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.

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. b-h, 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. j-l, 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, f-h, j-n). Each symbol represents one individual. All data are mean ± s.e.m and were analysed by two-tailed, unpaired Student’s t-test or paired t-test (d, g, h).

Extended Data Fig. 2. Tumor cells reinforce pro-tumorigenic 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 18h (n=3 per group). Data are representative of three independent experiments. e, Proliferation of CFSE-labelled 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 (10ng/ml) in the absence or presence of 0.25μg/ml anti-IL-4 and 0.25μg/ml anti-IL-13 neutralizing antibodies for 18h (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 18h (n=3 per group). Data are representative results of three independent experiments. h, Multiplex cytokine array was used to determine the concentration of IL13 (left) and IL4 (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. i-k, 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 18h (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 lμγ/ml of Tunicamycin, 1μM thapsigargin and CM for 16h (n=3 per group). Data are representative results of three independent experiments. Data are mean ± s.e.m. were analysed by two-tailed, unpaired Student’s t-test or one-way ANOVA with Tukey’s multiple comparison test (e).

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

a, Immunoblots of indicated proteins in control or XBP1-deficient BMDMs stimulated with or without 1 μM thapsigargin for 6h. Data are representative results of two independent experiments. b, Quantification of BODIPY staining in BMDMs generated from WT (XBP1^wt) or KO (XBP1^cKO) 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 XBP1^wt (n=5) and XBP1^cKO 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 XBP1^cKO mice treated with PBS or with anti-CSF1R as indicated in the methods; XBP1^wt PBS (n=13), XBP1^cKO PBS mice (n=12), XBP1^wt αCSF1R (n=12), XBP1^cKO 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 XBP1^wt PBS, XBP1^cKO PBS, XBP1^wt αCSF1R; n=9 for XBP1^cKO 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. h-k, 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. m-p, 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. q-t, 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 XBP1^wt (n=9 for B16-OVA and n=8 for MC38-OVA) or XBP1^cKO (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 analysed by two-tailed, unpaired Student’s t-test.

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

a, Immunoblots of indicated proteins in BMDMs expressing scramble or STAT3-targeting gRNAs treated with 10ng/ml IL-6 for 6h. 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 18h. 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 analysed by two-tailed, unpaired Student’s t-test.

Extended Data Fig. 5. β-glucosylceramide, rather than cholesterol, is released by tumor cells to mediate the pro-tumorigenic 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 18h (n=9 per group). Data are pooled from three independent experiments. i, Proliferation of CFSE-labelled 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 72h; T cells (n=6), WT (n=8), KO (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. l-m, 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 analysed by two-tailed, unpaired Student’s t-test (a-f), paired Student’s t-test (g, j, l-m), one-way ANOVA with Sidak’s multiple comparison test (i), one-way ANOVA with Tukey’s multiple comparison test (k).

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-labelled 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 72h (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 LPCAT3^fl/fl (WT) and LysM-Cre LPCAT3^fl/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 LPCAT3^fl/fl (WT) and LysM-Cre LPCAT3^fl/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 analysed 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).

Figure 1. The TME promotes lipid accumulation in pro-tumorigenic TAMs.

a,b, Representative histogram (left) and quantitative plot of the geometric mean fluorescence intensity (MFI) (right) of BODIPY (a) and Filipin III (indicated as Filipin) (b) in splenic macrophages or tumor-associated macrophages (TAMs) of YUMM1.7 melanoma-bearing mice (n=9 per group in a; n=5 per group in b). c, Representative electron microscope images (left) and quantification (right) of lipid droplet formation in sorted TAMs and splenic macrophages (n=23 per group), Scale bar, 1μM. d, Representative histogram (left) and quantitative plots of the MFI (right) of BODIPY staining in TAMs gated based on ARG1 expression (n=9 per group). e, Representative electron microscope images (left) and quantitative plot (right) of ER extension in splenic macrophages and TAMs of YUMM1.7 melanoma-bearing mice (n=10 per group), scale bar, 200nm. Data are pooled from two independent experiments (a-b, d). Data derived from one electron microscopy experiment (c, e). Each symbol represents one individual. All data are mean ± s.e.m and were analysed by two-tailed, unpaired Student’s t-test.

Figure 2. The TME activates IRE1/sXBP1 in pro-tumorigenic TAMs.

a, qPCR analysis of mRNA expression of indicated genes in splenic macrophages (n=9) and TAMs (n=8) isolated from YUMM1.7 melanoma-bearing mice. b, Representative histogram (left) and quantitative plot of the percentage (right) of sXBP1⁺ cells among splenic macrophages and TAM of YUMM1.7 melanoma-bearing mice (n=9 per group). c, Representative histogram (left) and quantitative plot of the MFI (right) of BODIPY staining in TAMs gated based on sXBP1 expression (n=9 per group). d, Representative histogram (left) and quantitative plot of the percentage (right) of sXBP1⁺ cells among ARG1⁺ and ARG1^- TAMs (n=9 per group). e, Violin plot and box plot show distributions of ER stress scores in anti-tumorigenic and pro-tumorigenic macrophages from different human and murine cancer types. Data are pooled from at least two independent experiments. Each symbol represents one individual. All data are mean ± s.e.m and were analysed by two-tailed, unpaired Student’s t-test (a-d), one-tailed unpaired Student’s t-test (e). Box plot where whiskers represent the 5 and 95th percentile values, box limits represent the 25th and 75th percentiles, black line represents the median (e).

Figure 3. Tumor cells drive pro-tumorigenic polarization in BMDMs via IRE1.

a,b, BMDMs were cultured in regular culture medium (Ctrl) or in presence of YUMM1.7 melanoma cell-conditioned medium (CM) for 18h. Flow cytometry analysis was performed to analyse lipid content with n= 4 per group (a) or quantitative analysis of the expression of indicated genes with n= 3 per group (b). Data are representative results of three independent experiments. c, Immunoblots of indicated proteins in BMDM treated with indicated conditions and mouse embryonic fibroblasts (MEFs) treated with Tunicamycin (TM) or Nelfinavir (NFR) at the indicated doses as positive controls. Data are representative results of two independent experiments. d, qPCR analysis of mRNA expression of indicated genes in BMDMs stimulated with control medium (Ctrl), YUMM1.7 CM in the presence of 50μM STF081030 (CM+STF) or control vehicle (CM). Data are representative results of three independent experiments with n= 3 per group. e, Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labelled T cells activated with anti-CD3 and anti-CD28 alone or in co-culture with BMDM previously exposed to CM or CM in presence of 50μM STF for 24h, in a ratio 2:1 (n=3 per group). Data are representative of three independent experiments. f, qPCR analysis of mRNA expression of indicated genes in control or IRE1-deficient BMDMs transduced with YUMM1.7 CM. Data are pooled from two independent experiments from four independent repeats (n=5 or 6 per group). g, qPCR analysis of mRNA expression of indicated genes in BMDM treated with tunicamycin, thapsigargin or YUMM1.7 CM for 16h. Data are representative results of three independent experiments with n= 3 per group. All data are mean ± s.e.m. and were analysed by two-tailed, unpaired Student’s t-test (a-b, d, f-g) or one-way ANOVA with Tukey’s multiple comparison test (e).

Figure 4. Deletion of XBP1 in TAMs suppresses tumor growth.

a, qRT-PCR analysis of indicated genes in Cas9-expressing BMDM transduced with retrovirus expressing gRNA targeting control or XBP1 sequence and exposed to Ctrl or CM (n=6 per group). Data are pooled from two independent experiments and repeated four times. b, Proliferation of CFSE-labelled T cells activated with anti-CD3 and anti-CD28 alone or in co-culture with BMDM isolated from wild type (XBP1^wt) or XBP1^cKO mice and previously exposed to CM in a ratio 2:1 (n=3 per group). Data are representative of three independent experiments. c,d, Tumor growth (c) and tumor weight (d) of YUMM1.7-OVA melanoma from XBP1^wt (n=5) or XBP1^cKO (n=6) mice. Data are representative of three independent experiments. e, Representative plots (left) and percentages of iTAMs and mTAMs cells among CD11b+ myeloid cells in tumor-bearing XBP1^wt (n=6) and XBP1^cKO (n=9) mice. Data are pooled from two independent experiments. Each symbol represents one individual. All data are mean ± s.e.m. and were analysed by two-tailed, unpaired Student’s t-test (a, c-e) or one-way ANOVA with Tukey’s multiple comparison test (b).

Figure 5. Activation of IRE1-STAT3 signal supports CM-induced polarization.

a, qPCR analysis of mRNA expression of indicated genes in BMDMs stimulated with YUMM1.7 CM in the presence of 50μM STF081030 (CM+STF) or vehicle (CM) BMDMs transduced with retrovirus expressing empty vector (EV) or sXBP1(sXBP1) as indicated in method; (n=6 per group for EV-CM, EV-CM-STF, XBP1-Ctrl and XBP1-CM-STF), EV-Ctrl (n=5), XBP1-CM (n=5). Data are pooled from two independent experiments and repeated four times. b, Immunoblots of BMDMs treated with CM for indicated duration. Data are representative results of three independent experiments. c, qPCR analysis of mRNA expression of indicated genes in BMDMs stimulated with control medium or YUMM1.7 CM (n=5 per group). BMDMs were transduced with retrovirus expressing control or STAT3-targeting gRNAs. Data are pooled from two independent experiments and repeated four times. d, Immunoblots of indicated proteins in BMDMs treated with indicated treatments. Ctrl: control medium; CM: YUMM1.7 conditioned medium; IL6: IL-6 treatment (10ng/ml); IL10: IL-10 treatment (10ng/ml). Anti-IL-6/IL-10 antibodies were added alone or in combination (0.25μg/ml anti-IL-6 and 0.25μg/ml anti-IL-10). Data are representative results of three independent experiments. e, qPCR analysis of mRNA expression of indicated genes in BMDMs treated with CM in the absence or presence of 0.25μg/ml anti-IL-6 and anti-IL-10 antibodies (n= 3 per group). Data are representative results of three independent experiments. f, Representative images (left) and quantification (right) of proximity ligation assay (PLA) of BMDM exposed to Ctrl or CM for 18h, scale bar 10μM. Data are representative of two independent experiments: Ctrl (n=22), CM (n=35). g, Immunoblots (left) and quantification (right) (n=3 per group) of indicated proteins in control or IRE1-targeting gRNA expressing BMDMs stimulated with control medium (Ctrl) or YUMM1.7 CM (CM). Data are representative results of three independent experiments. All data are mean ± s.e.m. and were analysed by two-tailed, unpaired Student’s t-test (a, c, e, f) or one-way ANOVA with Sidak’s multiple comparison test (g).

Figure 6. Mincle-dependent glucosylceramide sensing pathway tailors macrophage activation.

a-c, Immunoblots of indicated proteins (a and b) and qPCR analysis of indicated genes (c) in BMDMs treated with control medium (Ctrl), CM or CM without lipids for the indicated time points (a) or for 18h (b and c) (n=3 sample per group in (c)). Data are representative results of three independent experiments. d, Heatmap of mRNA expression of pro-tumorigenic genes, upon exposure of CM or CM without lipids, in BMDMs (n=3 per group). e, Quantitative plot of Mincle protein expression in BMDMs treated with control medium (Ctrl) or CM (n=3). Data are representative results of three independent experiments. f, Proliferation of CFSE-labelled T cells activated with anti-CD3 and anti-CD28 alone or in co-culture with BMDM previously treated with CM or with CM in presence of 5μg/ml anti-Mincle antibody in a ratio 2:1 for 72h (n=3). Data are representative of three independent experiments. g,h, qPCR analysis of indicated genes (g) (n=5 or 6) and immunoblots of indicated proteins (left) with quantification (right) (n=3) (h) in BMDMs cultured with control medium (Ctrl) or CM in the absence or presence of 5μg/ml αMincle antibody. Data are pooled from two independent experiments and repeated four times in (g) and three times in (h). i, immunoblot of indicated proteins in YUMM1.7 cells shCTRL or shUGCG. j, qPCR analysis of indicated genes in BMDMs exposed to CM derived from YUMM1.7 shCTRL or YUMM1.7 shUGCG (n=6). Data are pooled from two independent experiments and repeated four times. k, immunoblots of indicated proteins (left) and quantification (right) of BMDMs cultured with CM from shCTRL or shUGCG YUMM1.7 (n=3). l-n, Tumor growth curve (l) and tumor weight (m) of YUMM1.7 shCTRL and YUMM1.7 shUGCG tumors in co-engrafted mice (n=10). Ratio of iTAMs/mTAMs from the indicated tumors of tumor-bearing mice (n=10) (n). Data are pooled from two independent experiments. Each symbol represents one individual. Data are mean ± s.e.m. and were analysed by two-tailed (c, e, g, j), unpaired Student’s t-test or paired t-test (m-n) and one-way ANOVA with Tukey’s multiple comparison test (f, h,) and RM one-way ANOVA with Sidak’s multiple comparison test (k).

Figure 7. CM causes reshuffling of lipid composition and saturation of ER membrane.

a, MFI of Filipin III staining in BMDMs treated with CM or CM in presence of simvastatin (10μM) or αMincle antibody (5μg/ml) (n=3). b, qPCR analysis of indicated genes in BMDMs stimulated with control medium (Ctrl) or CM in presence of simvastatin (10μM) alone or with αMincle antibody (5μg/ml) (n=3). Data are representative results of at least two independent experiments. c-e, LC-MS/MS lipidomics performed on the endoplasmic reticulum membrane isolated from BMDMs exposed to control medium (Ctrl) or CM (n=4 per group). The ratio of the absolute value of whole phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species (c), the ratio PC/PE of single lipid species with polyunsaturated fatty acid chain (d), and the pie plot (left panel) and quantification result (right panel) showing the percentage of polyunsaturated fatty acid (PUFA) of PC species and their distribution compared to monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA) (e). Data are pooled results of four independent replicates. f-h, LC-MS/MS lipidomics has been performed on the endoplasmic reticulum membrane isolated from empty vector (EV) or LPCAT3 overexpressing BMDMs treated with CM (n=4 per group). The ratio of the absolute value of whole phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species (f), the ratio PC/PE of single lipid species with polyunsaturated fatty acid chain (g), and the pie plot (left panel) and quantification result (right panel) showing the percentage of polyunsaturated fatty acid (PUFA) of PC species and their distribution compared to monounsaturated fatty acid (MUFA) and saturated fatty acid (SFA) (h). Data are pooled results of four independent replicates. i, Quantitative plot of ER extension detected by electron microscopy in empty vector (EV) or LPCAT3 overexpressing BMDMs stimulated with control medium (Ctrl) or CM (n=14 per group). j-l, qPCR analyses of mRNA expression of indicated genes (j and l) (n=5 or 6) and immunoblot and quantification of indicated proteins (k) in EV or LPCAT3 overexpressing BMDMs stimulated with control medium (Ctrl) or CM (n=3). Data are representative results of three independent experiments. Data are mean ± s.e.m. and were analysed by two-tailed, unpaired Student’s t-test (a-j, i) and one-way ANOVA with Sidak’s multiple comparison test (k). Box plot where whiskers represent the min and max values, box limits represent the min and max values, black line represents the median (i).

Figure 8. LXR agonist reduces tumor burden and hampers TAM survival in a LPCAT3-dependent manner.

a-d, qPCR of mRNA expression of indicated genes (a, b and d) and immunoblots (left) and quantification (right) of indicated proteins (c) in BMDMs stimulated with control medium (Ctrl) or CM in the absence or presence of GW3965 (3μM) (n=3). Data are representative results of three independent experiments. e, Proliferation of CFSE-labelled T cells activated with anti-CD3 and anti-CD28 alone of in co-cultured with BMDM previously treated with CM or with CM in presence of GW3965 in a ratio 2:1 for 72h (n=6). Data are pooled of three independent experiments. f,g, Tumor growth (f) and tumor weight (g) of YUMM1.7-OVA melanoma treated with either control vehicle or GW3965 (n=10 per group). Data are pooled from 2 independent experiments. h-l, Lethally irradiated C57BL/6 mice were transplanted with WT (LPCAT3 ^fl/fl) or KO (LysM-Cre LPCAT3^fl/fl) BM cells. 7 weeks post bone marrow transplantation, mice were engrafted with YUMM1.7-OVA melanoma cells and then treated with control vehicle or GW3965. Tumor growth (h) and tumor weight (i) of YUMM1.7-OVA melanoma were monitored (WT+Vehicle: n=9; WT+GW3965: n=10; KO+Vehicle: n=10; KO+GW3965: n=9). On day 18 post tumor engraftment, the percentage of total TAMs (j), iTAMs (k) and MFI of sXBP1 in iTAMs (l) in YUMM1.7-OVA melanomas were measured by flow cytometry. Data are pooled from two independent experiments. Each symbol represents one individual. All data are mean ± s.e.m and were analysed by two-tailed, unpaired Student’s t-test (a-b, d, f-l) and one-way ANOVA with Tukey’s multiple comparison test (c, e).