Abstract
BackgroundThe incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world. The tumour has a high proportion of desmoplastic stroma and is correlated with a worse prognosis when cancer associated myofibroblasts (CAFs) are present. Recent studies showed promising results after liver transplantation (LTx) in non-resectable early stage CCA. Mycophenolic acid (MPA) and the mTor inhibitor Everolimus are used to prevent organ rejection but recently were shown to exhibit an antiproliferative effect on CCA-cells. Little is known about the influence of immunosuppressive drugs on tumour cell proliferation and migration after paracrine stimulation by CAFs. Moreover, it is still unknown, which signaling pathways are activated following these specific cell-cell interactions.MethodsCCA cell lines HuCCT1 and TFK1 were utilized for the study. CAFs were derived from resected CCA cancer tissue. Cell viability was measured by the crystal violet assay and tumour cell invasion was quantified using a modified co-culture transmigration assay. Semiquantitative cytokine-expression was measured using a cytokine-array. Protein expression and phosphorylation of ERK, STAT3 and AKT was determined by Western-blot analysis.ResultsCCA cells treated with MPA exhibited a dose related decrease in cell viability in contrast to Cyclosporine A (CSA) treatment which had no effect on cell viability. Everolimus significantly inhibited proliferation at very low concentrations. The pro-invasive effect of CAFs in co-culture transmigration assay was significantly reduced by Everolimus at a concentration of 1nM (p = 0.047). In contrast, MPA and CSA showed no effect on tumour cell invasion. Treatment of CAFs with 1nM Everolimus showed a significant reduction in the expression of IL 8, IL 13, MCP1, MIF and Serpin E1. CCA-cells showed significant increases in phosphorylation of ERK, STAT3 and AKT under the influence of conditioned CAF-media. This effect was suppressed by Everolimus.ConclusionsThe secretion of proinflammatory cytokines by CAFs may lead to increased activation of JAK/STAT3-, ERK- and AKT-signaling and increased migration of CCA-cells. Everolimus abrogates this effect and inhibits proliferation of CCA-cells even at low concentrations.LTx for non-resectable early stage CCA is currently performed in several clinical studies. Consistent with a role for common immunosuppressants in inhibiting tumour cell-proliferation and -invasion, our study indicates that a combination of standard therapies with Everolimus and MPA is a promising therapy option to treat CCA following LTx.
Highlights
The incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world
Effect of immunosuppressive and cytostatic agents on tumour cell-viability To study the effect of the different drugs, we first treated the two tumour cell lines and cancer associated myofibroblasts (CAFs) with variable concentrations of Cyclosporine A (CSA), Everolimus and Mycophenolic acid (MPA)
MPA and CSA showed no significant influence on cell viability of CAFS in a dose dependent manner except for the test dosage of 10 μM at 24 h with MPA treatment (p < 0.05, unpaired t-test) (Fig. 3e and f)
Summary
The incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world. Recent studies showed promising results after liver transplantation (LTx) in non-resectable early stage CCA. In most cases the tumour is non resectable at the time of diagnosis leaving only palliative treatment options which have low survival rates [2,3,4,5]. Published results from the latest clinical studies have indicated 5-year survival rates between 71 and 82 % for non-resectable early stage CCA [6]. In the above mentioned studies the recipients were treated with neo-adjuvant therapy based on the Mayo protocol [8, 9] In this protocol only patients with locally non-resectable early stage CCA or arising CCA in the setting of underlying primary sclerosing cholangitis (PSC) were included
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