Lung cancer is the leading cause of cancer-related death worldwide. The majority of patients are diagnosed at advanced and currently incurable stages. Non-small cell lung cancer (NSCLC) represents around 85% of cases. Alterations in the DNA damage response (DDR) and resulting genomic instability (GI) contribute to NSCLC etiology and progression. However, their therapeutic exploitation is disappointing. Circulating tumor cells (CTCs) that dissociate from the primary tumor or its metastases harbor distinct biological properties that allow them to travel through circulation and colonize distant organs. Further insight into CTC biology and identification of their vulnerabilities would provide a rationale for the targeting of the most aggressive tumor clones that fuel metastatic progression.We hypothesized that the DDR and genome integrity maintenance dysfunctions are critical processes in CTC metastatic potency and NSCLC progression. The main aim of my PhD project was to perform a molecular and functional characterization of NSCLC CTCs to elucidate the mechanistic basis of their tumorigenic potential and identify novel therapeutic targets. Using CTCs from 55 patients with advanced-stage NSCLC, our laboratory has established four CTC-derived xenografts (CDX) (GR-CDXL1, GR-CDXL2, GR-CDXL3 and GR-CDXL4) and three CDX-derived cell lines. Preliminary analyses have shown that the four CDX recapitulated patient tumor histology and response to platinum-based chemotherapy, which validated the clinical relevance of our models. To determine the extent to which the CDX was representative of the corresponding patient tumor biopsy, whole-exome sequencing (WES) was performed on the CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4), CDX-derived cell lines, corresponding patient tumor biopsy and single CTCs. The four major goals of my thesis were as follows: (i) identification of genomic alterations and in-depth comparative genomic analysis of the CDX, CDX-derived cell lines, corresponding patient tumor biopsy and CTCs using WES analysis data, (ii) functional characterization of DDR activity and chromosomal instability (CIN) events in the CDX-derived cell lines, (iii) in vitro drug assays targeting DDR defects, (iv) 3D modeling of the metastatic potency of our CDX-derived cell lines in ovo in the chick embryo chorioallantoic membrane (CAM) and in vivo in immunodeficient mice and pharmacological targeting of metastasis.Genomic analysis by WES has shown considerable mutational landscape similarity between the CDX, the cell lines, the patient biopsies and single CTCs. WES and reconstruction of phylogenetic trees of the CDX and the CDX-derived cell lines revealed truncal alterations in key DDR and genome integrity-related genes prevalent across models and assessed as therapeutic targets in vitro, in ovo and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to bi-allelic BRCA2 mutation and FANCA deletion and unrepaired DNA lesions post-mitosis. GR-CDXL1 cells were sensitive to PARP inhibitor (PARPi) olaparib, despite chemoresistance, which challenges the current clinical rationale claiming that chemosensitive NSCLC patients should respond to PARPi. Targeting CIN through centrosome clustering inhibition in GR-CDXL3 impeded tumor growth in ovo and in vivo. In GR-CDXL4, olaparib sensitivity was dictated by SLFN11 overexpression, which also corresponded with increased neuroendocrine marker expression at patient disease progression, suggesting a predictive value of SLFN11 in histological transformation of NSCLC into SCLC.This study unravels distinct DDR profiles as a central mechanism underpinning CTC metastatic potency. It also suggests SLFN11 overexpression as a potential biomarker of sensitivity to PARPi in NSCLC independently of BRCAness. Overall, our models provide a robust platform for drug testing of DDR-targeted strategies to expand patient categories that may benefit from precision medicine in metastatic NSCLC.