Circulating tumour cells (CTCs) are detected in many cancers, including non-small cell lung cancer (NSCLC) where they have prognostic significance. We hypothesised that tumour architecture disruption during radiotherapy (RT), might release tumour cells into the peripheral circulation. Some of these CTCs could be viable and cause metastasis. We used γ-H2AX, a biomarker for radiation-induced double strand breaks (DSBs), as a “radiation signature” to identify CTCs entering the circulation from irradiated tumours.
We first separated RT-released CTCs by FACS based on their “radiation signature” from serial blood samples in 10 NSCLC patients given palliative RT with daily fractions of 3-4 Gy. The sorted CTCs were identified by their cytokeratin-positivity, CD45-negativity and morphology.
We subsequently visualised CTCs directly by microscopy of buffy coat cytospin preparations of 7 patients labelled with various markers. Quantitative analysis revealed different patterns of CTC release and clearance after different fractions with the greatest CTC rise seen most commonly after fraction 1. CTCs were present as single cells and clumps. In one case, the cell number after the first fraction increased by 326% over the pre-RT level, with a peak of 112 CTCs per ml blood. The increase and pattern of γ-H2AX foci in CTC nuclei confirmed that they derived from the irradiated volume. Apoptotic tumour cells and necrotic clumps were observed in increased numbers but many CTCs appeared viable as they formed γ-H2AX foci in response to ex-vivo 3 Gy irradiation. Furthermore, during the course of RT, many CTCs with cytologically confirmed tumour morphology were found to have undergone epithelial-mesenchymal transition (EMT) as indicated by loss of cytokeratin positivity and gain of EMT markers.
These findings are highly clinically significant because we can observe the fate of cells released from irradiated tumours, and potentially could modify RT schedules to minimize viable tumour cell dissemination.