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Clinical Trial
. 2018 Sep 15;78(18):5243-5258.
doi: 10.1158/0008-5472.CAN-18-0413. Epub 2018 Jul 16.

IL1 Receptor Antagonist Controls Transcriptional Signature of Inflammation in Patients With Metastatic Breast Cancer

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Free PMC article
Clinical Trial

IL1 Receptor Antagonist Controls Transcriptional Signature of Inflammation in Patients With Metastatic Breast Cancer

Te-Chia Wu et al. Cancer Res. .
Free PMC article

Abstract

Inflammation affects tumor immune surveillance and resistance to therapy. Here, we show that production of IL1β in primary breast cancer tumors is linked with advanced disease and originates from tumor-infiltrating CD11c+ myeloid cells. IL1β production is triggered by cancer cell membrane-derived TGFβ. Neutralizing TGFβ or IL1 receptor prevents breast cancer progression in humanized mouse model. Patients with metastatic HER2- breast cancer display a transcriptional signature of inflammation in the blood leukocytes, which is attenuated after IL1 blockade. When present in primary breast cancer tumors, this signature discriminates patients with poor clinical outcomes in two independent public datasets (TCGA and METABRIC).Significance: IL1β orchestrates tumor-promoting inflammation in breast cancer and can be targeted in patients using an IL1 receptor antagonist. Cancer Res; 78(18); 5243-58. ©2018 AACRSee related commentary by Dinarello, p. 5200.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

K. Palucka is a consultant/advisory board member for Swedish Orphan Biovitrum AB. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.
In situ IL1β expression in breast cancer tumors. A, IL1β in supernatants of breast cancer tumor fragments (T) and in macroscopically noninvolved surrounding tissue (ST); mean and median secretion of indicated cytokine in pg/mL. Wilcoxon sign-rank test. B, ILiβ concentrations plotted in relation to histopathologic tumor stage. N, number of tissue samples from different patients with indicated disease stage. Welch t test. C, Top, whole-section scan of representative breast cancer tumor (ER+PR+HER2). CD11c (green), IL1β (red), cytokeratin (blue) expression. Bottom, zoomed area illustrating coexpression of individual markers as indicated. D, Quantitative histocytometry see (Materials and Methods) analysis of immunofluorescence staining on breast cancer tissue. The nonparametric Wilcoxontest. E, RNAtranscripts visualized in breastcancertumorsectionswith QuantiGeneViewRNA ISH tissueassaykit. Human PTPRC(CD45) and human IL1B are green and red, respectively.
Figure 2.
Figure 2.
IL1β induces TSLP production from breast cancer cells. A, IL1β, TSLP, IFNγ, and IL13 in supernatants of breast cancer samples. The logarithm of four cytokine measurements (IL1β, TSLP, IL13, IFNγ: left to right at x-axis and top to bottom at y-axis) was taken for total 145 patients and the dependency between any pair of cytokine measurements was tested under the null hypothesis of independence among the four measurements. Log-likelihood and its Bartlett correction were performed and P values for both methods were significantly low (P < 0.001). Histogram plots indicated distribution of the cytokines expression among all patients. Pairwise correlations were tested among 6 pairs of cytokine measurements. Scatter plots indicated the correlation pairwise. Using Pearson and Spearman correlation, all P values were found significant (P < 0.025). B, Heatmap generated on the basis of the log-transformed four cytokine measurements using R package “pheatmap” with “ward.D2” method. Patients were split into four groups based on the “ward.D2” clustering method. The levels of cytokines were color coded as indicated. C, RNA transcripts visualized in breast cancer tumor sections with QuantiGene ViewRNA ISH. Human KRT8 and human TSLP are green and red, respectively. D, MDA-MB-231 cells were treated with medium alone, 10 ng/mL of IL1β, IL1α, TNFα, or IL6 for the indicated time course. TSLP mRNA level by quantitative real-time PCR normalized to internal control GAPDH. Bars show the mean ± SEM for triplicate wells from a representative experiment. *, P < 0.05; **, P < 0.0i; ***, P < 0.000i. E, ChIP by using anti-RNA polymerase II was performed. ChIP-qPCR analysis on lfTSLP (black) and sfTSLP (white) genes from MDA-MB-231 cells with IL1β or IL1β blocking for 1 hour. Percentage of input summarized from three experiments.`
Figure 3.
Figure 3.
IL1β production in DCs is caspase-1 and contact dependent. A, Scatter plots with line of best fit and Spearman correlation (r) of IL1B with NLRP3, NLRC4, caspase-1 (CASP1), caspase-8 (CASP8), and caspase-11 (SFRS2IP) expression in TCGA dataset (870 patients). B and C, MDA-MB-231 breast cancer cells and cDCs were cocultured in chamber wells for 18 hours, in the presence of caspase-1 inhibitor or DMSO. The percentage of HLA-DR+ cells showing expression of pro-IL1β (B) or that of mature IL1β (C). D, MDA-MB-231 (MDA) or Hs578T (HS) breast cancer cells cocultured with blood monocytes (Mono), MDDCs, cDCs, or monocyte-derived macrophages (macroph.) in regular tissue culture wells (Contact) or Transwell to separate two types of cells in culture for 48 hours. IL1β levels in supernatants by Luminex. Values are plotted as mean ± SEM from triplicate experiments. Welch t test was used. n.s., not significant.
Figure 4.
Figure 4.
IL1β production in cDCs and monocytes is triggered by TGFβ. A, cDCs were cocultured with MDA-MB-231 breastcancercellsorHs578T breast cancer cells for 16 hours. Intracellular IL1β expression in gated viable cells by FACS. B, Summary of the percentage of IL1β+ cDCs. Each dot represents one experiment. C, Surface expression of activated TGFβ1 by flow cytometry in breast cancer cell lines and nonmalignant cells. D, MDA-MB-231 cells were cocultured with DCs for 48 hours, in presence of different doses of TGFβR kinase inhibitor or anti-TGFβ neutralizing antibody, DMSO, or isotype control, respectively. Histograms of IL1B transcription levels analyzed by quantitative RT-PCR, normalized to GAPDH. Bars show the mean ± SEM for triplicate wells from a representative experiment. Kruskall- Wallis test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. n.s., not significant. E, IL1β level in supernatants by Luminex. Values are plotted as mean ± SEM (independent t test was used). F, Same conditions as in E, but over a 16-hour culture period and with the readout being fraction of total DCs being IL1β+. Intracellular staining of culture with anti-IL1β antibody by flow cytometry. Each dot represents one experiment. G, MDA-MB-231 cells were cocultured with cDCs for 48 hours in presence of different doses of TAK1 inhibitor or DMSO. IL1β levels in the supernatants after 48 hours of coculture by Luminex. Each dot represents one experiment. H, cDCs were cocultured with MDA-MB-231 cells in presence or absence of anti-TGFβ neutralizing antibody plus TGFβR kinase inhibitor (TGFβ blocking) for 60 minutes; pTAK1 was detected by specific staining and analyzed on FACS.
Figure 5.
Figure 5.
IL1 and TGFβ mediate tumor-promoting type 2 cytokines in humanized mouse model. Hs578T breast tumor-bearing NOD/SCIDβ2–/– mice were reconstituted with MDDCs and autologous T cells. Mice were treated with either: (i) anti-TGFβ neutralizing antibody on days 3, 6, and 9; (ii) anakinra daily starting on day 3; or (iii) controls such as isotype and PBS. A, Scheme of experimental mouse model. B, Kinetics of tumor growth from multiple experiments. Number of mice in each group is indicated. C, Breast tumor fragments were harvested at day 16 after tumor implantation and stimulated for 16 hours with PMA and ionomycin. Cytokines were measured by Luminex. D, Cell suspensions stained for IL13 and IFNγ expression by FACS. Representative plots from three different mice. E, TSLP concentration by Luminex in tumor only versus PBS control versus anakinra group. F, IL1β concentration by Luminex in isotype control versus anti-TGFb neutralizing antibody group.
Figure 6.
Figure 6.
Anakinra modulates transcriptional signature in the blood. A, Hierarchical clustering of the 288 differentially expressed genes identified using mixed model analysis, between healthy controls (HC) and patients with breast cancer at any time point during the study. The expression of genes was averaged from all patients in each time point. B, patient baseline; RI, patient run-in (two weeks after anakinra only); EOM, end of month after anakinra plus chemotherapy. B, Box plots representing the log2-transformed expression of a subset of genes. Horizontal lines, median. Boxes represent the first and third quartiles (25th and 75th percentile). Whiskers extend to the highest or lowest values within 1.5-fold of the interquartile range. C, Genes differentially expressed between any time point posttreatment and patient baseline (B; P <> 0.05).
Figure 7.
Figure 7.
Basal subtype of breast cancer is linked with high IL1β and anakinra-dependent signature. A, IL1B transcript expression in different breast cancer subtypes annotated in the METABRIC database. N, number of samples per subtype. Kruskal–Wallis test was P < 0.001. B, The gene sets controlled by anakinra treatment in the blood were used in unsupervised analyses of transcriptional datasets from invasive BrCa tumors in the METABRIC datasets. Two clusters were formed on the basis of gene expression and assigned as red and black clusters. C, IL1B transcription in two clusters in both datasets. D, Number of patients in each cluster among different subtypes of breast tumors.

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