How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial–mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal–epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal–epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal–epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.
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The microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE96754 and the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012942; the mass spectrometry data is further available in Supplementary Table 2. The human clinical breast cancer data are not available in GEO; these were derived from the TCGA Research Network and the dataset derived from this resource that supports the findings of this study is available in https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Breast%20Cancer%20(BRCA). The human breast cancer data were also derived from the NKI-295 dataset47, which is available at https://xenabrowser.net/datapages/?cohort=Breast%20Cancer%20. Finally, the human breast cancer data from the EMC-MSK dataset is available in the publication by Bos and colleagues69. The source data relevant for the clinical data analyses performed in this study are available in Supplementary Table 9. Unprocessed western blot images are provided as Supplementary Fig. 9. The source data supporting the findings of this study are provided in Supplementary Table 9. All protocols, cell lines and reagents are available from the corresponding author on request.
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We thank L. M. Wakefield for providing the SORE6–mCherry stemness reporter, R. Nusse for providing the 7×TCF–GFP Wnt reporter, G. Laevsky for assistance with microscopy and C. DeCoste for assistance with flow cytometry. This work was supported by fellowships from the NIH (grant no. F31CA192461) and NJCCR to M.E., the National Institutes of Health NHLBI (grant no. PO1 HL107146), the Program of Excellence in Glycosciences and the Team Jobie Fund to R.S., and grants from the Susan G. Komen Foundation (grant no. SAC160067), Glycomimetics Inc., Brewster Foundation, Department of Defense (grant no. BC123187) and the National Institutes of Health (grant no. R01CA141062) to Y.K. This research was also supported by the Preclinical Imaging, Genomic Editing and Flow Cytometry Shared Resources of the Rutgers Cancer Institute of New Jersey (grant no. P30CA072720).
Integrated supplementary information
(a) Schematic representation of the comparative E-selectin binding assay. GFP-labelled experimental cells cocultured with RFP-labelled control cells are probed with E-selectin-IgG or isotype IgG (as the baseline control for each cell line) and quantified by flow cytometry using AL647 conjugated anti-IgG. This yields a ratio of AL647 signal between GFP+ and RFP+ cells with each normalized to their isotype IgG control as a quantitative measurement of relative E-selectin binding ability. (b) Flow cytometry histogram showing binding of MDA-MB-231-Fut cells to E-selectin-IgG chimera or isotype IgG, or to E-selectin-IgG at the presence of EDTA or GMI-1271 as negative controls. Data representative of 5 independent experiments. (c) E-selectin binding to isogenic derivatives (GFP+) compared to parental MDA-MB-231 cells (RFP+). Median intensity ratios were calculated using (SELE-GFP/IgG-GFP)/(SELE-RFP/IgG-RFP). n = 6 (Parent, SCP6, LM2, SCP32, SCP2, BM2), n = 3 (SCP38) or n = 7 (SCP25) independent biological replicates. Student’s t-test, two-sided, compares respective cell derivative to the parental cell line. (d) Representative figure of a flow cytometry histogram obtained from E-selectin binding to the isogenic MDA-MB-231 series using MDA-MB-231–RFP as an internal control. Data representative of independent biological replicates from c. (e) Total RNA from bone marrow and lungs in naïve and 6 h LPS-treated NOD/SCID mice was analysed for Sele (normalized by CD31) mRNA expression levels by qPCR. n = 3 technical replicates representative of 3 independent experiments. (f) Total pooled RNA from human bone marrow and lung tissues was analysed for Sele (normalized by CD31) mRNA expression levels by qPCR. n = 3 technical replicates. Experiment performed once from pooled RNA samples. (g) BLI quantification of bone metastasis burden following intracardiac injection of the M1a cell line into WT or Sele−/− SCID mice. n = 7 (KO), n= 9 (WT). Mann–Whitney U test, two-sided. (h) Representative BLI and X-ray images of bone metastasis in WT and Sele−/− NOD/SCID mice. White arrows indicate osteolytic lesions. Data representative of median signal from (h). (i, j) Quantification of osteloytic area (i) and the number of osteolytic lesions (j) in the hindlimbs of animals. n = 14 (KO) or 18 (WT) hindlimbs/group. Mann–Whitney U test, two-sided. Data representative of two independent experiments (g–j). (k) Primary tumour growth of M1a cells implanted orthotopically in WT or Sele−/− NOD/SCID mice. n = 7 mice/group. Student’s t-test, two-sided. (l) BLI quantification of lung metastasis burden following intravenous injection of LM2 cells into WT and Sele−/− NOD/SCID mice. n = 10 (KO), n = 7 (WT). Student’s t-test, two-sided. (m) Representative BLI and gross morphological images of lungs from LM2-injected mice. Data representative of median signal from l. (n) Macrometastatic nodes counted on the surface of the lungs of the LM2-injected WT or Sele−/− NOD/SCID mice. n = 10 (KO), n = 6 (WT). Student’s t-test, two-sided. (o) BLI quantification of lung metastasis burden following intravenous injection of M1a cells into WT and Sele−/− NOD/SCID mice. n = 10 (KO) or 9 (WT) mice/ group, Student’s t-test, two-sided. (p) Macrometastatic nodes counted on the lungs of the M1a-injected WT or Sele−/− NOD/SCID mice. n = 8 (KO), n = 7 (WT). Student’s t-test, two-sided. Experiment performed once (k–p). Bar graph data represent mean ± s.e.m. Boxplot data represent median, interquartile range, and spikes to upper and lower adjacent values.
Supplementary Figure 2 Quantification of α1–3 Fut expression and activity in MDA-MB-231 and M1a cells.
(a) Total RNA from MDA-MB-231 cells stably expressing each α1-3Fut was analysed for Fut3-7,9 expression by qPCR. n = 3 technical replicates. (b) Proliferation rates of MDA-MB-231-Fut3-7,9 were compared to vector control cells by the EZQuant assay. n = 3 biological replicates. Student’s t-test, two-sided. No statistically significant difference (p>0.05) between Fut groups vs Vector group. (c) Total RNA from M1a cells expressing each α1-3Fut was analysed for Fut3-7,9 expression by qPCR. n = 3 technical replicates. (d) Comparative flow cytometry analysis of E-selectin binding to stable α1-3 Fut-expressing M1a cells using SUM159-RFP as an internal control. Data representative of 4 independent experiments. (e, f) Quantification of osteloytic area (e) and the number of osteolytic lesions in the hind limbs of Nu/Nu mice receiving an intracardiac injection of M1a cells expressing each Fut enzyme (f). n = 12 (Fut4, Fut6), 10 (Fut3, Fut5, Fut9), 6 (Fut7), or 20 (Vector) hindlimbs. Mann–Whitney U test, two-sided. (g) RNA-Seq counts of each Fut enzyme’s first exon extracted from the TCGA RNA-seq clinical dataset in tumour (T) compared to adjacent normal (N) tissue. n = 955 patients. Data represent median, interquartile range and spikes to upper and lower adjacent values. (h) Isolation of subpopulations of MDA-MB-231 cells with high or low E-selectin binding ability by FACS. (i) E-selectin binding levels in sorted cell lines were quantified by comparative flow cytometry analysis after five passages. n = 3 biological replicates, Student’s t-test, two-sided. (j) Total RNA of poorly metastatic MDA-MB-231 and SCP6 cell lines compared to highly bone metastatic SCP25 and lung metastatic LM2 sublines was analysed for endogenous levels of each α1-3 Fut by qPCR. Fut5/7/9 were undetectable in each cell line. n = 3 technical replicates, Data represent mean ± s.e.m.
(a) Schematic of the rationale and protocol used for the extraction and identification of N-glycosites. Both the MDA-MB-231 and M1a cell lines contain potential substrates for Fut-mediated fucosylation to create sLeX, a tetrasaccharide glycosylation necessary for E-selectin binding. To identify these E-selectin ligands, the extracellular surface of MDA-MB-231 or M1a cells was oxidized with sodium metaperiodate to form hydrazide-reactive carbonyl groups from the cis-diols contained in the carbohydrates groups of membrane glycoproteins. Biotin-hydrazide was covalently linked to these oxidized glycoproteins, which were then purified by streptavidin affinity chromatography under denaturing conditions. Specific n-glycosylated residues were identified by sequential digestion first with trypsin to remove non-glycosylated peptides, followed by specific release of glycosylated peptides with PNGase F, and subsequently by MS/MS identification. (b) Proliferation assays were performed over 48 h on M1a or BM2 cells incubated with varying concentrations of D,L-Threo PDMP, Tunicamycin, or 1-Deoxymannojirimycin and cell viability was quantified with EZQuant. n=4 (Tunicamycin, PDMP) or n=3 (1-DMJ) biological replicates per concentration. Data represent mean ± s.e.m. (c) The comparative flow cytometry assay was performed on BM2 and M1a cells incubated with non-toxic doses of each indicated metabolic inhibitor for 48 h. Cells were harvested and mixed with untreated MDA-MB-231–RFP as the internal control followed by E-selectin-IgG probing. n=3 biological replicates. Student’s t-test, two-sided. (d) M1a cells expressing each Fut enzyme were harvested, split into three samples and probed with E-selectin-IgG with PBS, GMI-1271 (80 μg/mL) or EDTA (20 mM). These were combined with SUM159-RFP cells probed with E-selectin-IgG in the absence of inhibitory agents as internal control and the comparative flow cytometry assay was conducted. The bottom panel represents the same experiment conducted with control IgG as negative controls for both test M1a-Fut and control SUM159-RFP cell lines. Dashed lines represent median binding values in the positive control condition. Data representative of 4 independent experiments (b-d). Data represent mean ± s.e.m.
(a) Cellular membrane integrity measured by DAPI uptake following oxidation with 10 mM Sodium (Meta) periodate was analysed by flow cytometry. (b, c) Biotin-hydrazide-conjugated metaperiodate-oxidized cells probed with Streptavidin-AL647 were then permeabilized to label with DAPI and were imaged by immunofluorescence (b) and quantified by flow cytometry (c). Scale bars represent 10 μm. Data representative of 3 independent experiments (a-c). (d) BLI quantification of bone metastasis burden after intracardiac injection of CD44-KO or control BM2 cell lines into Nu/Nu mice. n = 6 (CD44), n = 8 (control). Mann–Whitney U test, two-sided. (e) Survival data was tabulated when mice became moribund and used for Kaplan–Meier survival curve analysis. n = 6 (CD44) n = 8 (control). Cox’s proportional hazards model, two-sided. Experiment performed once (d-e). (f) Representative precursor ion extracted ion chromatograms (XICs) for the doubly-charged deamidated Glg1 tryptic peptide, LNLTTDPK. XICs and respective peak areas were calculated in Skyline from the data-dependent MS analysis of N-glycopeptide enrichment experiments in M1a and MDA-MB-231 cells. XICs correspond to the summed intensities of the first three precursor isotope ions (resolving power of 100,000 @ 400 m/z) as function of time. Insets, mass spectrum showing the isotope envelope of deamidated LNLTTDPK extracted at the apex of the XIC traces. The monoisotopic ion is labelled with the measured m/z. Data representative of 4 independent biological replicates. (g) mRNA levels of three main Glg1 splice variants were measured by qPCR in the sorted MDA-MB-231 cells with different E-selectin binding ability. n = 3 technical replicates. (h) mRNA expression of the same variants was also tested in the MDA-MB-231, SCP6, SCP25, and LM2 cells. n = 4 technical replicates. (i) Total RNA from MDA-MB-231 or BM2 cells expressing Glg1 variant 1 or variant 3 was analysed for Glg1 expression. n = 4 technical replicates. (j) Western blot of MDA-MB-231 and BM2 cells with stable ectopic expression of Glg1 variant 3. (k) Western blot of BM2 cells with or without CRISPR–Cas9-mediated knockout of Glg1. Data representative of 3 independent experiments (j, k). (l) Comparative flow cytometry analysis of E-selectin binding was conducted on the indicated cell lines using MDA-MB-231–RFP as an internal control. Binding levels were normalized and compared to respective controls. n = 5 independent biological replicates. Student’s t-test, two-sided. Data represent mean ± s.e.m. Unprocessed original scans of the blots in j and k are shown Supplementary Fig. 9.
(a) Total lysate and membrane fractions from M1a cells were probed by western blot for Glg1 expression. Experiment performed once. (b) Immunoblot of M1a cell membrane lysates using E-selectin-IgG as a probe. (c) Immunoblot using anti-Glg1 as a probe of total cell lysates, immunoprecipitation supernatants, and E-selectin-immunoprecipitated proteins from each M1a cell line. Data representative of 2 independent experiments (b, c). Unprocessed original scans of the blots are shown Supplementary Fig. 9.
(a) qPCR quantification of Glg1 mRNA following stable shRNA-mediated knockdown of Glg1 in BM2 cells. n = 3 technical replicates. (b, c) BLI quantification of bone metastasis burden after intracardiac injection of control or Glg1-KO BM2 cells (b, n= 9 mice/group) or Glg1-KO M1a cells (c, n= 9 (Control) or 7 (Glg1-KO1) mice/group) into Nu/Nu mice. Mann–Whitney U test, two-sided. (d) Representative image of BLI and X-ray images from M1a Glg1-knockout-injected mice. Data representative of median signal from c. White arrows indicate osteolytic lesions. (e, f) The number of lesions/ hindlimb (e) and average osteolytic area (f) were quantified from X-rays of M1a Glg1 knockout-injected mice. n = 18 (Control) or 14 (Glg1-KO1) hindlimbs per group. Mann–Whitney U test, two-sided. Data representative of two independent experiments (b-f). (g) qPCR analysis of Glg1 expression in control or Glg1-knockdown LM2 cells. n = 4 technical replicates. (h) BLI quantification of lung metastasis burden following intravenous injection of control and Glg1-knockdown LM2 cells into Nu/Nu mice. n = 8 (shGlg1-1) or 9 (shGlg1-2, Control) mice/group. Student’s t-test, two-sided, demonstrated non-significance at all time points (p>0.05). (i) Survival data was tabulated when mice became moribund and was used for Kaplan–Meier survival curve analysis which demonstrated non-significance for all curves, Cox’s proportional hazards model, two-sided. n = 8 (shGlg1-1) or 9 (shGlg1-2, Control) mice/group. Data represent mean ± s.e.m. (j) Kaplan–Meier survival curve of breast cancer patients in the NKI-295 dataset stratified by median expression level of Glg1 and Fut3/Fut6 RNA. n = 295. Cox’s proportional hazards model, two-sided. (k) Kaplan–Meier organ-specific metastasis-free survival curves of ER- breast cancer patients in the EMC-MSK dataset stratified by median expression level of Glg1 and Fut3/Fut6 mRNA. n = 244. Cox’s proportional hazards model, two-sided.
(a) Confocal imaging of bone metastasis of Nu/Nu mice after intra-iliac artery injection of GFP-labelled SCP28 cells. Mice were labelled with a retro-orbital injection of 10 μg anti-CD31 prior to euthanasia. Scale bars represent 50 μm. (b) Confocal imaging of BM2-GFP bone lesions in Nu/Nu mice stained for E-selectin and Glg1. Scale bars represent 100 μm (c) Confocal imaging of E-selectin-induced intensity changes in Keratin-14 expression in BM2 cells after culturing on E-selectin compared to IgG-coated plates (10 μg/mL) for 24h. Scale bars represent 20 μm. Data representative of 3 independent experiments (a-c). (d, e) GSEA of multiple EMT-related signatures, including Blick EMT1 (d), Claudin-low (GSE18229), Sarrio-EMT (GSE8430), and Luminal (GSE22446) (e), in the ranked gene list of BM2 cells cultured in E-selectin vs. IgG-coated plates. Single mRNA isolation used for each condition in each cell line. p and q statistics by GSEA software, n = 108 gene sets queried. (f) Western blot of N-cadherin and Slug from M1a cells bound to IgG or E-selectin-coated plates (10 μg/mL) for 24h. Data representative of 2 independent experiments. (g, h)Confocal imaging of bone lesions from BM2-GFP injected mice stained for E-cadherin (g) and EpCam (h). Scale bars represent 100 μm (g) and 50 μm (h). Data representative of >5 independent biological replicates. Unprocessed original scans of the blots in f are shown Supplementary Fig. 9.
Supplementary Figure 8 E-selectin binding-induced Wnt signalling and E-selectin is critical to prostate cancer bone metastasis.
(a) Confocal imaging of ex vivo bone sections from Nu/Nu mice that developed bone metastasis following intracardiac injection of BM2-TGC cells. Live animal labelling with retro-orbital anti-CD31 was conducted to visualize vasculature. Scale bars represent 50 μm. Data representative of 5 biological replicates. (b) Western blot of analysis of hDkk1-Flag expression in BM2-TGC cells expressing Dkk1–FLAG or the control vector. Data representative of 3 independent experiments. (c) Flow cytometry measurement of Wnt activation using the BM2-TGC reporter cell line expressing either Dkk1–FLAG or empty vector seeded over IgG or E-selectin-treated plates (10 μg/mL), or treated with L Cell Wnt3a-conditioned media vs control-conditioned media. Wnt inhibitors ICG-001 (25 μM), LF3 (50 μM), and Dkk1 were tested against each condition during 48h of culture. Data representative of 3 independent experiments. (d) BLI quantification of bone metastasis burden after intracardiac injection of BM2 cells into Nu/Nu mice followed by 7 days of treatment with either LF3 (40 mg/kg body mass, n =15 mice) or vehicle (n = 12 mice). Mann–Whitney U test, two-sided. Data representative of 2 independent experiments. (e, f) Total number of lesions per hindlimb (e) and total osteolytic area (f) were quantified between treatments. n = 24 (Vehicle) or 30 (LF3) hindlimbs/ group. Mann–Whitney U test, two-sided. Data represent mean ± s.e.m. (g) Flow cytometry measurement of E-selectin-IgG binding to ob1, a bone metastatic derivative of DU145 prostate cancer cell line. Data representative of 3 independent experiments. (h) Ex vivo bioluminescence of hindlimbs from WT and Sele−/− NOD/SCID mice injected with ob1 cells. n = 20 (KO) or 28 (WT) hindlimbs/group. Mann–Whitney U test, two-sided. Data represent mean. (i) Kaplan–meier survival plot of WT and Sele−/− mice injected with ob1 cells. n = 11 (KO) or 8 (WT) mice/group. Cox’s proportional hazards model, two-sided. Data in h and i representative of 2 independent experiments. (j) Confocal imaging of ob1 cells plated 24h on E-selectin- or IgG-coated (10 μg/mL) ibidi plates stained for EpCAM. Scale bar represents 10μm. Data representative of 3 independent experiments. (k) Immunofluorescence staining of E-selectin, Ki67, and Glg1 in FFPE-bone sections from a bone metastasis sample from a prostate cancer patient. Scale bar represents 50 μm. Data representative of 13 sections from 2 biopsy specimens. Unprocessed original scans of the blots in b are shown Supplementary Fig. 9.
Unprocessed scans of the non-quantitative western blots provided for the indicated figure panels.
Supplementary Figures 1–9, Supplementary Table titles/legends, Supplementary Video 1 title/legend
Spontaneous bone metastasis rates from M1a–Fut primary tumour.
Mass spectrometry identification of candidate Fut substrates in MDA-MB-231 and M1a cells.
E-selectin ligand candidate list from N-glycan profiling.
Kaplan–Meier analysis of candidate E-selectin ligands.
Genes contained in the non-canonical MET signature.
Key characteristics of the cell lines used in this study.
List of all oligonucleotide sequences used in this study.
List of all antibodies used in this study.
Statistics source data.
Spinning-disc microscopy of BM2-TGC cells seeded over control IgG, E-selectin-IgG or a poly-HEMA non-adherent condition.
About this article
Nature Reviews Cancer (2019)