Background: Circulating tumour cell (CTC) number is a prognostic biomarker in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). However, it is recognised that CTCs are heterogeneous and a better appreciation of CTC biology is required to reveal avenues of therapeutic intervention in metastatic lung cancer. CTC immunofluorescent assays were developed to evaluate two proposed mechanisms of tumour cell dissemination: epithelial to mesenchymal transition (EMT) and vasculogenic mimicry (VM). Methods: An immunofluorescent assay was developed to profile ISET filtered CTCs for the expression of epithelial and mesenchymal markers (cytokeratins and vimentin). This assay was evaluated in a pilot cohort of NSCLC and SCLC patient blood samples. In an index NSCLC patient, a CTC derived explant (CDX) tumour was generated from the same blood draw allowing comparison between CTCs in the donor blood sample and the tumour arising from tumorigenic CTCs. As VM had not been reported in SCLC, SCLC patient biopsies were evaluated from 41 limited stage patients to demonstrate VM vessels (Periodic Acid Schiff (PAS)+/CD31-) and correlate with clinical outcomes. A second assay was developed to profile SCLC CTCs for co-expression of cytokeratins and VE-cadherin (a biomarker of VM) and applied to blood samples from 38 SCLC patients (8 limited stage, 30 extensive stage). In vitro and in vivo studies were performed using VE-cadherin positive and negative SCLC cell lines (H446 and H446 shRNA VE-cadherin knock down) to determine if VE-cadherin played a functional role in the formation of VM networks. Results: Sub-populations of CTCs which were epithelial (cytokeratins+/vimentin-), mesenchymal (cytokeratins-/vimentin+) and mixed epithelial/mesenchymal (cytokeratins+/vimentin+) were identified in all NSCLC and SCLC samples studied. In the index NSCLC patient from whom CTCs generated a CDX, the cytokeratins+/vimentin+ expression profile was consistent between CTCs and CDX tumour. In 41 limited stage SCLC patients, VM was identified for the first time and was associated with shorter patient survival, consistent with the adverse prognosis reported in association with VM in other cancer types. A second immunofluorescent assay identified sub-populations of CTCs with a putative 'VM phenotype' (cytokeratins+/VE-cadherin+) in 37/38 SCLC patients. In pre-clinical studies, VE-cadherin was shown to be necessary for formation of VM networks in Matrigel and for the formation of PAS+/CD31- VM structures in vivo in xenografts. VM vessels were also observed across a panel of CDX tumours that were a superior model to study VM compared to the typically small, necrotic primary tumour biopsies. Conclusion: Sub-populations of lung cancer CTCs were detected and classified based upon their markers of EMT (cytokeratins and vimentin) and VM (VE-cadherin), two putative mechanisms leading to tumour cell dissemination in lung cancer. A better understanding of mechanisms of EMT and VM in lung cancer, via study of CTCs, has the potential to reveal novel therapeutic avenues. CTCs could provide a minimally invasive predictive biomarker.
Date of Award | 1 Aug 2016 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Caroline Dive (Supervisor) & Fiona Blackhall (Supervisor) |
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- circulating tumour cells
- lung cancer
Mechanisms of tumour cell dissemination in lung cancer - a study of circulating tumour cells
Metcalf, R. (Author). 1 Aug 2016
Student thesis: Phd