Targeting metastasis-initiating cells through the fatty acid receptor CD36 (original) (raw)

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Acknowledgements

Research in the laboratory of S.A.B. for this project is supported by the European Research Council (ERC), the Government of Cataluña (SGR grant), the Fundación Botín and Banco Santander, through Santander Universities, and Worldwide Cancer Research. We would like to thank the Beug Stiftung Foundation for their support. S.M. was supported by a La Caixa International PhD fellowship. A.A. was supported by an EU Cofound postdoctoral fellowship. L.D.C. was supported by the Spanish ‘Ministerio de Educación y Ciencia’ (SAF2013-48926-P) and the European Commission’s 7th Framework Program 4DCellFate grant number 277899. We thank the Vall D´Hebron Research Institute Tumor Biobank for their assistance with the human samples. We also thank R. Wong for the Ln-7 cell line and J. Zuber for the PMSCV-Luc2-PGKneo-Ires GFP vector. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). We thank V. Raker for manuscript editing.

Author information

Authors and Affiliations

  1. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, 08028, 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, Barcelona, 08003, Spain
    Stefania Mejetta & Luciano Di Croce
  3. Department of Dermatology, IMIM, Hospital del Mar, Barcelona, 08003
    Agustí Toll
  4. Department of Oral and Maxillofacial Surgery, Vall D´Hebron Hospital, Barcelona, Universitat Autònoma de Barcelona, Barcelona, 08035, Spain
    Juan Antonio Hueto & Coro Bescós
  5. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, 08010, Spain
    Luciano Di Croce & Salvador Aznar Benitah
  6. Universitat Pompeu Fabra (UPF), Barcelona, 08002, Spain
    Luciano Di Croce

Authors

  1. Gloria Pascual
  2. Alexandra Avgustinova
  3. Stefania Mejetta
  4. Mercè Martín
  5. Andrés Castellanos
  6. Camille Stephan-Otto Attolini
  7. Antoni Berenguer
  8. Neus Prats
  9. Agustí Toll
  10. Juan Antonio Hueto
  11. Coro Bescós
  12. Luciano Di Croce
  13. Salvador Aznar Benitah

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.

Corresponding author

Correspondence toSalvador Aznar Benitah.

Ethics declarations

Competing 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.

Additional information

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

Extended data figures and tables

Extended Data Figure 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. a–c, 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.

Source data

Extended Data Figure 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).

Extended Data Figure 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.

Source data

Extended Data Figure 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.

Source data

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.

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.

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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.

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 b–d, 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.

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Extended Data Figure 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.

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Extended Data Figure 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.

Extended Data Figure 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.

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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.

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.

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Pascual, G., Avgustinova, A., Mejetta, S. et al. Targeting metastasis-initiating cells through the fatty acid receptor CD36.Nature 541, 41–45 (2017). https://doi.org/10.1038/nature20791

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