Critical steps for circulating exosomes analyses · Dr. Hector Peinado´s Group Microenvironment...
Transcript of Critical steps for circulating exosomes analyses · Dr. Hector Peinado´s Group Microenvironment...
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Héctor Peinado Selgas, PhD.Head, Microenvironment and Metastasis GroupSpanish National Cancer Research Centre (CNIO)
Critical steps for circulating exosomes analyses
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Where are we? Exosomes in liquid biopsy
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Exosomes in liquid biopsy
Exosomes
Adapted from Int. J. Mol. Sci. 2017, 18(2), 264;
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Use of exosomes to detect tumor mutation heterogeneity
Exosomes
Adapted from Amirouchene-Angelozzi N, Cancer Discov. 2017
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Limitations: Extracellular vesicles = exosomes
Oncosomes
CytoplastExomere
Adapted from Cell Mol Life Sci. 2011 Aug;68(16):2667-88.
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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%
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Potential tumor exosomal markers for diagnosis (so far)
Sometimes… I see tumor-derived exosomes
Transl Cancer Res 2018;7(Suppl 2):S226-S242
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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
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Use of circulating exosomes in melanoma
Alvaro Gonzalez, Salvador Martin-Algarra, Clínica Navarra
Protocol: ExoquickDetection: ELISAOrigin: Serum-Frozen
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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
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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
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Detection of DNA in circulating exosomes
Thakur et. al. Cell Research (2014) 24:766–769.
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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
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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.
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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.
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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