Enrichment of circulating head and neck tumour cells using spiral microfluidic technology

Abstract

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC.

Figure 1. Spiral microfluidic chip setup.

Figure 2. The mean diameter and standard deviation of 5 human cell lines and normal human lymphocytes.

Figure 3

(i) The mean % recovery and standard deviation for spiked cell lines (n = 3). (ii, a) Presentation of spike-in and recovered tumour cells FaDu cells into blood of a normal healthy volunteer. Tumour cells: cytokeratin (red), DAPI (blue) White blood cells: CD45 (green), DAPI (blue). Scale bar represents 10 μm (b) RFP-labelled tumour cells from whole blood. Scale bar represents 50 μm.

Figure 4

Day 24, Bioreactor grown (A) FaDu spheroid (B) MDA-MD-468 (cell cluster). (C) MDA-MD-468 Spiral sorted single and cell clusters (D) 600x image of Spiral sorted cell cluster. Scale bar represents 10 μm.

Figure 5. Box plot summary indicating the range of CTCs (pan-CK + CD45-DAPI + ) cells/10 ml.

The box plot presents the median and all individual data points. NHV; normal healthy volunteers.

Figure 6. Patient “micro_01, micro_06”; CTC clusters detected in the blood.

(a) 100x magnification showing the CTC cluster in the top of the cytospot. 1000x images of (b) DAPI (c) Cytokeratin/DAPI composite (d) DAPI (e) Cytokeratin (f) Cytokeratin/DAPI composite. Scale bar represents 50 μm. Figure 6 continued. Patient “micro_12”; CTC doublet detected in blood. (g) DAPI (h) Cytokeratin/DAPI composite (i) EGFR/DAPI composite. Patient “micro_03” single CTCs detected in blood. (j) DAPI (k) Cytokeratin/DAPI composite (l) EGFR/DAPI composite. Scale bar 10 μm (g–i). Scale bar 50 μm (j–l).

Figure 7. EGFR gene amplification in CTCs derived from head and neck cancer patients.

Dual colour FISH assay probes for EGFR (red) and chromosome seven (CEP7, green). Row 1 shows individual stains for DAPI, CEP7, EGFR and the composite image (DAPI, CEP7, EGFR). Row 2 shows further patient CTC composite images showing EGFR amplification. Scale bar represents 10 μm.

Figure 8. A FDG-PET study from a patient presenting with CTCs and had progressive disease to the lungs showing an 8 × 5 mm nodule in the left upper lobe.