Expression profiles of cell populations purified from human CRC (4 ways)
ABSTRACT: The survival of isolated metastatic cells and expansion into macroscopic tumour has been recognized as a limiting step for metastasis formation in several cancer types yet the determinants of this process remain largely uncharacterized. In colorectal cancer (CRC), we identify a transcriptional programme in tumour-associated stromal cells, which is intimately linked to a high risk of developing recurrent disease after therapy. A large proportion of CRCs display mutational inactivation of the TGF-beta pathway but paradoxically they are characterized by high TGF-beta production. In these tumours, TGF-beta instructs a transcriptional programme in stromal cells, which confers a high risk of developing metastatic disease. We purified by FACS CD31(+), CD45(+), FAP(+) and Epcam(+) populations from fresh CRC samples and assessed their gene expression profiles Freshly obtained tumours from 6 CRC patients treated at Hospital del Mar or Hospital Clinic (Barcelona, Spain) were minced and incubated with Collagenase IV (100 U mL-1). Enzymatic reaction was stopped by adding 10% FBS, single cells were collected by sequential filtering and resuspended in ammonium chloride (0.15M) to lyse erythrocytes. Cells were stained with FAP unconjugated rabbit antibody. After two washes with HBSS, cells were stained with an APC conjugated anti-rabbit antibody; anti-hEPCAM/TROP1-FITC, anti-CD31-PE and anti-CD45-PE-Cy7 conjugated antibody. Dead cells were labelled with Propidium Iodide. Fluorescence Activated Cell Sorting (FACS) was used to separate 2000 cells from each population; [CD45(+), EPCAM(-), CD31(-), FAP(-)], [CD45(-) EPCAM(+), CD31(-), FAP(-)], [CD45(-), EPCAM(-), CD31(+),FAP(-)] and [CD45(-), EPCAM(-), CD31(-), FAP(+)].