Background & aims: Cholangiocarcinoma (CCA) is a very aggressive cancer showing high cancer stem cells (CSCs) presence. Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumours. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). Approach & results: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5, CD90, EpCAM, CD133, CD13) and primary cell cultures were prepared. DCLK1 expression was analysed in CCA cell cultures by real-time quantitative polymerase chain reaction (RT-qPCR), Western Blot and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS-Assay, cell population doubling time), apoptosis and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and RT-qPCR. DCLK1 serum concentration was analysed by enzyme-linked immunosorbent assay (ELISA). We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared to unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, decreased colony formation capacity and colony size in both iCCA and pCCA compared to untreated cells. In situ analysis confirm that DCLK1 is present only in tumours, but not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of iCCA (high), pCCA (high), HCC (low) and cirrhotic (low) patients, but it was almost undetectable in healthy controls. Conclusion: DCLK1 characterizes a specific CSC-subpopulation of iCCACD133+ and pCCALGR5+ and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.
Nevi, L., Di Matteo, S., Carpino, G., Zizzari, I., Safarikia, S., Ambrosino, V., et al. (2021). DCLK1, a putative novel stem cell marker in human cholangiocarcinoma. HEPATOLOGY, 73(1), 144-159 [10.1002/hep.31571].
DCLK1, a putative novel stem cell marker in human cholangiocarcinoma
Ambrosino, Valeria;Massironi, Sara;Invernizzi, Pietro;
2021
Abstract
Background & aims: Cholangiocarcinoma (CCA) is a very aggressive cancer showing high cancer stem cells (CSCs) presence. Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumours. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). Approach & results: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5, CD90, EpCAM, CD133, CD13) and primary cell cultures were prepared. DCLK1 expression was analysed in CCA cell cultures by real-time quantitative polymerase chain reaction (RT-qPCR), Western Blot and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS-Assay, cell population doubling time), apoptosis and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and RT-qPCR. DCLK1 serum concentration was analysed by enzyme-linked immunosorbent assay (ELISA). We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared to unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, decreased colony formation capacity and colony size in both iCCA and pCCA compared to untreated cells. In situ analysis confirm that DCLK1 is present only in tumours, but not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of iCCA (high), pCCA (high), HCC (low) and cirrhotic (low) patients, but it was almost undetectable in healthy controls. Conclusion: DCLK1 characterizes a specific CSC-subpopulation of iCCACD133+ and pCCALGR5+ and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.
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