title: Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.
abstract: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX).
  
  
  
  CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient.
  
  
  
  The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype.
  
  
  
  This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Morrow
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: F
authors.initials: F
authors.lastname: Trapani
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: R L
authors.initials: RL
authors.lastname: Metcalf
authors.affiliation: Tumour Genomics Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bertolini
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: C L
authors.initials: CL
authors.lastname: Hodgkinson
authors.affiliation: RNA Biology Group, University of Manchester, Manchester.
authors.forename: G
authors.initials: G
authors.lastname: Khandelwal
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: P
authors.initials: P
authors.lastname: Kelly
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: M
authors.initials: M
authors.lastname: Galvin
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: L
authors.initials: L
authors.lastname: Carter
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: K L
authors.initials: KL
authors.lastname: Simpson
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: S
authors.initials: S
authors.lastname: Williamson
authors.affiliation: Computational Biology Support Team, Cancer Research UK Manchester Institute, University of Manchester, Manchester.
authors.forename: C
authors.initials: C
authors.lastname: Wirth
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: N
authors.initials: N
authors.lastname: Simms
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: L
authors.initials: L
authors.lastname: Frankliln
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: K K
authors.initials: KK
authors.lastname: Frese
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: D G
authors.initials: DG
authors.lastname: Rothwell
authors.affiliation: The Christie NHS Foundation Trust, Manchester.
authors.forename: D
authors.initials: D
authors.lastname: Nonaka
authors.affiliation: RNA Biology Group, University of Manchester, Manchester.
authors.forename: C J
authors.initials: CJ
authors.lastname: Miller
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK.
authors.forename: G
authors.initials: G
authors.lastname: Brady
authors.affiliation: The Christie NHS Foundation Trust, Manchester; Institute of Cancer Sciences, University of Manchester, Manchester; Cancer Research UK Lung Cancer Centre of Excellence, Manchester, UK.
authors.forename: F H
authors.initials: FH
authors.lastname: Blackhall
authors.affiliation: Clinical and Experimental Pharmacology Group, University of Manchester, Manchester, UK; Cancer Research UK Lung Cancer Centre of Excellence, Manchester, UK. Electronic address: caroline.dive@cruk.manchester.ac.uk.
authors.forename: C
authors.initials: C
authors.lastname: Dive
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics*; D063646:Carcinogenesis / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002945:Cisplatin / Q000008:administration & dosage; D004268:DNA-Binding Proteins / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000473:pathology; D051379:Mice; D009154:Mutation; D009360:Neoplastic Cells, Circulating / Q000187:drug effects / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000473:pathology; D000068437:Pemetrexed / Q000008:administration & dosage; D014157:Transcription Factors / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C504003:TTF1 protein, human; D014157:Transcription Factors; D000068437:Pemetrexed; D002945:Cisplatin
keywords: circulating tumour cells; epithelial to mesenchymal transition (EMT); non-small-cell lung cancer; patient-derived circulating tumour cells explants (CDX); preclinical therapeutics