Abstract

The presence of disseminated tumour cells (DTCs) in bone marrow is predictive of poor metastasis-free survival of patients with breast cancer with localized disease. DTCs persist in distant tissues despite systemic administration of adjuvant chemotherapy. Many assume that this is because the majority of DTCs are quiescent. Here, we challenge this notion and provide evidence that the microenvironment of DTCs protects them from chemotherapy, independent of cell cycle status. We show that chemoresistant DTCs occupy the perivascular niche (PVN) of distant tissues, where they are protected from therapy by vascular endothelium. Inhibiting integrin-mediated interactions between DTCs and the PVN, driven partly by endothelial-derived von Willebrand factor and vascular cell adhesion molecule 1, sensitizes DTCs to chemotherapy. Importantly, chemosensitization is achieved without inducing DTC proliferation or exacerbating chemotherapy-associated toxicities, and ultimately results in prevention of bone metastasis. This suggests that prefacing adjuvant therapy with integrin inhibitors is a viable clinical strategy to eradicate DTCs and prevent metastasis.

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Code availability

Codes used in this study (for example, ImageJ macros for image analysis) are freely available from the corresponding author upon reasonable request.

Data availability

RNA-seq data that support the findings of this study have been deposited in the GEO under accession code GSE119153. MS data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012002. Source data for Figs. 1c,f–h, 2b,c,e,g–i,k,m, 3a,b,e,h, 4g,i, 5c,e–g,i–m, 6b,e,h,j,l and 7b,f,h,j, and Supplementary Figs. 1c–e, 3a–l, 4b,c, 5b,d,e,g,h,j and 6a–d have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank the following people: G. Merlino and C.-P. Day for providing C.FVB-Tg(GNrhr-luc/EGFP)L8Mrln/LmwJ mice; A. Putnam for providing human umbilical vein endothelial cells; S. Beronja for providing murine targeted shRNA constructs; P. Paddison for providing the mVenus-p27K vector; C. Morse for instruction on bilateral ovariectomy; S. Hingorani for in-depth discussions about preclinical trial design; and P. Lampe for instruction regarding antibody purification. They are also thankful to S. Beronja for his critical feedback on this work. This study was catalysed by start-up funds provided by the Fred Hutchinson Cancer Research Center and a grant from the Cuyamaca Foundation (to C.M.G.), and supported to its completion by an Era of Hope Award from the Department of Defense (DoD) Breast Cancer Research Program (BCRP; W841XWH-15-1-0201 to C.M.G. and K.C.H.), a grant from the Breast Cancer Research Foundation (IIDRP-17-001 to C.M.G.), a grant from the National Breast Cancer Coalition’s Artemis Project for Metastasis Prevention, a Physical Sciences Oncology Project Grant from the NIH/NCI (U54CA193461-01 to C.M.G.), and by the Comparative Medicine, Antibody Technology, Experimental Histopathology and Genomics Shared Resources of the Fred Hutchinson/University of Washington Cancer Consortium (P30 CA015704). P.S.N.’s laboratory is supported by the Prostate Cancer Foundation and NIH/NCI grants P50CA097186, U54CA224079 and R01CA165573, and by the DoD awards PC170431 and PC160662. K.C.H.’s laboratory is also supported by a grant from the NIH/NCI (R33CA183685). A.D. is supported by a fellowship from the Terri Brodeur Breast Cancer Foundation, C.A.G. is supported by a postdoctoral fellowship from the Susan G. Komen Foundation, J.D. is supported by a Postdoctoral Breakthrough award by the DoD’s BCRP, A.R.L. was supported by a Cardiovascular Pathology Training Grant from the NIH (5T32HL007213-38) and is currently supported by a fellowship from the NIH/NCI (1F31CA228424-01), and S.B.C. is supported by a Cellular and Molecular Biology Training Grant from the NIH (T32GM007270).

Author information

Author notes

  1. These authors contributed equally: Patrick Carlson, Arko Dasgupta, Candice A. Grzelak, Jeanna Kim.

Affiliations

  1. Public Health Sciences Division/Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    • Patrick Carlson
    • , Arko Dasgupta
    • , Candice A. Grzelak
    • , Jeanna Kim
    • , Ryann E. Shor
    • , Erica T. Goddard
    • , Jinxiang Dai
    • , Emma M. Schweitzer
    • , Andrea R. Lim
    • , Sarah B. Crist
    •  & Cyrus M. Ghajar
  2. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    • Alexander Barrett
    •  & Kirk C. Hansen
  3. Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    • Ilsa M. Coleman
    • , Peter S. Nelson
    •  & Cyrus M. Ghajar
  4. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    • Ilsa M. Coleman
    •  & Peter S. Nelson
  5. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, WA, USA

    • Andrea R. Lim
    •  & Sarah B. Crist
  6. Department of Pathology, University of California, San Diego, La Jolla, CA, USA

    • David A. Cheresh
  7. Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA

    • David A. Cheresh
  8. Department of Medicine, University of Washington, Seattle, WA, USA

    • Peter S. Nelson
  9. Department of Urology, University of Washington, Seattle, WA, USA

    • Peter S. Nelson
  10. Department of Pathology, University of Washington, Seattle, WA, USA

    • Peter S. Nelson

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Contributions

P.C., A.D., C.A.G., J.K., R.E.S., E.T.G., J.D., E.M.S., A.R.L. and S.B.C. performed experiments and analysed data. I.M.C. and P.S.N. conducted all procedures related to RNA sequencing, and analysed and interpreted resulting data. A.B. and K.C.H. conducted all ECM-targeted MS, and analysed and interpreted the resulting data. D.A.C. provided critical reagents. D.A.C., P.S.N. and K.C.H. provided scientific insight. C.M.G. conceived the study, conducted experiments, analysed and interpreted data, and wrote the manuscript. All authors read and provided feedback on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Cyrus M. Ghajar.

Integrated supplementary information

  1. Supplementary Figure 1 Dose-dense chemotherapeutic regimens select for perivascular disseminated tumour cells in tibia.

    Tibia from the experiment detailed in Fig. 1a were stained and whole-mounted. Tile scans encompassing the full area of the bone and the full Z-depth that could be imaged using this method were acquired. n = 228 DTCs were analyzed across three tibia from three AC-treated mice, and a) binned in histogram format or b) dot-plotted as individual distances to the nearest blood vessel (red), megakaryocyte (gray) or osteoblast (black). c-e) Average of these distances to the nearest c) blood vessel, d) megakaryocyte and e) osteoblast for mice that underwent treatment with vehicle (n = 286 DTCs analyzed) or dose-dense AC (n = 228). *** P < 0.0001; all P values obtained via unpaired, two-tailed t test. f-h) the percentage of DTCs on (i.e., with a plotted distance of zero) h) blood vessels, i) megakaryocytes and j) osteoblasts in tibia of mice that underwent treatment with vehicle or dose-dense AC. For c-e, centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.

  2. Supplementary Figure 2 Endothelium does not bias mesenchymal stem cells towards or away from osteo- and adipo-genic lineages in bone marrow MVNs.

    a) Gene set enrichment analysis of GO “Osteoblast Differentiation” genes and b) Hallmark “Adipogenesis” genes in bone marrow MVNs and bone marrow stroma (derived from analysis of (n = 2 biologically independent samples). The enrichment statistic was computed using the Weighted Kolmogorov Smirnov test, and controlled for multiple testing by false discovery rate. Neither indicate significant enrichment in bone marrow MVNs. P-values and FDR q values listed atop figure. Follow-up staining of bone marrow stroma (MSC) and bone marrow MVNs (MSC+EC) for calcium deposits and lipid droplets via c) the von Kossa method and d) Oil Red O stain. Representative images from n = 3 independent experiments shown.

  3. Supplementary Figure 3 Microvascular endothelium protects basal breast tumour cells from other therapies and luminal/ER+ breast tumour cells from doxorubicin.

    T4-2 cells were seeded on bone marrow stroma or on bone marrow MVNs, as described in Fig. 2a, and treated with increasing doses of 5-FU or lapatinib. Dose response of T4-2 ‘DTCs’ to 5-FU on bone marrow a) stroma and b) MVNs (n = 3 independent experiments). NS denotes no significance, whereas * P = 0.020 and ** P = 0.005 by one-way ANOVA and Dunnett’s multiple comparison test, comparing the indicated dose to vehicle treatment. The percentage of TUNEL-positive cells was quantified across n = 3 independent experiments for c) stroma and d) MVNs. NS, no significance, *** P < 0.0001 by one-way ANOVA and Dunnett’s post-test compared to vehicle treatment. Dose response of T4-2 cells to lapatinib on bone marrow e) stroma and f) MVNs (n = 5 independent experiments). NS, no significance, * P = 0.038, ** P = 0.0038 by one-way ANOVA and Dunnett’s post-test, compared to vehicle treatment. g) The percentage of TUNEL-positive cells was quantified across n = 3 independent experimentsfor g) stroma and h) MVNs. NS, no significance, *** P < 0.0001 when comparing the condition indicated to vehicle by one-way ANOVA and Dunnett’s post-test. Dose response of MCF-7 breast tumour ‘DTCs’ to doxorubicin on bone marrow i) stroma and j) MVNs (n = 5 independent experiments). NS, no significance, * P = 0.0373, *** P = 0.0007 when comparing the indicated condition(s) to vehicle (0 nM) by one-way ANOVA and Dunnett’s post-test. TUNEL intensity within YFP+ tumour cells/cell clusters was quantified on bone marrow k) stroma and l) MVNs across n = 3 independent experiments. *** P < 0.0001. Indicated P values obtained by unpaired two-tailed t test. For TUNEL analyses, the number of cells (predominantly single cells and clusters of 2–4 cells) analyzed across n = 3 independent experiments is stated within each panel. For a-l, centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.

  4. Supplementary Figure 4 IGF-1 triggers quiescent breast tumour cells to re-enter the cell cycle.

    Top: To assess the effect of IGF-1 on the phenotype of perivascular breast tumour cells, bone marrow MVNs were primed with 300 ng/mL IGF-1 at days 10, 12 and 15 (vs. vehicle control) a) Representative images of quiescent, microvascular-associated T4-2 cells at Day 10 (left), and after priming with vehicle or with 300 ng/mL IGF-1 at Day 17 (middle). Cultures were fixed and stained at this time point to analyze the proliferative status of tumour cells (right). Scale bars, 50 μm. White arrowhead denotes a Ki67+ tumour cell within an IGF-1 primed culture. b) Tumour cell outgrowth between day 10 and day 17 after priming with vehicle or IGF-1, normalized to vehicle control (n = 5 independent experiments). ** P = 0.0016 by unpaired, two-tailed t test. c) Percentage of Ki67+ DTCs/DTC cluster at day 17 after priming with vehicle or 300 ng/mL IGF-1. n = 31 (Vehicle) and n = 32 (Primed) DTCs were analyzed across n = 3 independent experiments. *** P = 0.0002 by unpaired, two-tailed t test. For b-c, centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.

  5. Supplementary Figure 5 Knockdown studies confirm that integrin inhibition functions specifically in the tumour cell compartment to induce chemosensitization, without affecting cell survival.

    a) Representative immunoblots of integrin-β1 and the nuclear envelope proteins Lamin A/C (used as a loading control). ‘K’ depicts the Kaleidoscope molecular weight marker. Lanes 1 and 3 are lysates of independently generated T4-2 cells transduced with non-targeting shRNA (shNT). Lanes 2 and 4 are lysates of independently generated T4-2 cells transduced with shRNA targeting ITGB1 (shITGB1, clone 391001). b) Densitometry from n = 3 independent experiments, normalized to the band intensity of shNT control. *** P < 0.0001 by unpaired, two-tailed t test. shNT and shITGB1 T4-2 cells were seeded on bone marrow MVNs as depicted in Fig. 2a and analyzed 12-days later. c) Representative images of bone marrow MVNs at this time point. Scale bar, 200 μm. d) Number of clusters and e) Tumour cell outgrowth at day 12, to assess the effect of ITGB1 knockdown on tumour cell phenotype in the absence of doxorubicin (n = 5 independent experiments). ** P = 0.0039. P values obtained by unpaired, two-tailed t test. Cultures were treated at days 12 and 15 with 2500 nM doxorubicin, then fixed and stained two days later to assess DTC viability. f) Representative images of TUNEL stained shNT and shITGB1 DTCs on bone marrow MVNs. Scale bar, 20 μm. White arrowhead, TUNEL-positive tumour cell. g) Tumour cell outgrowth over the treatment period (n = 5 independent experiments). * P = 0.050 by unpaired two-tailed t test. The total percentage of h) TUNEL-positive tumour cells, i) TUNEL-positive single DTCs, and j) TUNEL-positive cells within 2–4 cell clusters was analyzed across n = 116 (shNT) and n = 124 (shITGB1) total cells/clusters across three independent experiments. *** P < 0.0001 by unpaired, two-tailed t test. For b, d-e, g-h, j centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.

  6. Supplementary Figure 6 Integrin inhibition does not exacerbate chemotherapy-induced toxicities.

    Mice treated as described in Fig. 6d were a) weighed weekly. Body weights of treated mice did not differ significantly over the course of treatment or after one week, as assessed by multiple unpaired t tests and correcting for multiple comparisons using the Holm-Sidak method. Cohort size: IgG+AC: 11 animals; AIIB2+AC: 10 animals; AIIB2+LM709+AC: 9 animals. b) Bone marrow vascular density was determined from pan-laminin staining femurs of athymic nude females from the study described in Fig. 6c (Cohort 1). Data were not statistically significant, as determined by Kruskall-Wallis test and Dunn’s post-test to correct for multiple comparisons. c) Spleens from these mice were also weighed to grossly account for changes in immune cell content. Splenic weights did not differ between treatment conditions (but all were substantially lower than mice treated only with isotype control antibody), as assessed by one-way ANOVA and Dunnett’s post-test. For (b) and (c), Cohort size: IgG+AC: 4 animals; AIIB2+AC: 4 animals; AIIB2+LM709+AC: 3 animals. d) The effect of AIIB2 and/or AC therapy on immune cell content within bone marrow of uninoculated, immune-competent FVB mice was quantified by flow cytometery to determine whether AIIB2 affected specific immune cell populations. n = 3 animals were analyzed per treatment. No significant differences in viable CD45+ leukocytes were observed. *P = 0.029 and **P = 0.0096 when comparing the number of CD11b+ cells in IgG+AC and AIIB2+AC treated mice to untreated mice. **P = 0.0011 and ***P = 0.0008 when comparing the number of B220+ cells in IgG+AC and AIIB2+AC treated mice to untreated mice; *P = 0.033 when comparing the number of CD3+ cells in IgG+AC treated mice to untreated mice (comparing CD3+ cells in AIIB2+AC vs. untreated mice yielded P = 0.073). In each case, comparing IgG+AC and AIIB2+AC treatments yielded P values >0.983. All comparisons were made using one-way ANOVA and Dunnett’s post-test. For a-d, centre line represents the mean, error bars the s.e.m. Statistical tests and P values are indicated. Source data are provided in Supplementary Table 1.

  7. Supplementary Figure 7 Unprocessed immunoblots to assess shRNA-mediated knockdown of VWF and integrin β1.

    a) Full representative immunoblot of VWF and nuclear envelope proteins Lamin A/C (used as a loading control). Lane 1 is from lysate of HUVEC transduced with non-targeting (NT) shRNA. Lane 2 is from lysate of HUVEC transduced with shRNA targeting VWF (VWF, clone 643). Lane 3 is from lysate of HUVEC transduced with shRNA targeting VWF (VWF, clone 620). Results are representative of those obtained from n = 3 independently generated lysates per condition. Densitometry from these three independent experiments is shown in Fig. 5b. b) Full representative immunoblots of integrin-β1 and the nuclear envelope proteins Lamin A/C. ‘K’ depicts the Kaleidoscope molecular weight marker. Lanes 1 and 3 are independently generated lysates from T4-2 cells transduced with non-targeting shRNA (shNT). Lanes 2 and 4 are independently generated lysates from T4-2 cells transduced with shRNA targeting ITGB1 (shITGB1, clone 391001). Results are representative of those obtained for n = 3 independently generated lysates per condition. Densitometry from these three independent experiments is shown in Supplementary Figure 5b.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7, and legends for Supplementary Tables and Supplementary Videos.

  2. Reporting Summary

  3. Supplementary Table 1

    Statistics source data.

  4. Supplementary Table 2

    Primary tumour, bone marrow area, and DTC measurements related to Fig. 6l.

  5. Supplementary Table 3

    Details on antibodies used for immunofluorescent staining, immunoblotting, flow cytometry and blocking integrin function.

  6. Supplementary Table 4

    Sequence information for shRNA clones.

  7. Supplementary Video 1

    Apoptosis of p27-positive and p27-negative breast tumour cells in response to doxorubicin on bone marrow stroma.

  8. Supplementary Video 2

    Apoptosis of p27-negative breast tumour cells in response to doxorubicin on bone marrow microvascular niche culture.

  9. Supplementary Video 3

    Death of a breast tumour cell cluster containing a p27-positive and a p27-negative breast tumour cell in response to doxorubicin on a bone marrow microvascular niche culture.

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DOI

https://doi.org/10.1038/s41556-018-0267-0