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Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGF-β2

Nature Cancer volume 2, pages 327–339 (2021)Cite this article

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Abstract

In the bone marrow (BM) microenvironment, where breast cancer (BC)-disseminated tumor cells (DTCs) can remain dormant for decades, NG2+/Nestin+ mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here we reveal that periarteriolar BM-resident NG2+/Nestin+ MSCs can also instruct BC DTCs to enter dormancy. NG2+/Nestin+ MSCs produce transforming growth factor (TGF)-β2 and bone morphogenetic protein (BMP)7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase, results in p27 induction. Genetic depletion of MSCs or conditional knockout of TGF-β2 in MSCs using an NG2-CreER driver led to bone metastatic outgrowth of otherwise dormant p27+/Ki67− DTCs. Also, patients with estrogen receptor-positive BC without systemic recurrence displayed higher frequency of TGF-β2 and BMP7 detection in the BM. Our results provide direct proof that hematopoietic stem cell dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2+/Nestin+ MSC niche homeostasis may result in a break from dormancy and BC bone relapse.

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Fig. 1: Depletion of NG2+/Nestin+ MSCs awakens dormant DTCs in the BM.
Fig. 2: Dormant to proliferative shift of BM E0771-GFP cancer cells upon depletion of NG2+/Nestin+ MSCs.
Fig. 3: NG2+/Nestin+ MSCs are as a source of pro-dormancy factors TGF-β2 and BMP7 in the BM.
Fig. 4: NG2+/Nestin+ MSCs activate TGF-β2 and BMP7 growth inhibitory signaling in cancer cells.
Fig. 5: Conditional KO of TGF-β2 in NG2+/Nestin+ MSCs awakens dormant DTCs in the BM.
Fig. 6: Schematic representation of the model by which HSC niches induce dormancy of BC DTCs in the BM.

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Data availability

The data generated during the current study that support the reported findings are available in the manuscript, including in the provided Source Data files and from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Aguirre-Ghiso laboratory for helpful discussions and the assistance of the Dean’s Flow Cytometry CoRE, Microscope CoRE and Small Animal Imaging CoRE at BioMedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai. We are grateful to C. Prophete (Frenette’s laboratory) for mouse husbandry. We thank M. Rypdal, A.V. Pladsen and O.C. Lingjærde for preparing the BC DTC material and data. The BC DTC work in Oslo has received funding from the Norwegian Cancer Association and Norwegian Health Region South-East (H.G. Russnes and B. Naume). Grant support was received from the National Institutes of Health (NIH)/National Cancer Institute (NCI) CA109182, CA216248, CA218024, CA196521, The V-Foundation and the Samuel Waxman Cancer Research Foundation Tumor Dormancy Program to J.A.A.G.; HL069438, DK056638, DK116312 and DK112976 to P.S.F.; Portuguese Foundation for Science and Technology (SFRH/BD/100380/2014) to A.R.N.; MD/PhD program of the University of Lyon and the Ecole Normale Supérieure of Lyon to E.R.; Susan G. Komen Career Catalyst Research (CCR18547848), NCI Career Transition Award (K22CA196750), NIH (R01CA244780) and Tisch Cancer Institute NIH Cancer Center Grant (P30-CA196521) to J.J.B.C.; Instituto Serrapilheira/Serra-1708-15285 to A.B. and NIH (HL126705, CA218578, R01HL145064) and American Heart Association (17GRNT33650018) to M.A.

Author information

Author notes

  1. Alexander Birbrair

    Present address: Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, Brazil

Authors and Affiliations

  1. Division of Hematology and Oncology, Department of Medicine and Department of Otolaryngology, Department of Oncological Sciences, Black Family Stem Cell Institute, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Ana Rita Nobre, Emma Risson, Deepak K. Singh, Julie F. Cheung & Julio A. Aguirre-Ghiso

  2. Abel Salazar Biomedical Sciences Institute, University of Porto, Porto, Portugal

    Ana Rita Nobre

  3. Université de Lyon, Lyon, France

    Emma Risson

  4. Division of Hematology and Oncology, Department of Medicine, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Julie S. Di Martino & Jose Javier Bravo-Cordero

  5. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA

    Jiapeng Wang, John Johnson & Mohamad Azhar

  6. Division of Surgery and Cancer Medicine, Department of Oncology, Oslo University Hospital, Oslo, Norway

    Hege G. Russnes

  7. Division of Laboratory Medicine, Department of Pathology, Oslo University Hospital, Oslo, Norway

    Hege G. Russnes

  8. Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY, USA

    Alexander Birbrair & Paul S. Frenette

  9. Division of Cancer Medicine, Department of Oncology, Oslo University Hospital, Oslo, Norway

    Bjorn Naume

  10. Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Bjorn Naume

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Contributions

A.R.N. designed, planned and conducted experiments, analyzed data and wrote the manuscript; E.R., D.K.S. and J.F.C. conducted experiments; J.S.d.M. and J.J.B.C. helped with optimizing the imaging experiments; J.W., J.J. and M.A. provided necessary mouse model and samples and TGF-β2 expertise input; H.G.R. and B.N. provided human data; A.B. and P.S.F. provided mouse models and BM expertise input; P.S.F. and J.A.A.G. designed experiments, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Julio A. Aguirre-Ghiso.

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Competing interests

J.A.A.G. is a scientific co-founder of, scientific advisory board member and equity owner in HiberCell and receives financial compensation as a consultant for HiberCell, a Mount Sinai spin-off company focused on the research and development of therapeutics that prevent or delay the recurrence of cancer.

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Extended data

Extended Data Fig. 1 Control of depletion of NG2+/Nestin+ MSCs and awakening of dormant PyMT-CFP DTCs in the BM..

a, 7-week old NG2-CreERiDTR mice (NG2-CreER- or +) were daily injected with tamoxifen for 5 days, followed by 1-day rest and 2 days with diphtheria toxin (DT). 24 h later 2 × 105 E0771-GFP cells were intra-cardiac injected and 2 weeks later mice were euthanized. b,c, FACS plots (b) and quantifications (c) confirming the depletion of NG2+/Nestin+ MSCs (CD45−Ter119−CD31−PDGFRα+CD51+) in NG2-CreERiDTR mice upon TAM and DT treatments compared with WT mice (n = 5 WT and 5 iDTR mice, median, 2-tailed Mann–Whitney U-test, p = 0.008). d-g, Detection and characterization of MMTV-PyMT-CFP cells in BM flushes by FACS (n = 5 WT and 5 iDTR mice). e, Incidence of bone metastasis (>1000 GFP+ DTCs/106 BM cells) 2 weeks after cancer cell injections (2-tailed Fisher’s exact test). f, Number of MMTV-PyMT-CFP cancer cells per million BM cells (median, 2-tailed Mann–Whitney U-test). g, Percentage of Ki67high E0771-GFP cancer cells (median, 2-tailed Mann–Whitney U-test). *p≤0.05 (p-values indicated above). n.s. not significant.

Source data

Extended Data Fig. 2 Pro-inflammatory cytokine status, vessel permeability and BM DTC seeding in NG2+/Nestin+ MSCs-depleted mice.

a, Levels of pro-inflammatory cytokines in BM supernatant of WT and NG2-CreERiDTR mice (n = 13 WT and 11 iDTR mice, from 2 independent experiments, median and interquartile range, 2-tailed Mann–Whitney tests, *p≤0.05, n.s. not significant, n.d. not detected). b-d, NG2-CreERiDTR mice (NG2-CreER- or +) were daily i.p. injected with tamoxifen for 5 days followed by a rest day and 2 i.p. injections of DT. E0771-GFP cells were intra-cardiac injected and 24 h after 70 K Dextran-TexasRed was injected 15 minutes prior euthanasia. c, Representative images of Dextran extravasation in perfused bones. Arrows, E0771-GFP cells. d, Number of E0771-GFP cells detected after 1 week of in vitro expansion of the BM aspirates collected 24 h after injection into mice (n = 5 WT and 5 iDTR mice, median). e,f, NG2-CreERiDTR mice (NG2-CreER- or +) were daily i.p. injected with TAM for 5 days, followed by intra-cardiac injection of E0771-GFP cancer cells. Cells were allowed to disseminate and extravasate for 72 h followed by 2 i.p. injections of DT. f, Number of E0771-GFP cells/million BM cells (n = 5 WT and 5 iDTR mice, median and 2-tailed Mann–Whitney tests, p=0.015). *p≤0.05 (p-values indicated above), n.s. not significant.

Source data

Extended Data Fig. 3 Effect of NG2+/Nestin+ MSCs, TGFβ2 and BMP7 on signalling pathways and growth cancer cells.

a, 3D Matrigel assay used to track solitary cell to cluster growth. Single cells were plated on top of Matrigel in low density and the percentage of single cells, doublets and clusters was quantified 4 days after. b,c, Co-culture of human HNSCC PDX-derived T-HEp3 (b, n = 4 independent experiments, 4 wells per condition, mean and SEM, 2-tailed Mann–Whitney tests p=0.04) and mouse BC MMTV-PyMT (c, n = 2 independent experiments, 4 wells per condition, mean) cells with sorted Nestin-GFP- and Nestin-GFP+ MSCs for 4 days. d-k, E0771 cells were treated every day for 4 days with TGFβ2, BMP7 or bone marrow conditioned media (BM-CM) of TGFβ2+/+ or TGFβ2+/- mice. d and h. Percentage of cancer cells in a single cell, doublet or cluster state with the indicated treatments (d, n = 5 independent experiments, 4 wells per condition each, mean and SEM, 2-tailed Mann–Whitney tests, p=0.03 and 0.005; h, n = 4 independent experiments, 4 wells per condition each, mean and SEM, 2-tailed Mann–Whitney tests, p=0.000005, 0.0001, 0.002 (Ct vs. TGFβ2+/+: CS, doublets and clusters respectively) and 0.02 (TGFβ2+/+ vs. TGFβ2+/- clusters)). e-g and i-k. Percentage of positive cells for c-Cas-3, p-ATF2 and p27 upon the indicated treatments (n = 2 independent experiments, 4 wells per condition each, mean, minimum and maximum). l, Western blots for the indicated antigens detected in E0771 cells treated for 24 h with the TGFβ2, BMP7 and different BM-CM preparations. Molecular weight markers in kDa. *p≤0.05, p-values indicated above.

Source data

Extended Data Fig. 4 Effect of TGFβ2 and BMP7 on signalling pathways (using ELK, p38 and p27 sensors) and MSCs in E0771 cancer cell growth.

a-d, T-HEp3 cells with ERK, p38 and p27 activity biosensors were reversed transfected with control siRNA or siRNAs for TGFBRIII and BMPRII followed by 24-hour treatments with TGFβ2 and BMP7. a, TGFBRIII and BMPRII mRNA levels 48 h after transfection with the indicated siRNAs (n = 3 independent experiments, mean, minimum and maximum, 2-tailed Mann–Whitney tests, p=0.01 (TGFbR3) and 0.002 (BMPR2)). b–d, Quantification of the T-HEp3-biosensors activity (n = 3 independent experiments each, mean, minimum and maximum, 2-tailed Mann–Whitney tests, p=0.05). e, Percentage of E0771 cells in a single cell (p = 0.002), doublet or cluster (p = 0.005) state after co-culture with Control (passaged) or revitalized (r) MSCs (n = 4 independent experiments, 4 wells per condition per experiment, mean and SEM, 2-tailed Mann–Whitney tests). f, qPCR of TGFβ1, TGFβ2 and BMP7 from Control and rMSCs (n = 2). *p≤0.05, p-values indicated above.

Source data

Extended Data Fig. 5 NG2-CreERTGFβ2 mouse model controls.

a, TGFβ1 mRNA levels in NG2+/Nestin+ MSCs (sorted using CD45−Ter119−CD31−PDGFRα+CD51+ markers) in NG2-CreERTGFβ2 mice upon TAM treatments compared with WT mice (n = 4 independent experiments). b, TGFβ1, TGFβ2 and BMP7 mRNA levels in MSCs (CD45−CD31−Ter119−PDGFRα+CD51+), osteo-progenitor (CD45−Ter119−CD31−ALCAM+, OPCs) and endothelial (CD45−Ter119−CD31+vEcad+, ECs) cells from NG2-CreERTGFβ2 mice upon TAM treatments compared with WT mice (n = 2 independent experiments). *p≤0.05, p-values indicated above.

Source data

Supplementary information

Reporting Summary

Supplementary Fig. 1

FACS strategy and gates used to sort BM cells. a, FACS strategy and gates used to sort CD45−CD31−Nestin-GFP− or +/bright BM cells. b, FACS strategy and gates used to sort MSCs (CD45−CD31−Ter119−PDGFRa+CD51+, MSCs), osteo-progenitor cells (CD45−Ter119−CD31−ALCAM+, OPCs) and endothelial cells (CD45−Ter119−CD31+vEcad+, ECs) from WT mice.

Supplementary Video 1

Movie of a typical sonogram used to conduct imaging-guided injection of cells into the left ventricle of the heart. This methodology ensures intracardiac injection efficiency, minimizing variability of injections on BM DTC burden analysis.

Source data

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Unprocessed western blots from Extended Data Fig. 3.

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Raw numerical data behind graphs from Extended Data Fig. 4.

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Raw numerical data behind graphs from Extended Data Fig. 5.

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Nobre, A.R., Risson, E., Singh, D.K. et al. Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGF-β2. Nat Cancer 2, 327–339 (2021). https://doi.org/10.1038/s43018-021-00179-8

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  • DOI: https://doi.org/10.1038/s43018-021-00179-8

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