Atomic force microscopy combined with microfluidics for label-free sorting and automated nanomechanics of circulating tumor cells in liquid biopsy

Liquid biopsies are expected to advance cancer management, and particularly physical cues are gaining attention for indicating tumorigenesis and metastasis. Atomic force microscopy (AFM) has become a standard and important tool for detecting the mechanical properties of single living cells, but studies of developing AFM-based methods to efficiently measure the mechanical properties of circulating tumor cells (CTCs) in liquid biopsy for clinical utility are still scarce. Herein, we present a proof-of-concept study based on the complementary combination of AFM and microfluidics, which allows label-free sorting of individual CTCs and subsequent automated AFM measurements of the mechanical properties of CTCs. With the use of a microfluidic system containing contraction-expansion microchannels, specific cancer cell types were separated and harvested in a marker-independent manner. Subsequently, automated AFM indentation and force spectroscopy experiments were performed on the enriched cells under the precise guidance of the label-free identification of cells using a deep learning optical image recognition model. The effectiveness of the presented method was verified on three experimental sample systems, including mixed microspheres with different sizes, a mixture of different types of cancer cells, and a mixture of cancer cells and blood cells. The study illustrates a feasible framework based on the integration of AFM and microfluidics for non-destructive and efficient nanomechanical phenotyping of CTCs in bodily fluids, which offers additional possibilities for the clinical applications of AFM-based nanomechanical analysis and will also benefit the field of mechanobiology as well as cancer liquid biopsy.