Tumour exosomal cemip protein promotes cancer cell colonization in brain metastasis

ABSTRACT The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the

contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances

cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone

metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by

pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and

inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by _Ptgs2_, _Tnf_ and _Ccl/Cxcl_, known to promote brain vascular remodelling and metastasis.

CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that

targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS EXOSOMAL MIR-155-5P DERIVED FROM GLIOMA STEM-LIKE CELLS

PROMOTES MESENCHYMAL TRANSITION VIA TARGETING ACOT12 Article Open access 19 August 2022 HYPOXIA LUAD H1975 CELL-DERIVED EXOSOMAL MIR-671-3P PROMOTES ANGIOGENESIS VIA REGULATING KLF2-VEGFR2

AXIS Article Open access 16 April 2025 EXOSOMES DERIVED FROM HYPOXIC GLIOMA DELIVER MIR-1246 AND MIR-10B-5P TO NORMOXIC GLIOMA CELLS TO PROMOTE MIGRATION AND INVASION Article 14 January 2021

DATA AVAILABILITY RNA-seq raw data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE136628. Mass spectrometry raw data have

been deposited in ProteomeXchange with the primary accession code PXD015210. The mass spectrometry processed data of MDA-MB-231 parental (parental), brain-tropic (231-BR (BrT1) and 831

(BrT2)), lung-tropic (4175 (LuT1) and 4173 (LuT2)) and bone-tropic (1833 (BoT1)) exosomes (Fig. 1c) are available in Supplementary Table 2. The processed RNA sequencing data from Fig. 4d and

Supplementary Tables 4, 5, 6, 7, for murine BrECs and microglia cells isolated from ex vivo brain slices treated with PBS, 231 BrT1 WT, 231 BrT1 CEMIP KO1 and KO2 exosomes, are available as

Supplementary Table 3. The processed patient data from Fig. 5 and Supplementary Fig. 5 are available as Supplementary Table 8. Unprocessed scans and replicates for all immunoblots presented

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(2016). Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M. Ginsberg, G. Marra, P. Raju and T. Milner for reagents and expert advice; L. Nogues Vera, M. Teixeira and S.

Grass for help in the laboratory; M. Schaeffer for proofreading; T. Zhang and K. Gyan for help with bioinformatics analysis; L. Cohen-Gould, the MSKCC Molecular Cytology Core Facility and

K. Uryu for imaging counselling; T. Miller and F. Fang at the MSKCC Flow cytometry Core Facility, and T. Baumgartner at the Weill Cornell Medicine Flow Cytometry Core, as well as R. Bowman,

for expert cell sorting; and the MSKCC Gene Editing and Screening Core Facility for molecular cloning and gene editing advice. We gratefully acknowledge support from the following funding

sources: the National Cancer Institute (CA169538 to D.L. and CA232093 to D.L.), the US Department of Defense (W81XWH-13-1-0427 to D.L.), the Breast Cancer Research Foundation (to D.L. and

C.M.G.), the Champalimaud Foundation, the Daedalus Fund for Innovation (Weill Cornell Medicine, to D.L.), the Children’s Cancer and Blood Foundation, the Pediatric Oncology Experimental

Therapeutics Investigator’s Consortium Foundation, the Nancy C. and Daniel P. Paduano Foundation, the Eileen and James A. Paduano Foundation, the Sohn Foundation, the Hartwell Foundation,

the Manning Foundation, the Thompson Foundation, the Malcolm Hewitt Wiener Foundation and the Tortolani Foundation. G.R. has been supported by a Peter Oppenheimer Fellowship, awarded by the

American Portuguese Biomedical Research Fund, and by the Fundação para a Ciência e a Tecnologia from Portugal. A.H. was supported by a Susan Komen Foundation for the Cure Fellowship. H.P. is

supported by grants from MINECO (SAF2014-54541-R), Fundación Fero, Asociación Española Contra el Cáncer and Worldwide Cancer Research. C.M.G. is supported by a US Department of Defense

Breast Cancer Research Program Era of Hope Scholar Award (W81XWH-15-1-0201), the US National Cancer Institute (CA193461-01), the National Breast Cancer Coalition’s Artemis Project and the

Pink Gene Foundation. AUTHOR INFORMATION Author notes * These authors contributed equally: Gonçalo Rodrigues, Ayuko Hoshino, Candia M. Kenific, Irina R. Matei. AUTHORS AND AFFILIATIONS *

Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill

Cornell Medicine, New York, NY, USA Gonçalo Rodrigues, Ayuko Hoshino, Candia M. Kenific, Irina R. Matei, Loïc Steiner, Daniela Freitas, Han Sang Kim, Ilana Scandariato, Irene Casanova-Salas,

 Haiying Zhang, Alberto Benito-Martin, Linda Bojmar, Yonathan Ararso, Katharine Offer, Quincey LaPlant, Weston Buehring, Huajuan Wang, Xinran Jiang, Héctor Peinado, Maria de Sousa & 

David Lyden * Graduate Program in Areas of Basic and Applied Biology, Abel Salazar Biomedical Sciences Institute, University of Porto, Porto, Portugal Gonçalo Rodrigues & Maria de Sousa

* International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Tokyo, Japan Ayuko Hoshino * JST, PRESTO, Tokyo, Japan Ayuko Hoshino * Swiss Institute for

Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Loïc Steiner & Etienne Meylan * i3S-Institute for Research and

Innovation in Health, University of Porto, Porto, Portugal Daniela Freitas * IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal Daniela

Freitas * Instituto de Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Porto, Portugal Daniela Freitas * Yonsei Cancer Center, Division of Medical Oncology, Departments of

Internal Medicine, and Pharmacology, Yonsei University College of Medicine, Seoul, Korea Han Sang Kim * Samuel J. Wood Library, Weill Cornell Medicine, New York, NY, USA Peter R. Oxley *

Public Health Sciences Division/Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Jinxiang Dai & Cyrus M. Ghajar * Ansary Stem Cell Institute,

Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY, USA Chaitanya R. Badwe, Tyler M. Lu & Shahin Rafii * Woman’s Malignancies Branch, Center

for Cancer Research, National Cancer Institute, Bethesda, MD, USA Brunilde Gril & Patricia S. Steeg * Proteomics Resource Center, The Rockefeller University, New York, NY, USA Milica

Tešić Mark, Brian D. Dill & Henrik Molina * Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine and Infertility, Weill Cornell Medicine (WCM), New York, NY, USA Tyler

M. Lu * Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Yuan Liu & David R. Jones * Thoracic Oncology Service, Memorial Sloan Kettering Cancer Center,

New York, NY, USA Joshua K. Sabari & Charles M. Rudin * Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Sandra J. Shin, Navneet Narula, Paula

S. Ginter & David Pisapia * Breast Medicine Service, Department of Medicine, Memorial Sloan Kettering Cancer Centre, New York, NY, USA Vinagolu K. Rajasekhar * Orthopaedic Service,

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA John H. Healey * Systems Oncology Group, Champalimaud Research, Champalimaud Centre for the Unknown, Lisbon,

Portugal Bruno Costa-Silva * Department of Pathology, University of California, San Diego, La Jolla, CA, USA Shizhen Emily Wang * Department of Cardiothoracic Surgery, Weill Cornell

Medicine, New York, NY, USA Nasser Khaled Altorki * Microenvironment and Metastasis Laboratory, Department of Molecular Oncology, Spanish National Cancer Research Center (CNIO), Madrid,

Spain Héctor Peinado * Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA Jacqueline Bromberg * Department of Medicine, Weill Cornell Medicine, New York, NY,

USA Jacqueline Bromberg Authors * Gonçalo Rodrigues View author publications You can also search for this author inPubMed Google Scholar * Ayuko Hoshino View author publications You can also

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publications You can also search for this author inPubMed Google Scholar * David Lyden View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS G.R.

designed the experimental approach, performed the experimental work, analysed the data, coordinated the project and wrote the manuscript. A.H. performed PT growth and exosome education in

vivo studies, cancer cell proliferation in vitro studies, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. C.M.K. generated CEMIP OE, performed

molecular cloning work and genetic manipulation of cancer cells, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. I.R.M. performed brain slice ex

vivo FACS analysis and exosome education in vivo studies, cancer cell culture and exosome isolation, coordinated the project, and wrote and reviewed the manuscript. L.S. performed brain

slice ex vivo experimental work, tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and quantification, cancer cell invasion in vitro studies, western blot

analysis, cancer cell culture and exosome isolation, and contributed to figure panel assembly. D.F. performed density gradient exosome isolation, characterization and analysis, western blot

analysis and cancer cell culture. H.S.K. and P.R.O. performed RNA sequencing data analysis. I.S. performed tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and

quantification, cancer cell culture and exosome isolation. I.C.-S. performed western blot analysis and assisted in analysis of human data. J.D., C.R.B. and T.M.L. performed in vitro BrEC

experimental work and FACS analysis. M.T.M., B.D.D. and H.M. performed exosome mass spectrometry and proteomic data analysis. A.B.-M. assisted in mouse studies and FACS analysis. H.W. and

X.J. assisted in tissue processing and immunostaining, cancer cell culture and exosome isolation. L.B., K.O., Q.L., Y.A., W.B. and H.W. received and processed human samples. Y.A., W.B. and

H.W. assisted in the maintenance of mouse colonies. Y.L., J.K.S., S.J.S., N.N., J.H.H., N.K.A., C.M.R., D.R.J. and D.P. provided human samples. V.K.R. generated and provided the N2LA human

lung cancer cell line. S.E.W. provided the 231-HM human breast cancer cell line. B.G. and P.S.S. provided the 231BR human breast cancer cell line and gave feedback on the project. S.R.

provided endothelial cell expertise and reagents. D.P. coordinated human studies and contributed to the experimental design. D.P., N.N. and P.S.G. analysed the human data. E.M. and H.Z. read

the manuscript and gave feedback on the project. B.C.-S., H.P., C.M.G. and J.B. contributed to hypothesis discussion, experimental design, data interpretation and project coordination.

M.d.S. coordinated the project, contributed to hypothesis discussion, experimental design, data interpretation and wrote the manuscript. D.L. conceived the hypothesis, led the project,

interpreted the data and wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Maria de Sousa, David Pisapia or David Lyden. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 BRAIN SLICE MODEL TO STUDY THE ROLE OF TUMOUR EXOSOMES IN METASTATIC COLONIZATION. A, Illustration of organotropic metastatic

derivatives1,2,3,4,5,6 of MDA-MB-231 breast cancer cell model (parental (gray) and brain (purple), lung (orange), and bone (green) metastatic) and respective cell-derived exosomes analyzed.

Schematic of the brain slice _ex vivo_ model optimized for the study of exosome-mediated cancer cell brain colonization. B, Schematic of the brain slice model used to study the effects of

exosome pre-treatment on the brain microenvironment and cancer cell phenotypes (left, cancer cell number—whole brain slice mount; and right, cancer cell invasion—brain slice transversal

section). Invading cells (white arrows) inside the region of interest, denoted by the blue square, are comprised of all cancer cells below the first layer of brain cells on the top of the

brain slice. C, Representative whole slice fluorescence images of BrT1 GFP+ cells growing on top of brain slices pre-treated with exosomes or PBS. D, Left, representative immunofluorescence

microscopy images of proliferating Ki-67+ BrT1 GFP+ cells invading brain slices pre-treated with exosomes. White and red arrows indicate invading Ki-67- or Ki-67+ cells, respectively. Right,

quantification of Ki-67+ invading cancer cell number. E, Left, representative fluorescence images of 231 parental mCherry+ cells growing on top of brain slices pre-treated with exosomes or

PBS. Right, quantification of 231 parental mCherry+ cell number. F, Left, immunoblot of CEMIP, small EV and exosomal markers (HSP70, Syntenin-1 and CD81) and ACTB in fractions obtained by

OptiPrep™ density gradient ultracentrifugation7 of BrT1 exosomes. Right, densitometry quantification of protein expression in the initial exosome population (_Input_) and across fractions

with different density, given in arbitrary units [a.u.]. Small EV and exosome-containing fractions are shown between dashed lines. The number of cells per FOV are averages ± SEM, from _n_ =

3, 4 (D) and _n_ = 3 (E) individual brain slices, scoring two fields per slice. A representative experiment of three (D, E) or four (F) independent biological replicates is shown. Brain

slice images (C) are representative of three independent biological replicates. Brain slice sections are stained with DAPI (blue), and dotted blue lines delineate the top and bottom limits

of the brain slice (D). Scale bars, 500µm (C) and 100µm (D, E). Error bars depict mean ± SEM. _P_ values were calculated by ANOVA (E) or two-sided Student’s t-test (D). See Supplementary

Fig. 6 for unprocessed blots. See Supplementary Table 1 for statistics source data. SUPPLEMENTARY FIGURE 2 EXOSOMAL CEMIP MODULATES THE BRAIN VASCULAR NICHE TO SUPPORT METASTASIS. A, Left,

immunoblot of CEMIP expression in cell and exosomal protein extracts from BrT1 WT and BrT1 CEMIP knockout (KO1 and KO2) cells. Immunoblotting for exosomal markers (Syntenin-1 and CD81) and

ACTB is shown below. Right, densitometry quantification of CEMIP normalized to the CEMIP expression in BrT1 WT exosomes. CEMIP expression was normalized to ACTB expression for each sample.

B, Transmission electron microscopy (TEM) of BrT1 WT and BrT1 CEMIP-KO1 and -KO2 exosomes. C, Size distribution and protein content analysis of BrT1 WT and BrT1 CEMIP-KO1 and -KO2 exosomes.

Exosome size (mode, nm) and number were evaluated by NanoSight particle tracking. Protein content per exosome ([particle]/[protein]) was assessed by factoring in the protein concentration.

D, Quantification of BrT1 WT and 231 BrT1 CEMIP-KO1 and -KO2 GFP+ cell number on top of brain slices. E, Left, representative fluorescence microscopy image of BrT1 GFP+ cells growing on top

of the brain slice. Right, representative fluorescence microscopy image of BrT1 GFP+ cells invading the brain slice in transversal section. F, Quantification of proliferation of BrT1 WT and

BrT1 CEMIP-KO1 and -KO2 cells _in vitro_ over three days. G, Quantification of transwell Matrigel invasion of BrT1 WT and BrT1 CEMIP-KO1 and -KO2 cells _in vitro_ over 24 hours. H,

Quantification of BrT1 KO2 GFP+ cells on top of brain slices pre-treated with exosomes or PBS. The number of cells per FOV are averages ± SEM, from _n_ = 8, 9, 9 (D), _n_ = 9, 7, 9, 9 (H)

individual brain slices, scoring two fields per slice; and the number of invading cells per FOV are averages ± SEM, from _n_=3 individual transwell cultures (G), scoring a representative

field per transwell membrane. One of three (A - C, D, H) independent biological replicates is shown. Graphs depicting _in vitro_ proliferation and invasion (F and G) display three

independent biological replicates. TEM images and immunofluorescence brain slice images (B and E) are representative of three independent biological replicates. Scale bars, 200 nm (B), and

100 µm (E). Error bars depict mean ± SEM. _P_ values were calculated by ANOVA (C - D, and F - H). See Supplementary Fig. 6 for unprocessed blots. See Supplementary Table 1 for statistics

source data. SUPPLEMENTARY FIGURE 3 CEMIP LOSS DOES NOT AFFECT PRIMARY TUMOUR GROWTH OR _IN SITU_ GROWTH IN THE BRAIN. A, Left, quantification of brain metastatic _in situ_ growth in mice

intracranially injected with BrT1 WT or BrT1 CEMIP-KO cells. Cranial bioluminescence signal (Total photon flux—photons/second (p/s)) in mice over 3-weeks post-intracranial injection of

GFP-labelled BrT1 WT or BrT1 CEMIP-KO luciferase-positive cells. Right, representative IVIS image of brain signal at week 3. B, Quantification of primary tumour growth8 in mice injected with

BrT1 WT or BrT1 CEMIP-KO cells. One experiment with _n_=5 mice per experimental group was performed (A, B). See Supplementary Table 1 for statistics source data. SUPPLEMENTARY FIGURE 4

EXOSOMAL CEMIP AFFECTS BREC BIOLOGY AND INDUCES VASCULAR REMODELING. A, Schematic of the brain slice model setup for the study of exosome interaction with brain microenvironment resident

cells. B, Representative fluorescence image of BrT1 exosomes (green) interacting with CD31+ BrECs (red) _in vivo_ 24 hours post-intracardiac injection of labelled exosomes. C, Representative

fluorescence image of BrT1 exosomes (green) and associated extravasated rhodamine-labelled Dextran (red) in the brain 24 hours post-intracardiac injection of labelled exosomes (right,

enlarged inset). D, Schematic of the ETF assay setup for studying exosome-dependent vascular network formation by isolated BrECs9,10. E, Left, immunoblot of CEMIP expression in cells and

exosomes of 231 parental Control and 231 parental CEMIP overexpressing (OE) models generated11,12. ACTB and the exosomal marker CD81 are shown below. Right, densitometry quantification of

CEMIP expression is normalized to expression in 231 parental Control exosomes. CEMIP expression was normalized to ACTB expression for each sample. F, Quantification of junction (top) and

isolated segment (bottom) number in the vasculature formed upon pre-treatment with exosomes or PBS. G, Left, representative fluorescence image of tumour vasculature (red) in BrT1 brain

metastases (white). Right, quantification of metastatic tumour and normal vessel diameter in brains from mice injected intracardiacally with BrT1 WT, BrT1 CEMIP-KO1 or -KO2 cells. H,

Schematic of the brain slice model setup for studying exosome-induced gene expression changes in stromal cells of the brain. Brain slices were pre-treated with BrT1 WT, BrT1 CEMIP-KO1 or

-KO2 cell-derived fluorescently-labelled exosomes. I, Flow cytometry analysis of exosome uptake. Percentage of exosome-positive (Exo+) endothelial (CD45_−_ CD31+) and microglial (CD45+

CD11blow CD49dlow)13 cells in brain slices is shown. J, Representative confocal images of the adhesion of fluorescently-labelled BrT1 WT, BrT1 CEMIP-KO1 or -KO2 exosomes with endothelial

cells (CD31+) in the brain slice. Arrows indicate co-localization of exosomes (green) with endothelial cells (red). For _in vivo_ experiments, _n_=4 mice were analyzed per group (G).

Individual vessel diameter was obtained from the average of three measurements along the extension of the vessel. Metastatic tumour and normal brain vascular diameters were scored in up to 5

individual metastatic lesions across two sagittal sections from different brain areas per individual presenting brain metastases (G). The number of junctions and isolated segments per FOV

are averages ± SEM, from _n_ = 5, 7, 8, 7, 8, 8 individual µ-slide angiogenesis chamber wells (F), scoring a representative field per µ-slide well. A representative experiment is shown from

three independent biological replicates (E - F). Graphs depicting endothelial and microglial cell exosome uptake and tumour vasculature diameter (I and G) display the average of three and

two independent biological replicates, respectively. Immunofluorescence images of _in vivo_ exosome uptake by BrECs, vascular leakiness and confocal images of the interaction and BrEC

exosome uptake in the brain slice (B, C and J) are representative of three independent biological replicates. Scale bars, 50µm (B), 50μm and 100μm (C), 100µm (G) and 75µm (J). Error bars

depict mean ± SEM. _P_ values were calculated by ANOVA (F, G, I). See Supplementary Fig. 6 for unprocessed blots. See Supplementary Table 1 for statistics source data. SUPPLEMENTARY FIGURE 5

CEMIP IS A BIOMARKER OF BRM AND IS PRESENT IN EXOSOMES FROM PATIENTS. A, Kaplan-Meier survival curve for brain metastasis patients depicting time to last follow up (LFU) or death from time

of primary tumour diagnosis based on low (green) or high (red) CEMIP expression in brain metastatic tumour. B, Representative image of CEMIP expression in 231 parental and BrT1 cells _in

vitro_ by immunohistochemistry. C, Immunoblot of CEMIP expression in exosomes collected from culture of human brain and bone metastatic tissue explants resected from patients. ACTB was used

as loading control. Marker indicated by m. D, Immunoblot of CEMIP expression in exosomes collected from culture of human non-small cell lung cancer primary tumour tissue resected from

patients. ACTB was used as a loading control. IHC images (B) and immunoblots (C, D) are representative of one experiment. Scale bars, 100µm (B). _P_ values was calculated by Log-rank

(Mantel-Cox) test (A). See Supplementary Fig. 6 for unprocessed blots. See Supplementary Table 1 for statistics source data. SUPPLEMENTARY FIGURE 6 UNPROCESSED WESTERN BLOTS CONTINUED.

Western blot replicates for Fig. 1e (Replicate C is shown in figure). Sample order is the same as specified in respective manuscript figure. Western blot replicates for Supplementary Fig. 1f

(Replicate B is shown in figure). Sample order is the same as specified in respective manuscript figure. Western blot replicates for Supplementary Fig. 2a (Replicate A is shown in figure).

Sample order is the same as specified in respective manuscript figure. Western blot replicates for Supplementary Fig. 4e (Replicate A is shown in figure). Sample order is the same as

specified in respective manuscript figure. Western blots for Supplementary Fig. 5c. Sample order is the same as specified in respective manuscript figure. Western blots for Supplementary

Fig. 5d. Sample order is the same as specified in respective manuscript figure. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures 1–6 and table titles/legends.

REPORTING SUMMARY SUPPLEMENTARY TABLE 1 Statistics source data. SUPPLEMENTARY TABLE 2 Mass spectrometry data. SUPPLEMENTARY TABLE 3 RNA sequencing data. SUPPLEMENTARY TABLE 4 Heatmap of

significant genes differentially expressed in brain endothelial and microglial cells following BrT1 exosome treatment, relative to the WT condition. SUPPLEMENTARY TABLE 5 Top significant IPA

canonical pathways - BrECs and microglia – 231 BrT1 exosomes and exosomal CEMIP specific – List of canonical pathways affected by 231 BrT1 exosome and exosomal CEMIP treatment in BrECs and

microglia. SUPPLEMENTARY TABLE 6 Heatmap of significant genes differentially expressed in brain endothelial and microglial cells following exosomal CEMIP treatment, relative to the WT

condition. SUPPLEMENTARY TABLE 7 Top 10 significant Gene Ontology - Biological Processes - BrECs and microglia – exosomal CEMIP specific - List of significant Biological Processes affected

by exosomal CEMIP treatment in BrECs and microglia. SUPPLEMENTARY TABLE 8 Patient data. SUPPLEMENTARY TABLE 9 Correlation of CEMIP expression in PTs and metastatic status. SUPPLEMENTARY

TABLE 10 Antibodies and primers. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Rodrigues, G., Hoshino, A., Kenific, C.M. _et al._ Tumour exosomal CEMIP

protein promotes cancer cell colonization in brain metastasis. _Nat Cell Biol_ 21, 1403–1412 (2019). https://doi.org/10.1038/s41556-019-0404-4 Download citation * Received: 30 May 2019 *

Accepted: 19 September 2019 * Published: 04 November 2019 * Issue Date: November 2019 * DOI: https://doi.org/10.1038/s41556-019-0404-4 SHARE THIS ARTICLE Anyone you share the following link

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