THU-457-DoubleCortin/Like Kinase 1 (DCLK1) expression characterized specific cancer stem cell subpopulations of human cholangiocarcinoma primary cell cultures where its inhibition exerts anti-neoplastic effects

Background and aims: Cholangiocarcinoma (CCA) is a very aggressive cancer with high chemoresistance. We demonstrated that CCA is enriched of Cancer Stem Cells (CSCs); this feature is associated with aggressiveness and drug resistance. Recently, DCLK1 was validated as a CSC marker in different gastrointestinal tumors. Our aims were to evaluate: i) DCLK1 expression and biological function in primary cell cultures of CCA subtypes iCCA (intrahepatic) and pCCA (perihilar) and; ii) DCLK1 expression in human CCA samples in situ and its serum concentration. Method: Primary cell cultures were prepared from surgical specimens of human iCCA and pCCA and CSCs were immunosorted for specific markers (LGR5, CD13, CD90, EpCAM, CD133). hBTSC and hHPSC physiological primary stem cell cultures were used as controls of iCCA and pCCA respectively. DCLK1 expression was analysed by RTqPCR, western blot and immunofluorescence. In functional studies, the effects of a selective DCLK1 inhibitor (LRRK2-IN-1, 72hrs of treatment) on cell proliferation (MTS Assay, population doubling time-PDT), apoptosis (AnnessinV-FITC/PI) and colony formation capacity were evaluated. DCLK1 gene expression in surgical resected CCA and healthy samples was evaluated by RT-qPCR. DCLK1 serum concentration was measured in CCA, HCC, cirrhotic and healthy patients by ELISA. Results: For the first time, we demonstrated DCLK1 mRNA and protein expression in iCCA and pCCA. An increased expression of DCLK1 in CCA was evidenced in association with other CSC markers and its highest expression was observed in specific subpopulations of CCA-CSCs (i.e. pCCALGR5+ and iCCACD133+). DCLK1 showed cytoplasmic localization in pCCALGR5+, iCCACD133+, unsorted pCCA and iCCA cell cultures. LRRK2-IN-1 (5 μM) added to CCA cultures increased PDT, decreased proliferation, colony formation capacity and colony size, and induced apoptosis in both iCCA and pCCA compared with controls (p < 0.01). LRRK2-IN-1 showed a dose-dependent antiproliferative effect (2.5μM-20 μM) by MTS assay with an IC50 of 9.61 μM in unsorted pCCA, 14.72 μM in unsorted iCCA, 4.51 μM in pCCALGR5+ and 9.61 μM in iCCACD133+ cells. Furthermore, LRRK2-IN-1 did not influence hBTSC and hHPSC primary cell cultures viability. DCLK1 gene expression was lower in healthy tissues than in specimens of iCCA and pCCA (p < 0.01). Interestingly, DCLK1 was detected in serum samples of iCCA and pCCA patients. DCLK1 serum levels were lower in cirrhotic and HCC patients compared to CCA patients (p < 0.05), but we have never observed DCLK1 protein into serum samples of healthy controls. Conclusion: In conclusion, DCLK1 expression characterizes specific CCA-CSC subpopulations and could represent a serum biomarker for CCA. DCLK1 inhibition exerts anti-neoplastic effects in primary CCA cell cultures