Abstract
MOTIVATION: Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales in vivo, and how these may affect detection and enumeration of rare CTCs from small blood samples. METHODS: We recently developed an optical instrument called "diffuse in vivo flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice. Here, we used DiFC to study short-term changes in CTC numbers in multiple myeloma and Lewis lung carcinoma xenograft models. We analyzed CTC detections in over 100 h of DiFC data, and considered intervals corresponding to approximately 1%, 5%, 10%, and 20% of the PB volume. In addition, we analyzed changes in CTC numbers over 24 h (diurnal) periods. RESULTS: For rare CTCs (fewer than 1 CTC per ml of blood), the use of short DiFC intervals (corresponding to small PB samples) frequently resulted in no detections. For more abundant CTCs, CTC numbers frequently varied by an order of magnitude or more over the time-scales considered. This variance in CTC detections far exceeded that expected by Poisson statistics or by instrument variability. Rather, the data were consistent with significant changes in mean numbers of CTCs on the timescales of minutes and hours. CONCLUSIONS: The observed temporal changes can be explained by known properties of CTCs, namely, the continuous shedding of CTCs from tumors and the short half-life of CTCs in blood. It follows that the number of cells in a blood sample are strongly impacted by the timing of the draw. The issue is likely to be compounded for multicellular CTC clusters or specific CTC subtypes, which are even more rare than single CTCs. However, we show that enumeration can in principle be improved by averaging multiple samples, analysis of larger volumes, or development of methods for enumeration of CTCs directly in vivo.