Utility of a liquid biopsy to identify intra-tumoural molecular heterogeneity in head and neck squamous cell carcinoma: the role of circulating tumour cells

BACKGROUND: Circulating tumour cells (CTCs) in the blood of cancer patients are a potentially rich source of biomarkers to guide cancer therapy, particularly when tumours cannot be sampled directly. The optimal method of CTC enrichment/isolation and subsequent downstream characterisation remains unclear. Current marker dependent platforms are biased towards specific CTC sub-groups, and CTC characterisation focuses on genomic characterisation, being unable to detect post-transcriptional modification. AIMS: The aims of this project were: i) develop blood sampling methods able to preserve key CTC parameters with minimal sampling handling, ii) optimise a marker-independent CTC enrichment technique, the Parsortix microfluidic platform, for use in head and neck squamous cell carcinoma (HNSCC), iii) investigate methods for direct proteomic characterisation of CTCs to detect relevant post translational modifications. Collectively these aims would streamline sample collection thus simplifying and enhancing national/international multi-centre CTC research studies and trials while allowing deeper characterisation of CTC biology. METHODS: A mock CTC model using the FaDu and SCC047 HNSCC cell lines spiked into healthy donor blood was developed and used to optimise the Parsortix platform and downstream immunofluorescence microscopy and flow cytometry characterisation. Subsequently, a mass cytometry antibody panel of 44 markers (including epithelial/EMT, proliferative, stemness, immune and phosphorylated signalling proteins) was optimised for staining quality on Transfix fixed cells, following Parsortix enrichment. Pre-treatment blood samples from HNSCC patients were prospectively recruited through the Accelerated2 sample collection platform. RESULTS: In cell line spiking experiments, non-fixative EDTA blood collection tubes (BCTs) enriched with Parsortix demonstrated a mean capture rate of 53.5%, across a range of 9-129 cells/ml spiked concentrations (n=13). Transfix fixation BCTs demonstrated significantly improved capture rates of spiked cells at 0hr, 24hr and 72hr timepoints, when compared to EDTA BCTs. Parsortix enrichment significantly altered the gene expression of unfixed cells, causing downregulation of genes associated with RNA and ribosomal/protein processing genes and upregulation of genes associated with oxidative stress and cell injury/apoptosis. Using immunofluorescence microscopy, CTCs were identified from HNSCC patient samples (enriched with Parsortix) w