Figure 1

A: normalised PRL-3 mRNA expression of colorectal (HCT-116, CaCo2, HT-29, DLD-1, LoVo, KM12C, KM12SM, SW480 and MH35-D2) and pancreatic cell lines (NP18-MP2 and NP18-MH5) cultured in DMEMF12 + 10% FBS or conditioned medium (CM) obtained from carcinoma-associated fibroblasts derived from a liver metastasis (CAFlm). Total RNA were retrieved from cellular pellets using TrizOL Reagent method. cDNA was obtained using recombinant ribonuclease inhibitor RNAse OUT and M-MLV retrotranscriptase. Quantitative real-time RT-PCR analyses were performed using the Light-Cycler 2.0 Roche System and LightCycler FastStart DNA Master SyBR Green I kit (Roche). For normalisation of PRL-3 expression levels we analysed expression of GAPDH. Oligonucleotides sequences are provided upon request. All of the experiments were performed in triplicate using two different retrotranscriptions. B: normalised PRL-3 mRNA expression of DLD-1 cells cultured in conditioned media (CM) derived from fibroblasts and carcinoma-associated fibroblasts. We obtained matched fibroblasts cultures from fresh surgical specimens from one patient with colorectal carcinoma with synchronous hepatic metastasis under the supervision of the Ethics Committee of the Hospital Universitari de Bellvitge. We isolated normal colonic fibroblasts (NCF) from the normal colonic mucosa at least 5 cm from the surgical margin, carcinoma-associated fibroblasts (CAFpt) from the primary tumour and carcinoma-associated fibroblasts (CAFlm) from the synchronous metastasis. We also obtained foreskin fibroblasts from another patient. All tissues were minced into small fragments and washed with Hanks Balanced Salt Solution (Gibco) and filtered with a 70 μm cell strainer (BD). Then fibroblasts and CAF's were cultured in Dulbecco's modified Eagle's F12 medium (Lonza), containing 10% fetal bovine serum (Gibco) and penicillin/streptomycin. After five passages, cells in confluence were harvested and cultures were used to make CM. Thus, fibroblasts or CAF's were grown to confluence in DMEMF12 containing 10% FBS. Then were washed twice in PBS and incubated for 48 h in DMEMF12 without FBS. Next, fibroblasts/CAFs cultures were washed twice in PBS and incubated for 48 h in DMEMF12 containing 10% FBS. CM was centrifuged 5 minutes at 3000 rpm and filtered in 22 μm units and stored at -80°C until use. Therefore, CM from activated myofibroblasts (CAFpt, CAFlm) produces a higher stimulation of mRNA PRL-3 than CM from normal colonic fibroblasts from the same patient. As a control we used DMEMF12 + 10% FBS. In all panels, error bars indicate standard deviation corresponding to three different experiments.