RNA isolation and species-specific primers design

For gene expression studies, mouse or PDX PBMC, with or without human CTCs, were lysed by adding 500 μl of Trizol (Invitrogen, Carlsbad, CA, USA). RNA quantity and purity were assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Fischer Scientific, Wilmington, DE, USA). Total RNA (~ 1 μg) was reverse-transcribed in a final volume of 20 μl using random hexamers and SuperScript II (Life Technologies, Carlsbad, CA, USA). For each gene, human-specific primers were designed using Primer-BLAST at the National Center for Biotechnology Information and verified using the Genome Browser UCSC In-Silico PCR tool; primers with sequence homologies between Homo sapiens and Mus musculus were excluded. If possible, primers that amplified regions spanning two or more exons or the 5′- or 3’-UTR regions of the transcript were selected, to avoid amplification of genomic sequences and operator or laboratory contamination (Additional file 2: Table S2). To validate species-specificity, all oligonucleotides were tested in RT-PCR on RNA pools from human cell lines (ZR-75-1, MCF-7, MDA-MB-231 breast cancer, and MDA-MB-435 melanoma cells) and Sv/129 mouse blood. One μl of cDNA was amplified using DreamTaq DNA Polymerase (Life Technologies), according to the manufacturer’s instructions. Single nested PCR was performed with 1 μl of a 1:10 diluted pre-amplification reaction, by using DreamTaq DNA Polymerase (Life Technologies). PCR products were then analyzed on a 2% low melting point agarose gel.