CTC collection and cell lysis

To isolate the CTCs, each blood sample was diluted in a 1:1 ratio with the HBSS buffer solution and incubated with 10% EpCAM MicroBeads (Miltenyi Biotec) with shaking at room temperature for an hour. Afterward, equal volumes of buffer solution and the blood samples containing the magnetically labeled CTCs were loaded onto the buffer inlet and sample inlet, respectively, before withdrawn from the chip at a flow rate of 2 ml/hour. After sample processing, 300 μl of buffer solution was loaded to both the sample and buffer inlets and withdrawn at the same flow rate to wash out remaining samples in the microchannels. Solution collected from the high-expression outlet (~500 μl) of each chip was injected into a 1.5 ml centrifuge microtube (Axygen) and set on the DynaMag-2 magnet (Thermo Fisher Scientific) for an hour. After the cells settled down, supernatant was pipetted out slowly without touching the bottom or the wall of the tube, leaving minimal solution (5 to 10 μl) containing CTCs. Alkaline lysis method adapted from a previous publication was used to lyse CTCs (25). Briefly, 10 μl of 200 mM KOH with 50 mM dithiothreitol was added to CTC solution and heated at 65°C for 10 min in a thermocycler. Lysate was then neutralized by 10 μl of 900 mM tris-HCl (pH 8.3) with 300 mM KCl and 200 mM HCl.