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 isenriched of Cancer Stem Cells (CSCs); this feature is associated withaggressiveness and drug resistance. Recently, DCLK1 was validated asa CSC marker in different gastrointestinal tumors.Our aims were to evaluate: i) DCLK1 expression and biologicalfunction in primary cell cultures of CCA subtypes iCCA (intrahepatic)and pCCA (perihilar) and; ii) DCLK1 expression in human CCAsamples in situ and its serum concentration.Method: Primary cell cultures were prepared from surgical specimens of human iCCA and pCCA and CSCs were immunosorted forspecific markers (LGR5, CD13, CD90, EpCAM, CD133). hBTSC andhHPSC physiological primary stem cell cultures were used as controlsof 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 oftreatment) on cell proliferation (MTS Assay, population doublingtime-PDT), apoptosis (AnnessinV-FITC/PI) and colony formationcapacity were evaluated. DCLK1 gene expression in surgical resectedCCA and healthy samples was evaluated by RT-qPCR. DCLK1 serumconcentration was measured in CCA, HCC, cirrhotic and healthypatients by ELISA.Results: For the first time, we demonstrated DCLK1 mRNA andprotein expression in iCCA and pCCA. An increased expression ofDCLK1 in CCA was evidenced in association with other CSC markersand its highest expression was observed in specific subpopulations ofCCA-CSCs (i.e. pCCALGR5+ and iCCACD133+). DCLK1 showed cytoplasmiclocalization in pCCALGR5+, iCCACD133+, unsorted pCCA and iCCA cellcultures. 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 withcontrols (p < 0.01). LRRK2-IN-1 showed a dose-dependent antiproliferative effect (2.5μM-20 μM) by MTS assay with an IC50 of9.61 μM in unsorted pCCA, 14.72 μM in unsorted iCCA, 4.51 μM inpCCALGR5+ and 9.61 μM in iCCACD133+ cells. Furthermore, LRRK2-IN-1did not influence hBTSC and hHPSC primary cell cultures viability.DCLK1 gene expression was lower in healthy tissues than inspecimens of iCCA and pCCA (p < 0.01). Interestingly, DCLK1 wasdetected in serum samples of iCCA and pCCA patients. DCLK1 serumlevels were lower in cirrhotic and HCC patients compared to CCApatients (p < 0.05), but we have never observed DCLK1 protein intoserum samples of healthy controls.Conclusion: In conclusion, DCLK1 expression characterizes specificCCA-CSC subpopulations and could represent a serum biomarker forCCA. DCLK1 inhibition exerts anti-neoplastic effects in primary CCAcell cultures