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)