Abstract
Although chemotherapy can stimulate antitumor immunity by inducing interferon (IFN) response, the functional role of tumor-associated macrophages in this scenario remains unclear. Here, we found that IFN-activated proinflammatory macrophages after neoadjuvant chemotherapy enhanced antitumor immunity but promoted cancer chemoresistance. Mechanistically, IFN induced expression of cytoplasmic long noncoding RNA IFN-responsive nuclear factor-κB activator (IRENA) in macrophages, which triggered nuclear factor-κB signaling via dimerizing protein kinase R and subsequently increased production of protumor inflammatory cytokines. By constructing macrophage-conditional IRENA-knockout mice, we found that targeting IRENA in IFN-activated macrophages abrogated their protumor effects, while retaining their capacity to enhance antitumor immunity. Clinically, IRENA expression in post-chemotherapy macrophages was associated with poor patient survival. These findings indicate that lncRNA can determine the dichotomy of inflammatory cells on cancer progression and antitumor immunity and suggest that targeting IRENA is an effective therapeutic strategy to reversing tumor-promoting inflammation.
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Data availability
RNA microarray data generated for this study have been deposited in the NCBI GEO database and are accessible through GEO accession nos. GSE134599, GSE134600 and GSE134601. The sequence data generated have been deposited in NCBI’s Sequence Read Archive database and are accessible through accession nos. PRJNA555730 (RNA), PRJNA555733 (RNA) and PRJNA555732 (DNA). The PRIDE database accession nos. for proteomics data reported in this paper are PXD022673 and PXD022674. Source data for Figs. 1–5 and 7 and Extended Data Figs. 2–10 are provided in Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank all the healthy donors and patients who volunteered their blood or tissue samples for this study. This work was supported by grants from the National Key Research and Development Program of China (2016YFC1302300 and 2017YFA0106300), the Natural Science Foundation of China (81621004, 81720108029, 81930081, 91940305, 91942309, 81672614, 81902699, 81802645 and 81860546), Guangdong Science and Technology Department (2017B030314026, 2019A1515011485, 2020B1212030004 and 2020B1212060018), Clinical Innovation Research Program of Bioland Laboratory (2018GZR0201004), Guangzhou Science Technology and Innovation Commission (201803040015), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019BT02Y198), Tip-top Scientific and Technical Innovative Youth Talents of Guangdong special support program (no. 2016TQ03R553) and China Postdoctoral Science Foundation (2018M640868, BX20190396 and 2019M663270). The research is partly supported by the Fountain-Valley Life Sciences Fund of the University of Chinese Academy of Sciences Education Foundation.
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E.S., S.S. and J.L. conceived the ideas and designed the research. J.L., L.L., J.C., J.L., W.Z., X.Z., J.L., X.C., L.Y., Y.X., F.C., D.H. and X.Z. performed the experiments. W.W., C.G., S.H., Z.Y., Z.L., L.Y., J.L., X.L., Q.Z. and X.M. provided external clinical samples. J.L., L.S., M.Z. and M.L. participated in study design. J.L., L.L., J.C., Q.L. and S.S. analyzed the data. J.L., L.L. and J.C. drafted the paper and figures. S.S. and E.S. revised the paper and figures. All authors reviewed the paper.
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Peer review information Nature Cancer thanks George Calin, Luca Cassetta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Infiltrated macrophages are associated with chemoresistance and poor prognosis.
a, Representative immunofluorescent images of CD68 and CD8 staining in human breast cancer before chemotherapy (upper) and after chemotherapy (lower). b, Representative immunofluorescent images of CD163 and CD8 staining in human breast cancer before chemotherapy (upper) and after chemotherapy (lower). a & b, Number of CD68+/CD163+ macrophages (green) or CD8+ T cells (red) per field was quantified by ImageJ and showed in Fig. 1b, Extended Data Fig. 1c. Complete remission (CR) or partial remission (PR) was classified as chemosensitive, while stable disease (SD) or progressive disease (PD) was chemoresistant. Scale bar, 50 μm. c, The counts of CD68+ macrophages in clinical breast tumor samples of internal cohort before and after neoadjuvant chemotherapy correlate with therapeutic efficacy. mean ± s.e.m., n = 409 patients, statistical significance was determined by two-sided one-way ANOVA with Tukey test. d & e, Kaplan-Meier survival curves for breast cancer patients with low (< 12 cells per field) and high CD163+ macrophage (≥ 12 cells per field) infiltration in the tumor samples before or after chemotherapy of internal cohort (d, n = 409 patients) and external cohort (e, n = 316 patients). f & g, Kaplan-Meier curves for disease-free survival of breast cancer patients with low (blue line) and high (red line) CD163+ macrophage infiltration in the biopsies of breast tumors of different subtypes before chemotherapy (f) and after chemotherapy (g) of two cohorts (ER positive, n = 325 patients; HER2 positive, n = 234 patients; Triple negative, n = 166 patients). d-g, P values were calculated with the two-sided log-rank test. h, Graphs depict the correlation between the counts of CD68+ macrophages and CD8+ T cells in breast cancer samples of internal cohort before (left) and after chemotherapy (right), P values were determined by two-tailed Pearson correlation coefficient test. i, Representative flow plots of the purified macrophages and CD8+ CTLs from breast tumor, > 90% purity of purified cells were confirmed.
Extended Data Fig. 2 Macrophages polarized by chemotherapy promote therapeutic resistance of breast cancer but enhance anti-tumor immunity.
a, left: flow cytometry measured transduction efficiency of CD8+ T cells transduced with a lentiviral vector encoding NY-ESO-1-TCR; right: western blot measured transduction efficiency of MCF-7 cells transduced with a lentiviral vector encoding NY-ESO-1. CD8+ T cells were obtained from the peripheral blood of breast cancer patients. The experiments were performed twice with similar results each. b, Representative flow plots of the purified macrophages and CD8+ T cells from PBMCs, > 90% purity of purified cells were confirmed. c, Schematic of tumor-conditioned macrophages (Tc-Mϕ) induced in vitro. Human peripheral blood derived macrophages (PBDMs) were cultured alone or co-cultured with tumor cells pretreated without chemotherapeutics (Tc-Mϕ) or with chemotherapeutics (chemo-macrophages, chemo-Mϕ). ADM, adriamycin; DTX, docetaxel. d, Representative flow plots showing ADM induced apoptosis of tumor cells cultured alone or co-cultured with PBDMs, Tc-Mϕ or chemo-Mϕ. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with ADM treated tumor cells. e, Electron microscopy of MCF-7, SKBR3 and MDA-MB-231 cells treated with ADM co-cultured with PBDMs, Tc-Mϕ or chemo-Mϕ. The experiment was performed twice with similar results. Scale bar, 5 μm. f, ADM (2 μg/ml) affected cell cycle arrest. mean ± s.e.m., n = 3 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with untreated tumor cells; ns, not significant by two-sided one-way ANOVA with Tukey test compared with ADM treated tumor cells. g, The expression of perforin (upper) and granzyme B (lower) in CD8+ T cells cultured alone or co-cultured with PBDMs, Tc-Mϕ or chemo-Mϕ. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test. h, Chemotaxis of CD8+ CTLs cultured alone or co-culture with PBDMs, Tc-Mϕ or chemo-Mϕ 16 hr after seeding in Boyden Chambers. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test.
Extended Data Fig. 3 Macrophages exhibit proinflammatory phenotype after neoadjuvant chemotherapy.
a, Schematic of animal experiments showing treatment of chemotherapeutics. b, Representative images of CD8 staining in the tumors of PyMT;C3 or PyMT;Csf1op mice after ADM treatment. The experiment was performed three times with similar results. Scale bar, 400 μm. c, Representative heatmaps for the differentially expressed genes (fold change > 3) in the macrophages isolated from paired breast tumor samples of four patients before and after neoadjuvant chemotherapy. The expression levels were shown in log2-transformed values. d, GSEA analysis of mRNA profiles revealed enrichment of Jak1-STAT1 and NF-κB target genes in the macrophages isolated from breast cancer samples obtained after chemotherapy and enrichment of IL-4-STAT6 target genes before chemotherapy. e, Quantitative RT-PCR for cytokine mRNA expression in the macrophages obtained before or after chemotherapy. mean ± s.e.m., n = 5 patients, statistical significance was determined by two-tailed Student’s t test. f, Representative images of IFNα and IFNβ staining (green) in the CD163+ macrophages (red) and CK+ tumor cells (white) of the clinical samples from breast cancer patients before and after neo-adjuvant chemotherapy. The experiment was performed three times with similar results. Scale bar, 200 μm. g, ELISA for IFNα produced by primary tumor cells and macrophages isolated from PyMT mice with or without ADM treatment. mean ± s.e.m., n = 3 independent experiments, statistical significance was determined by two-tailed Student’s t test compared with PBS treated group. h, ELISA for the cytokines IL-6, CXCL15, TNFα, IL-15, CXCL9 and CXCL10 produced by macrophages isolated from breast tumors in MMTV-PyMT mice treated with or without chemotherapy in the presence of IgG or IFNAR1 neutralizing antibody. mean ± s.e.m., n = 8 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with ADM and IgG treated mice.
Extended Data Fig. 4 Macrophages polarized by chemotherapy promote anti-tumor immunity via Jak1-STAT1 signaling.
a, ELISA for cytokines released by macrophages obtained following chemotherapy treated with or without Jak-STAT1 inhibitors (Ruxolitinib or Fludarabine) or NF-κB inhibitors (SC3060 or JSH23). mean ± s.e.m., n = 4 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with PBS treated macrophages. b, IC50 for the ADM-treated MCF-7 (upper) and E0771 (lower) co-cultured with Tc-Mϕ or chemo-Mϕ in the presence or absence of IL-15, CXCL9, CXCL10 or IL-6 neutralizing antibody. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with MCF-7 or E0771 cells co-cultured with untreated (-) chemo-Mϕ. c, Chemotactic assays for the CD8+ CTLs cultured alone or co-cultured with chemo-Mϕ in the presence or absence of CXCL9, CXCL10 or IL-6 neutralizing antibody. Scale bar, 200 μm. Quantitative data are shown as mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with CD8+ CTLs co-cultured with untreated (-) chemo-Mϕ. d, IC50 for the ADM treated (left) and DTX treated (right) MCF-7 cells co-cultured with PBDMs, Tc-Mϕ or chemo-Mϕ in the presence or absence of indicated inhibitors. SC3060 and JSH23, NF-κB inhibitors. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with MCF-7 cells co-cultured with untreated (-) chemo-Mϕ.
Extended Data Fig. 5 IRENA is a conserved lncRNA between humans and mice.
a, qRT-PCR for IRENA expression in macrophages obtained from breast cancer patients before or after chemotherapy. mean ± s.e.m., n = 5 different patients, statistical significance was determined by two-tailed Student’s t test. b, qRT-PCR for IRENA expression in macrophages obtained from PyMT mice treated by ADM or DTX. mean ± s.e.m., n = 8 independent experiments, statistical significance was determined by two-tailed Student’s t test compared with PBS treated group respectively. c, Diagram for the genomic location of human IRENA (left) and mouse IRENA (right). d & e, Genome browser (UCSC, http://genome.ucsc.edu/) depiction of IRENA and its conserved analogs in human (d) and mouse (e), IRENA is annotated as ENST00000623256 in the human assembly and ENSMUST00000136998 in the mouse assembly. f, Assessment of the protein-coding potential of IRENA. The experiment was performed twice with similar results. g & h, The best match of predicted short peptides and mass spectrometer detected peptides of human (g) and mouse (h). i, qRT-PCR showing expression of IRENA in different tissues or cells in MMTV-PyMT mice with or without chemotherapy treatment. mean ± s.e.m., n = 3 independent experiments. j, Northern blot for IRENA expression in the cytoplasm and nucleus of macrophages treated with IFNα. The experiment was performed twice with similar results. k, qRT-PCR for mouse IRENA expression in the macrophages obtained from mouse treated with IFNα or IFNβ in the presence or absence of IFNAR1-neutralizing antibody. mean ± s.e.m., n = 3 independent experiments, statistical significance was determined by two-tailed Student’s t test compared with the IgG group respectively. l, qRT-PCR showing estimated copy numbers of IRENA per macrophage in human and mouse. Equivalent molecules per cell were calculated based on the assumption that total RNA per macrophage is 10 pg.
Extended Data Fig. 6 Type I interferon enhances IRENA expression in macrophages via Jak1-STAT1-ISGF3 signal.
a, qRT-PCR for IRENA expression in macrophages treated with or without Jak-STAT1 inhibitors (Ruxolitinib or Fludarabine) or NF-κB inhibitors (SC3060 or JSH23). mean ± s.e.m., n = 3 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with IFNα or IFNβ treated macrophages without DMSO/inhibitors respectively. b, Luciferase reporter assays for the transcription activities of IRENA promoter region (−1881 to +83 nt relative to TSS of human IRENA, −1787 to +126 nt relative to TSS of mouse IRENA) or its deleting variants cloned upstream of the firefly luciferase coding region. Data were normalized to renilla luciferase and presented with respect to control vector (Vec) set to a value of 1. mean ± s.e.m., n = 3 independent experiments; statistical significance was determined by two-tailed Student’s t test. c, Diagram for the ISRE motif at the human and mouse IRENA promoter region. d, shRNA mediated knockdown efficiency of STAT1, STAT2, IRF9 or STAT3 in THP-1. The experiment was performed twice with similar results. e, Expression of IRENA in IFNα activated THP-1 cells treated by STAT1, STAT2, IRF9 and STAT3 shRNA. mean ± s.e.m., n = 4 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with the IFNα treated THP-1 cells. f & g, ChIP analysis for the binding of STAT1, STAT2 and IRF9 to IRENA promoter in macrophages treated with IFNα. mean ± s.e.m., n = 3 independent experiments, statistical significance was determined by two-tailed Student’s t test compared with the PBS treated cells.
Extended Data Fig. 7 IRENA promotes NF-κB activation but not Jak1-STAT1 activation in macrophages.
a, Efficiency of IRENA knockdown. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with untreated (-) chemo-Mϕ. b, Chemotactic assays for CD8+ T cells cultured alone or co-cultured with chemo-Mϕ with or without IRENA knockdown. Scale bar, 200 μm. Quantitative data are shown as mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with CD8+ CTLs co-cultured with untreated (-) chemo-Mϕ. c, Flow cytometry for perforin (upper) and granzyme B (lower) levels in the human CD8+ T cells co-cultured in presence of chemo-Mϕ with or without IRENA knockdown. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with untreated (-) chemo-Mϕ group. d, Tumoricidal effects of ESO CTLs affected by macrophages. CMFDA+PI− cells were designated as surviving tumor cells. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with ESO CTLs co-cultured with untreated (-) chemo-Mϕ. e, Upper, a representative western blot for total and phosphorylated Jak1, STAT1, IKK and IκBα in control or IFNα treated PBDMs with or without IRENA knockdown. Lower, relative phosphorylated IKK and IκBα protein levels quantified from n= 3 independent experiments using ImageJ. Protein levels were normalized using GAPDH as the loading control. mean ± s.e.m., statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with IFNα treated PBDMs. f, GSEA analysis revealed enrichment of NF-κB target genes in the PBDMs overexpressed IRENA compared with control group (vector).
Extended Data Fig. 8 PKR is bound by IRENA.
a & b, Mass spectrometry analysis for the peptides of IRENA-interacting PKR. c, Western blot for the total and phosphorylated PKR of PKR/IRENA two-overexpressed macrophages or control macrophages. The experiment was performed twice with similar results. d, RNA pulldown assay for the in vitro interaction of sequentially deleted mouse IRENA variants with mouse PKR, n = 3 independent experiments. Upper, sequentially deleted mouse IRENA variants; lower, western blot of mouse PKR pulled by mouse IRENA variants. e, Predicted secondary structure of mouse IRENA817–1032 truncation by Mfold. f, Upper, western blot for the total mouse PKR (mPKR) pulled down by indicated mutant mouse IRENA fragments. (mIRENA, full-length mouse IRENA; mIR817–1032: IRENA truncation mutant containing nt 817–1032; mIR817–1032mA: mutations of hairpin A of nt 817–1032; mIR817–1032mB: mutations of hairpin B of nt 817–1032; mIR817–1032mA+B: mutations of hairpin A and hairpin B of nt 817–1032. A representative blot from three independent experiments is shown. Lower, relative pulled PKR protein levels quantified using ImageJ. Protein levels were normalized using PKR of input. Results are shown as the mean ± s.e.m. g, Bio-layer interferometry assays for the in vitro interaction of indicated mutant mouse IRENA fragments with mouse PKR, n = 3 independent experiments. h, qRT-PCR for the IRENA undergone RNase protection assay. IRENA was incubated with PKR or IgG and subjected to RNase T1 digestion prior to qRT-PCR examination. The fold enrichment of IRENA-PKR/IgG is shown for every 10 nt region ranging from nt 465 to 895 of human IRENA. mean ± s.e.m., n = 3 independent experiments.
Extended Data Fig. 9 Enhanced anti-tumor immunity sustains in IRENA conditional knockout mice post-chemotherapy.
a, Orientation of IRENA and neighbor gene transcription (upper: human; lower: mouse). b & c, Schematic overviews for the strategy to generate an IRENAloxp/loxp allele (b) and macrophage-specific knockout by Csf1r-cre homologous recombination (c), PyMT;KO, PyMT;KO;Tg mice were generated by cross-fertilizing. d, Expression of Nkx2-2, IRENA and Pax1 in macrophages of IRENA knockout mice or wild type mice. e, IRENA expression in PyMT;Csf1r-cre;IRENAloxp/loxp (KO), PyMT;Csf1r-cre;IRENAloxp/loxp;IRENATg (Tg) mice or background PyMT (WT) mice treated with ADM or PBS. d & e, mean ± s.e.m., n = 5 independent experiments, nd, not detected, statistical significance was determined by two-tailed Student’s t test. f, Schematic overview for the strategy to generate mice with transgenic overexpression of IRENA from the ROSA26 locus. g, Representative images of TUNEL+ (green) apoptotic tumor cells in the tumor sections of mice with or without chemotherapy. The experiment was performed twice with similar results. Scale bar, 50 μm. h, Representative flow plots of the purified CD8+ CTLs > 90% purity of the populations from tumors of PyMT mice. i, Chemotactic assays for the mouse CD8+ T cells co-cultured with macrophages purified from the tumors of PyMT;KO, PyMT;KO;Tg and PyMT mice treated with or without chemotherapy. Scale bar, 200 μm. Quantitative data are shown as mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test. j-n, Representative flow plots for perforin (j), granzyme B (k), CD38 (l), CD69 (m) and IFNγ (n) levels of CD8+ T cells purified from the tumors of PyMT;KO, PyMT;KO;Tg and PyMT mice treated with or without chemotherapy. mean ± s.e.m., n = 5 independent experiments, statistical significance was determined by two-sided one-way ANOVA with Tukey test compared with ADM treated PyMT mice. o, Representative images of F4/80 staining (red) in the tumors of PyMT;C3 or PyMT;Csf1op mice with or without macrophage transfer. The experiment was performed twice with similar results. Scale bar, 400 μm.
Extended Data Fig. 10 High IRENA expression in macrophages is associated with poor clinical outcomes in breast cancer patients.
a, Correlation between RNA expression of IRENA and cytokines IL-6, IL-8, TNFα, IL-15, CXCL9 and CXCL10 in macrophages in the breast tumor samples obtained following chemotherapy, P values were determined by two-tailed Pearson correlation coefficient test, n = 26 patients. b, Representative images of FISH for IRENA and immunofluorescent staining for CD68 in the paraffin-embedded tumor sections of chemotherapy-sensitive or resistant breast cancer patients (IRENA, green; CD68, red). Scale bar, 50 μm. c, Number of IRENA+ macrophages per field correlated with therapeutic efficacy of internal cohort. mean ± s.e.m., statistical significance was determined by two-sided one-way ANOVA with Tukey test. d, Kaplan-Meier survival curves for breast cancer patients of two cohorts with low and high IRENA+ macrophage infiltration in three different subtypes. e, Kaplan-Meier survival curves for breast cancer patients with low and high IRENA+ macrophage infiltration in three grades (grade 1, grade 2 and grade 3). f, Kaplan-Meier survival curves for breast cancer patients with low and high IRENA+ macrophage infiltration in three stages (stage I, stage II and stage III). g, Kaplan-Meier survival curves for breast cancer patients with low and high IRENA+ macrophage infiltration stratified by Ki67 status (Ki67 < 15%, Ki67 ≥ 15%). d-g, P values were calculated with the two-sided log-rank test.
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Liu, J., Lao, L., Chen, J. et al. The IRENA lncRNA converts chemotherapy-polarized tumor-suppressing macrophages to tumor-promoting phenotypes in breast cancer. Nat Cancer 2, 457–473 (2021). https://doi.org/10.1038/s43018-021-00196-7
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DOI: https://doi.org/10.1038/s43018-021-00196-7
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