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Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity

Abstract

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

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Fig. 1: The TME promotes lipid accumulation in pro-tumorigenic TAMs.
Fig. 2: The TME activates IRE1/sXBP1 in pro-tumorigenic TAMs.
Fig. 3: Tumor cells drive pro-tumorigenic polarization in BMDMs via IRE1.
Fig. 4: Deletion of XBP1 in TAMs suppresses tumor growth.
Fig. 5: Activation of IRE1–STAT3 signal supports CM-induced polarization.
Fig. 6: Mincle-dependent glucosylceramide sensing pathway tailors macrophage activation.
Fig. 7: CM causes reshuffling of lipid composition and saturation of ER membrane.
Fig. 8: LXR agonist reduces tumor burden and hampers TAM survival in a LPCAT3-dependent manner.

Data availability

RNA-seq results are available in the Gene Expression Omnibus database under accession code (GSE166735). Other data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank L. Glimcher and G. Hotamisligil for providing XBP1flox/flox mice and D. Sancho for providing Mincle-deficient bone marrows. We thank T. Shimizu and J.-i. Miyazaki for providing plasmid LPCAT3-pCNX2. P.-C.H. is funded by the Swiss Institute for Experiment Cancer Research (ISREC 26075483), European Research Council Staring Grant (802773-MitoGuide), SNSF project grants (31003A_163204 and 31003A_182470), UNIL Interdisciplinary Grant, the Cancer Research Institute (CLIP investigator award and Lloyd J. Old STAR award) and Ludwig Cancer Research. J.W.L. is supported by NIH R01CA1932556. P.P. is supported by the New York University Abu Dhabi Research Enhancement Fund Swedish Research Council (Vetenskapsrådet), Swedish Cancer Society (Cancerfonden). D.M. is supported by French government grants managed by the French National Research Agency under the references ANR-11-LABX-0021, ANR-15-IDEX-0003 and ANR-19-CE14-0020. S.C.-C.H. is supported by Cancer Research Institute CLIP Investigator Award, the VeloSano Pilot Award, the Case Comprehensive Cancer Center American Cancer Society Pilot Grants and the Cleveland Digestive Diseases Research Core Center Pilot Grant (IRG91-022-19, IRG-16-186-21, 1P30DK097948). L.N.R. is supported by Immunology T32 Training Program (AI089474). J.I. is supported by SNSF R’Equip 316030_183377. F.M. is supported by Swiss Cancer league KFS-4230-08-2017.

Author information

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Authors

Contributions

G.D.C. and P.-C.H. designed the research. G.D.C, Y.-R.Y. and C.-H.T. performed in vivo experiments. G.D.C., Y.-R.Y. and X.L. performed in vitro experiments. H.G.-A. and J.I. performed lipidomic experiments and analysis. F.F. performed electron microscopy analyses. L.Z., M.F. and L.N.R. performed western blots. X.X. and P.P. performed computational analysis of RNA sequencing. A.J. and D.M. provided donor mice and bone marrow for bone marrow transplantation experiments. Z.X. and J.W.L. performed computational analysis of single-cell RNA sequencing of human and murine tumor cohorts. S.C.-C.H. and F.M. provided feedback and advice. G.D.C. and P.-C.H. wrote the manuscript.

Corresponding author

Correspondence to Ping-Chih Ho.

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Competing interests

P.-C.H. is scientific advisory for Elixiron Immunotherapeutics, Acepodia and Novartis. P.-C.H. also receives research support from Roche and Elixiron. J.W.L. is a paid advisor to Restoration Foodworks. The remaining authors declare no competing interests.

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Peer review information: Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 TAMs display high lipid content and ER stress responses in the inducible Braf/Pten melanoma model.

a, Representative histogram (left) and quantitative plot (right) of BODIPY C12 staining in splenic macrophages (n = 10) or tumor-associated macrophages (TAMs) (n = 11) from YUMM1.7 melanoma bearing mice. Data are pooled from two independent experiment. bh, Analysis of Braf/Pten tumor-bearing mice seven weeks after tamoxifen administration. b, c, Representative histogram (left) and quantitative plot (right) of BODIPY staining (b) and BODIPY C12 staining (c) in splenic macrophages or TAMs isolated from Braf/Pten tumor-bearing mice (n = 5 per group). d, Representative histogram (left) and quantitative plots (right) of BODIPY staining in TAMs gated based on ARG1 expression (n = 10 per group). e, qPCR analysis of mRNA expression of the indicated genes in splenic macrophages and TAMs isolated from Braf/Pten tumor-bearing mice (n = 4 per group). f, Representative histogram (left) and quantitative plot of the abundance (right) of sXBP1+ subset among splenic macrophages and TAMs in Braf/Pten tumor-bearing mice (n = 10 per group). g, Representative histogram (left) and quantitative plot (right) of BODIPY staining in TAMs gated based on sXBP1 expression (n = 10 per group). h, Representative histogram (left) and quantitative plot of the abundance (right) of sXBP1+ cells among ARG1+ and ARG1 TAMs (n = 10 per group). i, Gating strategy applied to define ARG1 + macrophages from spleen and tumor of YUMM1.7 tumor-bearing mice. jl, Representative histogram (j) and quantitative plot of BODIPY staining (k) or sXBP1 (l) in skin-resident macrophages or TAMs from Braf/Pten tumor-bearing mice (n = 10 per group). m, Representative histogram (left) and quantitative plot (right) of pPERK staining in splenic macrophages or tumor-associated macrophages (TAMs) (n = 9 per group) of YUMM1.7 melanoma bearing mice. Data are pooled from two independent experiments. n, qRT-PCR of the indicated genes from sorted splenic macrophages and TAMs (n = 8 per group) isolated from YUMM1.7 tumor-bearing mice. Data are representative of two independent experiments (b, c, e). Data are pooled from two independent experiment (a, d, fh, jn). Each symbol represents one individual. All data are mean ± s.e.m and were analyzed by two-tailed, unpaired Student’s t-test or paired t-test (d, g, h).

Source data

Extended Data Fig. 2 Tumor cells reinforce protumorigenic polarization in macrophages via an IL-4/IL-13 independent manner.

a, Representative histogram (left) and quantitative plot (right) of BODIPY FL C12 staining in BMDM cultured in DMEM (Ctrl) or YUMM1.7 CM (n = 4 per group). Data are representative of two independent experiments. b, c, Representative histogram (left) and quantitative plot (right) of ARG1 (b) and MRC1 (c) expression in BMDMs cultured in DMEM or CM (n = 3 per group). Data are representative of three independent experiments. d, qPCR analysis of mRNA expression of the indicated genes in BMDMs cultured in DMEM or CM for 18 h (n = 3 per group). Data are representative of three independent experiments. e, Proliferation of CFSE-labeled T cells activated with anti-CD3 and anti-CD28 alone or in co-culture with BMDM Naïve or previously exposed to CM in a ratio 2:1 (n = 6). Data are pooled of two independent experiments. f, qPCR analysis of Arg1 mRNA expression in BMDMs treated with IL-4 and IL-13 (10 ng/ml) in the absence or presence of 0.25μg/ml anti-IL-4 and 0.25μg/ml anti-IL-13 neutralizing antibodies for 18 h (n = 3 per group). g, qPCR analysis of mRNA expression of indicated genes in BMDMs treated with CM in the absence or presence of 0.25μg/ml anti-IL-4 and 0.25μg/ml anti-IL-13 neutralizing antibody for 18 h (n = 3 per group). Data are representative results of three independent experiments. h, Multiplex cytokine array was used to determine the concentration of IL-13 (left) and IL-4 (right) in CM from YUMM1.7 and MEF cells. Stand0 to Stand7 show the increased concentration detected by the standard provided by the kit (n = 4). Data are representative of two independent experiments. ik, Representative histogram (left) and quantitative plot (right) of BODIPY staining (i), and protein expression of ARG1 (j), and MRC1 (k) in BMDMs stimulated with regular culture medium (Ctrl) or CM from YUMM1.7 (CM) or MEF (CM MEF) (n = 3 per group). Data are representative results of three independent experiments. l, qPCR analysis of indicated genes in BMDMs exposed to CM or Tunicamycin for 18 h (n = 3 per group). Data are representative results of three independent experiments. m, Quantification of BODIPY staining in BMDMs treated with CM in the absence or presence of 50μM STF083010; Ctrl (n = 7), CM (n = 7), CM-STF (n = 6). Data are pooled from three independent experiments. n, Immunoblots of indicated proteins in BMDM transduced with retrovirus expressing scramble or IRE1-targeting gRNAs. o, qPCR analysis of BIP and sXBP1 mRNA expression in BMDMs treated with 1μγ/ml of Tunicamycin, 1μM thapsigargin and CM for 16 h (n = 3 per group). Data are representative results of three independent experiments. Data are mean ± s.e.m. were analyzed by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey’s multiple comparison test (e).

Source data

Extended Data Fig. 3 XBP1 supports protumorigenic polarization in response to cancer-derived stimuli.

a, Immunoblots of indicated proteins in control or XBP1-deficient BMDMs stimulated with or without 1μM thapsigargin for 6 h. Data are representative results of two independent experiments. b, Quantification of BODIPY staining in BMDMs generated from WT (XBP1wt) or KO (XBP1cKO) mice cultured stimulated with regular culture medium (Ctrl) or YUMM1.7 CM (n = 12 per group). Data are pooled from three independent experiments. c, Percentages of TAMs (F4/80+ CD11b+ Gr1-) among CD45+ cells in melanomas from tumor-bearing XBP1wt (n = 5) and XBP1cKO mice (n = 6). Data are representative results of three independent experiments. d, e, Tumor growth (d) and tumor weight (e) of YUMM1.7-OVA melanoma from WT and XBP1cKO mice treated with PBS or with anti-CSF1R as indicated in the methods; XBP1wt PBS (n = 13), XBP1cKO PBS mice (n = 12), XBP1wt αCSF1R (n = 12), XBP1cKO αCSF1R (n = 13). Data are pooled from three independent experiments. f, percentages of TAMs (F4/80+ cells gated on CD11b + Gr1-) among CD45 + cells in the experiment showed in d, e (n = 8 per group for XBP1wt PBS, XBP1cKO PBS, XBP1wt αCSF1R; n = 9 for XBP1cKO αCSF1R). Data are pooled from two independent experiments. g, Representative plots of iTAMs and mTAMs populations in tumor and spleen of YUMM1.7 tumor-bearing. hk, Representative histograms (up) and quantitative plots (down) of PDL1 (h), MHCII (i), sXBP1 (j) and ARG1 (k) expression in iTAMs and mTAMs from YUMM1.7 tumor-bearing mice (n = 6). Data are pooled from two independent experiments. l, Representative plots of iTAMs and mTAMs populations in tumor and spleen of Braf/Pten melanoma-bearing mice. mp, Representative histograms (up) and quantitative plots (down) of PDL1 (m), MHCII (n), sXBP1 (o) and ARG1 (p) expression in iTAMs and mTAMs from Braf/Pten melanoma-bearing mice (n = 5). Data are representative of two independent experiments. Each symbol represents one individual. qt, Tumor growth (q) and tumor weight (r) of B16-OVA melanoma and tumor growth (s) and tumor weight (t) of MC38-OVA colon adenocarcinoma in XBP1wt (n = 9 for B16-OVA and n = 8 for MC38-OVA) or XBP1cKO (n = 10 for B16-OVA and n = 7 for MC38-OVA) mice. Data are pooled from two independent experiments. Data are mean ± s.e.m. were analyzed by two-tailed, unpaired Student’s t-test.

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Extended Data Fig. 4 STAT3 is required for CM-induced protumorigenic polarization.

a, Immunoblots of indicated proteins in BMDMs expressing scramble or STAT3-targeting gRNAs treated with 10 ng/ml IL-6 for 6 h. Data are representative results of two independent experiments. b, c, Immunoblots of indicated proteins (b) and qPCR analysis of mRNA expression of indicated genes (c) in BMDMs treated with CM in the presence of vehicle (CM) or 10μM Stattic (CM + Stattic) (n = 6 for Ctrl and n = 5 for CM and CM + Stattic). Data are representative results of three independent experiments. d, Multiplex cytokine array was used to determine the concentration of IL-10 (left) and IL-6 (right) in CM from YUMM1.7 and MEF cells. Stand0 to Stand7 show the increased concentration detected by the standard provided by the kit (n = 4). Data are representative results of two independent experiments. e, Immunoblot of indicated proteins in BMDMs treated with control media (Ctrl), cancer cell conditioned media (CM) or CM plus 50μM STF081030 for 18 h. Data are representative of two independent experiments. f, Immunoblot of BMDMs treated with control vehicle (Ctrl), cancer cell conditioned media (CM) or tunicamycin (1μg/ml; Tuni.) for the indicated time points. Data are representative of two independent experiments. All data are mean ± s.e.m and were analyzed by two-tailed, unpaired Student’s t-test.

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Extended Data Fig. 5 β-glucosylceramide, rather than cholesterol, is released by tumor cells to mediate the protumorigenic polarization in macrophages.

a, b, Quantification results of Cholesterol (a) (n = 6 per group) and fatty acids (b) (n = 4 per group) on YUMM1.7 CM prior (Yumm1.7) and after treatment with lipid removal reagent (Y1.7 w.o. Lipids). Data are pooled from two independent experiments. c, Immunoblot of YUMM1.7 cells stably transduced with lentivirus expressing short hairpin RNA against scramble or HMGCR sequence. d, qPCR analysis of indicated genes in BMDMs exposed to CM isolated from YUMM1.7 shCTRL or from YUMM1.7 shHMGCR (n = 3). Data are representative of three independent experiments. e, qPCR analysis of mRNA expression of indicated genes in BMDMs treated with CM in the absence or presence of 1μg/ml α-CD36 antibody (n = 3 per group). Data are representative results of two independent experiments. f, Quantification of BODIPY staining in BMDM cultured with regular culture medium (Ctrl) or with YUMM1.7 CM in the absence or presence of 5μg/ml α-Mincle antibody; Ctrl (n = 9), CM (n = 9), CMα-Mincle (n = 8). Data are pooled from three independent experiments. g, h, Immunoblot and quantification of the indicated proteins (g) (n = 4 per group) and qPCR analysis of mRNA expression of ARG1 and MRC1 (h) of WT or Mincle-KO BMDMs exposed to regular culture medium (Ctrl) or or with YUMM1.7 CM for 18 h (n = 9 per group). Data are pooled from three independent experiments. i, Proliferation of CFSE-labeled T cells activated with anti-CD3 and anti-CD28 alone or co-cultured with WT or Mincle-KO BMDMs previously treated with CM in a ratio 2:1 for 72 h; T cells (n = 6), WT (n = 8), ΚΟ (n = 9). Data are pooled from three independent experiments. j, Quantification result of indicated β-glucosylceramide levels from CM derived from YUMM1.7 shCTRL and YUMM1.7 shUGCG cells (n = 3 per group). k, qPCR analysis of the indicated genes in BMDMs treated with Ctrl or CM derived from YUMM1.7 shCTRL and YUMM1.7 shUGCG cells alone or in presence of αMincle antibody (5μg/ml) (n = 3). Data are representative of two independent experiments. lm, Quantification result of indicated β-glucosylceramide levels in serum and tumor interstitital fluid (TIF) isolated from YUMM1.7 melanoma-bearing mice (l) or Braf/Pten melanoma-bearing mice (m) (n = 5 per group). All data are mean ± s.e.m and were analyzed by two-tailed, unpaired Student’s t-test (af), paired Student’s t-test (g, j, lm), one-way ANOVA with Sidak’s multiple comparison test (i), one-way ANOVA with Tukey’s multiple comparison test (k).

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Extended Data Fig. 6 Macrophage-specific ablation of LPCAT3 abolishes GW3965 anti-tumor responses.

a, b, qPCR analysis of mRNA expression of sXBP1 (a) and immunoblot and quantification (b) of indicated proteins in WT or LPCAT3-KO BMDMs exposed to regular culture medium (Ctrl) and YUMM1.7 CM in absence or presence of 3μM GW3965 (n = 9 per group for qPCR and n = 3 per group for immunoblots). Data are pooled from three independent experiments c, Proliferation of CFSE-labeled T cells activated with anti-CD3 and anti-CD28 alone or co-cultured with WT or LPCAT3-KO BMDMs previously treated with YUMM1.7 CM in the absence or presence of GW3965 in a ratio 2:1 for 72 h (n = 3 per group). Data are representative of three independent experiments. d, Illustration of experimental design for bone marrow transplantation. e, qPCR analysis of exon 3 of LPCAT3 gene in LPCAT3fl/fl (WT) and LysM-Cre LPCAT3fl/fl (KO) mice (n = 15). f, Bone marrow was isolated from WT and KO chimeric tumor-bearing mice and the abundance of indicated immune cells was measured by flow cytometry (n = 4). g, Percentage of mTAMs among CD11b+ tumor-infiltrating myeloid cells from YUMM1.7-OVA melanoma treated with either control vehicle or GW3965 in mice transplanted with BM cells from LPCAT3fl/fl (WT) and LysM-Cre LPCAT3fl/fl (KO) mice (WT + Vehicle: n = 9; WT + GW3965: n = 10; KO + Vehicle: n = 10; KO + GW3965: n = 9). Data are pooled from two independent experiments. Each symbol represents one individual. All data are mean ± s.e.m and were analyzed by two-tailed, unpaired Student’s t-test (a, e-g), RM one-way ANOVA with Bonferroni’s multiple comparison test (b), and ordinary one-way ANOVA with Tukey’s multiple comparison test (c).

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Di Conza, G., Tsai, CH., Gallart-Ayala, H. et al. Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity. Nat Immunol 22, 1403–1415 (2021). https://doi.org/10.1038/s41590-021-01047-4

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