Exosomes in NSCLC as a new tool for biomarkers searching
Elena Duréndez Sáez Laboratorio de Oncología Molecular, Fundación de Investigación del Hospital General Universitario de Valencia.
Unidad Mixta TRIAL-FIHGUV, Centro de Investigación Príncipe Felipe.
BACKGROUND
·Problems in clinical practice: -Lack of screening methods. - > 70- 75% of patients are diagnosed in advanced stages. - Difficulties in obtaining sufficient amount of tumor tissue for exhaustive molecular characterization.
Liquid Biopsy: A new approach for the molecular analysis in NSCLC
Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016
Why?: Limitations of Tissue Biopsy
BACKGROUND
TUMOR PROGRESSION
IMMUNE MODULATION
METASTASIS
·Small membranous vesicles (50-150nm)
Objectives: To analyze the differences in exosomes cargo secreted by lung tumorspheres (3D) and more differentiated tumor cells (2D) as well as from patients’ plasma.
Exosomes in Cancer
Cancer Stem Cell (CSC)
Tumor-derived Exosomes Modified. Reclusa P, et al. Transl Lung Cancer Res. 2016
BACKGROUND
·Playing a key role in NSCLC
NSCLC commercial cell lines
Primary cell cultures
Female, ADC
Male, SCC
NSCLC PLASMA SAMPLES
PHASE I PHASE II
HAVERST CELL CULTURE MEDIA SUPERNATANT
Herreros-Pomares A, et al. Cell Death and Disease. 2019
3D (Tumorespheres)
Enriched in CSCs population
2D (Monolayer)
Exosomes
500g x 5’ 3000g x 15’
10.000g x 30’
MATERIALS & METHODS
IMMUNOBLOT ELECTRON MICROSCOPY NTA
(Nanosight)
NSCLC commercial cell lines
Primary cell cultures
Female, ADC Male, SCC
NSCLC PLASMA SAMPLES
3D (Tumorespheres)
2D (Monolayer)
Exosomes Characterization
NSCLC Exosomes size around 100-150 nm
RESULTS: Characterization
SW900 H1975 H358 PC9 H1650 A549 H460 H827 H2228
GENDER MALE FEMALE MALE FEMALE
MALE MALE
MALE
FEMALE FEMALE
HISTOLOGY SCC ADC ADC ADC
ADC ADC
Large cell carcinoma
ADC ADC
MUTATIONS KRAS (G12V)
EGFR (L858R+T790M)
KRAS (G12S)
EGFR (DEL.19)
EGFR (DEL.19)
KRAS (G12C)
KRAS p.Q61H
PI3K p.E545K
EGFR (DEL.19) EML4-ALK REARRANGEMENT
Blue dots: Mutant alleles. Red dots: Wild-type alleles.
Yellow dots: Non-amplificated regions. Green dots: Amplicons that have both copies (WT and mutant).
0,5 ml of plasma
• Mutations detected in Exosomes from NSCLC cell cultures :
• Mutations detected in Exosomes from NSCLC plasma samples: dPCR
High sensitivity Different % of mutant fractions
Working optimization with lower input
volume than used in cfDNA (2ml)
PHASE I
MAF: 33.3 %
Camps and Jantus-Lewintre Lab. Pending publication
PHASE II
RESULTS: Exosomes cargo
EGFR L858R
MAF: 7.062 %
*MAF: Mutant allelic fraction
• Mutations detected in Exosomes from NSCLC plasma (0,5ML): EGFR T790M Limit of detection:
0.01%
RESULTS : Exosomes cargo
MAF: 17,634 %
KRAS G13D KRAS G12V
MAF: 44,628%
MAF: 2,299%
*MAF: Mutant allelic fraction
METHOD GO ID DESCRIPTION p-value
Enrichr GO: 1905475 Regulation of protein localization to membrane 0.006
Enrichr GO:0032148 Activation of protein kinase B activity 0.017
Enrichr GO:0032731 Activation of Interleukin-1 beta production 0.018
Enrichr GO:0051781 Stimulation of cell division 0.03
METHOD GO ID DESCRIPTION p-value
Enrichr GO:0034446 Substrate adhesion-dependent cell spreading 0.003
Enrichr GO:0000904 Cell morphogenesis involved in differentiation 0.013
Enrichr GO:0007229 Integrin-mediated signaling pathway 0.016
Enrichr GO:0007187 G-protein coupled receptor signaling pathway 0.028
Pathways overexpressed in 3D-derived Exosomes
Pathways overexpressed in 2D-derived Exosomes p<0.05
DIFFERENTIALLY EXPRESSED GENES (mRNA)
p<0.01
TRANSCRIPTOMIC ANALYSIS
Clariom D Human Assay
Collaboration: Dr. J. Paramio (CIEMAT)
Dr. Eva Serna (UV)
3D-Exosomes
2D-Exosomes
Camps and Jantus-Lewintre Lab. Pending publication
3D vs 2D-derived Exosomes PHASE I
RESULTS : Exosomes cargo
Phase II on going…
ADC vs SCC-derived Exosomes
AD
C
SCC
HIGHEST OVEREXPRESSION IN ADC
P- value (ADC vs SCC)
Fold-Change (ADC vs SCC)
XAGE1B 3.77E-05 4.25
p<0.01
Gallach S, et al. Oncotarget. 2017
Camps and Jantus-Lewintre Lab. Pending publication
HIGHEST OVEREXPRESSION IN SCC
P- value (SCC vs ADC)
Fold-Change (SCC vs ADC)
CABYR 2E-05 4.72
PHASE I Phase II on going…
DIFFERENTIALLY EXPRESSED GENES (mRNA)
RESULTS : Exosomes cargo
Validation in HGUV NSCLC cohort
cDNA RNA
Normal tissue
Tumor tissue
RNA Isolation
Reverse Transcription
ACTB CDKN1B
GUSB
Normalization
Relative gene expression Pfaffl formula
Data analysis Hydrolysis probe
186 NSCLC patients
RESULTS: Biomarkers validation
XAGE1B CABYR
cDNA RNA
Normal tissue
Tumor tissue
RNA Isolation
Reverse Transcription
ACTB CDKN1B
GUSB
Normalization
Relative gene expression Pfaffl formula
Data analysis Hydrolysis probe
186 NSCLC patients
RESULTS : Biomarkers validation
ADC group XAGE1B
49.63 months
NR
p = 0.013
CABYR
Validation in HGUV NSCLC cohort
TAKE HOME MESSAGE
EVs are a source of biomarkers that act modulating key signaling pathways in tumor development.
Differences in the cargo of EVs have been observed between: i) lung-tumourspheres (3D) and more differentiated tumor cells (2D) ii) ADC and SCC-derived exsomes.
Hence, EVs could be used in NSCLC as a new tool to study the molecular characteristics of
the tumor, providing tumor information in cases where it is not possible to obtain a tissue sample.
EVs cargo can reflect the molecular signatures of the tumor cells from which they were secreted.
Molecular Oncology Lab
Supported by GV/2018/026, PI18/00266, & Asociación Española Contra el Cáncer (AECC Valencia)
Dr. Carlos Camps Dra. Eloisa Jantus
Dra. Silvia Calabuig Dra. Macarena Ferrero
Sandra Gallach Eva Escorihuela
Marais Mosqueda
Elena Duréndez Andrea Moreno Susana Torres
Alejandro Herreros Ning Dong
Feiyu Zhang
ACKNOWLEDGEMENTS
Collaborators
Dr. Jesús Paramio (CIEMAT) Dra. Eva Serna (UV)
Dr. Rafael Sirera (UPV) Dra. Laura Muinelo (IDIS)
Dr. Alexandre de la Fuente (CHUS)