Targeting genome integrity dysfunctions impedes metastatic potency in non-small cell lung cancer circulating tumor cell-derived explants.

DNA damage and genomic instability contribute to non-small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity-related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity-related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

Circulating tumor cell copy-number heterogeneity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors.

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1st and 3rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK+/cytokeratins-, 46 (56%) ALK-/cytokeratins+ and 4 (5%) ALK+/cytokeratins+. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.

Tumor Evolution and Therapeutic Choice Seen through a Prism of Circulating Tumor Cell Genomic Instability.

Circulating tumor cells (CTCs) provide an accessible tool for investigating tumor heterogeneity and cell populations with metastatic potential. Although an in-depth molecular investigation is limited by the extremely low CTC count in circulation, significant progress has been made recently in single-cell analytical processes. Indeed, CTC monitoring through molecular and functional characterization may provide an understanding of genomic instability (GI) molecular mechanisms, which contribute to tumor evolution and emergence of resistant clones. In this review, we discuss the sources and consequences of GI seen through single-cell analysis of CTCs in different types of tumors. We present a detailed overview of chromosomal instability (CIN) in CTCs assessed by fluorescence in situ hybridization (FISH), and we reveal utility of CTC single-cell sequencing in identifying copy number alterations (CNA) oncogenic drivers. We highlight the role of CIN in CTC-driven metastatic progression and acquired resistance, and we comment on the technical obstacles and challenges encountered during single CTC analysis. We focus on the DNA damage response and depict DNA-repair-related dynamic biomarkers reported to date in CTCs and their role in predicting response to genotoxic treatment. In summary, the suggested relationship between genomic aberrations in CTCs and prognosis strongly supports the potential utility of GI monitoring in CTCs in clinical risk assessment and therapeutic choice.

CTC-Derived Models: A Window into the Seeding Capacity of Circulating Tumor Cells (CTCs).

Metastasis is the main cause of cancer-related death owing to the blood-borne dissemination of circulating tumor cells (CTCs) early in the process. A rare fraction of CTCs harboring a stem cell profile and tumor initiation capacities is thought to possess the clonogenic potential to seed new lesions. The highest plasticity has been generally attributed to CTCs with a partial epithelial-to-mesenchymal transition (EMT) phenotype, demonstrating a large heterogeneity among these cells. Therefore, detection and functional characterization of these subclones may offer insight into mechanisms underlying CTC tumorigenicity and inform on the complex biology behind metastatic spread. Although an in-depth mechanistic investigation is limited by the extremely low CTC count in circulation, significant progress has been made over the past few years to establish relevant systems from patient CTCs. CTC-derived xenograft (CDX) models and CTC-derived ex vivo cultures have emerged as tractable systems to explore tumor-initiating cells (TICs) and uncover new therapeutic targets. Here, we introduce basic knowledge of CTC biology, including CTC clusters and evidence for EMT/cancer stem cell (CSC) hybrid phenotypes. We report and evaluate the CTC-derived models generated to date in different types of cancer and shed a light on challenges and key findings associated with these novel assays.

Circulating tumor cells (CTCs) for the noninvasive monitoring and personalization of non-small cell lung cancer (NSCLC) therapies.

Growing evidences for tumor heterogeneity confirm that single-tumor biopsies frequently fail to reveal the widespread mutagenic profile of tumor. Repeated biopsies are in most cases unfeasible, especially in advanced cancers. We describe here how circulating tumor cells (CTCs) isolated from minimally invasive blood sample might inform us about intratumor heterogeneity, tumor evolution and treatment resistance. We also discuss the advances of CTCs research, most notably in molecularly selected non-small cell lung cancer (NSCLC) patients, highlighting challenges and opportunities related to personalized therapy.