Isolation and Characterization of CTCs and EVs

Eighteen milliliters of EDTA blood was collected and CTCs were isolated in duplicate from 5 ml of whole blood by positive immunomagnetic selection (AdnaTest EMT-2/StemCell Select^TM, QIAGEN) [13 (link)]. CTC-depleted blood remaining after positive immunomagnetic selection [20 (link)] as well as the remaining blood (not used for CTC isolation) were centrifuged and stored. EVs were isolated from pre-filtered plasma by affinity-based binding to a spin column [13 (link), 21 (link)]. Subsequently, the total RNA was isolated and purified (exoRNeasy Kit, QIAGEN). The mRNA was isolated from the CTC lysates and from the vesicular RNA eluates by Oligo(dT)₂₅ beads [13 (link)]. The supernatant remaining from the CTC lysates after incubation with the Oligo(dT)₂₅ beads, called the mRNA-depleted CTC lysate, was used to isolate the gDNA using the AllPrep DNA/RNA Nano Kit prototype (QIAGEN) [14 (link)]. cfDNA was isolated by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN) using plasma from CTC-depleted blood [20 (link)]. Buffy coat DNA and normal tissue DNA that was available (from 18 of the 26 patients) was used as matched germline control. Detailed protocols are available in Additional file 2.