Abstract
Cancer cells frequently alter their lipids to grow and adapt to their environment1,2,3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.
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Data availability
Proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD052718. scRNA-seq data has been deposited to the NCBI Gene Expression Omnibus and can be accessed with the accession number GSE270660. Source data are provided with this paper.
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Acknowledgements
The authors thank all Birsoy laboratory members for their thoughtful suggestions; members of the Rockefeller University Genomics Resource Center, Proteomics Resource Center and the Flow Cytometry Resource Center for their assistance. M.S. is a Howard Hughes Medical Institute (HHMI) Gilliam Fellow. G.U. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2431-21). We thank Cold Spring Harbor Laboratory (CSHL) Cancer Center Imaging Core Facility supported by NCI Cancer Center Support grant 5P30CA045508. S.B. acknowledges funding from The Oliver S. and Jennie R. Donaldson Charitable Trust, The G. Harold and Leila Y. Mathers Foundation, STARR Cancer Consortium (I13-0052) and The Mark Foundation for Cancer Research (20-028-EDV). K.B. is supported by the NIH/NIDDK (R01 DK123323-01), NIH/NCI (5U54CA261701-03), the Reem-Kayden award, Black Family Metastasis Research Center and Mark Foundation Emerging Leader Award. Data were generated by the Proteomics Resource Center at The Rockefeller University (RRID:SCR_017797) using instrumentation funded by the Sohn Conferences Foundation and the Leona M. and Harry B. Helmsley Charitable Trust.
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Contributions
K.B. and M.S. conceived of the project, designed the experiments, and wrote the manuscript with input from S.B. M.S. performed most of the experiments. G.U. helped with IFNγ sensitivity, proliferation and subcutaneous tumour experiments, and performed the hydrodynamic tail vein injections and luciferase flux assays. B.U. and R.W. helped with the in vivo CRISPR screens and coculture assays. A.C. performed flow cytometric immune profiling experiments. V. Shah performed the scRNA-seq analysis. H.A. performed the lipidomic analyses. P.B. performed tissue staining and analysis of IFNGR1 expression in tumours. V. Subramanyam performed survival and CIBERSORT analyses. H.-W.Y. performed orthotopic pancreas tumour injections. A.K. helped with bulk RNA-seq analysis. S.H. performed proteomic analyses. H.G. and G.D.V. provided input.
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K.B. is a scientific advisor to Nanocare Pharmaceuticals and Atavistik Bio. M.S. is an employee of Lime Therapeutics. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Cancer cell sphingolipid availability mediates immune evasion.
a. Cell count of red blood cells (RBC, left), white blood cells (WBC, middle), lymphocytes (LYM, right), neutrophils (NEU), monocytes (MONO), eosinophils (EOS), and basophils (BAS) in untreated C57BL/6 J mice (pink) and myeolablated C57BL/6 J mice on the day of tumor injections (d0, 36 h after radiation) and at the endpoint (d18). Mean ± SEM; n = 3 mice/ group. b. Ranks of differential gene scores between C57BL/6 J and myeloablated mice (x-axis) or NSG mice (y-axis). The highlighted genes are among the top 60 scoring genes and are involved in sphingolipid biosynthesis. c. Schematic of wildtype HY15549 cells grown in C57BL/6 J or NSG mice for 12 days before tumor collection and lipid extraction. Lipid abundances were determined by LC-MS analysis (right). d. Abundance of ceramide-derived lipid species in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels (left) or to total protein (right). Mean ± SEM, n = 3 mice/ group. e. Abundance of hexosyl-1-ceramides in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels. Mean ± SEM, n = 3 mice/ group. f. Abundance of hexosyl-2-ceramides in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels. Mean ± SEM, n = 3 mice/ group. g. Abundance of glucosylceramides in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels. Mean ± SEM, n = 3 mice/ group. h. Abundance of sphingosines in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels. Mean ± SEM, n = 3 mice/ group. i. Abundance of sulfatides in HY15549 cells grown in C57CBL/6 J versus NSG mice. Lipid abundance is normalized to cholesterol levels. Mean ± SEM, n = 3 mice/ group.
Extended Data Fig. 2 Loss of Sptlc1 or Sptlc2 depletes ceramide-derived lipid species.
a. Immunoblot analysis of SPTLC1 (top) and SPTLC2 (bottom) expression in wildtype HY15549 cells or knockout and cDNA-expressing clonal pairs. GAPDH is used as a loading control. b. Abundance of ceramide-derived lipid species in empty vector parental, Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells. Abundance is normalized to cholesterol levels of each sample (left) or to protein (right). Mean ± SEM, n = 2 biological replicates/ cell line. c. Abundance of ceramide species in empty vector parental, Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 2 biological replicates/ cell line. d. Abundance of hexosyl-1-ceramide species in empty vector parental, Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 2 biological replicates/ cell line. e. Abundance of sphingomyelin species in empty vector parental, Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 2 biological replicates/ cell line. f. Abundance of ceramide-derived lipid species in empty vector parental, Sptlc2-KO (KO) and Sptlc2-AB (AB, blue) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 2 biological replicates/ cell line. g. Abundance of ceramide-derived lipid species in Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells grown as tumors in C57BL/6 J mice for 12 days. Abundance is normalized to cholesterol levels of each sample (left) or to protein (right). Mean ± SEM, n = 2 biological replicates/ cell line.
Extended Data Fig. 3 Loss of SPT impairs tumor growth in immunocompetent mice.
a. Weights of Sptlc1_sg6-KO (KO, pink) and Sptlc1_sg6-AB (AB, blue) HY15549 tumors grown in C57BL/6 J, NSG, or Rag− mice corresponding to Fig. 1g (left). Weights of Sptlc1_sg2-KO (KO, pink) and Sptlc1_sg2-AB (AB, blue) HY15549 tumors grown in C57BL/6 J or NSG mice (middle). Weights of Sptlc2_sg2-KO (KO, pink) and Sptlc2_sg2-AB (AB, blue) HY15549 tumors grown in C57BL/6 J or NSG mice (right). Mean ± SEM; n = 6 (C57BL/6 J) or 7 (NSG, Rag1−) mice/ group. b. Weights of HY15549 tumors formed from cells constitutively expressing shRNAs against LacZ (control), Sptlc1, or Sptlc2 and grown in C57BL/6 J mice. The tumors measured are shown below. On the right, immunoblot analysis of SPTLC1 (above) and SPTLC2 (below) in lysates from cells kept in culture or grown as tumors. Vinculin is used as a loading control. Mean ± SEM; n = 6 mice/ group. c. Weights of mixed population sgCTRL (gray) or sgSptlc1-KO (pink) HY15549 tumors grown subcutaneously in C57BL/6 J or NSG mice. Mean ± SEM; n = 5 mice/ group. Images of tumors are shown below. Scale bar = 1 cm. Immunoblot analysis of SPTLC1 is shown above. GAPDH is used as a loading control. d. Weights of Sptlc1-KO (KO, pink) and Sptlc1-AB (AB, blue) HY15549 tumors orthotopically grown in the pancreas of C57BL/6 J mice. Mean ± SEM; n = 8 mice/ group. Image of tumors is shown below. Scale bar = 1 cm. X marks tumors that were not detected at the endpoint.
Extended Data Fig. 4 Sphingolipid abundance mediates tumor control.
a. Schematic of the sphingolipid metabolism focused CRISPR screen. Syngeneic cancer cell lines derived from C57BL/6 J mice were transduced with the sphingolipid metabolism library and injected subcutaneously (SQ) into the flanks of C57BL/6 J (B6) or NSG mice. Tumors were collected, their genomic DNA extracted, and guide RNA (sgRNA) abundance was determined. N ≥ 3 mice/ group. b. Immunoblot analysis of SPTLC1 and SPTLC2 in HY15549 cells overexpressing empty vectors or Sptlc1 and Sptlc2 cDNA. GAPDH is used as a loading control. c. Volcano plot showing log2 fold difference in ceramide-derived lipid species between double-empty vector wildtype (dEV) or Sptlc1/Sptlc2 double-overexpression (dOE) HY15549 cells. d. Weights (top) and image (bottom) of double-empty vector wildtype (dEV) or Sptlc1/Sptlc2 double-overexpression (Sptlc1/2_dOE) HY15549 tumors grown in NSG mice. Mean ± SEM, n = 6 mice/ group, scale bar = 1 cm. e. Progression (left) and disease (right) free survival analysis of TCGA PDAC patients with high (blue) or low (pink) expression of SPTLC1, SPTLC2, and KDSR. N = 177. Error bands = 95% confidence interval.
Extended Data Fig. 5 Lysosomal sphingolipid salvage is sufficient to sustain cancer cell proliferation upon loss of de novo synthesis.
a. Cell doublings over time of Sptlc1 (left) or Sptlc2 (right) KO (KO, pink) and AB (AB, blue) HY15549 cells grown in vitro. Different shapes are used to distinguish distinct KO/AB clonal pairs. Mean ± SD; n = 3 biological replicates. b. Abundance of ceramide-derived lipid species in Sptlc1_sg6-KO (KO) left untreated or supplemented with sphingosine or ceramide, and Sptlc1_sg6-AB (AB, blue) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. c. Cell doublings of parental (EV, black), Sptlc1-KO (KO, pink), and KO HY15549 cells supplemented with sphingosine-1-phosphate (S1P, 1 µM, square), sphingosine (SP, 750 nM, triangle), C2-ceramide (Cer2, 5 µM, diamond), 3-ketodihydrosphingosine (KDS, 2 µM, square), C6-ceramide (Cer6, 1 µM, triangle), cholesterol (10 µM, square), arachidonate (10 µM, triangle), or palmitate (10 µM, diamond) and treated with increasing concentrations of Bafilomycin-A1. Mean ± SD; n = 3 biological replicates. d. Log2 fold change of ceramide-derived lipid species abundance in Sptlc1_sg6-KO (KO) HY15548 cells left untreated or treated with bafilomycin-a1 (Baf-A1) relative to Sptlc1_sg6-AB (AB) cells. N = 3 biological replicates. e. Abundance of ceramide-derived lipid species in Sptlc1_sg6-KO (KO), left untreated or treated with Bafilomycin-A,1and Sptlc1_sg6-AB (AB, blue) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. f. Abundance of hexosyl-1-ceramide species in Sptlc1_sg6-KO (KO), left untreated or treated with Bafilomycin-A,1and Sptlc1_sg6-AB (AB, blue) HY15549 cells. Abundance is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates.
Extended Data Fig. 6 Sphingolipid depletion promotes inflammation in the tumor microenvironment.
a. Differentially expressed genes used to annotate clusters (y-axis) of sequenced CD45+ cells. Average expression is color-coded, and circle size corresponds to the percentage of cells within the cluster expressing the gene (x-axis). b. Bar plot showing relative differences in leukocyte infiltration in Sptlc1-KO (KO) and Sptlc1-AB (AB) tumors. c. Distribution of interferon-γ (Ifng) expression across distinct CD45+ leukocyte populations isolated from Sptlc1-KO (pink) or Sptlc1-AB (blue) HY15549 tumors grown in C57BL/6 J mice. Data shown as mean ± SEM and analyzed by a two-sided Wilcoxon rank sum test with Benjamini-Hochberg correction. N = 5000 cells/ condition. Minima/maxima/center bounds are defined in the source data file. d. Flow analysis of relative proportions of NK, CD8+ T, and CD4+ T cells isolated from Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 tumors grown in C57BL/6 J mice. Mean ± SEM; n = 5 mice/ group. e. Mean fluorescence intensity (MFI) of CD44 in CD8+ T cells isolated from Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 tumors grown in C57BL/6 J mice. Mean ± SEM; n = 5 mice/ group. f. Flow analysis of relative proportions of CD44+/CD62L- CD4+ T cells isolated from Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 tumors grown in C57BL/6 J mice. Mean ± SEM; n = 5 mice/ group. g. MFI of CD44 in CD4+ T cells isolated from Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 tumors grown in C57BL/6 J mice. Mean ± SEM; n = 5 mice/ group. h. CIBERSORT analysis on TCGA expression data for PDAC tumors estimating the fraction of infiltrating NK cells in SPTLC1/SPTLC2/KDSRlow tumors (Low, pink) compared to SPTLC1/SPTLC2/KDSRhigh (High, blue) tumors. N = 177 tumors. Minima/maxima/center bounds are defined in the source data file. i. CIBERSORT analysis on TCGA expression data for PDAC tumors estimating the fraction of activated infiltrating NK cells in SPTLC1/SPTLC2/KDSRlow tumors (Low, pink) compared to SPTLC1/SPTLC2/KDSRhigh (High, blue) tumors. N = 177 tumors. Minima/maxima/center bounds are defined in the source data file. j. CIBERSORT analysis on TCGA expression data for PDAC tumors estimating the fraction of infiltrating CD8+ T cells in SPTLC1/ SPTLC2/ KDSRlow tumors (Low, pink) compared to SPTLC1/ SPTLC2/ KDSRhigh (High, blue) tumors. N = 177 tumors. Minima/maxima/center bounds are defined in the source data file. k. Histogram of IFNγ expression in NK cells monocultured or cocultured with Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 cells. l. Percent of IFNγ+ NK cells cocultured with Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 cells. Mean ± SEM; n = 3 biological replicates. m. MFI of IFNγ+ NK cells cocultured with Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 cells. Mean ± SEM; n = 3 biological replicates. n. Proliferation of OVA-null Sptlc1_sg6-KO (KO_EV, pink) or Sptlc1_sg6-AB (AB_EV, blue) HY15549 cells left untreated or cocultured with OT-1 CD8+ T cells at the indicated E:T ratios for 98 h. Mean ± SEM; n = 3 biological replicates. o. Weights and images of Sptlc1-KO HY15549 tumors grown in C57BL/6 J mice left untreated (NT), or treated with depleting antibodies for NK cells, CD8+ T cells, or both. Mean ± SEM; n = 4 mice/ group. Scale bar = 1 cm. X marks tumors that were not detected at the endpoint. Analyzed using a one-way ANOVA with Benjamini-Hochberg multiple test correction.
Extended Data Fig. 7 Interferon signaling pathways are activated in Sptlc1-KO cells in the presence of immune pressure.
a. PCA plot of RNA-seq from GFP+ Sptlc1_sg6-KO (KO) and Sptlc1_sg6-AB (AB) HY15549 cells grown C57BL/6 J, NSG mice, or cultured in vitro (left) or in vivo (right). b. PCA plot of RNA-seq from Sptlc1_sg6-KO (KO) and Sptlc1_sg6-AB (AB) HY15549 cells left untreated or cocultured with NK cells (2:1 E:T ratio for 5 h). c. Gene set enrichment analysis of genes significantly higher in Sptlc1_sg6-KO versus Sptlc1_sg6-AB HY15549 cells left untreated or cocultured with NK cells (2:1 E:T ratio for 5 h). GO terms are ranked by gene ratio, adjusted P-values (FDR, -log10) are color-coded, and the circle size corresponds to the total number of genes in the gene set. d. Second example of an immunoblot analysis of interferon-γ signaling pathway induction in Sptlc1_sg6-KO and Sptlc1_sg6-AB HY15549 cells left untreated or treated with increasing concentrations of IFNγ for 4 h. GAPDH is a loading control. e. Second example of an immunoblot analysis of interferon-γ signaling pathway induction in Sptlc1_sg6-KO and Sptlc1_sg6-AB HY15549 cells cocultured with NK cells at the indicated E:T ratios for 5 h. GAPDH is a loading control. f. Proliferation of double-empty vector wildtype cells (dEV, gray) and Sptlc1/Sptlc2 double-overexpression (dOE, blue) HY15549 cells treated with the indicated concentrations of IFNγ for 120 h. Mean ± SD; n = 3 biological replicates. g. Immunoblot of interferon-γ signaling pathway induction in double-empty vector wildtype cells (dEV) and Sptlc1/Sptlc2 double-overexpression (dOE) HY15549 cells left untreated or treated with increasing concentrations of IFNγ for 4 h. GAPDH is a loading control. h. Immunoblot of of Jak1 in Sptlc1-KO (KO), Sptlc1-AB (AB) and Sptlc1_sg6/Jak1-KO (sg_4, sg_8) HY15549 cells (top) and Sptlc1 in Sptlc1_sg6-KO (KO), Sptlc1_sg6-AB (AB) and Sptlc1_sg6/Ifngr1-KO (sg_9, sg_10) HY15549 cells (bottom). GAPDH is a loading control.
Extended Data Fig. 8 Sphingolipids impact plasma membrane expression of Ifngr1.
a. Immunoblot of whole cell and biotinylated plasma membrane protein lysates from Sptlc1_sg6-KO and Sptlc1_sg6-AB HY15549 cells used for proteomic analysis. E-cadherin (E-cad) is a control for membrane enrichment and GAPDH is a marker for whole cell proteins. b. Gene set enrichment analysis of the top 206 (PSM > 20) most abundant proteins detected by LC-MS in biotinylated plasma membrane protein lysates. Jensen Compartments Ontology terms are ranked by fold enrichment, adjusted P-values (FDR, -log10) are color-coded, and circle size corresponds to the total number of genes in the gene set. c. Log2 fold difference in whole cell proteins between Sptlc1_sg6-KO and Sptlc1_sg6-AB HY15549 cells versus -log10 P-value. d. Median fluorescence intensity of IFNGR1+ Sptlc1_sg6-KO (pink) or Sptlc1_sg6-AB (blue) HY15549 cells treated with 5 ng/mL interferon-γ for the indicated times. Mean ± SD, n = 3 biological replicates. e. Flow analysis of IFNGR1 (left) and median fluorescence intensity (right) of IFNGR1 in permeabilized Sptlc1_sg6-KO (pink) and Sptlc1_sg6-AB (blue) HY15549 cells left untreated or treated with 5 ng/mL interferon-γ for the indicated times. Mean ± SD, n = 3 biological replicates. f. Immunoblot of interferon-γ signaling pathway induction in Sptlc1_sg6-KO and Sptlc1_sg6-AB HY15549 cells left untreated or treated with IFNγ for the indicated times. GAPDH is a loading control. g. Immunocytochemistry analysis of IFNGR1 and CAV1 in Sptlc1-KO (KO) and Sptlc1-AB (AB) HY15549 cells left untreated (top) or treated with 5 ng/mL IFNγ for 24 h. Choleratoxin B (CTxB) is used as a plasma membrane and sphingolipid abundance marker. Scale bar = 10 µm. Quantification of plasma membrane bound IFNGR1 is on the right, n = 55 cells/ condition. h. Immunocytochemistry analysis of IFNGR1 in double-empty vector wildtype cells (dEV) and Sptlc1/Sptlc2 double-overexpression (dOE) HY15549 cells left untreated (top) or treated with 10 ng/mL IFNγ for 24 h. CTxB is used as a plasma membrane and sphingolipid abundance marker. Scale bar = 10 µm. Quantification of plasma membrane bound IFNGR1 is on the right, n = 50–54 cells/ condition. i. Flow analysis of whole cell and plasma membrane expression of IFNGR1 in double-empty vector cells (dEV, gray) and Sptlc1/Sptlc2 double-overexpression (dOE, blue) HY15549 cells left untreated (left) or treated with 10 ng/mL IFNγ (right) for 24 h. Mean ± SEM; n = 3 biological replicates.
Extended Data Fig. 9 De novo glycosphingolipid synthesis is limiting for cancer immune evasion.
a. Guide scores of genes encoding enzymes at each branchpoint of sphingolipid synthesis. Guide scores of the top 7% (pink) and bottom 20% (blue) IFNGR1+ HY15549 (top) and KP LUAD (bottom) cells transduced with the sphingolipid metabolism-focused sgRNA library are shown. b. Abundance of ceramide-derived lipid species in Ugcg_sg5-KO (KO) and Ugcg_sg5-AB (AB, blue) HY15549 cells. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. c. Abundance of hexosyl-1-ceramide species in Ugcg_sg5-KO (KO) and Ugcg_sg5-AB (AB, blue) HY15549 cells. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. d. Abundance of hexosyl-2-ceramide species in Ugcg_sg5-KO (KO) and Ugcg_sg5-AB (AB, blue) HY15549 cells. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. e. Flow analysis of plasma membrane glycosphingolipid levels measured by Choleratoxin B fluorescence (CTxB). f. Flow analysis of plasma membrane expression of IFNGR1 in sgCTRL-RFP and sgUgcg-RFP HY15549 cells. Mean ± SEM; n = 3 biological replicates. g. Weights of Ugcg_sg5-KO (KO, pink) and Ugcg_sg5-AB (AB, blue) HY15549 tumors grown in NSG mice. Tumors measured are shown below. Mean ± SEM, n = 4 mice/ group. h. Tumor weights of mixed population HY15549 cells expressing sgCTRL-RFP or sgUgcg-RFP grown in C57BL/6 J mice. Tumors measured are shown below. Mean ± SEM, n = 5 mice/ group.
Extended Data Fig. 10 Pharmacological depletion of glycosphingolipids impacts immune evasion.
a. Abundance of ceramide-derived lipid species in HY15549 cells left untreated (gray) or treated with 10 µM eliglustat for 24 h. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. b. Abundance of hexosyl-1-ceramide species in HY15549 cells left untreated (gray) or treated with 10 µM eliglustat for 24 h. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. c. Abundance of hexosyl-2-ceramide species in HY15549 cells left untreated (gray) or treated with 10 µM eliglustat for 24 h. Signal is normalized to cholesterol levels of each sample. Mean ± SEM, n = 3 biological replicates. d. Proliferation of wildtype HY15549 (left) or KP LUAD (right) cells left untreated (gray) or pretreated with eliglustat for 24 h (pink) and the indicated concentrations of interferon-γ (IFNγ) for 96 h. Mean ± SD; n = 3 biological replicates. e. Weights (left) and image (right) of wildtype KP LUAD tumors grown in C57BL/6 J mice on the indicated treatment regimens. Mean ± SEM; n = 6 (HY15549 −/−, +/−, −/+, KPLUAD −/+) or 7 (others) mice/ group; scale bar = 1 cm. Data were analyzed using a one-way ANOVA with Tukey’s multiple test correction. f. Survival analysis of TCGA PDAC patients with high (blue) or low (pink) expression of SPTLC1, SPTLC2, and KDSR. N = 177. Error bands = 95% confidence interval.
Supplementary information
Supplementary Table 1
List of sgRNAs and shRNAs.
Supplementary Data
Source data (lipidomics data).
Source data
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Soula, M., Unlu, G., Welch, R. et al. Glycosphingolipid synthesis mediates immune evasion in KRAS-driven cancer. Nature 633, 451–458 (2024). https://doi.org/10.1038/s41586-024-07787-1
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DOI: https://doi.org/10.1038/s41586-024-07787-1
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