Abstract
Purpose: Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone.
Experimental Design: Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell–derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control.
Results: In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05–3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26–3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation.
Conclusions: Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.
Translational Relevance
Our unique study using patient samples, humanized mouse models of bone metastasis, genetic manipulation of breast cancer cells, and in vitro modeling demonstrate multiple roles for tumor cell–derived IL1B in the process of breast cancer bone metastasis. These data are hugely significant to clinicians and basic scientists alike. Our results suggest that tumor cell–derived IL1B can be used as a biomarker to predict patients who are likely to experience breast cancer relapse and metastasis. In addition, we provide evidence that targeting IL1B signaling with the anti-IL1B antibody, canakinumab, or the IL1R antagonist, Anakinra, may provide therapeutic benefit to patients with tumors that express IL1B specifically by the malignant cells. Future research will establish whether tumor cell–derived IL1B can be used as a biomarker and therapeutic target for other cancers that metastasize to bone.
Introduction
There are over 508,000 deaths annually from breast cancer worldwide with around 75% of patients with advanced breast cancer developing bone metastasis (1). Although bone metastases are more common in estrogen receptor (ER)-positive (ER+) disease, ER-negative (ER−) tumors also metastasize to the skeleton. Once breast cancer has spread to the bones, the disease is incurable and existing treatments can only slow progression. Median survival is 2–3 years following diagnosis of skeletal involvement, hence new therapeutic approaches are needed to improve outcome for these poor prognosis patients. (2). Identification of molecules that drive tumor growth in bone will provide targets for developing such urgently needed novel therapies. Elevated tumor IL1B is associated with poor prognosis in a variety of cancers, including breast, prostate, colon, and lung (3). Importantly, recent data have demonstrated significantly reduced incidence of lung cancer in patients with atherosclerosis who have received the anti-IL1B antibody canakinumab (4). However, the mechanisms by which IL1B promotes cancer development and metastatic progression remains to be established.
We have recently identified a direct link between IL1B and breast cancer bone metastasis (2). In a small study using a tissue array of 150 human primary breast cancer samples from patients with stage II/III breast cancer with a median follow up of 84 months (AZURE trial; ref. 5), we found a significant correlation between IL1B expression and subsequent development of bone metastases (P < 0.0001; ref. 2). We have confirmed this link between IL1B and breast cancer bone metastasis in animal models by demonstrating increased IL1B expression in breast cancer cells that have been passaged in vivo to make them home to bone when injected directly into the bloodstream (2). Our previously published data, therefore, suggest that IL1B expressed by breast cancer cells makes them more aggressive and may play a role in the initiation of the metastatic process. IL1B also appears to play a crucial role in the development of overt metastases from disseminated tumor cells within the bone environment. Inhibiting IL1B binding to IL1 receptor 1 (IL1R1) inhibits metastases from tumor cells disseminated in bone and reduces growth of established bone metastasis in vivo (6). These findings suggest that IL1B also plays a role in the later stages of bone metastasis development in which breast cancer cells emerge from dormancy and proliferate in the bone microenvironment.
In this study, we have utilized in vitro and in vivo model systems in conjunction with patient samples to identify how tumor cell–derived IL1B drives the different stages of breast cancer progression and bone metastasis. We have also investigated the effect of targeting the IL1B pathway using compounds that are FDA approved for the treatment of rheumatoid arthritis, using the anti-human IL1B antibody canakinumab (canakinumab) and IL1R antagonist, Anakinra (Kineret), on in vivo models of spontaneous human breast cancer metastasis to human bone implants, to establish the potential of repurposing these drugs for the treatment of breast cancer bone metastasis.
Materials and Methods
Experimental models and subject details
Animals.
Experiments using human bone grafts were carried out in 10-week-old female NOD SCID mice, IL1B/IL1R1 overexpression bone-homing experiments used 6- to 8-week-old female BALB/c nude mice, effects of IL1B on the bone microenvironment used 10-week-old female C57BL/6 mice (Charles River), IL1R1 KO, or IL1B KO mice (7). Mice were maintained on a 12-hour light/dark cycle with free access to food and water. Experiments were carried out with home office approval under project license 40/3531 (University of Sheffield, Sheffield, United Kingdom).
Cell lines.
Human breast cancer MDA-MB-231-Luc2-TdTomato (Caliper Life Sciences), MDA-MB-231 (parental) MCF7, T47D (European Collection of Authenticated Cell Cultures; ECACC), MDA-MB-231 IV (2), as well as E0771 mouse mammary cancer cells (ECACC) bone marrow HS5 (ECACC) and human primary PRE-osteoblasts, OB1, (gift from M. Kruithof de Julio, University of Bern, Bern, Switzerland) were cultured in DMEM + 10% FCS (Gibco, Invitrogen). Cell lines purchased from commercial sources have been authenticated in house using short tandem repeat analysis of 10 loci. All cell lines were cultured in a humidified incubator under 5% CO2 and used at low passage >20 within 24 months of the last date of authentication.
Patient samples.
All human samples were obtained following written informed consent from patients and studies were conducted in accordance with the Declaration of Helsinki. IL1B and IL1R1 expression was assessed on tissue microarrays (TMA) containing primary breast tumor cores taken from 1,300 patients included in the clinical trial, AZURE (Sponsored by the University of Sheffield and approved as a United Kingdom national trial by the Clinical Trials Committee; controlled trials number ISRCTN79831382; ref. 5). These samples were taken from patients with stage II and III breast cancer, pretreatment between 2003 and 2006, without evidence of metastasis. Patients were subsequently randomized to standard adjuvant therapy with or without the addition of zoledronic acid for 5 years and followed up for 10 years (5). The TMAs were stained for IL1B and scored blindly, under the guidance of histopathologist, Prof. Andrew Hanby (Institute of Molecular Medicine, St. James' University Hospital, Leeds, UK), for IL1B/IL1R1 in the tumor cells or in the associated stroma. Tumor cell or stromal IL1B or IL1R1 was then linked to disease recurrence (any site) or disease recurrence specifically in bone (±other sites).
Human bone.
Human femoral bone was isolated from 60- to 70-year-old female patients who had undergone total hip replacement surgery for rheumatoid arthritis. Patients who had previous exposure to antiresorptive drugs or anti-IL1 treatments were excluded. All patients provided written, informed consent prior to participation in this study, which was carried out in accordance with the ethical guidelines set out in the Declaration of Helsinki. Human bone samples were collected under Human Tissue Act (HTA) license 12182, Sheffield Musculoskeletal Biobank (University of Sheffield, Sheffield, United Kingdom). Trabecular bone cores were prepared using an Isomet 4000 Precision saw (Buehler) with Precision diamond wafering blade (Buehler). Five-millimeter diameter discs were subsequently cut using a bone trephine before storing in sterile PBS at ambient temperature.
Method details
Transfection of tumor cells.
Human MDA-MB-231, MCF7, and T47D cells were stably transfected to overexpress IL1B or IL1R1 using plasmid DNA purified from competent E. Coli that have been transduced with an open reading frame plasmid containing human IL1B or IL1R1 (accession numbers NM_000576 and NM_0008777.2, respectively) with a C-terminal GFP tag (OriGene Technologies Inc.). Plasmid DNA purification was performed using a PureLink HiPure Plasmid Miniprep Kit (Thermo Fisher Scientific) and DNA quantified by UV spectroscopy before being introduced into human cells with the aid of Lipofectamine II (Thermo Fisher Scientific). Control cells were transfected with DNA isolated from the same plasmid without IL1B- or IL1R1-coding sequences.
In vitro studies.
In vitro studies were carried out with and without addition of 0–5 ng/mL recombinant IL1B (R&D Systems) ± 50 μmol/L IL1R antagonist (IL1Ra; Amgen).
Cells were transferred into fresh media with 10% or 1% FCS. Cell proliferation was monitored every 24 hours for up to 120 hours by manual cell counting using a 1/4002 hemocytometer (Hawkley) or over a 72-hour period using an Xcelligence RTCA DP Instrument (Acea Biosciences Inc). Tumor cell invasion was assessed using 6-mm transwell plates with an 8-μm pore size (Costar, Corning Incorporated) ± basement membrane (20% Matrigel, Invitrogen). Tumor cells were seeded into the inner chamber at a density of 2.5 × 105 for parental and MDA-MB-231 derivatives and 5 × 105 for T47D in DMEM + 1% FCS and 5 × 105 OB1 osteoblast cells supplemented with 5% FCS were added to the outer chamber. Cells were removed from the top surface of the membrane 24 and 48 hours after seeding and the cells that had invaded through the pores were stained with hematoxylin and eosin (H&E) before being imaged on a Leica DM7900 light microscope and manually counted.
Migration of cells was investigated by analyzing wound closure. Cells were seeded onto 0.2% gelatin in 6-well tissue culture plates (Costar, Corning Incorporated); once confluent, 10 μg/mL mitomycin C was added to inhibit cell proliferation and a 50-μm scratch made across the monolayer. The percentage of wound closure was measured at 24 and 48 hours using a CTR7000 inverted microscope and LAS-AF v2.1.1 software (Leica Applications Suite, Leica Microsystems). All proliferation, invasion, and migration experiments were repeated using the Xcelligence RTCA DP instrument and RCTA Software (Acea Biosystems Inc).
For coculture studies with human bone, 5 × 105 MDA-MB-231 or T47D cells were seeded onto tissue culture plastic or into 0.5-cm3 human bone discs for 24 hours. Media were removed and analyzed for the concentration of IL1B in the media by ELISA. For coculture with HS5 or OB1 cells, 1 × 105 MDA-MB-231 or T47D cells were cultured onto plastic along with 2 × 105 HS5 or OB1 cells. Cells were sorted by FACS (as described below) 24 hours later, counted, and lysed for analysis of IL1B concentration. Cells were collected, sorted, and counted every 24 hours for 120 hours.
In vivo studies.
To model human breast cancer metastasis to human bone implants, two human bone discs were implanted subcutaneously into 10-week-old female NOD/SCID mice (n = 10/group) under the anesthetic isoflurane. Mice received an injection of 0.003 mg vetergesic and Septrin was added to the drinking water for 1 week after bone implantation. Mice were left for 4 weeks before injecting 1 × 105 MDA-MB-231 Luc2-TdTomato, MCF7 Luc2, or T47D Luc2 cells in 20% Matrigel/79% PBS/1% toluene blue into the two hind mammary fat pads. Primary tumor growth and development of metastases was monitored weekly using an IVIS (Luminol) system (Caliper Life Sciences) following subcutaneous injection of 30 mg/mL d-luciferin (Invitrogen). On termination of experiments, mammary tumors, circulating tumor cells, serum, and bone metastases were resected. RNA was processed for downstream analysis by real-time PCR and the cell lysates were taken for protein analysis and whole tissue for histology as described previously (2, 8).
For therapeutic studies in NOD/SCID mice, placebo (control), 1 mg/mg/day IL1Ra, or 10 mg/kg/14 days canakinumab (Novartis Pharmaceuticals) were administered subcutaneously starting 7 days after injection of tumor cells. In BALB/c mice and C57BL/6 mice, 1 mg/kg/day IL1Ra was administered for 21 or 31 days or 10 mg/kg canakinumab was administered as a single subcutaneous injection. Tumor cells, serum, and bone were subsequently resected for downstream analysis.
Bone metastases were investigated following injection of 5 × 105 MDA-MB-231 GFP (control), MDA-MB-231 IV, MDA-MB-231 IL1B+, or MDA-MB-231 IL1R1+ cells into the lateral tail vein of 6- to 8-week-old female BALB/c nude mice (n = 12/group) or following intraductal injection of E0771 1.25 × 105 cells into the fourth and ninth mammary ducts of IL1B-KO or fl/fl control mice. Tumor growth in bones and lungs was monitored weekly by GFP imaging in live animals. Mice were culled 28 days after tumor cell injection at which point hind limbs, lungs, and serum were resected and processed for micro-CT (μCT), histology, and ELISA analysis of bone turnover markers and circulating cytokines as described previously (6).
Isolation of circulating tumor cells.
Whole blood was centrifuged at 10,000 × g for 5 minutes and the serum removed for ELISA assays. The cell pellet was resuspended in 5 mL of FSM lysis solution (Sigma-Aldrich) to lyse red blood cells. Remaining cells were repelleted, washed three times in PBS, and resuspended in a solution of PBS/10% FCS. Samples from 10 mice per group were pooled prior to the isolation of TdTomato-positive tumor cells using a MoFlow High Performance Cell Sorter (Beckman Coulter) with the 470-nm laser line from a Coherent I-90C tenable argon ion (Coherent). For gene expression analysis, TdTomato fluorescence was detected by a 555LP dichroic long pass and a 580/30 nm band-pass filter. Acquisition and analysis of cells was performed using Summit 4.3 software. After sorting, cells were immediately placed in RNA Protect Cell Reagent (Ambion) and stored at −80°C before RNA extraction. For counting numbers of circulating tumor cells, TdTomato fluorescence was detected using a 561 nm laser and an YL1-A filter (585/16 emission filter). Acquisition and analysis of cells was performed using Attune NxT software.
μCT imaging.
μCT analysis was carried out using a Skyscan 1172 X-ray–computed μCT scanner (Skyscan) equipped with an X-ray tube (voltage, 49 kV; current, 200 μA) and a 0.5-mm aluminum filter. Pixel size was set to 5.86 μm and scanning initiated from the top of the proximal tibia as described previously (9, 10).
Bone histology and measurement of tumor volume.
Bone tumor areas were measured on three, nonserial, H&E-stained, 5-μm histologic sections of decalcified tibiae per mouse using a Leica RMRB upright microscope and Osteomeasure software (Osteometrics Inc.) and a computerized image analysis system (9).
Western blotting.
Protein was extracted using a Mammalian Cell Lysis Kit (Sigma-Aldrich). Thirty micrograms of protein was run on 4%–15% precast polyacrylamide gels (Bio-Rad) and transferred onto an Immobilon-P nitrocellulose membrane (Millipore). Nonspecific binding was blocked with 1% casein (Vector Laboratories) before incubation with rabbit mAbs to human N-Cadherin (D4R1H; 1:1,000), E-Cadherin (24E10; 1:500), or Gamma-Catenin (2303; 1:500; Cell Signaling Technology) or mouse monoclonal GAPDH (ab8245; 1:1,000; Abcam) for 16 hours at 4°C. Secondary antibodies were anti-rabbit or anti-mouse horseradish peroxidase (HRP; 1:15,000) and HRP was detected with the Supersignal Chemiluminescence Detection Kit (Pierce). Band quantification was carried out using Quantity Once software (Bio-Rad) and normalized to GAPDH.
Gene analysis.
Total RNA was extracted using TRI reagent before being reverse transcribed into cDNA using High-capacity RNA-to cDNA Kit (Thermo Fisher Scientific). Relative mRNA expression of IL1B (Hs02786624), IL1R1 (Hs00174097), Caspase 1 (Hs00354836), IL1RN (Hs00893626), JUP (Hs00984034), N-Cadherin (Hs01566408), and E-Cadherin (Hs1013933) were compared with the housekeeping gene GAPDH (Hs02786624) and assessed using an ABI 7900 PCR System (Perkin Elmer) and TaqMan Universal Master Mix (all reagents were purchased from Applied Biosystems via Thermo Fisher Scientific). Fold change in gene expression between treatment groups was analysed by inserting Ct values into Data Assist V3.01 software (Applied Biosystems) and changes in gene expression were only analyzed for genes with a Ct value of ≤25.
IHC.
IHC for the endothelial cells was performed using a mouse-specific anti-CD34 antibody (MCA1825-GA: 1:50 dilution; Serotec) followed by a biotin-conjugated anti-rat secondary antibody (E0467 1:200) as described previously (Nutter and colleagues, 2014). Two nonserial sections per sample were scored prior to statistical analysis. TMAs were stained for IL1B (ab2105, 1:200 dilution, Abcam) and IL1R1 (ab59995, 1:25 dilution, Abcam) and scored blindly for IL1B/IL1R1 in the tumor cells or in the associated stroma. Tumor or stromal IL1B or IL1R1 was then linked to disease recurrence (any site) or disease recurrence specifically in bone (±other sites).
Biochemical analysis.
Serum concentrations of TRACP-5b and P1NP were measured using commercially available ELISA kits [Mouse TRAP Assay and Rat/Mouse P1NP Competitive Immunoassay Kit (Immunodiagnostic Systems)]. Concentrations of IL1B were analyzed in mouse bone marrow, human breast cancer cells, and in coculture systems using anti-mouse or anti-human Quantikine ELISA kits, as appropriate (R&D Systems), and according to the manufacturer's instructions.
Quantification and statistical analysis
For experiments using nonclinical material, group-wise comparisons were carried out using one-way independent ANOVA with Tukey multiple comparison test using GraphPad Prism software version 6.0. Statistical significance was defined as P ≤ 0.05. A Cox proportional hazards model, using the statistical software, was used to investigate the effect of IL1B in a multivariate model including number of involved lymph nodes, tumor grade, ER receptor status, tumor stage, and type of planned systemic therapy (endocrine therapy, chemotherapy, or both). Statistical significance was determined using two-sided P values <0.05.
Results
The IL1B pathway is upregulated during the process of human breast cancer metastasis to human bone
A mouse model of spontaneous human breast cancer metastasis to human bone implants was utilized to investigate how the IL1B pathway changes through the different stages of metastasis (ref. 11; Supplementary Fig. S1). Using this model, the expression levels of genes associated with the IL1B pathway increased in a stepwise manner at each stage of the metastatic process in both triple-negative (MDA-MB-231) and ER+ (T47D) breast cancer cells. Genes associated with the IL1B signaling pathway (IL1B, IL1R1, Caspase 1, and IL1Ra) were expressed at very low levels in both MDA-MB-231 and T47D cells grown in vitro and expression of these genes was not altered in primary mammary tumors from the same cells that did not metastasize in vivo (Fig. 1A).
In vivo model of spontaneous human breast cancer metastasis to human bone predicts a key role for IL1B signaling in breast cancer bone metastasis Two 0.5-cm3 pieces of human femoral bone were implanted subcutaneously into 8-week-old female NOD SCID mice (n = 10/group). Four weeks later, luciferase-labeled MDA-MB-231-luc2-TdTomato or T47D cells were injected into the hind mammary fat pads. Each experiment was carried out three separate times using bone from a different patient for each repeat. Histograms show fold change of IL1B, IL1R1, Caspase 1, and IL1Ra copy number (dCT) compared with GAPDH in tumor cells grown in vivo compared with those grown in a tissue culture flask (A, i); mammary tumors that metastasize compared with mammary tumors that do not metastasize (A, ii); circulating tumor cells compared with tumor cells that remain in the fat pad (A, iii), and bone metastases compared with the matched primary tumor (A, iv). Fold change in IL1B protein expression is shown in B and fold change in copy number of genes associated with EMT (E-Cadherin, N-Cadherin, and JUP) compared with GAPDH are shown in C (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; ^^^, P < 0.001 compared with naïve bone).
IL1B, IL1R1, and Caspase 1 were all significantly increased in mammary tumors that subsequently metastasized to human bone, compared with those that did not metastasize (P < 0.01 for both cell lines), leading to activation of IL1B signaling as shown by ELISA for active 17-kDa IL1B (Fig. 1B; Supplementary Fig. S2). IL1B gene expression increased in circulating tumor cells compared with metastatic mammary tumors (P < 0.01 for both cell lines). IL1B (P < 0.001), IL1R1 (P < 0.01), Caspase 1 (P < 0.001), and IL1Ra (P < 0.01) were further increased in tumor cells isolated from metastases in human bone compared with their corresponding mammary tumors, leading to further production of IL1B protein (Fig. 1; Supplementary Fig. S2). These data suggest that IL1B signaling may promote both initiation of metastasis from the primary site as well as development of breast cancer metastases in bone.
Tumor-derived IL1B promotes epithelial-to-mesenchymal transition and breast cancer metastasis
Expression levels of genes associated with tumor cell adhesion and epithelial-to-mesenchymal transition (EMT) were significantly altered in primary tumors that metastasized to bone compared with tumors that did not metastasize (Fig. 1C). We therefore generated IL1B-overexpressing cells (MDA-MB-231-IL1B+, T47D-IL1B+, and MCF7-IL1B+) to investigate whether tumor cell–derived IL1B is responsible for inducing EMT and metastasis to bone. Compared with the corresponding controls, all IL1B+ cell lines demonstrated increased EMT, exhibiting morphologic changes from an EMT phenotype (Fig. 2A) as well as reduced proliferation (Fig. 2B), reduced expression of E-cadherin, and Junction plakoglobin (JUP) and increased expression of N-Cadherin gene and protein (Fig. 2C and D). Wound closure (P < 0.0001 in MDA-MB-231-IL1B+; P < 0.001 in MCF7-IL1B+ and T47D-IL-1B+; Fig. 2E), migration, and invasion through Matrigel toward osteoblasts (P < 0.0001 in MDA-MB-231-IL1B+; P < 0.001 in MCF7-IL1B+ and T47D-IL1B+; Fig. 2G) were increased in tumor cells with elevated IL1B signaling (IL1B+) compared with their respective controls. These data imply that endogenous production of IL1B by tumor cells promotes EMT and promotes a more invasive and migratory phenotype.
Tumor-derived IL1B induces EMT in vitro. MDA-MB-231, MCF7, and T47D cells were stably transfected to express high levels of IL1B or scramble sequence (control) to assess effects of endogenous IL1B on parameters associated with metastasis. Increased endogenous IL1B resulted tumor cells changing from an EMT phenotype. A, The morphology of MDA-MB-231 and MCF7 cells before and after stable transfection with IL1B. B, Effects on proliferation of MDA-MB-231 and MCF7 cells over 70–72 hours. C and D show fold change in copy number and protein expression of IL1B, IL1R1, E-Cadherin, N-Cadherin, and JUP compared with GAPDH and B-Catenin, respectively. Ability of tumor cells to invade toward osteoblasts through Matrigel and/or 8-μm pores is shown in E and capacity of cells to migrate over 24 and 48 hours is shown using a wound closure assay in F. Data are shown as mean ± SEM (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).
Inhibition of IL1B signaling reduces spontaneous metastasis to human bone
As tumor cell–derived IL1B appeared to be promoting onset of metastasis through induction of EMT, we next investigated the effects of inhibiting IL1B signaling with an IL1Ra or the human antibody against IL1B, canakinumab, on primary tumor growth and spontaneous metastasis to human bone implants. Daily administration of IL1Ra had no effect on growth of MDA-Td cells in the mammary fat pad, whereas fortnightly administration of canakinumab resulted in significantly increased growth of the primary tumor (P = 0.003 compared with placebo and 0.006 compared with IL1Ra; Fig. 3A). In contrast, both IL1Ra and canakinumab reduced metastasis to human bone; metastases were detected in human bone implants from 7 of 10 control mice, 4 of 10 IL1Ra-treated mice, and 1 of 10 canakinumab-treated mice. Bone metastases isolated from IL1Ra and canakinumab treatment groups were also smaller than those detected in the control group (Fig. 3B) suggesting that specific inhibition of human IL1B produced by the tumor cells reduces metastasis. Numbers of cells detected in the circulation of mice treated with canakinumab or IL1Ra were significantly lower than those detected in the placebo-treated group: Only 3 tumor cells/mL were counted in whole blood from mice treated with either canakinumab or IL1Ra compared with 108 tumor cells/mL counted in blood from placebo-treated mice (Supplementary Fig. S4). These data suggest that inhibition of IL1 signaling prevents tumor cells from being shed from the primary site into the circulation.
Tumor cell–derived IL1B promotes spontaneous metastasis and bone colonization in vivo. In A and B, female NOD-SCID mice bearing two 0.5-cm3 pieces of human femoral bone received intra-mammary injections of MDA-MB-231Luc2-TdTomato cells. Starting 1 week after tumor cell injection, mice were treated with 1 mg/kg/day IL1Ra, 20 mg/kg/14 days canakinumab, or placebo (control; n = 10/group). All animals were culled 8 weeks following tumor cell injection. Effects on primary tumor growth (A) and bone metastases (B) were assessed in vivo and immediately postmortem by luciferase imaging and confirmed ex vivo on histologic sections. Data are shown as numbers of photons per second emitted 2 minutes following subcutaneous injection of d-luciferin. In C–E, 8-week-old female BALB/c nude mice were injected with control (scramble sequence) or IL1B-overexpressing MDA-MB-231 IL1B+ cells via the lateral tail vein. Tumor growth in bone and lung were measured in vivo by GFP imaging and confirmed ex vivo on histologic sections. Tumor growth in bone (C) and representative μCT images of tumor-bearing tibiae (D). The graph shows bone volume/tissue volume ratio indicating effects on tumor-induced bone destruction. E, Numbers and size of tumors detected in lungs by each of the cell lines. F, Spontaneous metastasis of E0771 cells from the fourth and ninth mammary glands to bone and lung in control and IL1B KO mice (*, P < 0.01; **, P < 0.001; ***, P < 0.0001; B, bone; L, lung and tumor areas are indicated with arrows).
Tumor cell–derived IL1B promotes bone homing and colonization of breast cancer cells
To assess whether metastasis was driven by IL1B from tumor cells or from the microenvironment, we used genetic manipulation to increase expression of IL1B in tumor cells alongside a mouse model from which IL1B has been genetically knocked out (IL1B KO). Injection of breast cancer cells into the tail vein of mice usually results in lung metastasis due to tumor cells becoming trapped in the lung capillaries. We have previously shown that breast cancer cells that preferentially home to the bone microenvironment following intravenous injection express high levels of IL1B, suggesting that this cytokine may be involved in tissue-specific homing of breast cancer cells to bone (2). In this study, intravenous injection of MDA-MB-231-IL1B+ cells into BALB/c nude mice resulted in significantly increased number of animals developing bone metastasis (75%) compared with animals injected with control cells (12%; P < 0.001; Fig. 3C). MDA-MB-231-IL1B+ tumors caused development of significantly larger osteolytic lesions in mouse bone compared with control cells (P = 0.03; Fig. 3D) There was a nonsignificant numerical reduction in lung metastases in mice injected with MDA-MB-231-IL1B+ cells compared with control cells (P = 0.16; Fig. 3E). Removal of IL1B from the microenvironment did not alter metastasis: Injection of E0771 cells into the mammary ducts of IL1B KO and control (IL1B fl/fl) mice resulted in metastasis in bone and lung (Fig. 3F). These data strengthen the hypothesis that endogenous IL1B rather than IL1B from the microenvironment promotes tumor cell homing and development of bone metastases.
Tumor cell–bone cell interactions further induce IL1B and promote development of overt metastases
Using assays specifically designed to amplify human mRNA, gene analysis data from our mouse model of human breast cancer metastasis to human bone implants suggested that the IL1B pathway was further increased when breast cancer cells are growing in the bone environment compared with metastatic cells in the primary site or in the circulation (Fig. 1A). We therefore investigated how IL1B production changes when tumor cells come into contact with bone cells and how IL1B alters the bone microenvironment to affect tumor growth (Fig. 4). Culture of human breast cancer cells into pieces of whole human bone for 48 hours results in increased secretion of IL1B into the media (P < 0.0001 for MDA-MB-231 and T47D cells; Fig. 5A). Coculture with human HS5 bone marrow cells revealed that increased IL1B concentrations originated from both the cancer cells (P < 0.001) and bone marrow cells (P < 0.001), with IL1B from tumor cells increasing approximately 1,000-fold and IL1B from HS5 cells increasing ∼100-fold following coculture (Fig. 4B).
Tumor cell–bone cell interactions stimulate IL1B production and cell proliferation. MDA-MB-231 and T47D human breast cancer cell lines were cultured alone or in combination with live human bone, HS5 bone marrow cells, or OB1 primary osteoblasts. A, The effects of culturing MDA-MB-231 or T47D cells in live human bone discs on IL1B concentrations secreted into the media. The effect of coculturing MDA-MB-231 or T47D cells with HS5 bone cells on IL1B produced from the individual cell types after cell sorting and the proliferation of these cells are shown in B and C. Effects of coculturing MDA-MB-231 or T47D cells with OB1 (osteoblast) cells on proliferation are shown in D. Data are shown as mean ± SEM (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).
IL1B in the bone microenvironment stimulates expansion of the bone metastatic niche. Effects of adding 40 pg/mL or 5 ng/mL recombinant IL1B to MDA-MB-231 or T47D breast cancer cells is shown in A and effects on adding 20 pg/mL, 40 pg/mL, or 5 ng/mL IL1B on proliferation of HS5 (bone marrow cells) or OB1 (osteoblasts) are shown in B and C, respectively. IL1-driven alterations of the bone vasculature was measured following CD34 staining in the trabecular region of the tibiae from 10- to 12-week-old female IL-1R1 knockout mice (D), BALB/c nude mice treated with 1 mg/mL/day IL-1Ra for 31 days (E), and C57BL/6 mice treated with 10 μmol/L IL1B neutralizing antibody for 4–96 hours (F). Data are shown as mean ± SEM (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).
It is hypothesized that when metastatic tumor cells arrive in the bone, they either reside in the vascular niche from which they may be stimulated to form overt metastasis through expansion of the associated vasculature or they are deposited in the bone marrow from which they home to the osteoblastic niche and become dormant (12–15). Stimulation of osteoblast proliferation is thought to stimulate proliferation of previously dormant tumor cells within this niche, thereby promoting onset of overt metastases (8, 12, 16). We therefore investigated the effects of exogenous IL1B and IL1B from tumor cells on proliferation of tumor cells, osteoblasts, and bone marrow cells as well as the effects of IL1B on CD34+ blood vessels. Coculture of HS5 bone marrow or OB1 primary osteoblast cells with breast cancer cells caused increased proliferation of all cell types [P < 0.001 for HS5, MDA-MB-231, or T47D (Fig. 4C) and P < 0.001 for OB1, MDA-MB-231, or T47D (Fig. 4D)]. Furthermore, administration of IL1B increased proliferation of HS5 (Fig. 5B) or OB1 (Fig. 5C) cells, but not breast cancer cells (Fig. 5A), suggesting that tumor cell–bone cell interactions promote production of IL1B that can drive expansion of the niche and stimulate the formation of overt metastases.
IL1B signaling was also found to have significant effects on the bone microvasculature. Preventing IL1B signaling in bone by knocking out IL1R1, pharmacologic blockade of IL1R with IL1Ra, or reducing circulating concentrations by administration of an anti-IL1B antibody reduced the average length of CD34+ blood vessels in the trabecular bone, where tumor colonization takes place (Wang and colleagues, 2014; P < 0.01 for IL1Ra and anti-IL1B–treated mice; Fig. 5C). These findings were confirmed by endomucin staining, which showed decreased numbers of blood vessels and blood vessels length in bone when IL1B signaling was disrupted (data not shown). ELISA analysis for endothelin 1 and VEGF showed reduced concentrations of both of these endothelial cell markers in the bone marrow in IL1R1 KO mice (P < 0.001 endothelin 1; P < 0.001 VEGF) and mice treated with IL1R antagonist (P < 0.01 endothelin 1; P < 0.01 VEGF) or canakinumab (P < 0.01 endothelin 1; P < 0.001 VEGF) compared with control (Supplementary Fig. S5). These data suggest that tumor cell–bone cell–associated increases in IL1B and high levels of IL1B in tumor cells may also promote angiogenesis, further stimulating metastases.
IL1B promotes osteoclast and osteoblast activity in vivo
High levels of bone turnover are associated with increased bone metastasis and it is hypothesized that interactions between tumor cells and bone cells instigate a vicious cycle whereby tumor cells stimulate osteoclast activity and bone resorption. Subsequent release of growth factors from the bone then stimulates growth of tumor cells (17). Bone turnover is primarily regulated by activity of osteoclast and osteoblasts; we therefore investigated the effects of IL1B signaling on the activity of these cell types (Supplementary Fig. S6). Mice that are globally deficient in IL1R1 have significantly increased trabecular bone volume (P < 0.0001; Supplementary Fig. S6A), decreased osteoclast activity (P < 0.0001), and decreased osteoblast activity (P < 0.0001) compared with control (IL1R1 fl/fl) mice (Supplementary Fig. S6B). Disrupting IL1B signaling by daily injection with 1 mg/kg IL1Ra or canakinumab every 14 days for 8 weeks in mice bearing human bone implants also led to reduced osteoclast (P < 0.001 for IL1Ra and P < 0.01 for canakinumab) and osteoblast (P < 0.0001 for IL1Ra and P < 0.001 for canakinumab) activity (Supplementary Fig. S6C). Interestingly, growing tumor cells that express high levels of IL1B in mouse bone (MDA-MB-231-IL1B+) resulted in increased osteoclast activity (P < 0.01) compared with control cells growing in the same environment, but osteoblast activity was not altered (Supplementary Fig. S6D). These data indicate that IL1B has profound effects on osteoclast and osteoblast activity in vivo and inhibiting bone turnover through anti-IL1 treatments may be a mechanism by which this therapeutic approach reduces bone metastases.
Tumor cell–derived IL1B predicts future breast cancer relapse in bone and other organs in clinical samples
To establish the relevance of our laboratory findings in a clinical setting, we investigated the correlation between IL1B and its receptor IL1R1 in patient samples. A total of 1,189 primary tumor samples (not previously included in our hypothesis-generating study; ref. 2) from patients with stage II/III breast cancer with no evidence of metastasis included in the randomized phase III AZURE trial evaluating standard adjuvant systemic therapy with or without the bisphosphonate, zoledronic acid (18), were stained for the 17-kDa form of active IL1B or IL1R1. Samples were scored separately for the expression of IL1B or IL1R1 in the tumor cells and the tumor-associated stroma. Patents were followed up for 10 years and correlation between IL1B/IL1R1 expression and distant recurrence or relapse in bone assessed using a Cox proportional hazards model. IL1B in tumor cells strongly correlated with distant recurrence at any site [P = 0.0016: HR = 2.09; confidence interval (CI), 1.26–3.48] or recurrence in bone at any time (P = 0.0387: HR = 1.85; CI, 1.05–3.26; Fig. 6). IL1B in the stroma did not correlate with future relapse nor did IL1R1 in the tumor or the stroma (Supplementary Table S1). Patients who had IL1B in their tumor cells and IL1R1 in the tumor-associated stroma were more likely to experience future relapse at a distant site (P = 0.042), compared with patients who did not have IL1B in their tumor cells, indicating that tumor-derived IL1B may not only promote metastasis directly, but also interact with IL1R1 in the stroma to promote this process.
Tumor-derived IL1B predicts future recurrence and bone relapse in patients with stage II and III breast cancer. A total of 1,189 primary breast cancer samples from patients with stage II and III breast cancer with no evidence of metastasis were stained for 17-kDa active IL1B. Tumors were scored for IL1B in the tumor cell population. Data shown are Cox model–predicted curves, allowing for other Cox model–included variables such as number of involved lymph nodes, representing the correlation between tumor-derived IL1B and subsequent recurrence at any site (A) or in bone over a 10-year time period (B).
Bone metastases are more prevalent in ER+ breast cancer, whereas ER− tumors are more likely to metastasize to lung. To investigate the prognostic value of IL1B in different patient subgroups, we analyzed the correlation between IL1B in tumor cells and future relapse in bone or any tissue for ER+ and ER− tumors separately. The link between IL1B and bone metastasis was significantly stronger in ER+ tumors (P = 0.02: HR = 2.09; CI, 1.11–3.83) than in ER− tumors (P = 0.65: HR = 0.71; CI, 0.16–3.09). However, no differences were observed between IL1B expression in tumor cells and recurrence at any site between patients with ER+ and ER− disease. Both groups showed a strong positive correlation with IL1B in tumor cells and subsequent distal metastases (HR = 2.05 for ER+ and 2.21 for ER−, respectively). Taken together, these data suggest that expression of IL1B by tumor cells is predictive of future development of metastases.
Discussion
Our data show that endogenous production of IL1B by breast cancer cells increases their metastatic potential in in vitro and in vivo models and is associated with increased bone recurrence in patients with breast cancer. Increased IL1B production was seen in ER+ and ER− breast cancer cells that spontaneously metastasized to human bone implants in vivo compared with nonmetastatic breast cancer cells (Fig. 1). The same link between IL1B and metastasis was made in primary tumor samples from patients with stage II and III breast cancer enrolled in the AZURE study (18) that experienced disease recurrence during a 10-year follow-up period. In a previous study, we have shown a link between IL1B in primary tumor samples and subsequent relapse in bone from 150 samples over a 5-year follow-up period (2). In this study, we were interested in assessing whether this correlation was associated specifically with IL1B produced by tumor cells or IL1B from the tumor environment. We therefore used an antibody to the active 17-kDa form of IL1B and scored IL1B in tumor cells and the stoma separately. Interestingly, expression of IL1B in tumor cells correlated with both relapse in bone and relapse at any site indicating that presence of this molecule is likely to play a role in metastasis in general. No correlation was observed between IL1B in the tumor microenvironment or IL1R1 expression, indicating that relapse in distant organs is influenced by tumor cell–derived IL1B. Further analysis revealed that correlation between IL1B expression in tumor cells and subsequent recurrence specifically in bone is highly significant in patients with ER+ tumors (P < 0.02), but this correlation was not observed in patients with ER− disease. In contrast, expression of IL1B in primary tumor samples was predictive of distal recurrence to any site in both ER+ and ER− disease. Bone metastases are more common from ER+ tumors and in our dataset of 1,189 patients, bone metastasis developed from 181 ER+ tumors and 29 ER− tumors. It is therefore likely that lack of correlation between IL1B and bone metastasis, observed in the ER− group, is due to the small number of patients in this group providing insufficient power for these calculations. A larger set of patients is required to test this hypothesis.
In agreement with the clinical data, genetic manipulation of breast cancer cells to artificially overexpress IL1B increased their migration and invasion capacities in vitro (Fig. 2). Furthermore, inhibition of IL1B signaling with the human-specific anti-IL1B antibody canakinumab or inhibition of the IL1R reduced the number of breast cancer cells shed into the circulation and reduced metastases in human bone implants (Fig. 3). Interestingly, administration of canakinumab significantly increased growth of MDA-Td cells in the mammary fat pad, whereas the IL1Ra had no effect. Daily administration of IL1Ra has previously been shown to reduce subcutaneous tumor growth of MDA-IV and MCF7 cells (6) as well as cause a small reduction in the growth of 4T1 cells injected into the mammary fat pad (6, 19). Data from both of these studies suggest that inhibition of IL1 does not exert direct cytotoxic effects on tumor cells, but inhibits tumor cell proliferation possibly by inhibiting the formation of new blood vessels (6, 19). In this study, both control and IL1Ra-treated tumors were small (>0.3 mm3) when mice were culled and therefore it is likely that tumors were too small for differences in growth to be detected (Fig. 3A). Until now, there have been no reports on the effects of canakinumab on tumor growth and metastasis. Data from patients with atherosclerosis who have received canakinumab demonstrate significantly reduced incidences of lung cancer; however, the study was not designed to investigate effects on existing cancer (4). Canakinumab is an antibody that specifically targets IL1B, whereas the IL1Ra inhibits both IL1B and IL1A, suggesting that IL1A may play a role in growth of the primary tumor, whereas IL1B is more prominent in driving the metastatic process.
Previous reports have focused on the role of exogenous IL1B in the progression of metastasis. Adding IL1B to MCF7 cells in culture in the presence of EGF or TNFa1 to MCF7 has been shown to promote EMT-related changes the actin cytoskeleton (20, 21) and induces an invasive phenotype (22). Our data are in agreement with these findings; tumor cells overexpressing IL1B and IL1R1-overexpressing cells stimulated with IL1B have a more mesenchymal phenotype and exhibit molecular changes associated with EMT including reduced JUP, a molecule whose reduction is strongly associated with tumor cell shedding into the blood (ref. 23; Fig. 2). However, we also show that to generate prometastatic effects, significantly higher doses of exogenous IL1B are required to achieve the same promigratory effects, as endogenous IL1B (5 ng/mL of exogenous IL1B exerts similar invasive and migratory effects as 10–20 pg/mL tumor-derived IL1B; Supplementary Fig. S3), suggesting that IL1B produced by the tumor cells is a more potent inducer of metastasis than exogenous sources. Spread of metastatic E0771 tumor cells from the primary site to bone or lung occurs in both IL1B KO and control animals (Fig. 3F). These data, taken with the absence of a correlation between IL1R1 in tumor cells and distant recurrence at any site (Supplementary Table S1) further imply that metastasis is stimulated by endogenous IL1B from the tumor cells rather than exogenous sources of IL1B acting upon the tumor cells.
Recent work has demonstrated that IL1B promotes IL17 expression from YδT cells causing expansion and polarization of neutrophils in mammary tumors that in turn promotes metastasis (24). The authors hypothesized that IL1B secreted from macrophages within the tumor is likely to be the key driver of this process. Canakinumab does not cross-react with mouse IL1B, and our in vivo models do not express T cells; therefore, although IL1B+ neutrophils are likely to be an important tumor promoter, it is clear that tumor-derived IL1B also plays a key role in driving the metastatic process. Retrospective analysis of our data show that IL1B+ immune cells were present at very low numbers in our patient samples and therefore it was not possible for us to investigate this hypothesis. However, patients who have IL1B in their primary tumors and IL1R1 in their tumor-associated stroma had increased breast cancer recurrence at distant sites. It is, therefore, likely that as well as promoting EMT, IL1B from tumor cells interacts with cells from the local environment, including immune cells, promoting a metastasis-inducing environment.
Once tumor cells leave their primary site, they home to secondary metastatic organs. In breast cancer, these are primarily bone, lung, and brain with bone being the most prominent (7). We have previously shown that a bone-seeking breast cancer cell line (MDA-IV) produces high concentrations of IL1B compared with parental MDA-MB-231 cells (2). Similarly, in a PC3 model of prostate cancer genetic overexpression of IL1B increased bone metastases from tumor cells injected into the heart, whereas genetic knockdown of this molecule reduced bone metastasis (25). In accordance with this, our current study shows that breast cancer cells engineered to overexpress IL1B (MDA-MB-231-IL1B+) have increased bone-homing capabilities. Importantly, all of the models described above have been designed to investigate bone metastasis and although our data show a strong link between IL1B expression and bone homing, it does not exclude IL1B involvement in metastasis to other sites.
Bone metastases occur when tumor cells are disseminated into the bone marrow and take up residence in the bone metastatic niche. This niche is thought to be made up of three interacting niches: the osteoblastic, vascular, and hematopoietic stem cell niche (reviewed by refs. 26, 27). There is increasing evidence from patient samples and model systems showing that stimulation of bone turnover and expansion of the bone metastatic niche promotes metastasis (8, 12, 13, 16, 28). Evidence for metastases in other organs predicts that proliferation of vascular endothelial cells and sprouting of new blood vessels may also promote proliferation of tumor cells in bone-driving metastases formation (14, 15, 29). We have previously shown that inhibiting IL1R signaling with the IL1R antagonist Anakinra prevents formation of overt metastasis from MDA-IV cells disseminated in bone by maintaining these cells in a state of dormancy (6). Recent evidence from mouse models of prostate cancer has also shown reduced bone metastasis from androgen receptor–positive prostate cancer cells disseminated in bone following administration of Anakinra (30). Shahriari and colleagues demonstrated that IL1B secreted by prostate cancer cells generated cancer-associated fibroblasts (CAF) in the skeleton via upregulation of S100A4 and COX2 (30). We have also demonstrated increased S100A4 in IL1B-overexpressing MDA IV breast cancer cells (2) and it is highly likely that IL1B produced by tumor cells induces changes in mesenchymal stem cells leading to the generation of CAFs that in turn provide a supportive environment for the growth of cancer cells in bone. Our new data clearly show that antimetastatic effects of Anakinra are not just due to reductions in CAFs, but due to inhibition of the activity of IL1B on the bone metastatic niche. Inhibition of IL1 signaling by treatments with canakinumab, IL1Ra, or genetic knockout of IL1R1 in vivo reduced bone turnover and blood vessel length in the area of bone most commonly colonized by breast cancer cells (the trabecular region of the metaphysis; ref. 12). This decreased blood vessel length is associated with decreased concentrations of endothelial cell growth factors endothelin 1 and VEGF (Supplementary Fig. S3). Therefore inhibiting IL1B signaling may also reduce expansion of the metastatic niche by inhibiting neovascularization (31–33). We established that direct contact between tumor cells, primary human bone samples, bone marrow cells, or osteoblasts promoted release of IL1B from both tumor and bone cells (Fig. 4). Exogenous IL1B did not increase tumor cell proliferation, even in cells overexpressing IL1R1 (data not shown). Instead, IL1B stimulated proliferation of bone marrow cells, osteoblasts, and blood vessel–induced proliferation of tumor cells (Fig. 4). It is therefore likely that arrival of tumor cells expressing high concentrations of IL1B will stimulate expansion of metastatic niche components and that contact between IL1B-expressing tumor cells and osteoblasts/blood vessels will drive tumor colonization of bone.
It is hypothesized that stimulation of osteoclast activity leading to increased bone resorption results in the release of growth factors into the local environment that in turn stimulates tumor growth (17). In vitro studies have previously reported that exposure of osteoclasts to IL1B stimulates osteoclastogenesis (34) and our new data confirm that this also occurs in vivo (Supplementary Fig. S6). Removal of IL1B signaling in IL1R1 KO mice or pharmacologic inhibition of IL1 signaling with IL1Ra or canakinumab significantly reduces osteoclast activity. Interestingly, canakinumab reduced osteoclast and osteoblast activity in mice bearing human bone implants, albeit to a lesser degree than Anakinra. Whether these effects are due to altered activity of osteoblasts and osteoclasts specifically in the human bone implants or direct effects of this human-specific antibody on mouse bone remains to be established. Taken together, our data suggest that in addition to other mechanisms, IL1B stimulated by tumor cell–bone cell interactions promotes osteoclast activity driving tumor progression at this site. Hence, inhibiting IL1B reduces bone metastases by blocking this process.
In conclusion, our data show that IL1B is a novel biomarker that can be used to predict risk of breast cancer relapse. IL1B activates different prometastatic mechanisms at the primary site compared with the metastatic site: Endogenous production of IL1B by breast cancer cells promotes EMT, invasion, migration, and organ-specific homing. Once tumor cells arrive in the bone environment, contact between tumor cells and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. These high concentrations of IL1B caused proliferation of the bone metastatic niche and increased osteoclastic bone resorption stimulating growth of disseminated tumor cells into overt metastases (see Supplementary Fig. S7). These prometastatic processes were inhibited by administration of anti-IL1B treatments. Therefore, targeting IL1B with canakinumab or an IL1Ra may represent a novel therapeutic approach for patients with breast cancer at risk of progressing to metastasis by preventing seeding of new metastases from established tumors and retaining tumor cells already disseminated in the bone in a state of dormancy.
Disclosure of Potential Conflicts of Interest
W.M. Gregory reports receiving speakers bureau honoraria from Abbvie and is a consultant/advisory board member for Celgene and Janssen. R.E. Coleman is an employee of prIME Oncology; reports receiving speakers bureau honoraria from Eisai and Amgen; holds ownership interest (including patents) in Inbiomotion; is a consultant/advisory board member for Boehringer Ingelheim, Astellas, and Amgen; and reports other remuneration in the form of travel support from Amgen. J.E. Brown reports receiving commercial research grants from Amgen and Bayer; reports receiving speakers bureau honoraria from Amgen and Novartis; and is a consultant/advisory board member for Amgen, Novartis, Bayer, Takeda, Sandoz, Roche, Bristol-Myers Squibb, and MSD. I. Holen reports receiving speakers bureau honoraria from Amgen. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: J.M. Wilkinson, J.E. Brown, I. Holen, P.D. Ottewell
Development of methodology: D.V. Lefley, A.M. Hanby, J.M. Wilkinson, J.E. Brown, P.D. Ottewell
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Tulotta, D.V. Lefley, K. Freeman, J.M. Wilkinson, X. Liu, S.M.J. Bradbury, L. Hambley, R.E. Coleman, J.E. Brown, P.D. Ottewell
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Tulotta, D.V. Lefley, W.M. Gregory, P.R. Heath, F. Nutter, A.R. Spicer-Hadlington, X. Liu, S.M.J. Bradbury, L. Hambley, J.E. Brown, P.D. Ottewell
Writing, review, and/or revision of the manuscript: C. Tulotta, W.M. Gregory, A.M. Hanby, P.R. Heath, J.M. Wilkinson, X. Liu, S.M.J. Bradbury, L. Hambley, M. Kruithof de Julio, R.E. Coleman, J.E. Brown, I. Holen, P.D. Ottewell
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.V. Lefley, P.R. Heath, F. Nutter, A.R. Spicer-Hadlington, X. Liu, V. Cookson, G. Allocca, P.D. Ottewell
Study supervision: P.D. Ottewell
Acknowledgments
The authors would like to thank the ECMC for supporting retrieval of clinical samples from the AZURE trial; Breast Cancer Now for supporting the salary of Dr. Victoria Cookson; Weston Park Cancer Charity for supporting technical input from Mrs. Alyson Evans; and Yorkshire Cancer Research for purchase of the IVIS imaging machine. We would also like to thank Professor Sheila Francis, University of Sheffield (Sheffield, United Kingdom) and Dr. Emmanuel Pinteaux, University of Manchester (Manchester, United Kingdom) for supplying the IL1R1 knockout mice. This work was funded by research grants from the Medical Research Council (MR/P000096/1) and UK and Weston Park Cancer Charity (CA142).
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.
Footnotes
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
- Received July 11, 2018.
- Revision received November 20, 2018.
- Accepted January 17, 2019.
- Published first January 22, 2019.
- ©2019 American Association for Cancer Research.
References
Volume 25, Issue 9
