Critical steps for circulating exosomes analyses

Transcription

1 Critical steps for circulating exosomes analyses Héctor Peinado Selgas, PhD. Head, Microenvironment and Metastasis Group Spanish National Cancer Research Centre (CNIO)

2 Where are we? Exosomes in liquid biopsy

3 Exosomes in liquid biopsy Exosomes Adapted from Int. J. Mol. Sci. 2017, 18(2), 264;

4 Use of exosomes to detect tumor mutation heterogeneity Exosomes Adapted from Amirouchene-Angelozzi N, Cancer Discov. 2017

5 Limitations: Extracellular vesicles = exosomes Oncosomes Exomere Cytoplast Adapted from Cell Mol Life Sci Aug;68(16):

6 Exosomes in liquid biopsy = tumor exosomes Representation of tumor-derived cargo <10% Proteins (receptors, oncoproteins ) RNA (mrna, smallrna, rrna ) DNA (ssdna, dsdna, retrotransposons) Lipids (sphingomyelin, ceramides ) Sugars (glycosilation) Metabolites

7 Potential tumor exosomal markers for diagnosis (so far) Sometimes I see tumor-derived exosomes Transl Cancer Res 2018;7(Suppl 2):S226-S242

8 Use of circulating exosomes in melanoma Retrospective series of plasma from Stage III melanoma patient (4 years followed up) Protocol: Ultracentrifugation Detection: WB Origin: EDTA-Frozen POD progression of disease, NED no evidence of disease Peinado et al. Nature Medicine. (18): (2012)

9 Use of circulating exosomes in melanoma Protocol: Exoquick Detection: ELISA Origin: Serum-Frozen Alvaro Gonzalez, Salvador Martin-Algarra, Clínica Navarra

10 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

11 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

12 Detection of DNA in circulating exosomes Thakur et. al. Cell Research (2014) 24:

13 ExoDNA represents the whole genome DNA WGS of human colon cancer cell line WGS of B16-F10 cancer cell line HCT116 gdna gdna ExoDNA HCT116 ExoDNA Regions below 10 Kb are the most represented 6951 regions represented 10Kb, 5 regions represented 100Kb Thakur, Peinado, Lyden Unpublished

14 ExoDNA reflects the mutational status of parental cells Melanoma exodna WT: Sk-Mel103, 146 & 147; V600E: Sk-Mel28, 133, 192 & 267 V, WT allele; E, mutant allele. Non-Small-Cell Lung Cancer cell exodna Thakur et. al. Cell Research (2014) 24:

15 Improving detection ExoNAs vs cfdna Overall survival (OS) per mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma Similar Sensibility Clin Cancer Res Jan 1;24(1):

16 Dr. Hector Peinado s Group Microenvironment and Metastasis Group Molecular Oncology Program, CNIO Madrid, Spain Susana García, PhD. Staff Scientist Marta Hergueta, PhD Postdoc Claudia Savini, PhD - Postdoc Laura Nogues, PhD Postdoc Ana Isabel Amor, Severo Ochoa PhD Student Lucia Robado de Lope, La Caixa PhD Student Teresa Gonzalez Muñoz FPU PhD Student Alberto Hernandez PhD Student Marina Mazariegos Tech Sara Sanchez-Redondo, Tech Alicia Teijeira, Ms. Student. Former Members: Alberto Benito Martin, PhD WCM, NY, USA Cristina Merino, Tech. - GSK, Madrid Angela Di Giannatale IRCCS Bambino Gesù, Rome 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