Abstract

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

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Data availability

Uncropped and replicate western blots that are not shown in the figures are provided in Supplementary Fig 9. The 4T1-EV data have been submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV180041; ref. 69). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository70 with the following dataset identifiers: PXD010362 (accession) and 10.6019/PXD010362 (DOI) for the 4T1 EV data and PXD010292 (accession) and 10.6019/PXD010292 (DOI) for the human EV data. The source data for all graphical representations are provided as Supplementary Tables 3 and 5. All other data supporting the findings of this study are available from the corresponding authors on request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank C. Rmili-Wyser, A. Bellotti, B. Torchia, D. Laoui (M.D.P.’s laboratory) and M. Duquette (R.K.J.’s laboratory) for help with some experiments; T. Kitamura (University of Edinburgh) for advice on lung colonization assays and for providing E0771-LG and E0771-LG:Fl cells and H.G. Augustin (DKFZ) for critical comments on the manuscript. The EPFL core facilities of flow cytometry (FCCF), histology (HCF) and bioimaging/optics platform (BIOp) are acknowledged for skilled technical assistance; R. Hamelin and M. Moniatte of the proteomics facility (PCF, EPFL) for performing LC–MS/MS on EVs; T.J. Chico for providing access to zebrafish lines in the aquarium at the University Sheffield and R. Klemke for the kind gift of CFP-MDA-MB-435 cells. This work was primarily funded by grants from the Swiss Cancer League (grant no. KFS-3007-08-2012), Swiss National Science Foundation (grant no. SNF 31003A-165963) and European Research Council (grant no. ERC EVOLVE-72505) to M.D.P. L.M.S. was supported by NIH grant no. KL2 TR001100. C.E.L. acknowledges support from Cancer Research UK (grant no. C11712/A13028), Yorkshire Cancer Research (grant no. S382) and Breast Cancer Now (grant nos 2016MayPR746 and 2016NovPCC003). M.L.I.-A. was supported by NIH (NCI 1R01CA197943). L.M.C. acknowledges support from a DOD BCRP Era of Hope Scholar Expansion Award (grant no. W81XWH-08-PRMRP-IIRA), Susan B Komen Foundation (grant no. KG110560) and Breast Cancer Research Foundation. A.B. was supported by Susan B Komen Foundation (grant no. CCR15224703). R.K.J. acknowledges support from the Ludwig Center at Harvard, National Foundation for Cancer Research and NCI (grant no. R35CA197743). J.W.P. was supported by the Wellcome Trust (grant no. 101067/Z/13/Z) and MRC (grant no. MR/N022556/1).

Author information

Author notes

  1. These authors contributed equally: Ioanna Keklikoglou, Chiara Cianciaruso.

Affiliations

  1. Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    • Ioanna Keklikoglou
    • , Chiara Cianciaruso
    • , Mario Leonardo Squadrito
    • , Caroline Baer
    • , Antonino Cassará
    • , Alan Guichard
    • , M. Luisa Iruela-Arispe
    •  & Michele De Palma
  2. MRC Centre for Reproductive Health, Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

    • Esra Güç
    •  & Jeffrey W. Pollard
  3. Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA

    • Laura M. Spring
    •  & Aditya Bardia
  4. Department of Oncology and Metabolism, Medical School, University of Sheffield, Sheffield, UK

    • Simon Tazzyman
    • , Lore Lambein
    •  & Claire E. Lewis
  5. Department of Cell, Developmental and Cancer Biology, Oregon Health and Sciences University, Portland, OR, USA

    • Amanda Poissonnier
    •  & Lisa M. Coussens
  6. Edwin L. Steele Laboratories, Department of Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA

    • Gino B. Ferraro
    •  & Rakesh K. Jain
  7. Department of Molecular Cell and Developmental Biology, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA, USA

    • M. Luisa Iruela-Arispe
  8. Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA

    • Lisa M. Coussens
  9. Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, NY, USA

    • Jeffrey W. Pollard

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Contributions

I.K. designed and performed most of the experiments, analysed and interpreted data and wrote the manuscript. C.C. designed and performed experiments, analysed and interpreted data and wrote the manuscript. E.G. and J.W.P. designed, performed and analysed experiments in Ccr2 KO mice. M.L.S. designed lentiviral vectors for gene KO, overexpression and reporter activity. A.C., C.B., A.G. and G.B.F. assisted with some experiments. A.P. and L.M.C. designed, performed and analysed experiments in MMTV-PyMT mice (OHSU cohort). S.T., L.L. and C.E.L designed, performed and analysed the zebrafish experiments. M.L.I.-A. designed and performed some experiments while on sabbatical in M.D.P.’s laboratory. L.M.S., A.B. and R.K.J. provided clinical samples and discussed and interpreted the results. All authors provided intellectual input, reviewed the data and the manuscript. M.D.P. designed, supervised and coordinated research, interpreted the data and wrote the manuscript.

Competing interests

L.M.S. reports consulting fees from Novartis. L.M.C. is a paid consultant for Cell Signaling Technologies, received reagent support from Plexxikon and NanoString Technologies and is a member of the Scientific Advisory Boards of Syndax Pharmaceuticals, Carisma Therapeutics and Verseau Therapeutics. A.B. reports consulting fees from Genentech/Roche, Immunomedics, Novartis, Pfizer, Merck, Radius Health, Spectrum Pharma and Taiho Pharma and received a research grant from Biothernostics. R.K.J. received honoraria from Amgen and consultancy fees from Merck, Ophthotech, Pfizer, SPARC, SynDevRx, and XTuit, owns equity in Enlight, Ophthotech, SynDevRx and serves on the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. M.D.P. reports honoraria from Merck and Sanofi/Regeneron Pharmaceuticals, received sponsored research grants from Hoffmann La-Roche, MedImmune and Deciphera Pharmaceuticals and serves on the Scientific Advisory Boards of Deciphera Pharmaceuticals and Genenta. The other authors declare no competing interests. Neither materials nor funding from the above organizations were used in this study.

Corresponding authors

Correspondence to Ioanna Keklikoglou or Michele De Palma.

Integrated supplementary information

  1. Supplementary Fig 1 PTX does not alter the physical features of mammary tumour-derived Evs.

    (a) Concentration (mean ± s.e.m.; n = 5 acquisitions of one sample/condition) and size distribution of EVs isolated from medium conditioned by CREMO- or PTX-treated 4T1 cells, determined by NTA. (b) Western blotting analysis of the indicated proteins in material recovered after low-speed (2,000 x g, 2K), medium-speed (4,600 x g, 4.6K) or high-speed (134,000 x g, 134K) centrifugation of medium conditioned by 4T1 cells treated as indicated. The experiment was performed once, but additional data on EVs isolated after high-speed centrifugation are also shown in Fig. 3d–f, h and Fig. 4c. (c) Correlation between EV protein content and EV concentration, measured by BCA and NTA, respectively, in EVs isolated from medium conditioned by 4T1 cells treated as indicated. Four serial dilutions and three technical replicates of one sample/condition were measured; the mean of technical replicates is shown. A simple linear regression function was used to determine the relationship between the two parameters. The amount of EVs contained in each EV dose administered to mice (15 μg) was determined by extrapolation of the data. (d) Representative TEM images of EVs isolated ex vivo from MMTV-PyMT tumours that had been treated in vivo with either CREMO or PTX (see Fig. 2d). Scale bars, 200 nm. The experiment was performed once. (e) Concentration (mean ± s.e.m.; n = 5 acquisitions of one sample/condition) and size distribution of MMTV-PyMT tumour-derived EVs obtained as in (d), determined by NTA. (f) Mode size (mean values ± s.d.; n = 3 independent EV preparations) of MMTV-PyMT tumour-derived EVs obtained as in (d), determined by NTA. (g) Western blotting analysis of CD9 and GAPDH in MMTV-PyMT tumour-derived cells and their EVs. The experiment was performed once. Source data are shown in Supplementary Table 5 and unprocessed blots in Supplementary Fig. 9

  2. Supplementary Fig 2 PTX enhances EV release from cancer cells in vitro and in tumour-bearing mice.

    (a) WST-1 assay-based viability of 4T1 cancer cells (mean ± s.d.; n = 6 independent cell cultures/condition) relative to untreated cells (UT). (b) mCh mean fluorescence intensity (MFI; mean ± s.e.m.; n = 3 independent cell cultures/condition) in medium conditioned by 4T1-mCh cells treated for 72h as indicated. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. (c) Protein content by BCA (left y axis) and concentration by NTA (right y axis) in EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from MDA-MB-231 cells treated for 72h with CREMO or PTX. Statistical analysis by unpaired two-tailed Student’s t-test. (d) Absolute mCh MFI (mean ± s.e.m.) in plasma of tumour-free (n = 6), 4T1 tumour-bearing (n = 10), and 4T1-mCh tumour-bearing Rag1-/- mice treated with CREMO (n = 12) or PTX (n = 14); two independent experiments combined (left panel). Statistical analysis as in (b). Right panel shows data after subtracting background fluorescence (ΔMFI; mean ± s.e.m.) and normalizing the results to tumour weight. Statistical analysis as in (c). (e) Correlation between tumour weight and mCh ΔMFI in plasma of mice shown in (d), right panel. The Pearson correlation coefficient (r) is indicated. (f) mCh ΔMFI (mean ± s.e.m.) in plasma of 4T1-mCh/HER2 tumour-bearing Rag1-/- mice (PBS, n = 7; PTX, n = 9; CREMO, n = 8). Statistical analysis as in (b). (g) Correlation between mCh MFI and protein concentration by BCA (left) or particle concentration by NTA (right) in EVs from medium conditioned by 4T1-mCh cells. Five (left) or four (right) serial dilutions and three technical replicates of one sample/condition were measured; the mean of technical replicates is shown. A simple linear regression function was used to extrapolate relationship between the two variables. (h) Western blotting analysis of the indicated proteins in EVs or EV-depleted, concentrated conditioned medium (CM) from 4T1, 4T1-mCh or PyMT-IK1-mCh cells. EVs and CM were recovered after high-speed (134,000 x g) centrifugation. Each experiment was performed once. (i) mCh MFI and protein concentration by BCA (left) or particle concentration by NTA (right) in EVs isolated from medium conditioned by 4T1-mCh cells. Six EV fractions were obtained by sucrose fractionation of purified EVs. Three technical replicates of each EV fraction/condition were measured; the mean of technical replicates is shown. (j) mCh MFI (mean ± s.e.m.) in plasma of 4T1 (n = 3) or 4T1-mCh (n = 7) tumour-bearing Rag1-/- mice. Plasma was incubated with or without proteinase K (PK). Statistical analysis as in (b). (k) mCh MFI (mean ± s.e.m.) of recombinant mCh incubated with or without PK in either water (n = 3 independent experiments/condition) or mouse plasma (n = 4). Data show percentage values over untreated (-PK) samples. Statistical analysis by two-way ANOVA with Sidak’s multiple comparison test. Source data are shown in Supplementary Table 5 and unprocessed blots in Supplementary Fig. 9

  3. Supplementary Fig 3 PTX increases cancer cell-derived EV release in a RAB27A-dependent and apoptosis-independent manner.

    (a, b) FACS of 4T1 cells treated for 48h with CREMO or PTX (n = 3 independent cell cultures/condition) in the presence of the pan-caspase inhibitor CAS-BIND Pro or DMSO (vehicle). Panel (a) shows representative FACS dot plots of cells stained for ANXA5 and 7AAD to identify early apoptotic (ANXA5+7AAD) cells. Panel (b) shows quantitative data (mean ± s.d.). Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. (c) mCh MFI (left y axis) and concentration by NTA (right y axis) of EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from 4T1-mCh cells treated for 72h with PTX in the presence of CAS-BIND Pro or DMSO (vehicle). Statistical analysis by unpaired two-tailed Student’s t-test. (d) Western blotting analysis of RAB27A and GAPDH in Rab27a-KO or WT 4T1-mCh cells. The experiment was performed once. (e-g) mCh MFI (left y axis) and concentration by NTA (right y axis) of EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from Rab27a-KO or WT 4T1-mCh cells treated as indicated. Statistical analysis as in (c). (h) Subcellular localization of mCh in 4T1-mCh cells treated as indicated. Panels above show representative images of cells stained with anti-beta-tubulin antibody (green) and DAPI (blue); mCh signal (magenta) was acquired as direct fluorescence. Scale bars, 10 μm. Panels below show ratio between membrane and cytoplasm-associated mCh signal (mean ± s.e.m.; n = 4 randomly selected image fields, each containing at least 120 cells, per condition). Statistical analysis as in (c). Note increased localization of mCh to plasma membranes after PTX in RAB27A-proficient cells. (i) mCh MFI (mean ± s.d.; n = 5 independent cell cultures/condition) of EVs from 4T1-mCh cells treated for 72h as indicated. Statistical analysis as in (c). (j) mCh MFI (left y axis) and concentration by NTA (right y axis) of EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from 4T1-mCh cells treated for 72h as indicated. Statistical analysis as in (c). (k) Protein content by BCA (left y axis) and concentration by NTA (right y axis) in EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from MDA-MB-231 cells treated for 72h as indicated. Statistical analysis as in (c). Source data are shown in Supplementary Table 5 and unprocessed blots in Supplementary Fig. 9

  4. Supplementary Fig 4 PTX promotes loading of ANXA6 into bona fide cancer cell-derived Evs.

    (a) Western blot analysis of the indicated proteins in 4T1-mCh EVs after fractionation by sucrose density gradient centrifugation. Note the association of ANXA6 with EV fractions with a density ranging from 1.12 to 1.17 g/ml. One representative experiment is shown of two performed. (b) Protein band intensity (n = 2 independent experiments, one of which is shown in (a) above) in the indicated EV fractions. For each protein, the relative signal intensity in each fraction is indicated as percentage of the total signal in all fractions. The line connects mean values. (c) ELISA assay showing the binding of antibodies against CD81, GP96 or ANXA6 to CREMO-EVs and PTX-EVs. Note the limited binding of an anti-ANXA6 antibody to either EV preparation after washing, compared to the anti-CD81 antibody. Results show one EV sample per condition and the mean of three technical replicates. (d) Western blotting analysis of ANXA6, GAPDH and CD81 in CREMO-EV and PTX-EV incubated with EDTA or PBS. Note that EDTA did not reduce ANXA6 signal in EVs, indicating intravesicular localization. One representative experiment is shown of three performed. (e) Viability of primary mouse bone marrow dendritic cells (BMDCs; mean ± s.d.; n = 3 independent cell cultures) treated as indicated for 72h. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. (f) Western blotting analysis of ANXA6, GAPDH and CD81 in CREMO-EV and PTX-EV isolated from BMDCs. One representative experiment is shown of two performed. (g) Viability of primary mouse embryonic fibroblasts (MEFs; mean ± s.d.; n = 4 independent cell cultures) treated as indicated for 72h. (h) Western blotting analysis of ANXA6, GAPDH and CD81 in CREMO-EV and PTX-EV isolated from MEFs. The experiment was performed once. (i) Western blotting analysis of ANXA6 and GAPDH in Anxa6-WT (parental) and Anxa6-KO 4T1 cells. The experiment was performed once. (j) Quantification of Oregon Green 488 PTX (left) and autofluorescent DOX (right) in EVs (mean ± s.d.; n = 3 independent cell cultures/condition) from Anxa6-WT or Anxa6-KO 4T1 cells. (k) Quantification of Oregon Green 488 PTX (left) and autofluorescent DOX (right) in EVs from MDA-MB-231, PyMT-IK1 and 4T1 cells. Data show mean values of three serial dilutions per sample. Source data are shown in Supplementary Table 5 and unprocessed blots in Supplementary Fig. 9

  5. Supplementary Fig 5 EV transfer to ECs induces a drug-independent pro-inflammatory response.

    (a) Representative confocal images of bEnd.3 cells either untreated or incubated for 24h with PTX-EVs (50 g/ml) from 4T1-mCh cells. Cells were stained with an anti-mCh antibody (red), AF488-conjugatd phalloidin (white) and DRAQ5 to reveal nuclei (cyan). Orthogonal projections from a z-stack acquisition are shown. Scale bars 10 μm. One image per condition is shown, representative of 3 independent experiments. (b) FACS analysis of bEnd.3 cells treated for 24h with increasing doses of EVs isolated from 4T1-mCh cells. One representative sample per condition is shown. (c, d) FACS analysis of mCh in bEnd.3 cells incubated with conditioned medium (CM; c) or purified mCh+ EVs (d) from 4T1-mCh (left panels) or PyMT-IK1-mCh (right panels) cells treated as indicated. mCh-negative 4T1 and PyMT-IK1 cells were used as controls. Data show mCh MFI (mean ± s.d.; n = 3 independent cell cultures/condition). Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. (e) FACS analysis of mCh in bEnd.3 cells incubated with conditioned medium (CM) of Anxa6-WT or Anxa6-KO 4T1-mCh cells treated as indicated. Data show mCh MFI (mean ± s.d.; n = 3 independent cell cultures/condition). Statistical analysis by two-way ANOVA with Sidak’s multiple comparison test. Source data are shown in Supplementary Table 5

  6. Supplementary Fig 6 ANXA6 induces NF-kB activation and Ccl2 upregulation in bEnd.3 cells.

    (a) Western blotting analysis of ANXA6 and GAPDH in Anxa6-WT and KO bEnd.3 cells. The experiment was performed once. (b) Western blotting analysis of ANXA6, mCh and beta-actin (ACTB) in untreated Anxa6-WT 4T1 and bEnd.3 cells, as well as untreated or EV-treated Anxa6-KO bEnd.3 cells. One representative experiment of two performed; additional data are shown in Fig. 7d. (c) Duolink staining of bEnd.3 cells. Upper panel shows ANXA6/p65 proximity expressed as number of dots/cell (mean ± s.e.m.; n = 8 randomly selected images with at least 12 cells each); unstained Anxa6-WT bEnd.3 cells (n = 3 randomly selected images with at least 12 cells each) were used as negative control. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. Bottom panels show representative images; nuclei are stained with DAPI (blue), actin cytoskeleton with phalloidin (red). Scale bar, 30 μm. (d) Duolink staining of Anxa6-KO bEnd.3 cells treated as indicated. Images show ANXA6/p65 proximity (white dots); nuclei are stained with DAPI (blue), actin cytoskeleton with phalloidin (red). Scale bars, 30 μm. Results are representative of two independent experiments. (e) NF-kB activity in bEnd.3 cells. Left panel shows the NF-kB reporter lentiviral vector (LV). Note that red fluorescence of LV-encoded mCh is much stronger than the red fluorescence of EV-transferred mCh. Therefore, EV-derived mCh does not affect quantification of NF-kB reporter activity. Right panel shows representative FACS dot plots of bEnd.3 cells either untransduced (UT) or transduced with the NF-kB reporter LV before incubation with or without TNFα for 48h. Transduced (mCh+) cells are gated and analysed for GFP expression. For quantitative data, see (f). (f) NF-kB activity determined by FACS analysis of transduced bEnd.3 cells incubated with or without TNFα for 24h. NF-kB activity is shown as GFP MFI in transduced (mCh+) cells (mean ± s.d.; n = 3 independent cell cultures/condition). Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test. (g-i) qPCR analysis of Ccl2 (g), Csf1 (h) and Cxcl12 (i) in bEnd.3 cells incubated with or without TNFα for 5h (mean ± s.d.; n = 3 independent cell cultures/condition). Statistical analysis as in (f). Source data are shown in Supplementary Table 5 and unprocessed blots are shown in Supplementary Fig. 9

  7. Supplementary Fig 7 Chemotherapy-elicited EVs display broad cellular tropism in tumour-bearing mice.

    (a) FACS analysis of mCh+CD45+CD11b+Ly6G macrophages/monocytes (mac/mono; mean ± s.e.m.; relative to viable lung-derived cells) in the lungs of 4T1 (n = 6) or 4T1-mCh (CREMO, n = 8; PTX, n = 9) tumour-bearing Rag1–/– mice treated as indicated. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. The right panels show representative FACS dot plots. (b) FACS analysis of mCh+CD45+CD11b+Ly6G mac/mono (mean ± s.e.m.; relative to viable lung-derived cells) in the lungs of 4T1 (n = 4) or 4T1-mCh/HER2 (PBS, n = 7; CREMO, n = 8; PTX, n = 9) tumour-bearing Rag1–/– mice treated as indicated. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. (c) qPCR analysis of Il6 in whole lung tissue (mean ± s.e.m.) of FVB/n mice that received either CREMO-EV (n = 10) or PTX-EV (n = 9). Statistical analysis by unpaired two-tailed Student’s t-test. (d) qPCR analysis of Il6 (mean ± s.e.m.) in FACS-sorted lung myeloid cells of FVB/n mice that received either CREMO-EV or PTX-EV (n = 5 mice per group, except Mac/CREMO-EV where n = 4). Statistical analysis by two-way ANOVA with Tukey’s multiple comparison test. (e) FACS analysis of mCh+CD31+CD45- liver ECs (mean ± s.e.m.; relative to viable liver-derived cells) in the livers of 4T1 (n = 6) or 4T1-mCh (CREMO, n = 7; PTX, n = 9) tumour-bearing Rag1–/– mice treated as indicated. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. The right panels show representative FACS dot plots. (f) Representative confocal immunofluorescence images of anti-CD31 EC (green) and anti-mCh (magenta) immunostaining of liver sections from 4T1-mCh tumour-bearing mice treated as in (e); nuclei are stained with DAPI (blue). Scale bars, 10 μm. Source data are shown in Supplementary Table 5

  8. Supplementary Fig 8 ANXA6 is detected in circulating EVs of breast cancer patients undergoing neoadjuvant therapy.

    LC-MS/MS quantification of ANXA6 in EVs isolated from the plasma of six breast cancer patients before chemotherapy (pre-treatment), after AC (anthracycline (DOX)/cyclophosphamide) and after PTX. The data show the number of exclusive ANXA6 peptides (left) and the quantitative values after normalization to the total proteins identified in the same area of the gel (right). Source data are shown in Supplementary Table 3

  9. Supplementary Fig 9 Unprocessed blots.

    Unprocessed and uncropped blots for all Western blotting data reported in the main and supplementary figures of this study

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–9 and Supplementary Table legends.

  2. Reporting Summary

  3. Supplementary Table 1

    Protein quantification in CREMO–EV and PTX–EV (n = 6 independent EV preparations per condition) isolated from 4T1 cells, determined by LC-MS/MS analysis.

  4. Supplementary Table 2

    Medical record of breast cancer patients.

  5. Supplementary Table 3

    LC-MS/MS analysis of EVs in the plasma of breast cancer patients.

  6. Supplementary Table 4

    List of antibodies used in flow cytometry (FC), immunofluorescence (IF), Western blotting (WB) and ELISA analyses.

  7. Supplementary Table 5

    Statistics Source Data.

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DOI

https://doi.org/10.1038/s41556-018-0256-3