OC.08.1 DOUBLECORTIN-LIKE KINASE 1 (DCLK1) IS A MARKER OF SPECIFIC SUBPOPULATIONS OF CANCER STEM CELLS (CSCS) IN HUMAN CHOLANGIOCARCINOMA (CCA) AND ITS INHIBITION EXERTS ANTI-CANCER EFFECTS

Cholangiocarcinoma(CCA) is an aggressive cancer with a low response to chemotherapeutics. Previously, we demonstrated that CCA is enriched of Cancer Stem Cells(CSCs); these features being associated with aggressiveness and drug resistance. In other solid tumours, DCLK1 has been demonstrated as a CSCs marker. The aim of this study was to evaluate in vitro the expression and the biological function of DCLK1 in mixed-intrahepatic-CCA(mixed-iCCA) and mucin-extrahepatic-CCA(mucin-eCCA). Surgical specimens of human CCA were enzymatically digested, immunosorted for specific CSC markers(LGR5, CD13, CD90, EpCAM, CD133) and, primary cell cultures were prepared. DCLK1 expression was analysed in primary CCA cell cultures by RT-qPCR, Western Blot(WB), immunofluorescence(IF) and ELISA. Functional studies were performed in immunosorted and unsorted cells by evaluating the effects of LRRK2-IN-1, a selective DCLK1 inhibitor, on cell proliferation(MTS Assay, Population Doubling Time-PDT), apoptosis(Annessin-V-FITC/Propidium Iodide) and colony formation capacity(Clonogenic Assay). RT-qPCR and WB analyses demonstrated an increased expression of DCLK1 in mucin-LGR5+-eCCA and mixed-CD133+-iCCA cells compared to unsorted cells(p<0.01). By IF, DCLK1 showed similar cytoplasmic localization in LGR5+, CD133+ cells and unsorted CCA cells. Very interestingly, DCLK1 was detected(ELISA) in the serum of CCA patients while it was almost undetectable in healthy controls. The DCLK1 inhibitor, LRRK2-IN-1 (5µM) added for 3 days in CCA cell cultures, markedly impaired cell proliferation and increased PDT, induced apoptosis, decreased colony formation capacity and colony size in both iCCA and eCCA(p< 0.01 vs untreated control cells). The analyses of dose-response curves demonstrated how the anti-proliferative effect(MTS Assay) of LRRK2-IN-1 is dose-dependent(2.5µM-20µM) with an IC50 of 9.61µM in unsorted mucin-eCCA, 14.72µM in unsorted mixed-iCCA, 4.51µM in mucin-LGR5+ and 9.61µM in mixed-CD133+ cells. In conclusion, DCLK1 expression characterizes specific CSC subpopulations of mixed-iCCA(CD133+) and mucin-eCCA(LGR5+) and its detection in CCA patients could represent a serum biomarker for CCA. Moreover, DCLK1 inhibition exerts anti-neoplastic effects in primary CCA cell cultures.