IL1β is a central regulator of systemic inflammatory response in breast cancer, but the precise regulatory mechanisms that dictate the overproduction of IL1β are largely unsolved. Here, we show that IL1β secretion is increased by the coculture of human monocyte–like cells and triple-negative breast cancer (TNBC) cells. In addition, macrophages robustly produced IL1β when exposed to the conditioned media of TNBC cells. Consistent with these observations, macrophage depletion decreased serum IL1β and reduced breast cancer progression in an orthotopic breast cancer mouse model. Profiling the secretome of human breast cancer cells revealed that the CD44 antigen was the most differentially released protein in basal conditions of TNBC cells. Antibody-mediated neutralization of CD44 abrogated IL1β production in macrophages and inhibited the growth of primary tumors. These results suggest IL1β-mediated oncogenic signaling is triggered by breast cancer cell membrane–derived soluble CD44 (sCD44) antigen, and targeting sCD44 antigen may provide an alternative therapeutic strategy for breast cancer treatment by modulating inflammatory tumor microenvironment.

Significance:

A novel positive feedback loop between IL1β and CD44 promotes TNBC malignant progression.

Tumor microenvironment is considered to exert a decisive effect on tumor progression (1). In addition to the cancer cells at a primary tumor site, there are various subsets of resident and infiltrated inflammatory cells including innate and adaptive immune cells, myeloid cells, and lymphoid cells in the tumor microenvironment (2, 3). These cells interplay with one another through complex and dynamic network of chemokines, cytokines, and growth factors (4–6). Tumor-associated inflammatory signaling and their microenvironmental crosstalk play pivotal roles in different stages of tumor development (7–10).

Inflammasomes are multiprotein complexes that promote inflammation through secretion of IL1β in response to microbial infection and endogenous damage-associated molecular patterns (DAMP), such as uric acid, ATP, high mobility group box 1, and the HSP70 and HSP90 (11). IL1β, one of the proinflammatory cytokines, engages innate immune responses through infiltration of inflammatory cells (e.g., neutrophils, macrophages, and monocytes) into infection sites (11). Because of its essential roles in innate immune responses, inflammasomes are indispensable for host defense against external infections and tissue damages. In addition to the inflammatory conditions, elevated levels of IL1β have been reported in various types of patients with cancer (7, 12). In breast cancer, it has been demonstrated that aberrant expression of IL1β and inflammasomes is closely associated with progressive and metastatic potential of breast cancer, resulting in poor prognosis (13–16). Nonetheless, the mechanism by which IL1β is released in breast tumor microenvironment remains elusive.

The connection between inflammation and cancer can be categorized into two pathways. One is inflammation-induced carcinogenesis, also known as the extrinsic pathway, and the other is cancer-associated inflammation or the intrinsic pathway (17, 18). Many human malignancies are related to inflammation-induced carcinogenesis. Examples are colitis-induced colorectal cancer (19), hepatitis B/C virus–mediated liver cancer (20), Helicobacter pylori–induced gastric cancer (21), liver fluke–associated cholangiocarcinoma (22, 23), and asbestos-associated mesothelioma (24). Compared with inflammation-induced carcinogenesis, the mechanism underlying cancer-associated inflammation in breast tumor microenvironment is not fully elucidated.

Here we report a novel mechanism responsible for IL1β production in the breast tumor microenvironment. Notably, triple-negative breast cancer (TNBC) cell–derived soluble CD44 (sCD44) antigen promotes IL1β secretion from macrophages. This finding suggests an importance of intercellular communication in tumor microenvironment between breast cancer cells and macrophages via sCD44 antigen–IL1β signaling axis as a novel immunotherapeutic target for better clinical outcomes of patients with TNBC.

Reagents

DMEM, RPMI1640, DMEM Nutrient Mixture F-12 (DMEM/F-12) medium, and FBS were purchased from Gibco BRL. Recombinant human (rh) IL1β and recombinant mouse (rm) IL1β were purchased from R&D Systems (201-LB and 401-ML). Clophosome and control liposomes were obtained from FormuMax Scientific Inc. (F70101C-NC). Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), ATP, apyrase, and dithiothreitol (DTT) were products of Sigma-Aldrich. d-Luciferin was purchased from Gold Biotechnology (LUCK). Brefeldin A was purchased from BD Biosciences. Primary antibody for IL1β was purchased from Cell Signaling Technology. Antibodies against apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), β-actin, E-cadherin, and N-cadherin were obtained from Santa Cruz Biotechnology. Antibodies for CD44 and mouse IgG2a isotype, and Cytokeratin were purchased from Novus Biologicals. Antibody against amyloid-β was obtained from Merck and Ki-67 was purchased from Abcam.

Cells and cell culture

MDA-MB-231 and MCF7 cells were obtained from Korean Cell Line Bank (KCLB) in 2013 and THP1 cells were obtained from KCLB in 2018. MCF10A and MDA-MB-468 cells were kindly provided by Prof. Marc Diederich [College of Pharmacy, Seoul National University, Seoul, Republic of Korea (South)] and Prof. Dong-Young Noh [College of Medicine, Seoul National University, Seoul, Republic of Korea (South)], respectively. The genetic identity of the cell lines was confirmed by short tandem repeat profiling and all cell lines were routinely tested for Mycoplasma contamination by PCR test method (iNtRON Biotechnology). All cell lines were maintained and used at the ≤ 25 passage number. Human breast cancer MDA-MB-231 and MDA-MB-468 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Human breast cancer MCF7 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Immortalized human benign breast epithelial MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 100 ng/mL cholera toxin, 20 ng/mL human EGF, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Human monocyte-like THP1 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 2 mmol/L l-glutamine, 25 mmol/L HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin. For inducing differentiation of monocyte-like THP1 cells to macrophages, the cells were seeded and incubated in 60-mm dishes at a density of 5 × 105/mL in medium containing 100 nmol/L PMA for 48 hours, followed by incubation in medium without PMA for another 24 hours. All cell lines were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2.

Human serum samples

Human serum samples from patients diagnosed with stage III breast cancer and healthy donors were obtained from Korea Institute of Radiological and Medical Sciences (KIRAMS) Radiation Biobank. Human serum samples were analyzed to measure levels of IL1β, IL18, IL6, TNFα, and CD44 by ELISA. This study was performed in accordance with the Declaration of Helsinki of the World Medical Association, and the study was approved by Institutional Review Board (IRB) of Seoul National University [Seoul, Republic of Korea (South); no. E1812/003-001]. Informed written consent for this study was waived by IRB approval.

Mouse orthotopic allograft (transplant) breast cancer model

All animal experiments were conducted on protocols approved by the Institutional Animal Care and Use Committee at Seoul National University [Seoul, Republic of Korea (South); SNU-180123-1-1]. Five-week-old female mice with BALB/c genetic background were purchased from Orient Bio Inc. The mice were acclimated for 1 week before use and maintained throughout the study in a controlled environment: 22 ± 2°C, 50% ± 5% relative humidity, and a 12-hour light/dark cycle.

For the orthotopic transplant breast cancer mouse model, 4T1-Luc cells or sh-IL1R1a 4T1 cells suspended in PBS were transplanted into fourth mouse mammary fat pad. Mice for each experiment were randomly assigned to groups. The primary tumor size was measured every 3–4 days using a caliper. The tumor volume was calculated as follows: (width2 × length)/2. For bioluminescence measurement, d-Luciferin (150 mg/kg) was administered intraperitoneally. Mice were then anesthetized with isoflurane (2%–3% isoflurane in oxygen flow rate of 1 L/minute). Bioluminescence images were obtained using the IVIS Spectrum in vivo imaging system (PerkinElmer) and the Living Image software. After 4–5 weeks of orthotopic transplantation, mice were euthanized by CO2 inhalation. Their blood samples were collected through cardiac puncture, and then, primary tumors and lung tissues were excised for measurement of tumor weight and assessment of lung metastasis.

ELISA

Sera from human and mouse, and supernatants from cell culture were analyzed using IL1β, IL18, IL6, TNFα, and CD44 ELISA kits according to the manufacturer's instructions. Human IL1β (ELH-IL1b), IL6 (ELH-IL6), TNFα (ELH-TNFa), and CD44 (ELH-CD44), and mouse IL1β (ELM-IL1b) ELISA kits were purchased from RayBiotech. Human IL18 (ab215539) ELISA kit was purchased from Abcam and mouse CD44 (LS-F8020) ELISA kit was obtained from LS Bio.

Tissue immunofluorescence staining

The dissected mouse primary tumor tissues were prepared for immunofluorescence analysis of the expression of IL1β, N-cadherin, E-cadherin, cytokeratin, Ki-67, and CD44. Four-micron–thick sections of 10% formalin-fixed, paraffin embedded tissues were placed on glass slides and deparaffinized three times with xylene and rehydrated through graded alcohol bath. The deparaffinized sections were heated by using microwave and boiled twice for 6 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval. The slides were stained with corresponding primary antibodies in 5% BSA at 4°C for overnight, and then washed and stained with secondary antibodies at room temperature for 1 hour. Nuclei were stained with 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes.

Flow cytometry

For preparation of macrophage and dendritic cells from mouse primary tumor, isolated mouse primary tumors were teased into small pieces by using a gentleMACS dissociator (Miltenyi Biotec), then digested with RPMI1640 medium containing digestion enzymes as manufacturer's instructions for 40 minutes. Red blood cells (RBC) were lysed with 1 mL of RBC lysis buffer (iNtRON Biotechnology) for 1 minute, and cells were separated using LSM (MP Biomedicals) gradient centrifugation. For macrophage isolation from spleen, spleen was smashed and ground onto the 100 μm nylon cell strainer (Corning Inc.), and the cells were resuspended in DMEM containing 10% FBS. For lysis of RBCs, the cells were incubated with 1 mL of RBS lysis buffer for 1 minute, and then the cells were separated using LSM gradient centrifugation. For Fc blocking, the cells were incubated with an anti-CD16/32 antibody in staining buffer containing 1% BSA and 0.1% sodium azide in PBS for 15 minutes. Specific antibodies for macrophage membrane (F4/80, CD11b, Ly6G, and Ly6C) and dendritic cell membrane (F4/80, CD11c, and MHCII) were added to samples and incubated for 1 hour. All antibodies for FACS analysis were purchased from BioLegend Inc. Cells were analyzed by a BD FACSCalibur, FACS Aria III or LSRFortessa (BD Biosciences). FlowJo software was used to analyze the data.

Coculture experiment

Human monocyte–like THP1 cells (6 × 105/mL) were seeded and differentiated in the bottom layer of 6-well culture plate, and a day after, breast cancer cells (5 × 105/mL) were seeded onto 0.4-μm porous insert layer (Corning Inc.). On day 4, both differentiated THP1 cells in the bottom layer and breast cancer cells in the insert layer were combined for coculture.

Western blot analysis

Cells were lysed in lysis buffer (150 mmol/L NaCl, 1% NP-40, 50 mmol/L Tris-HCl, pH 7.4, and protease inhibitors). The protein concentration was determined by using the Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Culture supernatant between 3 and 50 kDa was concentrated by Amicon ultracentrifugal filter units (Millipore). Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk for 1 hour at room temperature prior to incubation with appropriate primary antibodies. Subsequently, membranes were washed and incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibody. Following washing for three times, transferred proteins were detected with an Enhanced Chemiluminescence Detection Kit (Abclone).

Isolation and differentiation of bone marrow–derived macrophages and peritoneal macrophages

To obtain bone marrow–derived macrophages (BMDM), BM cells from femurs and tibias of 6- to 8-week-old BALB/c mouse were flushed out by cold PBS containing 2% heat-inactivated FBS. RBCs were lysed in RBC lysis buffer. After lysis RBCs, remaining cells were plated on sterile petri dishes and incubated for 3 days in DMEM containing 10% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 ng/mL M-CSF (BioLegend Inc.) at 37°C in a humidified atmosphere of 5% CO2. After 3 days, unattached cells were removed by changing medium containing 20 ng/mL M-CSF. To obtain peritoneal macrophages (PM), peritoneal cells were harvested from peritoneal cavity of 6- to 8-week-old BALB/c mouse by cold PBS containing 2% heat-inactivated FBS. RBCs were lysed in RBC lysis buffer. After lysis of RBCs, remaining cells were incubated in DMEM containing 10% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin for 3 hours at 37°C in a humidified atmosphere of 5% CO2. After 3 hours of incubation, unattached cells were removed by changing medium.

ASC oligomerization assay

For ASC oligomer cross-linking, cells were dissolved in lysis buffer (1% NP-40, 150 mmol/L KCl, and 20 mmol/L HEPES-KOH, pH 7.7) supplemented with protease inhibitors (0.1 mmol/L phenylmethylsulfonylfluoride, 1 μg/mL leupeptin, 11.5 μg/mL aprotinin, and 1 mmol/L sodium orthovanadate), followed by shearing 25 times through 20-gauge needle. The lysates were then centrifuged at 5,000 × g for 10 minutes at 4°C and the pellets were washed twice with PBS and resuspended in 500 μL PBS. The resuspended pellets were cross-linked with disuccinimidyl suberate (2 mmol/L) for 30 minutes and centrifuged at 5,000 × g for 10 minutes at 4°C. The cross-linked pellets were lysed in 20 μL 1 × SDS sample buffer, and boiled for 5 minutes at 95°C. The samples were separated by 10% nonreducing SDS-PAGE.

ASC speck formation assay

Human monocyte-like THP1 cells were seeded and differentiated at a density of 1.2 × 105/mL in a 4-chamber slide. Differentiated THP1 cells were treated with conditioned medium (CM) from breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF7) and immortalized human benign breast epithelial cells (MCF10A) CM for 12 hours. Chamber slides were fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then permeabilized with 0.5% Triton X-100 for 5 minutes at room temperature. Chamber slides were incubated with ASC primary antibody in PBST for overnight at 4°C and then washed and incubated with secondary antibody for 1 hour at room temperature. Nuclei were stained with 1 μg/mL of DAPI for 5 minutes. ASC specks were counted under the microscope (Nikon).

Real-time PCR

Total RNA was isolated from cells with TRIzol Reagent (Thermo Fisher Scientific) and 1 μg of RNA was reverse transcribed using the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega). Real-time quantitative PCR was performed on a 7500 Real-Time PCR instrument (Thermo Fisher Scientific) using the RealHelix Premier Quantitative PCR Kit (NanoHelix Co. Ltd). Dissociation curve analysis was performed to verify the identity of PCR products. Target gene expression was normalized to L32 or Actb. Data were analyzed using the comparative cycle threshold (ΔΔCt) method.

Breast cancer cell secretome analysis

The protein concentration of breast cancer cell secretome was measured using the BCA assay, and 100 μg of proteins were mixed with 8 mol/L urea at a 1:3 ratio. The final concentration of urea was set as 6 mol/L, and the mixture was sonicated for 30 minutes in 4°C. Then, DTT was treated for reduction of proteins to a final concentration of 10 mmol/L and incubated for 45 minutes at 37°C. Then iodoacetamide (Sigma-Aldrich) was treated for alkylation to the final concentration of 30 mmol/L and incubated in dark at room temperature. Trypsin was added to the samples (1:50, trypsin:sample) and incubated for overnight at 37°C.

Digested peptides were dried and resuspended in 0.1% formic acid in water and analyzed using the Q Exactive Mass Spectrometer coupled with the Waters ACQUITY UPLC system. For the proteome profiling analysis, the gradient was as follows (T min/% of solvent B): 0/5, 5/10, 100/40, 102/80, 112/80, 114/5, and 120/5. The peptides were eluted through a trap column, ionized through an EASY-spray column (50 × 75 μm ID) packed with 2 μm C18 particles at an electric potential of 1.8 kV. Full mass spectrometry data were acquired in a scan range of 400–2,000 T at a resolution of 70,000 at m/z 200, with an automated gain control target value of 1.0 × 106 and a maximum ion injection of 100 ms. The maximal ion injection time for MS/MS was set to 50 ms at a resolution of 17,500. Dynamic exclusion time was set to 30 seconds.

The MS2 spectra were searched with the MaxQuant (v. 1.5.1.2) against the Uniprot human database (containing 20,417 proteins). Carbamidomethylation of cysteine as a fixed modification and oxidation of methionine as variable modifications was used for each search. A false discovery rate cutoff of 1% was applied at the peptide spectrum match and protein levels. An initial precursor mass deviation up to 4.5 ppm and a fragment mass deviation up to 20 ppm were allowed. Protein identification required at least one peptide using the “razor plus unique peptides” setting in MaxQuant. Proteins were quantified using the XIC-based label-free quantification algorithm in MaxQuant.

Neutralization assay

CM of MDA-MB-231 and MDA-MB-468 was extracted for 24 hours. The CM was incubated with neutralizing antibodies (6 μg of anti-CD44 mAb and anti-amyloid-β mAb) for 6 hours in a rotator at room temperature. To remove antibody-bound proteins, CM was cutoff with 100 kDa of ultracentrifugal filter unit. PMA-differentiated THP1 cells were treated with under 100 kDa fraction of CM for 12 hours.

Wound-healing assay

MDA-MB-231 and MCF7 cells were seeded at the density of 3 × 105/mL into Ibidi Culture-Inserts (2 × 0.22 cm2) in 60-mm dishes. After 24-hour incubation, Ibidi Culture-Inserts were gently removed. Cells were treated with rhIL1β for another 48 hours. Wound width was measured under the microscope (Nikon).

Cell-cycle analysis

Cells were trypsinized, collected, and fixed with 70% EtOH for 24 hours. Then, the cells were washed with ice-cold PBS, and incubated in propidium iodide (PI) staining solution [1 mg/mL PI (Invitrogen), 0.2 mg/mL RNase A (Promega) and 0.1% v/v Triton X-100 in PBS] for 30 minutes at room temperature. PI-stained cells were analyzed on a BD FACSCalibur. FlowJo V10 software was used to analyze cell-cycle distribution.

Statistical analysis

All data are given as mean ± SD or mean ± SEM and are based on experiments performed at least three times. All statistical analysis and graphical displays were done with Prism software (GraphPad). Differences between two experimental conditions were analyzed using Student t test. Differences among more than three conditions were analyzed using ANOVA test. A value of P < 0.05 was considered statistically significant.

Correlation between IL1β and breast cancer development

As an initial approach to explore the inflammatory microenvironment in breast cancer, proinflammatory cytokines were analyzed in serum samples from healthy donors and patients diagnosed with stage III breast cancer (Supplementary Tables S1 and S2). Serum levels of representative proinflammatory cytokines (IL1β, IL18, IL6, and TNFα) were significantly elevated in patients with breast cancer compared with healthy controls (Fig. 1AD). The most striking difference was observed for IL1β with its mean serum concentration 40-fold higher in breast cancer patients than that in healthy individuals (Supplementary Fig. S1A). While the increased serum levels of IL1β were moderately correlated with elevated serum levels of IL18 and IL6, there was no such correlation between IL1β and TNFα in patients with breast cancer (Supplementary Fig. S1B–S1D). Next, we analyzed the serum IL1β in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (Fig. 1E). Consistent with finding in patients with breast cancer, the serum concentration of IL1β was significantly elevated in 4T1 tumor–bearing mice (191 ± 17.49 pg/mL) compared with non-tumor–bearing mice (29.8 ± 20.33 pg/mL). Moreover, expression of IL1β was markedly upregulated in primary tumor tissues of mice sacrificed at 5 weeks after inoculation of 4T1 cells compared with primary tumor tissues of mice sacrificed at 3 weeks after inoculation of 4T1 cells (Fig. 1F). These results provide evidence that elevated levels of IL1β are likely to be associated with breast cancer development and progression.

Figure 1.

Potential role of IL1β and IL1R1 signaling in breast cancer progression. Serum levels of IL1β (A), IL18 (B), IL6 (C), and TNFα (D) in healthy donors (n = 15) and patients with breast cancer (n = 15) were measured by ELISA. E, Serum levels of IL1β in nontumor–bearing mice (n = 5) and 4T1-BALB/c syngeneic orthotopic breast cancer mice (n = 5) were measured by ELISA. F, IL1β expression in mouse primary tumor tissues. Immunofluorescence staining of IL1β (red) and DAPI (blue) in mouse primary tumor tissues obtained at 3 and 5 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. Scale bar, 250 μm. G and H, 4T1-BALB/c syngeneic orthotopic breast cancer mice were subjected to intratumoral injection of rmIL1β (2 μg/kg) or saline biweekly for 3 weeks starting from 2 weeks after inoculation of 4T1-Luc cells (n = 4 for each group). G, IVIS images for visualizing primary tumors and quantification of bioluminescent. H, IVIS images for visualizing lung tissues and the incidence of lung metastasis. IO, 4T1 cells with/without knockdown of Il1r1a were orthotopically inoculated, and mice were euthanized at the end of the 5th week (n = 6 for each group). I, qRT-PCR for measurement of Il1r1a mRNA expression in 4T1 cells. J, Serum levels of IL1β in mice were measured by ELISA. K, Growth of primary tumors. L, Photograph and quantification of primary tumors. Representative photographs of lung metastatic foci and hematoxylin & eosin staining, incidence of lung metastasis (M), and quantification of lung metastatic foci (N). Red arrowheads, lung metastatic foci. Scale bar, 500 μm. O, Immunofluorescence staining of N-cadherin and E-cadherin in primary tumors. Scale bar, 100 μm. Data are presented as mean ± SEM (A–E, G, H, J–L, and N) and mean ± SD (I), analyzed by two-tailed Student t test (A–E, G, I, J, L, and N) or two-way ANOVA (K). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Figure 1.

Potential role of IL1β and IL1R1 signaling in breast cancer progression. Serum levels of IL1β (A), IL18 (B), IL6 (C), and TNFα (D) in healthy donors (n = 15) and patients with breast cancer (n = 15) were measured by ELISA. E, Serum levels of IL1β in nontumor–bearing mice (n = 5) and 4T1-BALB/c syngeneic orthotopic breast cancer mice (n = 5) were measured by ELISA. F, IL1β expression in mouse primary tumor tissues. Immunofluorescence staining of IL1β (red) and DAPI (blue) in mouse primary tumor tissues obtained at 3 and 5 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. Scale bar, 250 μm. G and H, 4T1-BALB/c syngeneic orthotopic breast cancer mice were subjected to intratumoral injection of rmIL1β (2 μg/kg) or saline biweekly for 3 weeks starting from 2 weeks after inoculation of 4T1-Luc cells (n = 4 for each group). G, IVIS images for visualizing primary tumors and quantification of bioluminescent. H, IVIS images for visualizing lung tissues and the incidence of lung metastasis. IO, 4T1 cells with/without knockdown of Il1r1a were orthotopically inoculated, and mice were euthanized at the end of the 5th week (n = 6 for each group). I, qRT-PCR for measurement of Il1r1a mRNA expression in 4T1 cells. J, Serum levels of IL1β in mice were measured by ELISA. K, Growth of primary tumors. L, Photograph and quantification of primary tumors. Representative photographs of lung metastatic foci and hematoxylin & eosin staining, incidence of lung metastasis (M), and quantification of lung metastatic foci (N). Red arrowheads, lung metastatic foci. Scale bar, 500 μm. O, Immunofluorescence staining of N-cadherin and E-cadherin in primary tumors. Scale bar, 100 μm. Data are presented as mean ± SEM (A–E, G, H, J–L, and N) and mean ± SD (I), analyzed by two-tailed Student t test (A–E, G, I, J, L, and N) or two-way ANOVA (K). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

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Tumor-progressive effects of IL1β

To further investigate potential involvement of IL1β in breast cancer progression, we first performed a wound-healing assay with two representative human breast cancer cell lines, MDA-MB-231 and MCF7 cells. As shown in Supplementary Fig. S2A, rhIL1β stimulated the proliferation of both MDA-MB-231 and MCF7 cells as evidenced by increased Ki-67 expression and the proportion of cells in the S-phase of the cell cycle (Supplementary Fig. S2B–S2E). Next, direct effects of IL1β on breast cancer progression were assessed by intratumoral injection of rmIL1β into primary tumor in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. Although there was a robust increase in the size of primary tumors by rmIL1β administration (Fig. 1G), the same treatment resulted in less pronounced enhancement in the pulmonary metastasis of breast cancer cells (Fig. 1H).

IL1β exerts its proinflammatory functions by binding to its receptor, IL1R1. To assess the involvement of IL1β–IL1R1 signaling in breast cancer cell growth and metastasis, Il1r1a knockdown murine mammary carcinoma 4T1 cells (Fig. 1I) were orthotopically transplanted into the 4th mammary fat pad of BALB/c mice. Although there was no significant difference between two groups in serum levels of IL1β (Fig. 1J), silencing of its receptor in 4T1 cells significantly retarded the growth (Fig. 1K), and reduced the size (Fig. 1L) of primary tumors as compared with those in the 4T1 sh-control cell inoculated group. In addition, knockdown of Il1r1a in 4T1 cells alleviated pulmonary metastasis of 4T1 cells and formation of lung metastatic foci (Fig. 1M and N). During metastasis, epithelial tumor cells lose their adhesion and polarity, and eventually acquire capability of cell motility to become mesenchymal stem cells through epithelial-to-mesenchymal transition (25). Epithelial and mesenchymal cells express high levels of E-cadherin and N-cadherin, respectively. Decreased expression of N-cadherin with concomitant increase in that of E-cadherin was observed in primary tumor tissues of the 4T1 sh-Il1r1a cell–inoculated group (Fig. 1O). Collectively, these results suggest that the IL1β–IL1R1 signaling axis in the breast tumor microenvironment has critical roles in breast cancer progression.

Cellular source of secreted IL1β in breast tumor microenvironment

Cells of the myeloid lineage, such as macrophages and dendritic cells, are largely recognized as major sources for releasing IL1β in the context of immune responses (11). However, cells outside of the myeloid compartment, including epithelial cells, are also able to release IL1β. For instance, IL1β secretion was observed in keratinocytes upon UV irradiation (26). To identify a cellular source of secreted IL1β in the breast tumor microenvironment, we first determined whether breast cancer cells themselves could release IL1β. Both human breast cancer (MDA-MB-231, MDA-MB-468, and MCF7) and immortalized human benign breast epithelial (MCF10A) cell lines barely expressed basal IL1B mRNA expression (Fig. 2A). Human monocyte-like THP1 cells, well-known for releasing IL1β upon LPS plus ATP stimulation, were used as a positive control. Furthermore, neither human breast cancer cells nor immortalized human benign breast epithelial cells released IL1β into culture supernatant (Fig. 2B). We further examined whether crosstalk between breast cancer cells and myeloid cells, such as macrophages and dendritic cells, could induce IL1β release in the breast tumor microenvironment. Herein, we focused on the interaction between breast cancer cells and macrophages due to 10 times more predominant population of macrophages than dendritic cells in primary tumors formed in the 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (Fig. 2C). Therefore, PMA-primed THP1 cells differentiated into macrophages were cocultured with human breast cancer cells to mimic mutual interaction in the breast tumor microenvironment using a transwell coculture system (Fig. 2D). Secretion of IL1β was dramatically increased by coculturing THP1 cells and TNBC cells (MDA-MB-231 and MDA-MB-468; Fig. 2E and F), and this secretion of IL1β was time-dependently enhanced (Fig. 2GJ). We next examined intracellular expression of pro- and cleaved-IL1β in both TNBC cells and THP1 cells to identify cellular origin of IL1β released in the coculture system. Protein expression of both pro- and cleaved-IL1β was not evident in MDA-MB-231 and MDA-MB-468 cells, but elevated in THP1 cells (Fig. 2KP), indicating that IL1β secretion in coculture supernatant was derived from THP1 cells, not from the breast cancer cells. To ensure that macrophages represent a major cellular source of secreted IL1β, which stimulates cancer progression in the breast tumor microenvironment, they were depleted by intraperitoneal injection of clodronate liposomes in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (Supplementary Fig. S3A and S3B). Injection of clodronate liposomes for 2 weeks starting from 2 weeks, after inoculation of 4T1 cells (Fig. 3A), significantly reduced the growth and the size of mouse primary tumors (Fig. 3B and C), as well as the serum levels of IL1β (Fig. 3D). However, there were no significant differences in the incidence of lung metastasis and the formation of lung metastatic foci upon macrophage depletion (Fig. 3E and F). In general, it takes over 1 month for breast cancer cells to metastasize to lung tissue in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model, but the in vivo experiment could not continue further due to a lethal condition caused by immunosuppression following macrophage depletion. Therefore, to elucidate the effects of macrophage depletion on lung metastasis in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model, we depleted macrophages for 2 weeks starting from 3 weeks after inoculation of 4T1 cells (Fig. 3G; Supplementary Fig. S3C). Although there were no significant differences in the growth and the size of mouse primary tumors (Fig. 3H and I), depletion of macrophages significantly alleviated lung metastasis of 4T1 cells (Fig. 3J and K). Consistent with the previous result, serum levels of IL1β were significantly reduced as a consequence of macrophage depletion in the 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (Fig. 3L). These data indicate that macrophage depletion made after propagation of orthotopic breast tumor is not sufficient to suppress the breast cancer growth, whereas the subsequent metastasis can be attenuated. Taken all together, the above findings strongly suggest that by interplaying with breast cancer cells, macrophages become competent to secrete IL1β in the breast tumor microenvironment.

Figure 2.

IL1β production measured in a coculture of THP1 cells and breast cancer cells. A, qRT-PCR for measurement of basal IL1B mRNA expression in human breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF7) and immortalized human benign breast epithelial cells (MCF10A). PMA-differentiated THP1 cells were primed with LPS (100 μg/mL) for 12 hours, followed by exposure to ATP (5 mmol/L) for 30 minutes. These cells were used as positive control for expression of IL1B. B, IL1β levels in CM of human breast cancer cells and immortalized human benign breast epithelial cells were measured by ELISA. C, Proportion of F4/80+CD11b+Ly6GLy6C macrophages and F4/80CD11c+MHCIIhigh dendritic cells in primary tumors of 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (n = 6). D–P, Coculture of PMA-primed THP1 cells with human breast cancer cells or immortalized human benign breast epithelial cells using the transwell coculture system. IL1β levels in coculture supernatant were measured by ELISA (E) and immunoblot analysis (F). G–J, Time-dependent secretion of IL1β in the supernatant from coculture of MDA-MB-231 cells and THP1 cells (G and H), and MDA-MB-468 cells and THP1 cells (I and J) was determined by ELISA and immunoblot analysis. K–P, Under the same condition, intracellular expression of pro- and cleaved-IL1β proteins was determined by immunoblot analysis (K and N) and quantified (L, M, O, and P). Data are presented as mean ± SD (A, B, E, G, I, L, M, O, and P), analyzed by one-way ANOVA (A, B, E, G, I, L, M, O, and P) or mean ± SEM (C), analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Figure 2.

IL1β production measured in a coculture of THP1 cells and breast cancer cells. A, qRT-PCR for measurement of basal IL1B mRNA expression in human breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF7) and immortalized human benign breast epithelial cells (MCF10A). PMA-differentiated THP1 cells were primed with LPS (100 μg/mL) for 12 hours, followed by exposure to ATP (5 mmol/L) for 30 minutes. These cells were used as positive control for expression of IL1B. B, IL1β levels in CM of human breast cancer cells and immortalized human benign breast epithelial cells were measured by ELISA. C, Proportion of F4/80+CD11b+Ly6GLy6C macrophages and F4/80CD11c+MHCIIhigh dendritic cells in primary tumors of 4T1-BALB/c syngeneic orthotopic breast cancer mouse model (n = 6). D–P, Coculture of PMA-primed THP1 cells with human breast cancer cells or immortalized human benign breast epithelial cells using the transwell coculture system. IL1β levels in coculture supernatant were measured by ELISA (E) and immunoblot analysis (F). G–J, Time-dependent secretion of IL1β in the supernatant from coculture of MDA-MB-231 cells and THP1 cells (G and H), and MDA-MB-468 cells and THP1 cells (I and J) was determined by ELISA and immunoblot analysis. K–P, Under the same condition, intracellular expression of pro- and cleaved-IL1β proteins was determined by immunoblot analysis (K and N) and quantified (L, M, O, and P). Data are presented as mean ± SD (A, B, E, G, I, L, M, O, and P), analyzed by one-way ANOVA (A, B, E, G, I, L, M, O, and P) or mean ± SEM (C), analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

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Figure 3.

Effects of macrophage depletion on IL1β secretion and breast cancer progression in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. AF, Macrophages were depleted from 2 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. For macrophage depletion, mice were injected with an intraperitoneal dose (1 mg/mouse) of clodronate liposomes or control liposomes three times a week (A and G). The depletion of macrophages was confirmed by flow cytometry analysis. A, Schematic representation of clodronate liposome injection (n = 4 for each group). B, Growth of primary tumors. C, Photograph of and quantification of primary tumors. D, Serum levels of IL1β were measured by ELISA. E, Photographs and the incidence of lung metastasis. F, Quantification of lung metastatic foci. Red arrowheads, lung metastatic foci. G–L, Macrophages were depleted from 3 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. G, Schematic representation of clodronate liposome injection (n = 5 for each group). H, Growth of primary tumors. I, Photograph and quantification of primary tumors. J, Representative photographs of lung metastatic foci and hematoxylin and eosin staining, incidence of lung metastasis. Yellow arrowheads, lung metastatic foci. K, Quantification of lung metastatic foci. Scale bar, 500 μm. L, Serum levels of IL1β were measured by ELISA. Data represent mean ± SEM, analyzed by two-tailed Student t test (C, D, F, I, K, and L), or two-way ANOVA (B and H). *, P < 0.05; ***, P < 0.001; n.s., nonsignificant.

Figure 3.

Effects of macrophage depletion on IL1β secretion and breast cancer progression in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. AF, Macrophages were depleted from 2 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. For macrophage depletion, mice were injected with an intraperitoneal dose (1 mg/mouse) of clodronate liposomes or control liposomes three times a week (A and G). The depletion of macrophages was confirmed by flow cytometry analysis. A, Schematic representation of clodronate liposome injection (n = 4 for each group). B, Growth of primary tumors. C, Photograph of and quantification of primary tumors. D, Serum levels of IL1β were measured by ELISA. E, Photographs and the incidence of lung metastasis. F, Quantification of lung metastatic foci. Red arrowheads, lung metastatic foci. G–L, Macrophages were depleted from 3 weeks after inoculation of 4T1 cells in a 4T1-BALB/c syngeneic orthotopic breast cancer mouse model. G, Schematic representation of clodronate liposome injection (n = 5 for each group). H, Growth of primary tumors. I, Photograph and quantification of primary tumors. J, Representative photographs of lung metastatic foci and hematoxylin and eosin staining, incidence of lung metastasis. Yellow arrowheads, lung metastatic foci. K, Quantification of lung metastatic foci. Scale bar, 500 μm. L, Serum levels of IL1β were measured by ELISA. Data represent mean ± SEM, analyzed by two-tailed Student t test (C, D, F, I, K, and L), or two-way ANOVA (B and H). *, P < 0.05; ***, P < 0.001; n.s., nonsignificant.

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Breast cancer cell-induced inflammasome activation in macrophages

It is well defined that inflammasome activation mediates IL1β production in immune cells. To determine whether inflammasome activation in macrophages could be triggered by breast cancer cell–derived soluble factors, we treated PMA-primed THP1 cells with CM from human breast cancer cells or immortalized human benign breast epithelial cells. In agreement with results from the coculture experiment (Fig. 2E and F), CM of MDA-MB-231 or MDA-MB-468 cell culture substantially enhanced the IL1β production in THP1 cells (Fig. 4A and B). Likewise, secretion of IL1β was also induced by CM from murine mammary carcinoma 4T1 cells treated with both murine BMDM and PM (Fig. 4C). Oligomerization and speck formation of ASC are read-outs for inflammasome activation (27, 28). Both events were markedly increased following treatment with CM derived from MDA-MB-231 or MDA-MB-468 cells (Fig. 4D and E). Moreover, THP1 cells treated with CM from both TNBCs exhibited the enhanced expression of the M1-type macrophage markers (Fig. 4F and G). These results indicate that secreted molecule(s) from breast cancer cells, especially triple-negative MDA-MB-231 and MDA-MB-468 cells, induce inflammasome activation, leading to IL1β production in macrophages.

Figure 4.

Inflammasome activation in macrophages induced by secreted soluble factors from breast cancer cells. A and B, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with CM of human breast cancer cells or immortalized human benign breast epithelial cells for 24 hours were measured by ELISA and immunoblot analysis. C, IL1β levels in culture supernatant of BMDM and PM treated with 4T1 CM for 24 hours were measured by ELISA. D, Immunoblot image of ASC oligomerization after cross-linking of pellets of PMA-primed THP1 cells treated with human breast cancer cell CM for 24 hours. E, ASC speck formation (yellow arrows) in PMA-primed THP1 cells treated with human breast cancer cell CM and immortalized human benign breast epithelial cell CM for 12 hours was determined by immunofluorescence staining. qRT-PCR for measurement of M1-type macrophage markers (iNOS, IL6, IL23, and IL1B) and M2-type macrophage markers (ARG1, PPARG, TGFB1, and CD36) in monocyte-like THP1 cells upon treatment with CM from MDA-MB-231 (F) and MDA-MB-468 (G) cells. Data are presented as mean ± SD (A, E, F, and G) analyzed by one-way ANOVA and mean ± SEM (C) analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Figure 4.

Inflammasome activation in macrophages induced by secreted soluble factors from breast cancer cells. A and B, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with CM of human breast cancer cells or immortalized human benign breast epithelial cells for 24 hours were measured by ELISA and immunoblot analysis. C, IL1β levels in culture supernatant of BMDM and PM treated with 4T1 CM for 24 hours were measured by ELISA. D, Immunoblot image of ASC oligomerization after cross-linking of pellets of PMA-primed THP1 cells treated with human breast cancer cell CM for 24 hours. E, ASC speck formation (yellow arrows) in PMA-primed THP1 cells treated with human breast cancer cell CM and immortalized human benign breast epithelial cell CM for 12 hours was determined by immunofluorescence staining. qRT-PCR for measurement of M1-type macrophage markers (iNOS, IL6, IL23, and IL1B) and M2-type macrophage markers (ARG1, PPARG, TGFB1, and CD36) in monocyte-like THP1 cells upon treatment with CM from MDA-MB-231 (F) and MDA-MB-468 (G) cells. Data are presented as mean ± SD (A, E, F, and G) analyzed by one-way ANOVA and mean ± SEM (C) analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

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Analysis of breast cancer cell secretomes

To identify the breast cancer cell–derived soluble factor(s) responsible for inflammasome activation and IL1β secretion in macrophages, we analyzed secretome of human breast cancer cell lines by LC/MS-MS using a label-free quantification approach. Total 1,560 human proteins were identified from CM of MDA-MB-231, MDA-MB-468, and MCF-7 human breast cancer cells (Supplementary Fig. S4A). Compared with other released protein fractions, the commonly released protein fraction (a) was strongly associated with the NFκB signaling pathway, which is responsible for IL1β expression (Supplementary Fig. S4B). To narrow down the candidate proteins, MDA-MB-231 CM was fractionated by molecular weight (MW; <50, 50–100, >100 kDa) using ultracentrifugal filter units. Among three fractions collected on the basis of MW, the 50–100 kDa fraction of MDA-MB-231 CM induced the highest secretion of IL1β from THP1 cells (Supplementary Fig. S4C). In addition, blocking protein transport from endoplasmic reticulum (ER) to Golgi apparatus by brefeldin A in MDA-MB-231 cells did not influence the secretion of IL1β from THP1 cells (Supplementary Fig. S5A), suggesting that the candidate proteins were not released through the ER-Golgi–mediated secretion mechanism.

Exosomes are cell-derived vesicles that are not released via the conventional ER-Golgi–mediated secretion pathway, and cancer cell–derived exosomes have a function in cell–cell communication using their contents (e.g., proteins, lipids, RNA species, and DNA; refs. 29–31). However, exosome-removed MDA-MB-231 CM by ultracentrifugation exerted no significant effects on IL1β secretion in THP1 cells (Supplementary Fig. S5B). Moreover, to exclude involvement of a well-known inflammasome activator, ATP (11), we utilized Apyrase to hydrolyze ATP in MDA-MB-231 CM. The hydrolysis of ATP in the MDA-MB-231-derived CM failed to alter the production of IL1β in THP1 cells (Supplementary Fig. S5C). Considering the MW information of protein candidates and the IL1β induction patterns of breast cancer cells, 7 protein candidates were narrowed down from the commonly released protein fraction ‘a’ of human breast cancer cell secretome analysis (Table 1).

Table 1.

Selected protein candidates from breast cancer cell secretome analysis.

Fold change
No.Accession No.Protein nameGene nameMDA-MB-231/MCF7MDA-MB-468/MCF7MW(kDa)
P16070 CD44 CD44 16.1 7.7 81.5 
P05067 Amyloid-beta A4Protein APP 6.0 3.0 86.9 
O00391 Sulfhydryl oxidase 1 QSOX1 5.5 1.0 82.5 
Q06481 Amyloid-like protein 2 APLP2 5.5 1.5 86.9 
Q14118 Dystroglycan DAG1 3.4 2.6 97 
P02787 Serotransferrin TF 2.4 1.7 77.0 
Q9H2G4 Testis-specific Y-encoded-like protein 2 TSPYL2 2.1 2.3 79.4 
Fold change
No.Accession No.Protein nameGene nameMDA-MB-231/MCF7MDA-MB-468/MCF7MW(kDa)
P16070 CD44 CD44 16.1 7.7 81.5 
P05067 Amyloid-beta A4Protein APP 6.0 3.0 86.9 
O00391 Sulfhydryl oxidase 1 QSOX1 5.5 1.0 82.5 
Q06481 Amyloid-like protein 2 APLP2 5.5 1.5 86.9 
Q14118 Dystroglycan DAG1 3.4 2.6 97 
P02787 Serotransferrin TF 2.4 1.7 77.0 
Q9H2G4 Testis-specific Y-encoded-like protein 2 TSPYL2 2.1 2.3 79.4 

Note: On the basis of the MW information and the differential IL1β induction patterns of breast cancer cells examined, 7 protein candidates were narrowed down from the commonly released protein fraction ‘a’ of human breast cancer cell secretome analysis (Supplementary Fig. S4A).

Involvement of soluble CD44 derived from breast cancer cells in IL1β secretion by macrophages

Among 7 protein candidates identified through the breast cancer cell secretome analysis, CD44 antigen was found to be the most differentially released protein in triple-negative MDA-MB-231 and MDA-MB-468 cells, compared with MCF7 cells (Table 1). CD44 is a single span transmembrane glycoprotein with multiple oncogenic functions, including tumor growth, metastasis, and chemoresistance in many cancers (32–34). Elevated levels of a soluble form of CD44 (sCD44) have been reported in the serum of patients with colon, renal, and gastric cancer (35, 36). In patients with breast cancer, significantly elevated serum concentrations of sCD44 were observed compared with healthy controls (Fig. 5A). Such increased serum levels of sCD44 correlated with increased serum levels of IL1β in patients with breast cancer (Fig. 5B). Moreover, a low survival rate was observed in patients with TNBC who had a high expression of CD44 (Fig. 5C). In line with this notion, TNBC cells released much larger amount of sCD44 in culture medium without stimulation compared with non-TNBC MCF7 cells and immortalized human benign breast epithelial MCF10A cells (Fig. 5D). To confirm that breast cancer cell–derived sCD44 was involved in IL1β secretion from macrophages, THP1 cells were treated with sCD44-neutralized CM from MDA-MB-231 and MDA-MB-468 cells. Neutralization of sCD44 by anti-CD44 mAb in the CM of MDA-MB-231 and MDA-MB-468 cells remarkably reduced IL1β secretion (Fig. 5E and F). However, antibody neutralization of another potential candidate protein, amyloid-β (Table 1) in the CM of the both human breast cancer cell lines failed to inhibit secretion of IL1β by THP1 cells (Fig. 5G and H). To determine the effects of CD44 on breast cancer progression in in vivo, CD44 neutralizing mAb and isotype antibody as a control were injected subcutaneously into nearby mouse primary tumor tissue, starting from 1 week after 4T1 cell inoculation in a 4T1-BALB/c orthotopic breast cancer mouse model (Fig. 5I). Although the neutralizing mAb against CD44 targeted both sCD44 and membrane-bound CD44, neutralization of CD44 markedly prevented the primary tumor growth (Fig. 5JN), and alleviated lung metastasis of 4T1 cells (Fig. 5O and P). Notably, serum levels of IL1β were also decreased by CD44 neutralizing mAb treatment (Fig. 5Q). On the basis of these findings, sCD44 released from breast cancer cells is most likely to be a bona fide stimulator of IL1β secretion by macrophages.

Figure 5.

Stimulation of IL1β secretion from macrophages by breast cancer cell–derived sCD44 antigen. A, Serum levels of sCD44 in healthy donors (n = 15) and patients with breast cancer (n = 15) were determined by ELISA. B, The correlation between serum levels of IL1β and sCD44 in patients with breast cancer was determined by Pearson correlation analysis. C, Survival rate of patients with TNBC depending on CD44 expression. D, CD44 levels in culture supernatant in human and mouse breast cancer cells and human normal breast epithelial cells were determined by ELISA. E and F, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with sCD44-neutralized MDA-MB-231 CM (E) or MDA-MB-468 CM (F) for 12 hours were measured by ELISA. MDA-MB-231 CM and MDA-MB-468 CM were treated with 6 μg of CD44 neutralizing mAb for 6 hours. G and H, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with amyloid β-neutralized MDA-MB-231 CM (G) or MDA-MB-468 CM (H) for 12 hours were measured by ELISA. MDA-MB-231 CM and MDA-MB-468 CM were treated with 6 μg of amyloid β neutralizing mAb for 6 hours. I–Q, CD44 was neutralized by anti-CD44 mAb in a 4T1-BALB/c orthotopic breast cancer mouse model. The anti-CD44 mAb or an isotype was injected subcutaneously every other day for 3 weeks into nearby primary tumor starting from 1 week after 4T1 cell inoculation. I, Schematic representation of clodronate liposome injection (n = 4 for each group). J, IVIS images for visualizing primary tumor. K, Quantification of bioluminescence of primary tumors. L, Primary tumor growth. M and N, Photograph (M) of and quantification of primary tumors (N). O, Representative photographs of lung metastatic foci and incidence of lung metastasis. P, Quantification of lung metastatic foci. Q, Serum levels of IL1β were measured by ELISA. Data are presented as mean ± SD (D–H) and mean ± SEM (A, K, L, N, P, and Q), analyzed by two-tailed Student t test (A, K, N, P, and Q), one-way ANOVA (D–H), or two-way ANOVA (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Figure 5.

Stimulation of IL1β secretion from macrophages by breast cancer cell–derived sCD44 antigen. A, Serum levels of sCD44 in healthy donors (n = 15) and patients with breast cancer (n = 15) were determined by ELISA. B, The correlation between serum levels of IL1β and sCD44 in patients with breast cancer was determined by Pearson correlation analysis. C, Survival rate of patients with TNBC depending on CD44 expression. D, CD44 levels in culture supernatant in human and mouse breast cancer cells and human normal breast epithelial cells were determined by ELISA. E and F, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with sCD44-neutralized MDA-MB-231 CM (E) or MDA-MB-468 CM (F) for 12 hours were measured by ELISA. MDA-MB-231 CM and MDA-MB-468 CM were treated with 6 μg of CD44 neutralizing mAb for 6 hours. G and H, IL1β levels in culture supernatant of PMA-primed THP1 cells treated with amyloid β-neutralized MDA-MB-231 CM (G) or MDA-MB-468 CM (H) for 12 hours were measured by ELISA. MDA-MB-231 CM and MDA-MB-468 CM were treated with 6 μg of amyloid β neutralizing mAb for 6 hours. I–Q, CD44 was neutralized by anti-CD44 mAb in a 4T1-BALB/c orthotopic breast cancer mouse model. The anti-CD44 mAb or an isotype was injected subcutaneously every other day for 3 weeks into nearby primary tumor starting from 1 week after 4T1 cell inoculation. I, Schematic representation of clodronate liposome injection (n = 4 for each group). J, IVIS images for visualizing primary tumor. K, Quantification of bioluminescence of primary tumors. L, Primary tumor growth. M and N, Photograph (M) of and quantification of primary tumors (N). O, Representative photographs of lung metastatic foci and incidence of lung metastasis. P, Quantification of lung metastatic foci. Q, Serum levels of IL1β were measured by ELISA. Data are presented as mean ± SD (D–H) and mean ± SEM (A, K, L, N, P, and Q), analyzed by two-tailed Student t test (A, K, N, P, and Q), one-way ANOVA (D–H), or two-way ANOVA (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

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Stimulation of sCD44 secretion from breast cancer cells by IL1β

To determine whether IL1β can influence the sCD44 expression, human breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF7) and immortalized human benign breast epithelial cells (MCF10A) were treated with rhIL1β. rhIL1β treatment induced sCD44 secretion from TNBC cells (MDA-MB-231 and MDA-MB-468; Fig. 6A). It has been known that sCD44 is produced by ectodomain cleavage of membrane-bound CD44 (34). Proteolytic cleavage of membrane-bound CD44 is mediated by various membrane-associated matrix metalloproteinases (MMP), such as ADAM10, ADAM17, and MT1-MMP (37). However, rhIL1β did not exert any significant effect to the mRNA expression of ADAM10, ADAM17, and MMP14 (Fig. 6BD), but increased expression of CD44 in human breast cancer MDA-MB-231 and MDA-MB-468 cells (Fig. 6EG).

Figure 6.

IL1β-induced sCD44 secretion from human breast cancer cells. A, sCD44 levels in culture supernatant were determined by ELISA. Human breast cancer cells and immortalized human benign breast epithelial cells were treated with rhIL1β (100 ng/mL) for 24 h. B–D, RT-qPCR for measurement of mRNA expression of ADAM10, ADAM17, MMP14 in human breast cancer cells and human normal breast epithelial cells. Human breast cancer cells and immortalized human breast epithelial cells were treated with rhIL1β (100 ng/mL) for 24 h. E and F, CD44 expression in MDA-MB-231 (E) and MDA-MB-468 (F) was examined by flow cytometry after 24 h of rhIL1β (10 ng/mL) treatment. The expression of CD44 was quantified based on mean fluorescence intensity (MFI). G, Immunofluorescence staining of CD44 (green) and DAPI (blue) in MDA-MB-231 and MDA-MB-468 cells. Human breast cancer cells were treated with rhIL1β (100 ng/mL) for 6 h. Scale bar, 100 μm. H, A proposed model for IL1β secretion from macrophages stimulated with breast cancer cell-derived sCD44. Data are presented as mean ± SD analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 6.

IL1β-induced sCD44 secretion from human breast cancer cells. A, sCD44 levels in culture supernatant were determined by ELISA. Human breast cancer cells and immortalized human benign breast epithelial cells were treated with rhIL1β (100 ng/mL) for 24 h. B–D, RT-qPCR for measurement of mRNA expression of ADAM10, ADAM17, MMP14 in human breast cancer cells and human normal breast epithelial cells. Human breast cancer cells and immortalized human breast epithelial cells were treated with rhIL1β (100 ng/mL) for 24 h. E and F, CD44 expression in MDA-MB-231 (E) and MDA-MB-468 (F) was examined by flow cytometry after 24 h of rhIL1β (10 ng/mL) treatment. The expression of CD44 was quantified based on mean fluorescence intensity (MFI). G, Immunofluorescence staining of CD44 (green) and DAPI (blue) in MDA-MB-231 and MDA-MB-468 cells. Human breast cancer cells were treated with rhIL1β (100 ng/mL) for 6 h. Scale bar, 100 μm. H, A proposed model for IL1β secretion from macrophages stimulated with breast cancer cell-derived sCD44. Data are presented as mean ± SD analyzed by two-tailed Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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IL1β is a potent proinflammatory cytokine released as a consequence of inflammasome activation in response to infection and injury, and plays fundamental roles in innate immunity (38). Upon activation of inflammasomes, IL1β triggers infiltration of inflammatory and immune cells into infected and damaged regions by inducing expression of adhesion molecules (e.g., intercellular adhesion molecule-1 and vascular cell adhesion molecules-1) in mesenchymal and endothelial cells (39), and release of inflammatory mediators (e.g., chemokines, and other cytokines; refs. 40–42). Although IL1β is involved in host defense mechanism, aberrant inflammasome activation, and consequently sustained production of IL1β are closely related to pathogenesis of autoimmune and inflammatory disorders (40). In addition to inflammatory diseases, many cancers have inflammatory characteristics in their tumor microenvironment. Therefore, attention has been focused on regulation of cancer progression by modulating inflammatory microenvironment. Breast tumor microenvironment is closely linked to cancer-associated inflammation, also called the intrinsic pathway (43). This study demonstrated tumor progressive potential of IL1β in a 4T1-BALB/c orthotopic breast cancer mouse model. Depletion of macrophages reduced serum levels of IL1β, and alleviated growth of primary tumor and lung metastasis. Although IL1β showed tumor supportive effects in this study, the roles of IL1β in tumor microenvironment can be varied depending on cell and organ types for its expression. Consistent with our current study, Wu and colleagues have reported that dendritic cell–derived IL1β in primary breast tumor microenvironment is associated with advanced disease stage (15). In another study, however, sustained IL1β-mediated systemic inflammatory responses prevent metastasis-initiating cancer cells from differentiating and colonizing at metastatic site (44).

In addition to cancer cells, various types of surrounding stromal cells comprise tumor microenvironment. These include myeloid cells, lymphoid cells, cancer-associated fibroblasts, and sometimes adipocytes (3). Although IL1β is mainly secreted by innate immune cells, nonprofessional immune cells are also able to release IL1β (45). For instance, substantial amount of IL1β was found to be secreted from human primary keratinocytes irradiated with UV (45). In this study, however, we excluded the possibility of human breast cancer cells to release IL1β because of substantially low expression of IL1B mRNA and undetectable amount of secreted IL1β in the culture medium of several human breast cancer cell lines. Innate immune cells, such as macrophages and dendritic cells, are well known for their capability of releasing IL1β in response to pathogen-associated molecular patterns or DAMPs. Both macrophages and dendritic cells also comprise a large portion of inflammatory tumor microenvironment except tumor cells. Wu and colleagues have reported that dendritic cells represent one of primary sources of IL1β secretion in the breast tumor microenvironment (15). In our 4T1-BALB/c orthotopic breast cancer mouse model, a much greater proportion of infiltrated and resident macrophages was observed in mouse primary tumor tissues than that of dendritic cells. Furthermore, macrophage depletion significantly reduced serum levels of IL1β as well as tumor growth and metastasis.

Through human breast cancer cell secretome analysis and a neutralizing experiment, we were able to identify sCD44, released by breast cancer cells, as a potential inducer of IL1β secretion by macrophages. CD44 is a nonkinase transmembrane glycoprotein that is expressed in connective tissues and BM as well as cancer cells. CD44 has been considered as one of the cancer stemness markers, and contributes to cancer cell proliferation, differentiation, migration, angiogenesis, and chemoresistance (33, 34, 46). In contrast to the well-known roles of cell-surface CD44 in cancer, the role of sCD44 in cancer development and progression has been poorly understood. High serum levels of sCD44 have been observed in several human malignancies, including colon cancer, breast cancer, gastric cancer, and liver cancer (36, 47–49). In our current study, high concentrations of sCD44 were also detected in the serum of patients with breast cancer compared with the healthy control. Notably, MDA-MB-231 and MDA-MB-468 TNBC cells released much larger amounts of sCD44 than non-TNBC cells. The existence of TNBC cells in the breast tumor microenvironment represents poor prognosis and aggressive behavior, which are characterized by a larger tumor size, a higher tumor grade, and enhanced dissemination to distant organs (50, 51). Therefore, increased secretion of sCD44 might contribute to an aggressive phenotype of TNBC in patients with breast cancer.

Although sCD44 is a key molecule responsible for breast cancer cell–driven IL1β secretion from macrophages in the breast tumor microenvironment, we do not exclude the possibility of involvement of other factors in this process. Indeed, a recent study has revealed that breast cancer cell–derived TGFβ induces IL1β secretion from dendritic cells (15). In addition, Wellenstein and colleagues reported WNT ligands-induced IL1β secretion (52). Deletion mutation of TP53 induced secretion of WNT ligands from breast cancer cells, and released WNT ligands stimulated IL1β secretion from tumor-associated macrophages, thereby driving systemic inflammation for breast cancer metastasis (52). Therefore, in addition to sCD44, inflammatory tumor microenvironment can be established by other soluble mediators (e.g., TGFβ and the WNT ligand) derived from other cancer cells.

Given the importance of IL1β and inflammasomes in tumor progression, several clinical and preclinical trials targeting IL1 have been attempted to treat breast cancer as well as other cancers malignancies. Of the approved IL1 blocking regimens, administration of anakinra (IL1 receptor antagonist) provided a significantly increased survival benefit for patients with metastatic colorectal cancer and pancreatic cancer (53, 54). In targeting metastatic breast cancer using anakinra with one of the chemotherapeutics (nab-paclitaxel, eribulin, or capecitabine), 2 of 11 patients showed a considerably reduced tumor size and IL1B gene expression in peripheral blood leukocytes (15). In addition, canakinumab, approved as an anti-IL1β neutralizing mAb, significantly reduced the incidence of lung cancer and showed 77% reduction in mortality of lung cancer in patients with atherosclerosis (55). Although IL1β targeting cancer therapy showed good therapeutic outcome in some cancers, there is still an increased risk of infection resulting from blockade of IL1β because of its fundamental roles in innate immunity. Therefore, precise timing and dosage of IL1β blocking agents should be determined before applied to patients with cancer. In this context, regulation of cancer cell–induced IL1β production might be a better option.

In conclusion, this work demonstrates that the breast cancer cell–derived soluble mediator, sCD44 induces IL1β secretion by macrophages in the breast tumor microenvironment, which promotes breast cancer progression and metastasis (Fig. 6H). Accordingly, targeting the sCD44–IL1β axis would be considered as an alternative promising strategy for the immunotherapy of breast cancer progression.

No potential conflicts of interest were disclosed.

Conception and design: J.-H. Jang, D.-H. Kim

Development of methodology: J.-H. Jang, J.W. Lee, K.P. Kim

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.-H. Jang, J.M. Lim, K.P. Kim

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.-H. Jang, J.M. Lim, S.J. Jeong, K.P. Kim

Writing, review, and/or revision of the manuscript: J.-H. Jang, D.-H. Kim, K.P. Kim, Y.-J. Surh

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.-H. Jang

Study supervision: K.P. Kim, Y.-J. Surh

This research was supported by the Global Core Research Center (GCRC) grant (no. 2011-0030001 to Y.-J. Surh) and the Bio & Medical Technology Development Program (no. 2019M3E5D3073567 to K.P. Kim) from the National Research Foundation (NRF) through Ministry of Science and ICT, Republic of Korea.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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