMs with PC3 cells induced a strong expression of macrophage migration inhibitory factor, a pleiotropic chemokine involved in carcinogenesis and aggressiveness in various cancers.32 These findings suggest that TAM-PC3 interactions establish a cytokine-rich microenvironment favoring tumor progression. Importantly, IL-6 expression was consistently elevated in TAMs, PC3 cells, and co-cultures, confirming its role as a key regulator.
Treatment of PC3 cells with IL-6 significantly enhanced proliferation in a dose-dependent manner, whereas wound-healing and invasion assays demonstrated increased motility after 48 h. These results are consistent with clinical data linking elevated serum IL-6 levels to CRPC and experimental evidence showing that IL-6 promotes cytoskeletal remodeling and motility via STAT3 activation.33,34 Together, these results confirm IL-6 as an autocrine and paracrine growth factor in the progression of prostate cancer.
Co-culture experiments revealed that TAMs significantly promoted PC3 cell proliferation, migration, and invasion. qRT-PCR analysis confirmed the upregulation of pro-tumorigenic genes, including TGF-β1, FOS, PXN, and CCND1, which are associated with proliferation, extracellular matrix interactions, and cell cycle progression.35 The strong induction of FOS, an immediate-early transcription factor linked to oncogenic programs, underscores the TAM-driven activation of tumorigenic pathways.36
To analyze the role of IL-6 signaling, PC3-TAM co-cultures were treated with WP1066, a selective STAT3 inhibitor, and/or an IL-6 antibody. WP1066 alone suppressed cell proliferation, whereas IL-6 neutralization had a minimal impact. However, the combined treatment significantly reduced proliferation, suggesting that IL-6 is an important activator of STAT3, although not the only one. Migration and invasion assays confirmed that both WP1066 and IL-6 neutralization attenuated TAM-induced motility. Gene expression analysis revealed that TAMs upregulated STAT3-regulated genes, such as TGF-α and FOS. These genes were suppressed by WP1066 alone or in combination with the IL-6 blockade.
Western blot analysis further confirmed that IL-6 induced the dose-dependent phosphorylation of JAK2 and STAT3 in PC3 cells, which is consistent with canonical IL-6 receptor signaling. Co-culturing with TAMs also increased the phosphorylation of both proteins, which was reduced by WP1066 and partially attenuated by IL-6 neutralization. These results suggest that although IL-6 is the dominant driver of STAT3 activation, other cytokines may contribute to the residual signaling. This redundancy highlights the complexity of tumor-immune crosstalk and may explain the limited clinical efficacy of IL-6 blockade as a monotherapy.
Several studies have demonstrated that factors derived from prostate cancer cells promote the differentiation of macrophages within the tumor microenvironment into TAMs.37, 38, 39 Furthermore, TAM-secreted IL-6 has been shown to have paracrine effects on prostate cancer cells by activating the JAK/STAT signaling pathway.40,41 However, these mechanisms have typically been investigated in isolation, which limits our comprehensive understanding of TAM-tumor cell interactions. To address this limitation, we co-cultured PC-3 cells with undifferentiated macrophages to determine the effect of prostate cancer-derived factors on macrophage differentiation. We also examined how IL-6 secreted by these differentiated M2 macrophages affects PC-3 cell proliferation and motility within the co-culture system. The significance of this study lies in the establishment of an in vitro co-culture model that closely mimics in vivo interactions between TAMs and prostate cancer cells. Using this system, we validated previous findings and revealed that, in addition to IL-6, multiple cytokines may mediate the crosstalk between macrophages and PC-3 cells.
In conclusion, our findings identify TAM-derived IL-6 as a central paracrine mediator of prostate cancer progression via activation of the JAK2/STAT3 signaling pathway. The proinflammatory and immunosuppressive hybrid phenotype of PC3-educated TAMs underscores their functional plasticity and ability to promote tumor progression via multiple mechanisms. From a therapeutic standpoint, dual inhibition of IL-6 and STAT3 may be a promising approach for disrupting macrophage-tumor interactions and attenuating metastatic potential in advanced prostate cancer. Future studies should evaluate combination regimens integrating STAT3 inhibition with TAM reprogramming or immunotherapy to achieve long-lasting tumor control.

Conflicts of interest
The authors declare no conflict of interest.