Targeting metastasis-initiating cells through the fatty acid receptor CD36

Journal name:
Nature
Volume:
541,
Pages:
41–45
Date published:
DOI:
doi:10.1038/nature20791
Received
Accepted
Published online
Corrected online
Corrected online

Abstract

The fact that the identity of the cells that initiate metastasis in most human cancers is unknown hampers the development of antimetastatic therapies. Here we describe a subpopulation of CD44bright cells in human oral carcinomas that do not overexpress mesenchymal genes, are slow-cycling, express high levels of the fatty acid receptor CD36 and lipid metabolism genes, and are unique in their ability to initiate metastasis. Palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36+ metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis in immunodeficient or immunocompetent orthotopic mouse models of human oral cancer, with no side effects. Clinically, the presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas, and inhibition of CD36 also impairs metastasis, at least in human melanoma- and breast cancer-derived tumours. Together, our results indicate that metastasis-initiating cells particularly rely on dietary lipids to promote metastasis.

At a glance

Figures

  1. CD44bright LRCs have lymphatic metastasis and lipid metabolism transcriptome signatures.
    Figure 1: CD44bright LRCs have lymphatic metastasis and lipid metabolism transcriptome signatures.

    a, LRC populations separated by flow cytometry from SCC-25-derived tumours. Three independent experiments, n = 6 mice per experiment. b, Percentage of dividing cells. *P < 0.05; two-tailed t-test; six independent experiments, n = 3 mice per experiment; data are mean ± s.e.m. c, Top ten disease categories upregulated in dye+ cells. d, Over-represented processes and signal transduction pathways in dye+ cells.

  2. Modulation of CD36 expression strongly affects metastatic penetrance and growth.
    Figure 2: Modulation of CD36 expression strongly affects metastatic penetrance and growth.

    a, b, Bioluminescence imaging (BLI) quantification (a) and frequency of tumours (b) in mice inoculated with tumour cells expressing empty vector (PLKO) or shRNA targeting Cd36. PLKO, n = 9 mice; shCD36#98, n = 7 mice; shCD36#99, n = 6 mice (*P = 0.003, Fisher’s exact test). Relative photon flux primary tumour, *P = 0.04 and metastasis, *P = 0.03 and **P = 0.006, two-tailed t-test; data are mean ± s.e.m. c, Representative haematoxylin and eosin staining of metastatic lymph nodes (LDs, lipid droplets). d, Immunostaining of lymph node metastases arising from cells expressing Cd36 shRNA. Inset, lipid droplets surrounding GFP+ CD44+ cells and showing free fatty acid content. Source data from mouse experiments are in Supplementary Information.

  3. CD36+ cells require fatty acid internalization to promote metastasis.
    Figure 3: CD36+ cells require fatty acid internalization to promote metastasis.

    a, BLI monitoring and frequency of tumours from mice fed either with high-fat diet (HFD) or control diet (CTD). PLKO, n = 5 mice; Cd36 shRNA, n = 5 mice. Primary tumour, P = 0.007 and P < 0.0001; metastasis, P = 0.01 and P = 0.04, two-tailed t-test. b, BLI monitoring and frequency of tumours generated from palmitic acid (PA)-treated cells. LN-Met, lymph node metastasis; PT, primary tumour. PLKO, n = 10 mice; Cd36 shRNA, n = 10 mice (*P = 0.05, **P = 0.03, ***P = 0.0001, Fisher’s exact test). BLI quantifications: primary tumour, *P = 0.01 and **P = 0.007; metastases, **P = 0.005, two-tailed t-test. Unt, control vector untreated; Ct, control vector treated with palmitic acid (PA) and Cd36 shRNA (shCD36) treated with palmitic acid (PA). c, BLI monitoring and frequency of tumours generated by primary OSCC cells transduced with wild-type (wt) CD36 (n = 10 mice) or CD36-K164A (n = 10 mice). *P = 0.03, Fisher’s exact test, and ****P < 0.0001, two-tailed t-test. BLI data in a–c are the mean ± s.e.m. Source data from mouse experiments are in Supplementary Information.

  4. CD36-positive cells initiate and promote OSCC metastasis.
    Figure 4: CD36-positive cells initiate and promote OSCC metastasis.

    a, Frequency of tumours generated by CD44bright CD36+ or CD44bright CD36 cells compared to the parental cell lines (P = 0.01, P = 0.009, two-tailed Fisher’s exact test). b, c, Frequency of metastasis-initiating cells (MIC) and tumour-initiating cells (TIC) for CD44bright CD36+, CD44bright CD36 or CD44bright populations determined by extreme limiting dilution analysis (ELDA). Goodness-of-fit tests, estimated slope= 0.643; likelihood ratio test of single-hit model, P = 0.01; and score tests of heterogeneity, P = 5 × 10−5. Source data from mouse experiments are in Supplementary Information. NS, not significant.

  5. CD36-blocking antibodies therapeutically inhibit metastasis of OSCC tumours.
    Figure 5: CD36-blocking antibodies therapeutically inhibit metastasis of OSCC tumours.

    a, b, Dose–response in BLI signal of tumours from mice treated daily with monoclonal JC63.1 (5, 10 or 20 μg; n = 7, 12 or 7 mice, respectively) or the IgA isotype control (n = 13 mice) for 3 weeks. **P = 0.008, IgA vs JC63.1 (10 μg) *P = 0.01 and IgA vs JC63.1 (5 μg) *P = 0.05, two-tailed t-test. Data are mean ± s.e.m. c, Frequency of tumour remission. d, Haematoxylin and eosin staining of metastatic lymph nodes of animals treated daily with JC63.1. Dashed line shows areas surrounded by lipid droplets. Source data from mouse experiments are in Supplementary Information.

  6. Orthotopically inoculated human oral squamous cell carcinomas contain a slow-cycling sub-population of CD44bright cells.
    Extended Data Fig. 1: Orthotopically inoculated human oral squamous cell carcinomas contain a slow-cycling sub-population of CD44bright cells.

    a, Overview of the tumorigenic and metastatic activities of the different OSCC cell lines injected into the tongues of NSG mice. b, Tumour development from mice injected with OSCC-pLuc-GFP cells (using the cell lines indicated). Tumour growth was monitored by bioluminescence imaging (BLI) over a four-week period. Data are given as the mean ± s.e.m. c, Frequency of metastases in the lymph nodes. ac, Detroit-562 cells, two independent experiments: exp. 1 n = 10 mice; exp. 2 n = 11 mice; VDH-02, n = 20 mice; VDH-01, n = 20 mice; VDH-00, n = 8 mice; SCC-25, three independent experiments: exp. 1 n = 13 mice, exp. 2 n = 17 mice, exp. 3 n = 7 mice; JHU-029, three independent experiments, n = 12 mice per experiment; FaDu, two independent experiments, exp. 1 n = 14 mice, exp. 2 n = 5 mice. d, Immunofluorescence analysis of in vitro cultured OSCC-RFP cells pulsed with DID and grown in 2D culture for 16 days. e, f, Flow cytometry analysis of dye-pulsed OSCC cells in vitro showing the kinetics of dye dilution. Data are given as mean fluorescence intensity. g, FACS strategy to FACS-sort CD44bright dye+, CD44bright dye and CD44dim cells from OSCC-pLucGFP oral tumours. Viable single cells were selected if GFP+ but negative for a lineage (Lin) cocktail of antibodies (H2KD, CD31 and CD45), to select human cells. GFP+ Lin cells were gated for CD44 and dye. Percentages from the total GFP+ Lin SCC-25 parental tumour are shown. h, Representative flow cytometry analyses to detect quiescent slow-cycling CSCs from OSCC cell lines. g, h, n = 8 animals per OSCC cell line. i, Global quantification of CD44bright dye+, CD44bright dye and CD44dim cells from OSCC-pLucGFP tumours reported in g and h. j, Immunofluorescence analysis of SCC-25-pLucGFP and JHU-029-pLucGFP primary tumours, collected five weeks after OSCC inoculation, to detect dye+ quiescent slow-cycling cancer stem cells (CSCs). Insets show a magnification of dye+ cells that co-localized with the CD44 marker. SCC-25, n = 5 tumours; JHU-029, n = 5 tumours. k, Percentage of dividing cells by flow cytometry analysis in the dye+, dye and CD44dim populations. Source data from mouse experiments are in Supplementary Information.

  7. Oral SCC label-retaining cells are defined by a lipid metabolism and metastasis transcriptome signature.
    Extended Data Fig. 2: Oral SCC label-retaining cells are defined by a lipid metabolism and metastasis transcriptome signature.

    a, Microarray analysis and heatmap of mRNA expression showing differentially expressed genes in dye+, dye and CD44dim cells. n = 4 biological replicates and 8 mice per replicate. b, Gene ontology (GO) analysis showing the top categories for diseases, biological processes and signal transduction pathways that were upregulated in the proliferative active (DID) as compared to LR-CSCs (DID+) populations. The resulting GO terms highlighted cell cycle–related categories. c, Over-represented genes in Dye+ and Dye populations. d, Lipid metabolism genes over-represented in dye+ cells. e, Gene ontology (GO) analysis showing top diseases and biological processes categories upregulated in the DID+ (LR-CSCs) and DID (proliferative) sorted populations from dye-pulsed Detroit-562 tumours analysed by microarrays. f, RT–qPCR validation by human-specific TaqMan gene expression assays of differentially expressed genes by microarray in the CD44+ DID+ and CD44+ DID populations. Data are given as relative expression levels. Human β-2-microglobulin was used as internal control gene. n = 5, *P < 0.05, **P < 0.005, two-tailed t-test. g, Gene expression overlapping analysis of the LR-CSC signatures from SCC-25 and Detroit-562 tumours showing the top represented common diseases and biological processes. Metastatic processes and lipid metabolism-related categories are highlighted in red. P = 2.10 × 10−49, hypergeometric test. h, Correlation between CD36 expression and DiD content for orthotopic transplants of SCC-25, JHU-029, Detroit-562, FaDu, VDH-00, VDH-01 and VDH-02 cells (n = 8 animals per cell line). Numbers indicate percentages from the total GFP+Lin OSCC parental tumour. Results are given as the mean ± s.e.m. (n = 7 OSCC orthotopic transplants; ***P = 0.0008, *P= 0.03, two-tailed t-test).

  8. LRCs correspond to CD36+ cells, and CD36 overexpression promotes metastatic initiation and progression.
    Extended Data Fig. 3: LRCs correspond to CD36+ cells, and CD36 overexpression promotes metastatic initiation and progression.

    a, CD36+ CD44bright OSCC cells detected by flow cytometry analysis of tumours from orthotopic transplants. Tumours were obtained from OSCC Detroit-562 (three independent experiments: exp. 1 n = 3, exp. 2 n = 3, exp. 3 n = 4 mice), JHU-029 (three independent experiments: exp. 1 n = 3, exp. 2 n = 3, exp. 3 n = 4 mice), SCC-25 (n = 8 mice), FaDu (n = 8 mice), VDH-00 (n = 8 mice), VDH-01 (n = 8 mice) and VDH-02 cells (n = 8 mice). Numbers indicate CD44bright CD36bright or CD44bright CD36low cells in the represented gate, expressed as percentages from the total GFP+ Lin OSCC parental tumour. Histograms show the correlation between CD36 expression and the DID content. The average counted events as a function of dye fluorescence intensity is reported for each population CD44bright CD36bright and CD44bright CD36low. b, BLI monitoring of tumours generated by SCC-25 cells (empty vector (EV) n = 7 and Cd36 overexpression (OE) n = 17 mice), or JHU-029 cells (EV n = 19 and Cd36 OE n = 24 mice), transduced with PMSCV-EV (empty vector) or Cd36-overexpression vector. Graphs show the frequency of developed tumours (SCC-25 ***P = 0.05, JHU-029 ***P = 0.03, Fisher exact test) and BLI signal quantifications (primary tumour, *P = 0.01 and ****P < 0.0001; metastasis, **P = 0.007, *P = 0.01, two-tailed t-test). Data are given as the mean ± s.e.m. c, d, Haematoxylin and eosin staining (c) and anti-human CD44 immunostaining (d) of lymph nodes isolated from animals reported in a (n = 5 animals per group). e, RT–qPCR analysis of OSCC parental and CD36OE cells. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, **P < 0.005, *P < 0.05, two-tailed t-test), data are given as the mean ± s.e.m. f, g, Flow cytometry analysis of OSCC tumours derived from PMSCV-EV or CD36OE cell transplants (n = 5 animals per group). Source data from mouse experiments are in Supplementary Information.

  9. Depletion of CD36 inhibits metastatic initiation and progression.
    Extended Data Fig. 4: Depletion of CD36 inhibits metastatic initiation and progression.

    a, BLI signal quantifications (*P = 0.01, two-tailed t-test) and frequency of developed tumours (*P = 0.04, two-tailed Fisher’s exact test) of PMSCV-EV and CD36–overexpressing tumours from VDH-00 primary cell line (PMSCV-EV, n = 7; CD36OE, n = 8). b, BLI monitoring of tumours from FaDu cell line transduced with either PLKO or shRNA CD36 (two independent experiments: exp1. and exp.2, n = 5 mice per group). Graphs show the frequency of developed tumours, and BLI signal quantification (metastasis lymph node, *P = 0.05; metastasis lung, **P = 0.002; two-tailed t-test). c, d, Flow cytometry analysis of tumours from OSCC cells transduced with PLKO or shRNA CD36#99. Numbers indicate the percentages of CD44bright CD36+, CD44bright CD36 or CD44dim cells in the represented gate (n = 6 animals per group). e, Relative RNA levels of CD36 in SCC-25 parental and shRNA CD36 cells, determined by RT–qPCR analysis using TaqMan gene expression assay. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, ****P < 0.005, two-tailed t-test). Data in a, b, e, are given as the mean ± s.e.m. f, Representative images of lungs from mice transplanted with PLKO or shRNACD36 FaDu cells (PLKO, n = 5 mice; shRNA CD36#99, n = 5 mice). g, Haematoxylin and eosin staining of metastatic lymph nodes from cells transduced with PLKO or shRNA Cd36. h, Representative haematoxylin-eosin staining of primary tumours from transplanted SCC-25 cells transduced with PLKO or Cd36 shRNA (n = 5 mice per group). Source data from mouse experiments are in Supplementary Information.

  10. CD36+ cells are defined by a lipid metabolism and metastatic signature, and require the fatty acid β-oxidation enzyme ACSL1 to promote metastasis.
    Extended Data Fig. 5: CD36+ cells are defined by a lipid metabolism and metastatic signature, and require the fatty acid β-oxidation enzyme ACSL1 to promote metastasis.

    a, b, Top categories for diseases (a) and biological process (b) upregulated in CD36+ CD44bright cells. c, Gene set enrichment analysis (GSEA) plot of CD36-associated signatures, highlighting strong enrichment for fatty acid metabolism. NES denotes normalized enrichment score. d, Comparative analysis of overlapping genes between CD36+ CD44bright and CD44bright DID+ upregulated signature, highlighting over-represented genes associated with lipid metabolism, cancer invasion and metastasis and transport and metabolism of nucleoside drugs. P = 1.359 × 10−16, hypergeometric test. e, Flow cytometry analysis of in vitro SCC-25 cells co-cultured with adipogenic OP-9 cells, showing the expression of three enzymes of fatty acid β-oxidation (ACADVL, ACADM and HADHA). Histograms show the average normalized number of events as a function of fluorescence intensity for the three enzymes (n = 2 biological replicates). f, BLI monitoring of tumours generated from OSCC cells transduced with either scrambled shRNA (SCR, n = 5 mice) or shRNA ACSL1#936 (n = 5 mice). Graphs show the frequency of developed tumours and the BLI signal quantification (**P = 0.001 and *P = 0.003, two-tailed t-test). g, Haematoxylin and eosin staining of metastatic lymph nodes from animals reported in f, showing the smaller metastases arising from Acsl1 shRNA transplants as compared to the control SCR (n = 5 animals per group). h, BLI monitoring of orthotopic transplants from CD36-overexpressing JHU-029 cells transduced with either control (SCR, n = 10 mice) or shRNA ACSL1#936 (n = 10 mice). Graphs show the BLI signal quantification (metastasis: *P = 0.03 and *P = 0.03, two-tailed t-test) and the frequency of developed tumours (CT vs OE-SCR *P = 0.03 and OE-SCR vs OE-shACSL1 *P = 0.04, Fisher exact test). i, Histogram shows the average normalized number of events as a function of CD36 fluorescence intensity. j, Relative RNA levels of OSCC cells reported in j, by RT–qPCR analysis. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, P = 0.03, two-tailed t-test). Data in f, h, j, are given as the mean ± s.e.m. Source data from mouse experiments are in Supplementary Information.

  11. CD36+ cells are stimulated by a high-fat diet or adipocyte-conditioned medium, and require the ability of CD36 to internalize fatty acids for their pro-metastatic potential.
    Extended Data Fig. 6: CD36+ cells are stimulated by a high-fat diet or adipocyte-conditioned medium, and require the ability of CD36 to internalize fatty acids for their pro-metastatic potential.

    a, Flow cytometry analysis of orthotopic transplants of Detroit-562 cells transduced with PLKO or shRNACD36#98 or #99, from mice fed with high-fat diet (HFD) or control diet (CD), analysed 4 weeks after OSCC injection. Numbers indicate CD44bright CD36+, CD44bright CD36 and CD44dim (differentiated) cells in the represented gate, expressed as percentages from the total GFP+ Lin OSCC parental tumour. n = 5 animals per group. b, Flow cytometry analysis of co-cultured SCC-25/OP-9, SCC-25/adipogenic OP9 or SCC-25/HNCAFS (head and neck cancer–associated fibroblasts) cells. Numbers indicate CD36+ cells in the represented gate, expressed as percentage. c, FACS analysis of co-cultured Detroit-562 or SCC-25 with OP9 (control) or adipogenic OP9, showing an increase in the percentage of CD36-positive cells in the adipogenic co-cultures. Numbers indicate CD44bright CD36+ and CD44bright CD36 from the total GFP+CD29 OSCC cells. d, CD36 mRNA relative expression levels, measured by RT–qPCR, from SCC-25 CD36 sorted cells either co-cultured with adipogenic OP9 (Ad.OP9) cells or not, or from SCC-25 CD36+ sorted cells co-cultured with Ad.OP9 cells. In bd, OSCC were co-cultured in vitro for 2 days. e, Flow cytometry analysis of OSCC cells co-cultured with adipogenic OP-9 cells or with 0.4 mM palmitic acid (PA). Histograms show the average normalized number of events as a function of CD36 and CD44 fluorescence intensity. f, cDNA and amino acid sequence of the CD36 receptor at the level of the point mutation introduced to generate the fatty acid-binding site mutant, CD36-K164A (left). Fatty acid uptake assay is shown for SCC-25 cells not transduced (as control, CT) or transduced with CD36wt (overexpressing wild-type CD36), shRNA Cd36 or CD36-K164A. g, BLI monitoring of transplants from SCC-25 cells overexpressing CD36wt (wild-type, n = 10) or CD36-K164A (n = 10). Frequency of developed tumours is expressed as percentage (*P = 0.02, Fisher exact test), and BLI signal quantification is expressed as the relative normalized photon flux (* P= 0.05, two-tailed t-test). Data are given as the mean ± s.e.m. h, FACS analysis of OSCC cells overexpressing either CD36 wild-type (wt) or mutant (Lys164mut). Histograms show the average normalized number of events as a function of CD36 and CD44 fluorescence intensity. Source data from mouse experiments are in Supplementary Information.

  12. Inhibition of CD36 results in metastatic lipotoxicity, and CD36+ cells are the only cells capable of initiating metastasis.
    Extended Data Fig. 7: Inhibition of CD36 results in metastatic lipotoxicity, and CD36+ cells are the only cells capable of initiating metastasis.

    a, Representative haematoxylin and eosin staining of metastatic lymph nodes from SCC-25-pLucGFP transplants with overexpressed wild-type CD36 or CD36-K164A. Dashed line denotes the areas surrounded by lipid droplets in the CD36-K164A-expressing cells. b, c, Caspase-3 immunostaining of the metastases reported in a and in Cd36 shRNA FaDu-pLucGFP metastatic lymph nodes, showing activated casp-3-positive apoptotic cells in the vicinity of droplets. d, Relative expression levels expressed as percentages of four populations, CD36+ CD44bright, CD36+ CD44dim, CD36 CD44bright and CD36 CD44dim, as determined by FACS analysis of the primary tumour and metastasis of the OSCC cell lines SCC-25, JHU-029, Detroit-562 and FaDu and the PDCs VDH-00, VDH-01 and VDH-02 (n = 4 biological replicates per cell line). e, Genes differentially expressed between CD36+ CD44bright and CD36+ CD44bright populations validated by RT–qPCR with human-specific TaqMan gene expression assays in SCC-25 EV (empty vector), SCC-25 CD36-overexpressing and SCC-25 Cd36 shRNA cells grown in vitro. Human β-2-microglobulin was used as internal control gene (n = 4 biological replicates, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed t-test). f, OSSC cells were co-cultured with adipogenic OP9 cells, FACS-sorted and injected into the oral cavity of NSG mice. g, FACS strategy to isolate CD36+ CD44bright, CD36 CD44bright and CD44bright cells from in vitro SCC-25 cells co-cultured with adipogenic OP-9 cells. Serial limiting dilutions of the different populations were injected immediately after FACS sorting. h, i, BLI monitoring (h) and primary tumour quantification (i) of mice injected with CD44bright CD36+ or CD44bright CD36 cells. Yellow arrows denote increased affinity in injected OSCC for the metastatic place, observed in some animals. j, k, Metastasis-initiating cell (MIC) frequency (j) and tumour-initiating cell (TIC) frequency (k) of the three different populations in g, as determined by ELDA software statistical analysis. Source data from mouse experiments are in Supplementary Information.

  13. CD36+ cells recapitulate the cellular and molecular heterogeneity of primary tumours and metastases when orthotopically transplanted.
    Extended Data Fig. 8: CD36+ cells recapitulate the cellular and molecular heterogeneity of primary tumours and metastases when orthotopically transplanted.

    a, Overview of experimental set-up. Detroit-562 cells co-cultured with adipogenic OP-9 cells were FACS-sorted to select the CD44bright and CD36+ CD44bright populations. Selected cells were then injected orthotopically into NSG mice. Tumours were collected after 4 weeks, and cells were isolated for gene expression analysis by microarray. b, CD36-associated signatures from lymph node metastases arising from CD36+ CD44bright or primary tumour CD44bright transplants, showing the top upregulated categories for diseases and biological processes. c, GSEA analysis of lymph node metastases from CD36+ CD44bright and primary tumours from CD44bright transplants. Ranked lists of primary tumour comparison versus top 300 genes of lymph node-Met sorted by fold change (FC) and ranked lists of lymph node-Met CD36+ comparison versus top 300 genes of primary tumour sorted by fold change (FC). Nominal P < 0.0001. All source data from mouse experiments are in Supplementary Information.

  14. Anti-CD36 neutralizing antibodies inhibit metastatic initiation, and cause metastatic regression of oral SCC.
    Extended Data Fig. 9: Anti-CD36 neutralizing antibodies inhibit metastatic initiation, and cause metastatic regression of oral SCC.

    a, BLI quantification of tumours from mice treated with anti-CD36 FA6.152 (anti-CD36 FA6.152, n = 3 mice; IgG1, n = 3 mice; **P = 0.004, two-tailed t-test). b, d, BLI monitoring of tumours from mice treated daily with anti-CD36 JC63.1 (anti-CD36: n = 5 mice; anti-IgA isotype control, n = 5 mice). Graphs show the BLI signal quantification (*P = 0.04, two-tailed t-test). c, Representative pictures of metastatic lymph nodes of animals treated daily with JC63.1 or IgA for 2.5 weeks. e, Activated caspase-3 immunostaining of metastatic lymph nodes of Detroit-562 transplants from mice treated with monoclonal anti-CD36 JC63.1 (10 μg per 100 μl), or with the IgA isotype control. f, BLI monitoring of immunocompetent C3H/HeJ mice treated daily with monoclonal JC63.1 or IgA. Graphs show BLI signals from tumours (*P = 0.05, two-tailed t-test). g, Fold change in metastasis BLI signal of the animals reported in d. h, Representative haematoxylin and eosin staining of liver, spleen, thymus and kidney of mice from f. No pathological differences related to anti-CD36 treatment were found (n = 10 animals per group). Data in a, d, f, g are given as the mean ± s.e.m.

  15. Expression of CD36 correlates with poor prognosis in several human tumours, and inhibition of CD36 inhibits metastasis of human melanoma and luminal A breast carcinoma cell lines.
    Extended Data Fig. 10: Expression of CD36 correlates with poor prognosis in several human tumours, and inhibition of CD36 inhibits metastasis of human melanoma and luminal A breast carcinoma cell lines.

    a, Correlation of CD36-associated signature expression or CD36 expression with overall and disease-free survival for patients. Red and green lines denote patients whose tumours expressed signatures or CD36 higher and lower than the median, respectively. b, BLI signals from metastasis developed in NSG mice injected with MCF-7 (PLKO, n = 10; Cd36 shRNA, n = 10 mice) and 501mel (PLKO, n = 10; Cd36 shRNA, n = 10 mice) cells (for breast MCF-7, *P = 0.04, two-tailed t-test and for melanoma 501mel, ***P = 0.0001 in liver metastasis and **P = 0.0003 in lung metastasis, two-tailed t-test). c, Relative proportion of developed metastases from mice in a (*P = 0.05, two-tailed Fisher’s exact test). d, BLI signals from primary tumours and relative blood and lung GFP RNA levels measured by qPCR analysis after intravenous injection of Detroit-562 and SCC-25 cells transduced with empty vector (control) or shRNA Cd36. Samples were collected immediately after injection (T-0h) and 12 and 48 h (T-12h and T-48h, respectively) after injection (n = 3 animals per time point in each of the groups; *P ≤ 0.05, two-tailed t-test). Data in b, d, are given as the mean ± s.e.m. e, GSEA of EMT genes in CD36+ and CD36 cells sorted from primary oral lesions (generated from CD44bright inoculated cells), or from lymph node metastases (generated from CD36+ CD44bright inoculated cells). CD36 cells express higher levels of EMT genes than CD36+ cells in both the primary lesion and lymph node metastases. Genes are ranked by t-statistic value. Enriched populations are indicated for each of the plots. Lower panels show the GSEA analysis of the same cohort of EMT genes compared between lymph node metastases and primary oral lesions within CD36+ cells or CD36 cells. Source data from mouse experiments are in Supplementary Information.

Change history

Corrected online 13 December 2016
The received date was corrected in the HTML.
Corrected online 04 January 2017
The Competing Interests statement and the Acknowledgements funding information were updated.

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Author information

Affiliations

  1. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain

    • Gloria Pascual,
    • Alexandra Avgustinova,
    • Mercè Martín,
    • Andrés Castellanos,
    • Camille Stephan-Otto Attolini,
    • Antoni Berenguer,
    • Neus Prats &
    • Salvador Aznar Benitah
  2. Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Dr. Aiguader 88, 08003 Barcelona, Spain

    • Stefania Mejetta &
    • Luciano Di Croce
  3. IMIM, Department of Dermatology, Hospital del Mar, 08003 Barcelona

    • Agustí Toll
  4. Vall D´Hebron Hospital, Barcelona, Department of Oral and Maxillofacial Surgery, Universitat Autònoma de Barcelona, Barcelona 08035 Spain

    • Juan Antonio Hueto &
    • Coro Bescós
  5. Catalan Institution for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain

    • Luciano Di Croce &
    • Salvador Aznar Benitah
  6. Universitat Pompeu Fabra (UPF), Barcelona 08002, Spain

    • Luciano Di Croce

Contributions

G.P. and S.A.B. designed all experiments. G.P. performed all experiments with the help of M.M. for the histological characterization of the lipotoxicity and A.C. for the analysis of the gene expression data. A.A. established the patient-derived cells and the oral cancer orthotopic method. C.S.-O.A. and A.B. performed statistical analyses. J.A.H., C.B. and A.T. provided the tumours from patients. S.M. established the dye protocol to detect LRCs. N.P. performed the histopathology analysis of the mice. L.D.C. analysed expression data. G.P. and S.A.B. wrote the manuscript.

Competing financial interests

The Institute for Research in Biomedicine in Barcelona has filed a provisional patent application that covers the application of inhibition of the fatty acid receptor CD3 by any method as an antimetastatic therapy against oral squamous cell carcinoma (European patent application number EP 2016/073208). Authors S.A.B., G.P., A.C. and M.M. are listed as inventors.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks A. Harris and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Orthotopically inoculated human oral squamous cell carcinomas contain a slow-cycling sub-population of CD44bright cells. (694 KB)

    a, Overview of the tumorigenic and metastatic activities of the different OSCC cell lines injected into the tongues of NSG mice. b, Tumour development from mice injected with OSCC-pLuc-GFP cells (using the cell lines indicated). Tumour growth was monitored by bioluminescence imaging (BLI) over a four-week period. Data are given as the mean ± s.e.m. c, Frequency of metastases in the lymph nodes. ac, Detroit-562 cells, two independent experiments: exp. 1 n = 10 mice; exp. 2 n = 11 mice; VDH-02, n = 20 mice; VDH-01, n = 20 mice; VDH-00, n = 8 mice; SCC-25, three independent experiments: exp. 1 n = 13 mice, exp. 2 n = 17 mice, exp. 3 n = 7 mice; JHU-029, three independent experiments, n = 12 mice per experiment; FaDu, two independent experiments, exp. 1 n = 14 mice, exp. 2 n = 5 mice. d, Immunofluorescence analysis of in vitro cultured OSCC-RFP cells pulsed with DID and grown in 2D culture for 16 days. e, f, Flow cytometry analysis of dye-pulsed OSCC cells in vitro showing the kinetics of dye dilution. Data are given as mean fluorescence intensity. g, FACS strategy to FACS-sort CD44bright dye+, CD44bright dye and CD44dim cells from OSCC-pLucGFP oral tumours. Viable single cells were selected if GFP+ but negative for a lineage (Lin) cocktail of antibodies (H2KD, CD31 and CD45), to select human cells. GFP+ Lin cells were gated for CD44 and dye. Percentages from the total GFP+ Lin SCC-25 parental tumour are shown. h, Representative flow cytometry analyses to detect quiescent slow-cycling CSCs from OSCC cell lines. g, h, n = 8 animals per OSCC cell line. i, Global quantification of CD44bright dye+, CD44bright dye and CD44dim cells from OSCC-pLucGFP tumours reported in g and h. j, Immunofluorescence analysis of SCC-25-pLucGFP and JHU-029-pLucGFP primary tumours, collected five weeks after OSCC inoculation, to detect dye+ quiescent slow-cycling cancer stem cells (CSCs). Insets show a magnification of dye+ cells that co-localized with the CD44 marker. SCC-25, n = 5 tumours; JHU-029, n = 5 tumours. k, Percentage of dividing cells by flow cytometry analysis in the dye+, dye and CD44dim populations. Source data from mouse experiments are in Supplementary Information.

  2. Extended Data Figure 2: Oral SCC label-retaining cells are defined by a lipid metabolism and metastasis transcriptome signature. (622 KB)

    a, Microarray analysis and heatmap of mRNA expression showing differentially expressed genes in dye+, dye and CD44dim cells. n = 4 biological replicates and 8 mice per replicate. b, Gene ontology (GO) analysis showing the top categories for diseases, biological processes and signal transduction pathways that were upregulated in the proliferative active (DID) as compared to LR-CSCs (DID+) populations. The resulting GO terms highlighted cell cycle–related categories. c, Over-represented genes in Dye+ and Dye populations. d, Lipid metabolism genes over-represented in dye+ cells. e, Gene ontology (GO) analysis showing top diseases and biological processes categories upregulated in the DID+ (LR-CSCs) and DID (proliferative) sorted populations from dye-pulsed Detroit-562 tumours analysed by microarrays. f, RT–qPCR validation by human-specific TaqMan gene expression assays of differentially expressed genes by microarray in the CD44+ DID+ and CD44+ DID populations. Data are given as relative expression levels. Human β-2-microglobulin was used as internal control gene. n = 5, *P < 0.05, **P < 0.005, two-tailed t-test. g, Gene expression overlapping analysis of the LR-CSC signatures from SCC-25 and Detroit-562 tumours showing the top represented common diseases and biological processes. Metastatic processes and lipid metabolism-related categories are highlighted in red. P = 2.10 × 10−49, hypergeometric test. h, Correlation between CD36 expression and DiD content for orthotopic transplants of SCC-25, JHU-029, Detroit-562, FaDu, VDH-00, VDH-01 and VDH-02 cells (n = 8 animals per cell line). Numbers indicate percentages from the total GFP+Lin OSCC parental tumour. Results are given as the mean ± s.e.m. (n = 7 OSCC orthotopic transplants; ***P = 0.0008, *P= 0.03, two-tailed t-test).

  3. Extended Data Figure 3: LRCs correspond to CD36+ cells, and CD36 overexpression promotes metastatic initiation and progression. (856 KB)

    a, CD36+ CD44bright OSCC cells detected by flow cytometry analysis of tumours from orthotopic transplants. Tumours were obtained from OSCC Detroit-562 (three independent experiments: exp. 1 n = 3, exp. 2 n = 3, exp. 3 n = 4 mice), JHU-029 (three independent experiments: exp. 1 n = 3, exp. 2 n = 3, exp. 3 n = 4 mice), SCC-25 (n = 8 mice), FaDu (n = 8 mice), VDH-00 (n = 8 mice), VDH-01 (n = 8 mice) and VDH-02 cells (n = 8 mice). Numbers indicate CD44bright CD36bright or CD44bright CD36low cells in the represented gate, expressed as percentages from the total GFP+ Lin OSCC parental tumour. Histograms show the correlation between CD36 expression and the DID content. The average counted events as a function of dye fluorescence intensity is reported for each population CD44bright CD36bright and CD44bright CD36low. b, BLI monitoring of tumours generated by SCC-25 cells (empty vector (EV) n = 7 and Cd36 overexpression (OE) n = 17 mice), or JHU-029 cells (EV n = 19 and Cd36 OE n = 24 mice), transduced with PMSCV-EV (empty vector) or Cd36-overexpression vector. Graphs show the frequency of developed tumours (SCC-25 ***P = 0.05, JHU-029 ***P = 0.03, Fisher exact test) and BLI signal quantifications (primary tumour, *P = 0.01 and ****P < 0.0001; metastasis, **P = 0.007, *P = 0.01, two-tailed t-test). Data are given as the mean ± s.e.m. c, d, Haematoxylin and eosin staining (c) and anti-human CD44 immunostaining (d) of lymph nodes isolated from animals reported in a (n = 5 animals per group). e, RT–qPCR analysis of OSCC parental and CD36OE cells. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, **P < 0.005, *P < 0.05, two-tailed t-test), data are given as the mean ± s.e.m. f, g, Flow cytometry analysis of OSCC tumours derived from PMSCV-EV or CD36OE cell transplants (n = 5 animals per group). Source data from mouse experiments are in Supplementary Information.

  4. Extended Data Figure 4: Depletion of CD36 inhibits metastatic initiation and progression. (663 KB)

    a, BLI signal quantifications (*P = 0.01, two-tailed t-test) and frequency of developed tumours (*P = 0.04, two-tailed Fisher’s exact test) of PMSCV-EV and CD36–overexpressing tumours from VDH-00 primary cell line (PMSCV-EV, n = 7; CD36OE, n = 8). b, BLI monitoring of tumours from FaDu cell line transduced with either PLKO or shRNA CD36 (two independent experiments: exp1. and exp.2, n = 5 mice per group). Graphs show the frequency of developed tumours, and BLI signal quantification (metastasis lymph node, *P = 0.05; metastasis lung, **P = 0.002; two-tailed t-test). c, d, Flow cytometry analysis of tumours from OSCC cells transduced with PLKO or shRNA CD36#99. Numbers indicate the percentages of CD44bright CD36+, CD44bright CD36 or CD44dim cells in the represented gate (n = 6 animals per group). e, Relative RNA levels of CD36 in SCC-25 parental and shRNA CD36 cells, determined by RT–qPCR analysis using TaqMan gene expression assay. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, ****P < 0.005, two-tailed t-test). Data in a, b, e, are given as the mean ± s.e.m. f, Representative images of lungs from mice transplanted with PLKO or shRNACD36 FaDu cells (PLKO, n = 5 mice; shRNA CD36#99, n = 5 mice). g, Haematoxylin and eosin staining of metastatic lymph nodes from cells transduced with PLKO or shRNA Cd36. h, Representative haematoxylin-eosin staining of primary tumours from transplanted SCC-25 cells transduced with PLKO or Cd36 shRNA (n = 5 mice per group). Source data from mouse experiments are in Supplementary Information.

  5. Extended Data Figure 5: CD36+ cells are defined by a lipid metabolism and metastatic signature, and require the fatty acid β-oxidation enzyme ACSL1 to promote metastasis. (635 KB)

    a, b, Top categories for diseases (a) and biological process (b) upregulated in CD36+ CD44bright cells. c, Gene set enrichment analysis (GSEA) plot of CD36-associated signatures, highlighting strong enrichment for fatty acid metabolism. NES denotes normalized enrichment score. d, Comparative analysis of overlapping genes between CD36+ CD44bright and CD44bright DID+ upregulated signature, highlighting over-represented genes associated with lipid metabolism, cancer invasion and metastasis and transport and metabolism of nucleoside drugs. P = 1.359 × 10−16, hypergeometric test. e, Flow cytometry analysis of in vitro SCC-25 cells co-cultured with adipogenic OP-9 cells, showing the expression of three enzymes of fatty acid β-oxidation (ACADVL, ACADM and HADHA). Histograms show the average normalized number of events as a function of fluorescence intensity for the three enzymes (n = 2 biological replicates). f, BLI monitoring of tumours generated from OSCC cells transduced with either scrambled shRNA (SCR, n = 5 mice) or shRNA ACSL1#936 (n = 5 mice). Graphs show the frequency of developed tumours and the BLI signal quantification (**P = 0.001 and *P = 0.003, two-tailed t-test). g, Haematoxylin and eosin staining of metastatic lymph nodes from animals reported in f, showing the smaller metastases arising from Acsl1 shRNA transplants as compared to the control SCR (n = 5 animals per group). h, BLI monitoring of orthotopic transplants from CD36-overexpressing JHU-029 cells transduced with either control (SCR, n = 10 mice) or shRNA ACSL1#936 (n = 10 mice). Graphs show the BLI signal quantification (metastasis: *P = 0.03 and *P = 0.03, two-tailed t-test) and the frequency of developed tumours (CT vs OE-SCR *P = 0.03 and OE-SCR vs OE-shACSL1 *P = 0.04, Fisher exact test). i, Histogram shows the average normalized number of events as a function of CD36 fluorescence intensity. j, Relative RNA levels of OSCC cells reported in j, by RT–qPCR analysis. Human β-2-microglobulin was used as internal control gene (n = 3 biological replicates, P = 0.03, two-tailed t-test). Data in f, h, j, are given as the mean ± s.e.m. Source data from mouse experiments are in Supplementary Information.

  6. Extended Data Figure 6: CD36+ cells are stimulated by a high-fat diet or adipocyte-conditioned medium, and require the ability of CD36 to internalize fatty acids for their pro-metastatic potential. (569 KB)

    a, Flow cytometry analysis of orthotopic transplants of Detroit-562 cells transduced with PLKO or shRNACD36#98 or #99, from mice fed with high-fat diet (HFD) or control diet (CD), analysed 4 weeks after OSCC injection. Numbers indicate CD44bright CD36+, CD44bright CD36 and CD44dim (differentiated) cells in the represented gate, expressed as percentages from the total GFP+ Lin OSCC parental tumour. n = 5 animals per group. b, Flow cytometry analysis of co-cultured SCC-25/OP-9, SCC-25/adipogenic OP9 or SCC-25/HNCAFS (head and neck cancer–associated fibroblasts) cells. Numbers indicate CD36+ cells in the represented gate, expressed as percentage. c, FACS analysis of co-cultured Detroit-562 or SCC-25 with OP9 (control) or adipogenic OP9, showing an increase in the percentage of CD36-positive cells in the adipogenic co-cultures. Numbers indicate CD44bright CD36+ and CD44bright CD36 from the total GFP+CD29 OSCC cells. d, CD36 mRNA relative expression levels, measured by RT–qPCR, from SCC-25 CD36 sorted cells either co-cultured with adipogenic OP9 (Ad.OP9) cells or not, or from SCC-25 CD36+ sorted cells co-cultured with Ad.OP9 cells. In bd, OSCC were co-cultured in vitro for 2 days. e, Flow cytometry analysis of OSCC cells co-cultured with adipogenic OP-9 cells or with 0.4 mM palmitic acid (PA). Histograms show the average normalized number of events as a function of CD36 and CD44 fluorescence intensity. f, cDNA and amino acid sequence of the CD36 receptor at the level of the point mutation introduced to generate the fatty acid-binding site mutant, CD36-K164A (left). Fatty acid uptake assay is shown for SCC-25 cells not transduced (as control, CT) or transduced with CD36wt (overexpressing wild-type CD36), shRNA Cd36 or CD36-K164A. g, BLI monitoring of transplants from SCC-25 cells overexpressing CD36wt (wild-type, n = 10) or CD36-K164A (n = 10). Frequency of developed tumours is expressed as percentage (*P = 0.02, Fisher exact test), and BLI signal quantification is expressed as the relative normalized photon flux (* P= 0.05, two-tailed t-test). Data are given as the mean ± s.e.m. h, FACS analysis of OSCC cells overexpressing either CD36 wild-type (wt) or mutant (Lys164mut). Histograms show the average normalized number of events as a function of CD36 and CD44 fluorescence intensity. Source data from mouse experiments are in Supplementary Information.

  7. Extended Data Figure 7: Inhibition of CD36 results in metastatic lipotoxicity, and CD36+ cells are the only cells capable of initiating metastasis. (1,106 KB)

    a, Representative haematoxylin and eosin staining of metastatic lymph nodes from SCC-25-pLucGFP transplants with overexpressed wild-type CD36 or CD36-K164A. Dashed line denotes the areas surrounded by lipid droplets in the CD36-K164A-expressing cells. b, c, Caspase-3 immunostaining of the metastases reported in a and in Cd36 shRNA FaDu-pLucGFP metastatic lymph nodes, showing activated casp-3-positive apoptotic cells in the vicinity of droplets. d, Relative expression levels expressed as percentages of four populations, CD36+ CD44bright, CD36+ CD44dim, CD36 CD44bright and CD36 CD44dim, as determined by FACS analysis of the primary tumour and metastasis of the OSCC cell lines SCC-25, JHU-029, Detroit-562 and FaDu and the PDCs VDH-00, VDH-01 and VDH-02 (n = 4 biological replicates per cell line). e, Genes differentially expressed between CD36+ CD44bright and CD36+ CD44bright populations validated by RT–qPCR with human-specific TaqMan gene expression assays in SCC-25 EV (empty vector), SCC-25 CD36-overexpressing and SCC-25 Cd36 shRNA cells grown in vitro. Human β-2-microglobulin was used as internal control gene (n = 4 biological replicates, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed t-test). f, OSSC cells were co-cultured with adipogenic OP9 cells, FACS-sorted and injected into the oral cavity of NSG mice. g, FACS strategy to isolate CD36+ CD44bright, CD36 CD44bright and CD44bright cells from in vitro SCC-25 cells co-cultured with adipogenic OP-9 cells. Serial limiting dilutions of the different populations were injected immediately after FACS sorting. h, i, BLI monitoring (h) and primary tumour quantification (i) of mice injected with CD44bright CD36+ or CD44bright CD36 cells. Yellow arrows denote increased affinity in injected OSCC for the metastatic place, observed in some animals. j, k, Metastasis-initiating cell (MIC) frequency (j) and tumour-initiating cell (TIC) frequency (k) of the three different populations in g, as determined by ELDA software statistical analysis. Source data from mouse experiments are in Supplementary Information.

  8. Extended Data Figure 8: CD36+ cells recapitulate the cellular and molecular heterogeneity of primary tumours and metastases when orthotopically transplanted. (651 KB)

    a, Overview of experimental set-up. Detroit-562 cells co-cultured with adipogenic OP-9 cells were FACS-sorted to select the CD44bright and CD36+ CD44bright populations. Selected cells were then injected orthotopically into NSG mice. Tumours were collected after 4 weeks, and cells were isolated for gene expression analysis by microarray. b, CD36-associated signatures from lymph node metastases arising from CD36+ CD44bright or primary tumour CD44bright transplants, showing the top upregulated categories for diseases and biological processes. c, GSEA analysis of lymph node metastases from CD36+ CD44bright and primary tumours from CD44bright transplants. Ranked lists of primary tumour comparison versus top 300 genes of lymph node-Met sorted by fold change (FC) and ranked lists of lymph node-Met CD36+ comparison versus top 300 genes of primary tumour sorted by fold change (FC). Nominal P < 0.0001. All source data from mouse experiments are in Supplementary Information.

  9. Extended Data Figure 9: Anti-CD36 neutralizing antibodies inhibit metastatic initiation, and cause metastatic regression of oral SCC. (2,352 KB)

    a, BLI quantification of tumours from mice treated with anti-CD36 FA6.152 (anti-CD36 FA6.152, n = 3 mice; IgG1, n = 3 mice; **P = 0.004, two-tailed t-test). b, d, BLI monitoring of tumours from mice treated daily with anti-CD36 JC63.1 (anti-CD36: n = 5 mice; anti-IgA isotype control, n = 5 mice). Graphs show the BLI signal quantification (*P = 0.04, two-tailed t-test). c, Representative pictures of metastatic lymph nodes of animals treated daily with JC63.1 or IgA for 2.5 weeks. e, Activated caspase-3 immunostaining of metastatic lymph nodes of Detroit-562 transplants from mice treated with monoclonal anti-CD36 JC63.1 (10 μg per 100 μl), or with the IgA isotype control. f, BLI monitoring of immunocompetent C3H/HeJ mice treated daily with monoclonal JC63.1 or IgA. Graphs show BLI signals from tumours (*P = 0.05, two-tailed t-test). g, Fold change in metastasis BLI signal of the animals reported in d. h, Representative haematoxylin and eosin staining of liver, spleen, thymus and kidney of mice from f. No pathological differences related to anti-CD36 treatment were found (n = 10 animals per group). Data in a, d, f, g are given as the mean ± s.e.m.

  10. Extended Data Figure 10: Expression of CD36 correlates with poor prognosis in several human tumours, and inhibition of CD36 inhibits metastasis of human melanoma and luminal A breast carcinoma cell lines. (399 KB)

    a, Correlation of CD36-associated signature expression or CD36 expression with overall and disease-free survival for patients. Red and green lines denote patients whose tumours expressed signatures or CD36 higher and lower than the median, respectively. b, BLI signals from metastasis developed in NSG mice injected with MCF-7 (PLKO, n = 10; Cd36 shRNA, n = 10 mice) and 501mel (PLKO, n = 10; Cd36 shRNA, n = 10 mice) cells (for breast MCF-7, *P = 0.04, two-tailed t-test and for melanoma 501mel, ***P = 0.0001 in liver metastasis and **P = 0.0003 in lung metastasis, two-tailed t-test). c, Relative proportion of developed metastases from mice in a (*P = 0.05, two-tailed Fisher’s exact test). d, BLI signals from primary tumours and relative blood and lung GFP RNA levels measured by qPCR analysis after intravenous injection of Detroit-562 and SCC-25 cells transduced with empty vector (control) or shRNA Cd36. Samples were collected immediately after injection (T-0h) and 12 and 48 h (T-12h and T-48h, respectively) after injection (n = 3 animals per time point in each of the groups; *P ≤ 0.05, two-tailed t-test). Data in b, d, are given as the mean ± s.e.m. e, GSEA of EMT genes in CD36+ and CD36 cells sorted from primary oral lesions (generated from CD44bright inoculated cells), or from lymph node metastases (generated from CD36+ CD44bright inoculated cells). CD36 cells express higher levels of EMT genes than CD36+ cells in both the primary lesion and lymph node metastases. Genes are ranked by t-statistic value. Enriched populations are indicated for each of the plots. Lower panels show the GSEA analysis of the same cohort of EMT genes compared between lymph node metastases and primary oral lesions within CD36+ cells or CD36 cells. Source data from mouse experiments are in Supplementary Information.

Supplementary information

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  1. Supplementary Information (165 KB)

    This file contains full legends for Supplementary Tables 1-8 and Supplementary Tables 9-11.

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  1. Supplementary Tables (21.3 MB)

    This zipped file contains Supplementary Tables 1-8.

Additional data