critical steps for circulating exosomes analyses · dr. hector peinado´s group microenvironment...

Héctor Peinado Selgas, PhD.Head, Microenvironment and Metastasis GroupSpanish National Cancer Research Centre (CNIO)

Critical steps for circulating exosomes analyses

Where are we? Exosomes in liquid biopsy

Exosomes in liquid biopsy

Exosomes

Adapted from Int. J. Mol. Sci. 2017, 18(2), 264;

Use of exosomes to detect tumor mutation heterogeneity

Exosomes

Adapted from Amirouchene-Angelozzi N, Cancer Discov. 2017

Limitations: Extracellular vesicles = exosomes

Oncosomes

CytoplastExomere

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

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 <10%

Potential tumor exosomal markers for diagnosis (so far)

Sometimes… I see tumor-derived exosomes

Transl Cancer Res 2018;7(Suppl 2):S226-S242

Use of circulating exosomes in melanoma

Retrospective series of plasma from Stage III melanoma patient

(4 years followed up)

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

Peinado et al. Nature Medicine. (18):883–891 (2012)Protocol: UltracentrifugationDetection: WBOrigin: EDTA-Frozen

Use of circulating exosomes in melanoma

Alvaro Gonzalez, Salvador Martin-Algarra, Clínica Navarra

Protocol: ExoquickDetection: ELISAOrigin: Serum-Frozen

Caution: in need of protocol standardization

Collection: Fresh, Frozen

Material: Plasma, Serum, Other

Protocol: Ultracentrifugation, Filtration, SEC, Kit

Measurement: Nanosight, Zeta, …

Detection: WB, ELISA, Mesoscale

Material: Protein, miRNA, DNA

Or at least… specification

2019 – Between pre-clinical and clinical application

Problems we face:

• Tumor exosomes represent a MINORITY of circulating exosomes –

unbiased analysis NOT RECOMENDABLE

• Results depend on the methods – NO STANDARD (e.g fresh/frozen, tubes,

ELISA, WB …)

• Studies are not properly design (n, treatment, doses, material, timing)

• Use in diagnosis is feasible – MARKER NEEDED

• Application in response to therapy or specific mutation

Detection of DNA in circulating exosomes

Thakur et. al. Cell Research (2014) 24:766–769.

WGS of human colon cancer cell line

ExoDNA represents the whole genome DNA

HCT116 ExoDNA

HCT116 gDNA

Regions below 10 Kb are the most represented6951 regions represented 10Kb, 5 regions represented 100Kb

WGS of B16-F10 cancer cell line

gDNA

ExoDNA

Thakur, Peinado, Lyden Unpublished

ExoDNA reflects the mutational status of parental cells

Non-Small-Cell Lung Cancer cell exoDNA

Melanoma exoDNA

WT: Sk-Mel103, 146 & 147; V600E: Sk-Mel28, 133, 192 & 267“V”, WT allele; “E”, mutant allele.

Thakur et. al. Cell Research (2014) 24:766–769.

Improving detection– ExoNAs vs cfDNA

Similar Sensibility

Overall survival (OS) per mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma

Clin Cancer Res. 2018 Jan 1;24(1):181-188.

Dr. Hector Peinado´s GroupMicroenvironment and Metastasis Group

Molecular Oncology Program, CNIOMadrid, Spain

Susana García, PhD. – Staff ScientistMarta Hergueta, PhD – PostdocClaudia Savini, PhD - PostdocLaura Nogues, PhD – Postdoc Ana Isabel Amor, Severo Ochoa – PhD StudentLucia Robado de Lope, La Caixa – PhD StudentTeresa Gonzalez Muñoz – FPU PhD StudentAlberto Hernandez – PhD StudentMarina Mazariegos – TechSara Sanchez-Redondo, TechAlicia Teijeira, Ms. Student.

Collaborators:

Jasminka Boskovic, Mercedes Robledo, Javier Muñoz - CNIO

Pablo L. Ortiz-Romero, José Luis Rodríguez-Peralto – H12O,

Madrid, Spain

Piotr Rutkowsky, Marie Curie Oncology Center, Poland

Laura Santambrogio (Albert Einstein) Mary Sue Brady (MSKCC).

USA

Johan Skog, Mikkel Noerholm – Exosome Diagnostics

Former Members:Alberto Benito Martin, PhD – WCM, NY, USACristina Merino, Tech. - GSK, MadridAngela Di Giannatale – IRCCS Bambino Gesù, Rome