CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRasG12D; p53F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.

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Fig. 1: CD9 identification.
Fig. 2: CD9 marks TICs.
Fig. 3: CD9high cells recapitulate tumour heterogeneity.
Fig. 4: CD9 is required for efficient PDAC development.
Fig. 5: CD9 facilitates glutamine import into TICs.
Fig. 6: CD9 expression in human PDAC and model of CD9 function in PDAC.

Data availability

Microarray data supporting the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE121864. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD015439. Clinical data and normalized RSEM read count tables for pancreatic adenocarcinoma were downloaded from the Broad firehose data repository (https://gdac.broadinstitute.org/). Source data for Figs. 1 and 5 and Extended Data Figs. 1 and 57 are available online. All other relevant data are available from the corresponding author. Ethical consent for use of human biological samples is currently restricted to the Adult Stem Cell Laboratory at the Francis Crick Institute.

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Acknowledgements

We thank D. Saur for providing the Pdx1-Flp, Trp53 FRT and FSF-KRasG12D mice, I. Malanchi and M. Yuneva for comments on the manuscript, C. Cremona for help in preparing the manuscript, the Yuneva laboratory for advice on organoid metabolomics and the V-9302 inhibitor, R. Mitter and S. Boeing from the Bioinformatics and Statistics Service at the Francis Crick Institute, K. Miyado for the EGFP−mCD9 fusion construct, D. Sarker, A. Prachalias, C. Cotoi, Y. Zen and A. Zamalloa for coordinating and providing the human samples used in this study, technicians from the Biological Research Facility at the Francis Crick Institute, and all other Behrens laboratory members for their feedback. T.E. was supported by a doctoral clinical fellowship from a CRUK Accelerator Award (C422/A23614). This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001039), the UK Medical Research Council (FC001039) and the Wellcome Trust (FC001039).

Author information

R.M.M.F., V.M.-Y.W. and A.B. conceptualised this study and designed experiments. R.M.M.F., V.M.-Y.W., J.A., T.E., M.Z.T., N.L., D.F. and J.C. performed experiments. R.M.M.F., V.M.-Y.W., D.J.B., N.L., A.P.S., E.H., E.L.N. and J.I.M. analysed data. R.M.M.F. and V.M.-Y.W. wrote the manuscript and prepared figures. R.M.M.F., V.M.-Y.W., J.I.M. and A.B. reviewed and edited the manuscript. A.B. acquired funding and supervised this study. All authors approved the content of the manuscript. J.A., T.E., M.Z.T. and N.L. contributed equally.

Correspondence to Axel Behrens.

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Extended data

Extended Data Fig. 1 CD9 identification.

a, YFP stain of pancreatic ducts of R26-LSL-YFP; Ck19-CreER mice 2 weeks post-tamoxifen. Scale bar, 50 μm, inset 50 µm x 50 µm. b, Flow cytometry analysis and genotyping of unrecombined and recombined cells from KFCkY pancreas 2 weeks post-tamoxifen. Expected band sizes (base pairs) are indicated; see Source Data for uncropped gels. c, CD44 and CD133 stain in consecutive pancreatic sections of KFCkY mice 2 weeks post-tamoxifen. Non-responsive regions magnified. Black arrows show apical expression of CD133 in non-responsive cells. Scale bar, 50 µm. d, CD44 gating strategy using rat IgG2b isotype control on KFCkY pancreas 2 weeks post-tamoxifen. Related to Fig. 1d. e, Validation of the CD44 antibody using rat IgG2b isotype control on KFCkY tumours; quantification of CD44+ cells. f, Quantification of transformed/CD44+ YFP+ foci in pancreatic tissues of KFCkY mice two (n = 5) and four (n = 3 biologically independent animals) weeks post-tamoxifen. Dot plot shows mean, s.d. g, Human PDAC markers among upregulated genes in microarray. Fold changes relative to NT cells. h, Flow cytometry analysis of CD44 and CD9 expression on live, YFP+ cells from KFCkY pancreas 4 weeks post-tamoxifen. i, Quantification of live tumour cells (DAPIYFP+, n = 3 biologically independent animals) from (h). Bar chart shows mean, s.d., p = 0.0013, two-sided t test. j, CD9, E-cadherin and DAPI staining on pancreatic tissues of KFCkY mice 4 weeks post-tamoxifen. Scale bar, 50 µm, 10 μm (inset). k, CD9 gating strategy using rat IgG2a isotype control on live KPCY tumour cells. Related to Fig. 1i. l, Validation of the CD9 antibody using rat IgG2a isotype control in live KPCY PDAC organoids; quantification of CD9+ cells. All flow cytometry plots and immuno-staining data shown in Extended Data Fig. 1 are representative of at least three biologically independent animals/experiments. Source data Source data

Extended Data Fig. 2 CD9 marks TICs.

a, Organoid formation assay (organoid number) of CD9high and CD9low YFP+ tumor cells freshly isolated from KFCkY PDAC (n = 4 biologically independent animals). Dot plot shows mean, s.d., p value from two-sided Mann-Whitney test. Right, representative brightfield images of generated organoids 10 days after plating. Scale bar, 250 µm. b, CD9 expression dynamics of two representative KPCY organoid cultures. Organoid cultures were dissociated at confluency and replated. The percentage of CD9high cells was measured by flow cytometry over time as the cultures grew. c, Flow cytometry analysis of a representative KPCY organoid culture at 4 and 8 days of culture, stained for CD9 and the proliferation marker Ki67. Proliferation is higher at 4 than 8 days, and only CD9high cells actively proliferate in the culture at both timepoints. Flow cytometry plots shown in (b) and (c) are representative of at least three biologically independent samples. d, Tumourigenic capacity of CD9high versus CD9low tumour cells from KPCY PDAC-derived organoids. 200,000, 20,000, 2,000 or 200 sorted tumour cells were injected into the flanks of Nu/Nu mice and the number of weeks to nodule detection (4 mm3) was registered. e, Bar charts of percentage of mice injected with 2,000 (above) or 200 (below) sorted CD9high or CD9low tumour cells that developed tumours by the given week. Each bar shows cumulative data of all mice harbouring tumours at a given week (n = 6 for 2,000 cells, n = 4 for 200 cells). f, Fold difference in tumour volumes between the CD9low- and CD9high-derived tumours at each dilution; n = 3, 5, 6 and 4 biologically independent animals for 200,000-, 20,000-, 2,000- and 200-cell injections, respectively. Dot plot shows mean, s.d., p values from two-sided t tests. Source data

Extended Data Fig. 3 CD9 is required for efficient PDAC formation.

a, Organoid formation assay (total organoid area) of KPC organoids upon CD9 knockdown (representative of n = 3 biologically independent experiments). Dot plot shows mean, s.d., p values from two-sided t test. Related to Fig. 4b. b, Flow cytometry plot of HEK293T cells transiently transfected with EGFP control and EGFP-mCD9 vectors. The KMC8 antibody was used to specifically recognise murine CD9. c, Live confocal microscopy images showing cytoplasmic EGFP and predominantly plasma membrane EGFP-mCD9 localisation in primary KPC cells. Scale bar, 20 µm. Flow cytometry plots and fluorescence imaging shown in (b) and (c) are representative of three biologically independent samples. d, Organoid formation assay (total organoid area) of KPC organoids upon CD9 overexpression (n = 3 biologically independent experiments). Related to Fig. 4e. Dot plot shows mean, s.d., p < 0.0001, two-sided t test. e, Schematic of the EGFP control and EGFP-mCD9 overexpression system using an EF1α promoter. f, Organoid formation assay (organoid number) of KPC organoids upon CD9 overexpression using the EF1α vector (n = 2 biologically independent experiments). p = 0.001, two-sided t test. g, Organoid formation assay (total organoid area) of KPC organoids upon CD9 overexpression using the EF1α vector (n = 2 biologically independent experiments). p = 0.002, two-sided t test. Dot plots in (f) and (g) show mean, s.d. Source data

Extended Data Fig. 4 Characterisation of KPC CD9 knock-out model.

a, Flow cytometry histogram of cells isolated directly from mouse pancreas of the indicated genotypes after gating on YFP+ cells. CD9 staining confirms heterozygous and complete knockout in CD9Δ/WT and CD9Δ/Δ cells, respectively. Flow cytometry plot is representative of three biologically independent samples. b, Cd9 mRNA levels measured by RT-qPCR in early passage (<5) KPC organoids of the indicated genotypes (n = 2 biologically independent animals per genotype); cells were isolated from endpoint tumours. Values were normalised to Gapdh and Actb, and fold changes calculated relative to CD9WT/WT cells. c, Representative histology of KPC pancreases at 8 weeks with indicated CD9 status (in the p53Δ/WT model, median survival of CD9WT/WT mice 17.4 weeks; n = 6, 4 and 8 biologically independent animals for CD9WT/WT, CD9Δ/WT and CD9Δ/Δ, respectively). d, Stacked bar graph showing quantification of precursor PanIN and PDAC lesions in KPC pancreases at 8 weeks from (c). KPC CD9Δ/WT mice exhibit later onset and/or slower tumor progression. e, Weight of subcutaneous grafts at endpoint after initial injection of 10,000 KPC CD9Δ/WT or KPC CD9Δ/Δ cells (n = 3 biologically independent animals, from organoid cultures derived from the p53Δ/Δ model at 4 weeks). P = 0.0252, two-sided t test. Dot plot shows mean, s.d. Source data

Extended Data Fig. 5 CD9 facilitates glutamine import into TICs.

a, Co-IP of transiently transfected Myc-hMCT1 (left) or Myc-mASCT2 (right) with EGFP-mCD9 after EGFP pull-down in HEK293T cells. Blots are representative of 3 independent experiments. See Source Data for uncropped blots. b, Flow cytometry plots co-staining CD9 with cell surface proteins involved in metabolism in KPC organoids. Representative of 3 independent experiments. c, Membrane co-localisation (arrowheads) of ASCT2 and EGFP-CD9 in KPC organoids by co-immunofluorescence. Scale bar, 20 µm; insert 20 by 20 µm. d, Immunofluorescence of ASCT on sorted CD9low and CD9high KPC cells. Scale bars, 20 µm. Representative of 3 independent experiments. e, Immunofluorescence of KPF-CD9 organoids after control vehicle or 4-OHT treatment. Representative of 2 independent experiments. Scale bars, 20 µm. Related to Fig. 5e. f, Immunofluorescence of ASCT2 in a KPCY mouse pancreas: non-transformed area (top) and transformed areas (bottom). Scale bars, 100 µm low mag, 20 µm high mag. Representative of at least 3 biologically independent animals. g, YFP, CD44, ASCT2 and DAPI staining on pancreatic tissue of KFCkY mice 1 week (top) and 4 weeks (bottom) post-tamoxifen. Scale bars, 20 µm. Representative of at least 3 biologically independent animals. h, Metabolic profiles of KPF-CD9 organoids (after control Adeno-EGFP (CD9F/WT) or Adeno-CRE-EGFP (CD9Δ/WT) infection) grown in organoid media containing 4 mM 13C-glutamine for 4 h. i, Some metabolites, including glycolytic intermediates, are equally abundant under the conditions in (h), but did not contain any detectable 13C label. Data in (h) and (i) represent one experiment carried out with six technical replicates. Due to technical limitations, statistics for metabolite abundance and label incorporation were performed on technical replicates separately using a two-way ANOVA corrected for multiple comparisons using the Holm-Sidak method, α = 0.05; bar charts show mean, s.d. Source data Source data

Extended Data Fig. 6 CD9 facilitates glutamine import into TICs and metabolic compensation in KPC mice.

a, Western blot showing overexpression of ASCT2 in organoids. Blot shown is representative of 2 independent experiments. See Source Data for uncropped blots. b, Organoid formation assay (total organoid area) of KPF-CD9 organoids after vehicle/Adeno-EGFP or 4-OHT/Adeno-CRE-EGFP treatment in the presence of basal or elevated (4 mM) levels of glutamine, and with control pLV or pLV ASCT2 overexpression (n = 3 biologically independent experiments). Related to Fig. 5h. Black P values (CD9F/WT versus CD9Δ/WT) calculated using two-way ANOVA corrected for multiple comparisons, Holm-Sidak method, α = 0.05. Blue P values (within CD9Δ/WT conditions) calculated using two-way ANOVA corrected for multiple comparisons, Dunnett’s test, α = 0.05. Dot plot shows mean, s.d. c, Organoid formation assay (total organoid area) of CD9low KPC cells with control pLV or pLV ASCT2 overexpression (n = 3 biologically independent experiments). Related to Fig. 5i. P = 0.0091, two-sided t test. Dot plot shows mean, s.d. d, Histology of KPC pancreases at 4 weeks with indicated CD9 status (in the p53Δ/Δ model, median survival of CD9WT/WT mice 6.2 weeks). At this timepoint, all mice present with PDAC, regardless of CD9 status. Images are representative of at least 3 biologically independent animals. e, Slc1a5 mRNA levels measured by RT-qPCR in early passage (<5) KPC organoids of the indicated genotypes (n = 2 biologically independent animals per genotype); cells were isolated from endpoint tumors. Values normalised to Gapdh and Actb; fold changes calculated relative to CD9WT/WT cells. f, Indicated tetraspanin mRNA levels measured by RT-qPCR in early passage (<5) KPC organoids of the indicated genotypes (n = 2 biologically independent animals per genotype); cells were isolated from endpoint tumours. Values normalised to Gapdh and Actb; fold changes calculated relative to CD9WT/WT cells. Source data Source data

Extended Data Fig. 7 CD9 expression in human PDAC.

a, Co-IP of endogenous CD9 with endogenous ASCT2 after ASCT2 pull-down in the human pancreatic cancer cell line PANC-1. Input, left, 10% polyacrylamide gel; Co-IP, right, 20% polyacrylamide gel to separate bands from IgG heavy and light chains (asterisks). Blot representative of 2 independent experiments. See Source Data for uncropped blots. b, Co-immunofluorescence for CD9 and ASCT2 on human PDAC, showing punctate CD9 at the plasma membrane, with high levels of ASCT2 in CD9high cells; scale bars, 20 µm. Data are representative of 3 biologically independent human samples. c, Flow cytometry plots co-staining CD9 with other PDAC CSC cell surface proteins in primary human PDAC organoid culture. CD9 gate separates CD9high cells from bulk; gates on the x-axis separate marker-positive from marker-negative cells (based on fluorescence minus one controls). Plots are representative of 3 biologically independent primary organoid lines. Source data

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Wang, V.M., Ferreira, R.M.M., Almagro, J. et al. CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth. Nat Cell Biol 21, 1425–1435 (2019) doi:10.1038/s41556-019-0407-1

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