- Open Access
Interleukin-32θ inhibits tumor-promoting effects of macrophage-secreted CCL18 in breast cancer
- Thu-Huyen Pham†1,
- Yesol Bak†1,
- Taeho Kwon2,
- Sae-Bom Kwon1,
- Jae-Wook Oh3,
- Jong-Hyung Park4,
- Yang-Kyu Choi4,
- Jin Tae Hong5Email author and
- Do-Young Yoon1Email authorView ORCID ID profile
© The Author(s). 2019
- Received: 17 March 2019
- Accepted: 20 May 2019
- Published: 24 May 2019
Tumor-associated macrophages can promote breast cancer metastasis by secreting cytokines and growth factors. Interleukin (IL)-32θ, a newly identified IL-32 isoform, was previously shown to down-regulate various proinflammatory factors of macrophages. Here, we report the presence of IL-32θ in breast cancer tissues and evaluate its effects on macrophage-regulated breast cancer metastasis.
RT-qPCR was used to analyze the mRNA expression of IL-32θ, Chemokine (C-C motif) ligand 18 (CCL18) in breast cancer tissues. In vitro cell-based experiments using IL-32θ-expressing MDA-MB-231 cells were conducted to examine the effects of IL-32θ on metastasis and its molecular signaling. In vivo xenograft, immunohistochemistry, and optical imaging models were generated to support in vitro and clinical findings.
The clinical data displayed opposite expression patterns of CCL18 and IL-32θ mRNA in macrophage-infiltrated breast tumor tissues compared with those in the other tissues tested. In MDA-MB-231 cells, IL-32θ overexpression attenuated migration, invasion, tumor-promoting factors, and increased epithelial markers levels upon treatment with conditioned media from THP-1-derived macrophages. Additionally, IL-32θ expression in a xenograft model led to a remarkable decrease in tumor size and macrophage-stimulated tumor promotion. This inhibition was mediated through a direct interaction with protein kinase C-δ (PKCδ), subsequently eliminating the downstream factors STAT3 and NF-κB. Blocking CCL18 during co-culture of macrophages and breast cancer cells reduced the levels of breast cancer progression-related factors and PKCδ downstream signaling suggesting CCL18 as the main macrophage-secreted factors triggering the signaling pathway inhibited by IL-32θ.
Our findings demonstrate a novel role of IL-32θ as an intracellular modulator to suppress macrophage-promoted breast cancer progression by targeting CCL18-dependent signaling.
- Breast cancer metastasis
Breast cancer is the most common cancer in females worldwide, and is also the leading cause of cancer-related death in the majority of countries . Tumor progression is the process by which tumor cells acquire more aggressive and malignant characteristics, allowing them to invade microenvironments and subsequently migrate to distant organs [2, 3]. In this process, epithelial-mesenchymal transition (EMT) is one of the key events that allows tumor cells to switch to mesenchymal phenotypes to facilitate their migration, invasion, and metastasis . This tumor metastasis and acquired resistance to tumor therapy is a result of the interaction between cancer cells and the tumor microenvironment, leading to the secretion of various factors that target cancer cells and manipulate their promotion [5–7]. Therefore, inhibition of these interactions can serve as a therapeutic approach in cancer.
Macrophages are the most abundant immune cells in the tumor microenvironment, which can occupy up to 50% of the entire tumor mass , and have been associated with poor outcomes in various carcinomas . Macrophages can be classified into M1 and M2 macrophages, which polarize into the respective forms in response to an environmental change. M2 macrophages facilitate angiogenesis, tissue remodeling , and promote breast cancer progression by secreting angiogenic factors and breast tumor mitogens . Tumor-associated macrophages (TAMs) are a type of M2 macrophages, and breast cancer TAMs display an alternative phenotype that promotes tumor invasion and metastasis . Further, cancer cells can educate macrophages to enhance tumor development and metastasis . Numerous studies have determined the relationship between breast cancer and macrophages, and cancer therapies targeting both breast cancer cells and macrophages are of great interest given their potential in the clinical setting.
Interleukin (IL)-32 was first reported as natural killer transcript 4 located on human chromosome 16p13.3 . IL-32 has various isoforms due to alternative splicing , although the role of each isoform in disease remains controversial . Among the IL-32 isoforms, our group discovered both IL-32θ and IL-32 small fragment  and reported the functions of IL-32θ in inflammation and cancer [16–18]. In the present study, we aimed to investigate the role of IL-32θ in the breast cancer microenvironment and to determine whether IL-32θ could suppress macrophage-induced breast cancer progression, and to explore the underlying molecular mechanisms.
mRNA extraction from breast cancer tissue
The biospecimens including breast tissues (n = 90) and sera (n = 55), and the characteristic information of breast cancer patients used in this study were provided by the Biobank of Chonnam National University Hwasun Hospital (Hwasun-gun, Korea) and Korea University Guro Hospital (Seoul, Korea). RNA was extracted from frozen tissues using a homogenizer and TRI Reagent® (Ambion, Austin, TX), and then cDNA was synthesized using the M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA) according to manufacturer’s instructions.
Cell culture and treatment
The MDA-MB-231 cell line (ATCC® HTB-26™, Manassas, VA) was cultured in DMEM (Hyclone Laboratories, Logan, UT). The human monocytic cell line THP-1 (Korean Cell Line Bank, Seoul, Korea, KCLB-40202) was cultured in RPMI-1640 (HyClone). Both mediums were supplemented with 10% heat-inactivated fetal bovine serum (MilliporeSigma, Burlington, MA), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C/5% CO2. To generate the conditioned media (CM), THP-1 cells were stimulated with 100 nM phorbol ester (PMA) (MilliporeSigma) for 48 h, the non-attached cells were washed with phosphate buffered saline (PBS) followed by addition of fresh culture media, and these cells were then incubated for another 24 h. The CM was collected and centrifuged to remove the remaining cells.
Generation of the IL-32θ-overexpressing cell line
We transfected MDA-MB-231 cells with the pcDNA3.1 (+)-6 × Myc-IL-32θ vector or pcDNA3.1 (+)-6 × Myc-empty vector, as described previously  and refer as MDA-MB-231-IL-32θ and MDA-MB-231-EV cells, respectively. In brief, the cells were seeded into 6-well plates (1 × 105 cells/well) and transfected with 3 μg of vector using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA). Afterwards, the cells were selected using medium containing 700 μg/ml G-418 (Duchefa Biochemie BV, Haarlem, The Netherlands) for two weeks. G-418-resistant colonies were then pooled and expanded.
Migration and invasion assays
For the migration assay, cells (5 × 105 cells/mL) were seeded onto the upper chambers of 24-well transwell plates (Corning Inc., Corning, NY) in serum-free DMEM. For the invasion assay, each transwell chamber was coated with 30 μl of Matrigel (Corning) before breast cancer cells were added to the top chamber. The lower chamber contained 500 μl macrophage-derived CM. Cells migrated or invaded for 24 h at 37 °C/5% CO2, and non-migrated or non-invaded cells were removed from the chamber interior by a cotton swab. Attached cells to the lower surface of the chamber were stained using Diff-Quick Kit (Sysmex, Kobe, Japan). Migrated or invaded cells were quantitated by dissolving stained cells in 100 μl of 10% acetic acid and then the mixture was transferred to a 96-well plate for colorimetric reading at 620 nm.
Quantitative reverse transcription PCR (RT-qPCR)
The mRNA expression levels in breast cancer cells were detected by RT-PCR for IL-32θ and RT-qPCR for other target genes. Total RNA was isolated using Easy-BLUE (iNtRON Biotechnology, SungNam, Korea), then reverse transcription was performed. qPCR was conducted using SensiFAST™ SYBR NO-ROX Kit (BIOLINE, London, UK). Samples were analyzed using the primer sets listed in Additional file 1: Table S1. Transcript levels were quantitated using the -ΔCt method (Ct = fluorescence threshold value; −ΔCt = Ct GAPDH – Ct target gene).
Enzyme-linked immunosorbent assay (ELISA)
Cells were cultured in the absence or presence of CM for 24 h, and then the culture media were replaced by fresh media for another 24 h. The cell culture supernatants were collected and analyzed using ELISA kits (R&D Systems, Minneapolis, MN) for human IL-1β, CCL5, CCL18, GM-CSF according to manufacturer’s instructions.
Immunoblotting and immunoprecipitation
For nuclear and cytoplasmic fractionation, cells were collected and fractionated using the NE-PER kit (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. For immunoprecipitation, cell lysates were mixed with specific antibodies and then pulled down by protein G-agarose beads. Samples were subjected to 10% SDS–PAGE before being transferred to PVDF membranes (MilliporeSigma). The membranes were blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.05% Tween-20 followed by primary antibody incubation at 4 °C overnight. After washing, horseradish peroxidase-conjugated IgG antibodies were added, and the membranes allowed to incubate for 1 h. Western blot was visualized using a chemiluminescence detection kit (Advanstar, Cleveland, Ohio) and detected by EZ-capture MG protein imaging system (ATTO, Tokyo, Japan). Specific antibodies used include those against Myc-tag, Flag-tag and phosphotyrosine-STAT3 (MilliporeSigma); IκBα, p-IκBα, p65, p50, PARP, and E-cadherin (Cell Signaling Technology, Danvers, MA); STAT3, COX-2, GAPDH (Santa Cruz Biotechnology, Dallas, TX); and anti-CCL18 neutralizing antibody (Abcam, Cambridge, MA). The monoclonal antibody KU-32-52 to detect IL-32 was prepared as previously described . The raw data of western blot results can be seen in Additional file 2.
Cells (3 × 105 cells/well) were seeded in a 6-well plate, cultured overnight, and then treated with or without CM for 24 h. MMP-9 activity in the supernatant was assayed as previously described . Gel staining was conducted with InstantBlue™ (MilliporeSigma) for 30 min in the dark. Areas of gelatinolytic degradation appeared as transparent bands on the blue background.
Cells were seeded on coverslips and incubated overnight. The attached cells were fixed, and permeabilized with cold acetone before blocking with 0.1% bovine serum albumin in PBS at room temperature (RT). Primary antibodies were added (1:100) to the coverslip incubating at 4 °C overnight. After washing with PBS, the coverslips were incubated with secondary antibodies at (1:200). Nuclei staining was performed by exposing to 4, 6-diamidino-2-phenylindole (1:2000) (MilliporeSigma) for 20 s. The stained cells were visualized using an upright fluorescence microscope (Olympus, Tokyo, Japan).
Xenograft model and optical imaging
All animal procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC No. KU17008) of Konkuk University. MDA-MB-231-EV and MDA-MB-231-IL-32θ cells (5 × 106 cells) were subcutaneously injected with Matrigel into the flanks of 5-week-old female athymic BALB/c nude mice (Nara Bio, Seoul, Korea). After 35 days, the tumors were harvested from euthanized mice. The tumor tissues were fixed with 10% formalin buffer, embedded in paraffin, and sectioned in 3 μm thickness for use in immunohistochemical analyses. Tumor volume was calculated using the formula V(mm3) = (shortest side2 × longest side)/2. For pre-and intra-operative tumor localization in the real-time resection, we conducted an in vivo tumor localization assay using the IRDye®-800CW 2-DG (2-deoxy-D-glucose) optical probe (LI-COR Biosciences, Lincoln, NE). Tumor localization was detected using optical imaging, particularly in the near-infrared fluorescence range. The tumorigenicity of MDA-MB-231-EV and MDA-MB-231-IL-32θ cells was assayed by intravenous injection of 1 × 106 cells resuspended in PBS into nude mice (n = 5 per group).
Formalin-fixed, paraffin embedded tumor tissue sections from mice were immersed in citrate buffer and boiled for 4 min in a microwave to retrieve antigens. Endogenous peroxidase activity was blocked with 3% (v/v) H2O2 for 10 min. Non-specific binding sites were blocked with 1% BSA for 30 min. Sections were incubated with the appropriate primary antibodies at 4 °C overnight and then the appropriate secondary antibodies for 1 h at RT. Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA) was used as a substrate, and the sections were then counterstained with hematoxylin (MilliporeSigma).
Chi-square or Fisher’s exact test was used to evaluate the relationship between IL-32θ expression and clinicopathological status. The mRNA expression in tumor tissues and protein secretion in breast cancer patients’ sera were analyzed by Mann-Whitney U test. Student’s t-test were used to compare the two groups in in vitro and in vivo experiments. Statistical analyses were performed using GraphPad Prism software version 5.0. All p-values were two-sided, and p < 0.05 was interpreted as being statistically significant.
Association between tumor IL-32θ mRNA levels and breast tumor characteristics
Association of IL-32θ mRNA expression and clinical characteristics of breast cancer patients
n = 90
n = 35
n = 55
Estrogen receptor (ER)
Progesterone receptor (PR)
Human epidermal growth factor receptor 2 (HER-2)
Luminal A (ER+ PR+/− HER-2- Ki67 low)
Luminal B (ER+ PR+/− HER-2+/Ki67 high)
Basal-like (ER- PR- HER-2- EGFR+/Ki67 high)
HER2-enriched (ER-PR-HER-2+ Ki67 high)
Opposing expression patterns of IL-32θ and CCL18 in breast tumor tissues
IL-32θ reduces macrophage-regulated EMT, invasion, and migration in breast cancer cells in vitro
IL-32θ directly interacts with PKCδ to subsequently inhibit NF-κB and STAT3 pathways in vitro
Blocking CCL18 signaling downregulates pro-malignancy factors and the PKCδ downstream pathway
IL-32θ inhibits tumor formation of breast cancer cells in vivo
Macrophages, a major component of the tumor microenvironment, can initiate and support the tumor progression and metastasis by secreting a range of growth factors, cytokines, and chemokines . IL-32 was found to not only target cancer cells but might also target the tumor microenvironment . Recent reports showed the correlation and different functions of IL-32 and its isoforms to various cancer diseases. As an example, IL-32γ can inhibit colon cancer cell growth by targeting NF-κB and STAT3 pathways  while another isoform, IL-32β, stimulates the migration of breast cancer cells through VEGF-STAT3 , and is involved in the increase of glycolysis under hypoxic conditions which supports cancer cell growth . Given these data, it appears that the effects of IL-32 on tumor development depend on both its isoforms and cancer types; however, the exact mechanisms remain unclear. Our previous data on IL-32θ, a recently discovered isoform, demonstrated its inhibition ability in macrophage differentiation , macrophage-secreted factors [16, 18, 19], and in colon cancer progression by regulating self-renewal and EMT . In this study, three isoforms, IL-32θ, IL-32β, and IL-32γ, were detected at different mRNA levels in 90 breast tumors. IL-32β exhibited the strongest expression which was compatible with its protumor effects reported in breast cancer  while IL-32γ was rarely expressed compared to the IL-32θ isoform (Additional file 1: Fig. S2a-c). Given this, the current study attempted to discover the role of IL-32θ in breast cancer progression and its tumor microenvironment. Our clinical data showed that IL-32θ expression was associated with the negativity of ER, PR, and HER-2, and with triple negative related breast cancer types. Based on this point, we chose MDA-MB-231 cells, a highly aggressive, basal-like breast cancer cells with triple negative background , together with PMA-treated THP-1 macrophage cells to mimic the interaction between macrophages and cancer cells within the tumor microenvironment and evaluate the role of IL-32θ on this interaction in vitro. This basal-like cell line is associated with both a poor prognosis and clinical outcome, due to its aggressiveness and high rate of metastasis . We determined that the EMT phenotypic changes of MDA-MB-231 cells caused by stimulation of CM from THP-1 macrophages could be inhibited by IL-32θ. Moreover, invasion and migration rates were remarkably reduced in IL-32θ-expressing cells after 24 h treatment with CM, suggesting that IL-32θ could be a potential factor inhibiting macrophage-induced breast cancer progression. The interaction between macrophages and breast cancer cells has been reported to increase levels of various tumor promoting factors such as COX-2, and MMP-9 which, in turn, supports the breast malignancy and an increase of TAM density in the tumor microenvironment [23, 26, 28, 35]. In agreement with these reports, the present study indicated that IL-32θ downregulated COX-2, MMP-9, and E-cadherin expression in breast cancer cells stimulated by macrophages demonstrating a modulatory role of IL-32θ in breast cancer development.
In addition, the precise mechanism by which IL-32θ reduces the effects of macrophage on breast cancer progression was addressed based on previous studies detailing that IL-32θ interacted directly with PKCδ to subsequently decrease STAT3 or NF-κB signaling in PMA-activated THP-1 cells [16, 19]. In line with this theory, the present study showed a direct interaction between IL-32θ and PKCδ in breast cancer cells. Especially under CM treatment condition, IL-32θ inhibited phosphorylation of IκBα plus STAT3, and nuclear translocation of NF-κB and STAT3 in MDA-MB-231 cells (Fig. 3b). Moreover, interfering PKCδ signaling with rottlerin, a PKCδ inhibitor, resulted in additive effects with IL-32θ in the decrease of STAT3 phosphorylation and IκBα degradation. Due to the fact that PKCδ mRNA expression was found to be significantly higher in ER-positive compared with ER-negative tumors , we applied this model on another breast cancer cell line with an estrogen-dependent background, MCF-7. However, IL-32θ could not reduce any signal activated by macrophage CM in MCF-7 cells (Additional file 1: Figure S3a-b). Since MCF-7 represents epithelial-like cells and MDA-MB-231 represents mesenchymal-like cells, it is suggested that IL-32θ seemed to effectively modulate the breast cancer with EMT-associated macrophages, which is essential for metastasis. The lack of PKCδ activation in MDA-MB-231 in the non-stimulated condition disappeared when MDA-MB-231 was co-cultured with macrophage CM. Further studies are necessary to define the association between IL-32θ and mesenchymal-like cells but not epithelial-like cells. In any case, these findings demonstrated that IL-32θ targeted the interaction between macrophage and mesenchymal-like breast cancer, and there requires a specific macrophage-secreted factor to trigger PKCδ signaling in breast cancer which was inhibited by IL-32θ.
During the investigation of the IL-32θ-regulated signaling upstream factors, CCL18 was considered as a potential activator due to its presence in the THP-1 macrophage CM, and the inverse expression between IL-32θ and CCL18 in breast tumor tissues infiltrated with CD206+ macrophages. GM-CSF secreted from breast cancer cells activates macrophages to become CCL18-expressing TAM-like cells, which reciprocally supports GM-CSF secretion and furthers EMT of breast cancer cells . Moreover, only GM-CSF significantly induced the production of TAM-related cytokines, and GM-CSF was found in CM from MDA-MB-231 cells but not MCF-7 cells . Consistent with this study, our study found a decrease in the amount of GM-CSF secretion in IL-32θ-expressing MDA-MB-231 cells which might be stimulated by CCL18 from macrophages. Further, IL-32θ did not suppress endogenous GM-CSF in MDA-MB-231 cells due to a lack of PKCδ activation and interaction. The transcription factors STAT3 and NF-κB were also reported as the downstream factors regulated by PKCδ in cancer cells [37, 38]. In line with the idea that CCL18 is a stimulator of PKCδ signaling, our data demonstrated that blocking CCL18 signaling suppressed the expression of the PKCδ downstream factors STAT3 or NF-κB as well as various cancer-related factors. These results supported the idea that macrophage-secreted CCL18 might act as a stimulator of PKCδ signaling regulated by IL-32θ.
The present study also provides the first in vivo evidence of the suppressive function of IL-32θ in breast cancer. A xenograft mouse model of MDA-MB-231-IL-32θ cells showed an increase of E-cadherin-positive cells, suggesting that IL-32θ reversed the effects on EMT, whereas STAT3 and NF-κB-positive cells were much more abundant in the absence of IL-32θ. Another imaging model in which breast cancer cells were activated by macrophage CM to become more aggressive also supported that IL-32θ could reduce the tumor localization clearly compared to the MDA-MB-231-EV group. These in vivo results are in accordance with the in vitro and clinical data demonstrating that IL-32θ acts via PKCδ signaling to regulate the effects of macrophage-soluble factors on breast cancer cells.
The small population of patients’ data collected recently does not allow us to perform a survival analysis to assess the relationship between IL-32θ and the survival rate of breast cancer patients. Moreover, during studying about the effects of IL-32θ on breast cancer cell proliferation, we have found that Bcl-2, an anti-apoptotic factor which has been proposed as a prognostic marker , was totally repressed by IL-32θ in vitro (data not shown). However, no significant change between two cell lines could be seen in the expression levels of the late apoptotic markers after 72 h from flow cytometry results (data not shown). Thus, it is necessary to study different types of cell death to understand by which mechanism IL-32θ may affect the cell death. Finally, although there are some aspects described above to be considered, these will be the subjects of ongoing studies.
The biospecimens and characteristic data used for this study were provided by the Biobank of Chonnam National University Hwasun Hospital (Hwasun, Korea) and Korea University Guro Hospital (Seoul, Korea), members of the Korea Biobank Network.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (2018R1A2B2001225).
TP and YB performed most of the experiments, analyzed the results and wrote the manuscript. TK conducted the optical imaging experiments. SK established the stable cell line. JO supported in vitro materials and contributed to edit the manuscript. JP and YC participated in xenograft experiments. DY and JTH supervised the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The protocol using biospecimens and related data in this study was approved by the Institutional Review Board (7001355–201704-E-047) of Konkuk University, and all subjects provided informed consent. All animal procedures were conducted according to the guidelines and approved by the Institutional Animal Care and Use Committee (IACUC No. KU17008) of Konkuk University.
Consent for publication
The authors declare that they have no competing interests.
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