exosomes: composition,biogenesis and role as intercellular messengers in...

Exosomes: composition,biogenesis and role as intercellular messengers in cancer

Héctor Peinado Selgas, PhD. Head, Microenvironment and Metastasis Group

Spanish National Cancer Research Centre (CNIO)

Tumor Progression – Role of microenvironment

Extracellular vesicles = exosomes

Cell Mol Life Sci. 2011 Aug;68(16):2667-88.

Oncosomes

Zoller, M. Nature Reviews Cancer (2009)

- Exosomes -

Exosomes in liquid biopsy = tumor exosomes

Proteins (receptors, oncoproteins…) RNA (mRNA, smallRNA, rRNA…) DNA (ssDNA, dsDNA, retrotransposons) Lipids (sphingomyelin, ceramides…) Sugars (glycosilation) Metabolites

Representation of tumor-derived cargo

Tumor shed both cells and cellular components that could be used as surrogate markers of tumor burden, mutations, progression,

outcome and response to therapy

Liquid Biopsies

CTCs Circulating

Tumor Cells

ctNA Circulating

Tumor Nucleid Acids

Extracellular Vesicles

Samples: Blood, plasma, serum, CSF, urine, saliva

Use of extracellular vesicles in liquid biopsy

Exosomes in liquid biopsy

Standarization is crucial

Isolation Methods

Sucrose

Size exclusion (Exo Spin)

Immunoaffinity (CD9-CD63)

Microfluidics

Exoquick

Ultracentrifugation

Sample

Plasma (EDTA-Heparin)

Serum

Urine

CSF

Saliva

Hour-Clinic

Ice-RT

Quantification

Nanosight

Izon

Protein

RNA

DNA

Fast, Simple, Reliable, Specific for tumors and Sensible

Not quite there! We learn as we go

Exosome

Microvesicles

Molecular Cargo in Extracellular Vesicles

dsDNA Thakur et al 2014 Kahlert et al 2014

c-MET MIF Integrins

Peinado et al 2012 Costa da Silva et al 2015 Hoshino et al 2015.

Role of tumor-secreted exosomes during tumor progression

Vlassov et al. BBA, 2012. 1820, 7: 940-948 Pant et al. Biochem. Pharmacol. 2012. 83, 11:1484-1494

Proteins (receptors, oncoproteins…) RNA (mRNA, smallRNA, rRNA…) DNA (ssDNA, dsDNA, retrotransposons) Lipids (sphingomyelin, ceramides…) Ions, pH, etc…

New circulating markers

Horizontal transfer Of molecules

Influence in tumor microenvironment

TUMOR

Tumor-derived exosomes as surrogate markers of cancer

Circulating exosomes isolated from plasma of melanoma patients

Circulating exosomes (100nm)

Total number of exosomes in plasma

Control I II III IV0

5000

10000

15000

20000

Stage

Par

ticle

s (x

108 )

/ m

l of p

lasm

a

Peinado et al. Nature Medicine. (18):883–891 (2012)

Protein content in circulating exosomes is increased in metastatic Stage IV melanoma patients

Micrograms of protein / exosome

Stage IV melanoma patients


Characterization of exosomes – mass spectrometry

18

87

99

B16-F1 Low metastatic

B16-F10 Highly metastatic

Symbol Entrez Gene Name ACTC1 actin, alpha, cardiac muscle 1 ALB albumin APOA1 apolipoprotein A-I APRT adenine phosphoribosyltransferase BSG basigin (Ok blood group) CD63 CD63 molecule CD151 CD151 molecule (Raph blood group) CD2AP CD2-associated protein ENG endoglin ENO3 enolase 3 (beta, muscle) HSPA1A/HSPA1B heat shock 70kDa protein 1A ITGA5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) VLA-4 Integrin alpha4beta1 LUM lumican MET met proto-oncogene (hepatocyte growth factor receptor) MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor) PGAM1 phosphoglycerate mutase 1 (brain) PGK2 phosphoglycerate kinase 2 PLG plasminogen PPIA peptidylprolyl isomerase A (cyclophilin A) PSAP prosaposin RAB10 RAB10, member RAS oncogene family RAB14 RAB14, member RAS oncogene family RAB1A RAB1A, member RAS oncogene family RAP1A RAP1A, member of RAS oncogene family RAP1B RAP1B, member of RAS oncogene family Rasl2-9 RAS-like, family 2, locus 9 RHOC ras homolog family member C RHOG ras homolog family member G TMEM59 transmembrane protein 59 TSPAN4 tetraspanin 4 TSPAN6 tetraspanin 6 VTN vitronectin

High Metastatic Melanoma exosome signature


Melanoma signature – Circulating exosomes Control

TYRP2

Stage III Stage IV Stage I

VLA-4

GAPDH

Hsp90

Hsp70

Hsc70

TYRP2 VLA-4 HSP70 HSC70

TYRP2 expression in circulating exosomes is associated with Stage III melanoma patient progression

Retrospective series of plasma from Stage III melanoma patient (4 years followed up)

– POD – progression of disease , NED – no evidence of disease


MET is increased in melanoma patient exosomes


Increased Frequency of MET-expressing Hematopoietic and Non-hematopoietic Progenitor Populations in Melanoma Patient Blood

Stage I—III

CD45- C-KITlow/+

Control

Stage IV

0 10 3 10 4 10 5 0 10 3 10 4 10 5

26

0 10 3 10 4 10 5 0 10 3 10 4 10 5

0.6

0 10 3 10 4 10 5 0 10 3 10 4 10 5

3.3 0 10 3 10 4 10 5 0

10 3 10 4 10 5

0.6

0 10 3 10 4 10 5 0 10 3 10 4 10 5

5.8

0 10 3 10 4 10 5 0 10 3 10 4 10 5

35

C-K

IT

TIE

2

MET


20

40

60

80P < 0.05

Stage

% c

-KIT

low

/+ M

ET+

TIE

2+ c

ells


20

40

60

80P < 0.001

Stage

% c

-KIT

low

/+ M

ET+

cel

ls

Proposed model - Role of exosomes in metastasis

Increase of BMPC populations Pro-metastatic behavior

c-MET-driven

Circulating exosomes are diagnostic factors of melanoma

patients

Melanoma signature VLA4

TYRP2 MET

Hsp70


Analysis of circulating exosomes in breast cancer patients

Number of Samples

Stage

0 20

I 64

II 41

III 9

IV 6

Histology

IDC 96

ILC 12

DCIS 25

LCIS 3

Lymph node status

LN- 44

LN+ 27

Receptor status

ER+/Her2- 41

ER+/Her2+ 9

ER-/Her2+ 6

ER-/Her2- 13 n=140

Analysis of circulating exosomes in breast cancer patients

n=140

contr

ol

Stag

e 0

Stag

e I

Stag

e II

Stag

e III

Stag

e IV

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10*

******

*****

pro

tein

exo

(µg)

/ m

l of p

lasm

a

Role of exosomes in metastatic organotropism

Ayuko Hoshino, PhD

Bruno Costa da Silva, PhD

Role of exosomes in metastatic organotropism

Hoshino et al. Nature (2015) doi:10.1038/nature15756

Mass Spectrometry profiling in exosomes derived from organotropic models

Breast Cancer (831, 231Br) Melanoma (131/4-5B2, SB1B)

Brain-metastatic Models

Breast Cancer (4173, 4175, 4180) Melanoma (131/8-2L) Osteosarcoma (143B) Rhabdomyosarcoma (RD, CT10) Wilms´Tumor (CCG9911, CSL)

Lung-metastatic Models Uveal Melanoma Colorectal (HCT116, HT29, SW620) Osteosarcoma (BXPC-3, HPAF-II, MiaPaca, PANC1) Gastric Cancer (SNU1, SNU16)

Liver-metastatic Models


Integrins are the protein family most represented exosomes derived from organotropic models

Hoshino et al. Nature (2015) doi:10.1038/nature15756 α6β4 α6β1

αvβ5

Integrins in circulating exosomes can predict metastatic tissues

β4 integrin – lung metastasis αv integrin – liver metastasis

Proposed model: Tumor-secreted exosomes are determinants of metastasis organotropism


CNIO, Molecular Oncology Program Laboratory of Microenvironment and Metastasis

Lymph node spread

Innane immunity

Obesity

New Therapies

New Markers

Imaging

Angela Di Giannatale

Teresa Gonzalez

Silvia Campanario

Marina Mazariegos

Cristina Merino

Lucia Robado

Alberto Benito

Susana Garcia

Ana Amor

Marta Hergueta

Metastasis

Laura Nogues

Peinado´s Lab Microenvironment and Metastasis

Molecular Oncology Program CNIO

Madrid, Spain

Susana García, PhD. – Staff ScientistMarta Hergueta, PhD – PostdocAna Isabel Amor, Severo Ochoa – Grad Student Lucia Robado de Lope, La Caixa – Grad StudentSilvia Campanario – StudentTeresa Gonzalez Muñoz – StudentCristina Merino –TechMarina Mazariegos – TechAlberto Benito Martin, PhD – WCMCAngela DiGiannatale, MD – WCMC - Italy

Estrella Chavero, Res. Tech. Sergio Haro, Undergrad. Diego Barba, Undergrad Macarena Tello – Summer StudentWang, Bryan Zichang – Summer Student

Dr. David Lyden – WCMCDr. Bromberg - MSKCC

Dra. Soengas (CNIO, Spain)Dra. Ortega (CNIO, Spain)Dr. Martin (G. Marañon)Dr Quintela (CNIO)Dr. Olmos (CNIO)Dr. Anton, Pazo (Servet, Zaragoza)Dr. Lopez Guerrero (IVO)Drs Proulx, Detmar (Switzerland)Dr. Rutkowski (Poland)Dr. Mehrara (MSKCC)Dr. Rodriguez Peralto, Ortiz (H12O)Dr. Ghajar (FredHuthc)

exosomes: composition,biogenesis and role as intercellular messengers in...

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