DCLK1, a putative novel stem cell marker in human cholangiocarcinoma

Lorenzo Nevi; Sabina Di Matteo; Guido Carpino; Ilaria Zizzari; Samira Safarikia; Valeria Ambrosino; Daniele Costantini; Diletta Overi; Antonella Giancotti; Marco Monti; Daniela Bosco; Valerio De Peppo; Andrea Oddi; Agostino Maria De Rose; Fabio Melandro; Maria Consiglia Bragazzi; Jessica Faccioli; Sara Massironi; Gian Luca Grazi; Pierluigi Benedetti Panici; Paquale Bartomeo Berloco; Felice Giuliante; Vincenzo Cardinale; Pietro Invernizzi; Giuseppina Caretti; Eugenio Gaudio; Domenico Alvaro

doi:10.1002/hep.31571

DCLK1, a putative novel stem cell marker in human cholangiocarcinoma

Lorenzo Nevi, Sabina Di Matteo, Guido Carpino, Ilaria Zizzari, Samira Safarikia, Valeria Ambrosino, Daniele Costantini, Diletta Overi, Antonella Giancotti, Marco Monti, Daniela Bosco, Valerio De Peppo, Andrea Oddi, Agostino Maria De Rose, Fabio Melandro, Maria Consiglia Bragazzi, Jessica Faccioli, Sara Massironi, Gian Luca Grazi, Pierluigi Benedetti PaniciPaquale Bartomeo Berloco, Felice Giuliante, Vincenzo Cardinale, Pietro Invernizzi, Giuseppina Caretti, Eugenio Gaudio, Domenico Alvaro

Istituto Regina Elena

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Journal	Hepatology
DOIs	https://doi.org/10.1002/hep.31571
Publication status	E-pub ahead of print - Sep 25 2020

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Nevi, L., Di Matteo, S., Carpino, G., Zizzari, I., Safarikia, S., Ambrosino, V., Costantini, D., Overi, D., Giancotti, A., Monti, M., Bosco, D., De Peppo, V., Oddi, A., De Rose, A. M., Melandro, F., Bragazzi, M. C., Faccioli, J., Massironi, S., Grazi, G. L., ... Alvaro, D. (2020). DCLK1, a putative novel stem cell marker in human cholangiocarcinoma. Hepatology. https://doi.org/10.1002/hep.31571

DCLK1, a putative novel stem cell marker in human cholangiocarcinoma. / Nevi, Lorenzo; Di Matteo, Sabina; Carpino, Guido; Zizzari, Ilaria; Safarikia, Samira; Ambrosino, Valeria; Costantini, Daniele; Overi, Diletta; Giancotti, Antonella; Monti, Marco; Bosco, Daniela; De Peppo, Valerio; Oddi, Andrea; De Rose, Agostino Maria; Melandro, Fabio; Bragazzi, Maria Consiglia; Faccioli, Jessica; Massironi, Sara; Grazi, Gian Luca; Panici, Pierluigi Benedetti; Berloco, Paquale Bartomeo; Giuliante, Felice; Cardinale, Vincenzo; Invernizzi, Pietro; Caretti, Giuseppina; Gaudio, Eugenio; Alvaro, Domenico.

In: Hepatology, 25.09.2020.

Research output: Contribution to journal › Article › peer-review

Nevi, L, Di Matteo, S, Carpino, G, Zizzari, I, Safarikia, S, Ambrosino, V, Costantini, D, Overi, D, Giancotti, A, Monti, M, Bosco, D, De Peppo, V, Oddi, A, De Rose, AM, Melandro, F, Bragazzi, MC, Faccioli, J, Massironi, S, Grazi, GL, Panici, PB, Berloco, PB, Giuliante, F, Cardinale, V, Invernizzi, P, Caretti, G, Gaudio, E & Alvaro, D 2020, 'DCLK1, a putative novel stem cell marker in human cholangiocarcinoma', Hepatology. https://doi.org/10.1002/hep.31571

Nevi, Lorenzo ; Di Matteo, Sabina ; Carpino, Guido ; Zizzari, Ilaria ; Safarikia, Samira ; Ambrosino, Valeria ; Costantini, Daniele ; Overi, Diletta ; Giancotti, Antonella ; Monti, Marco ; Bosco, Daniela ; De Peppo, Valerio ; Oddi, Andrea ; De Rose, Agostino Maria ; Melandro, Fabio ; Bragazzi, Maria Consiglia ; Faccioli, Jessica ; Massironi, Sara ; Grazi, Gian Luca ; Panici, Pierluigi Benedetti ; Berloco, Paquale Bartomeo ; Giuliante, Felice ; Cardinale, Vincenzo ; Invernizzi, Pietro ; Caretti, Giuseppina ; Gaudio, Eugenio ; Alvaro, Domenico. / DCLK1, a putative novel stem cell marker in human cholangiocarcinoma. In: Hepatology. 2020.

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title = "DCLK1, a putative novel stem cell marker in human cholangiocarcinoma",

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.",

author = "Lorenzo Nevi and {Di Matteo}, Sabina and Guido Carpino and Ilaria Zizzari and Samira Safarikia and Valeria Ambrosino and Daniele Costantini and Diletta Overi and Antonella Giancotti and Marco Monti and Daniela Bosco and {De Peppo}, Valerio and Andrea Oddi and {De Rose}, {Agostino Maria} and Fabio Melandro and Bragazzi, {Maria Consiglia} and Jessica Faccioli and Sara Massironi and Grazi, {Gian Luca} and Panici, {Pierluigi Benedetti} and Berloco, {Paquale Bartomeo} and Felice Giuliante and Vincenzo Cardinale and Pietro Invernizzi and Giuseppina Caretti and Eugenio Gaudio and Domenico Alvaro",

year = "2020",

month = sep,

day = "25",

doi = "10.1002/hep.31571",

language = "English",

journal = "Hepatology",

issn = "0270-9139",

publisher = "John Wiley and Sons Inc.",

}

TY - JOUR

T1 - DCLK1, a putative novel stem cell marker in human cholangiocarcinoma

AU - Nevi, Lorenzo

AU - Di Matteo, Sabina

AU - Carpino, Guido

AU - Zizzari, Ilaria

AU - Safarikia, Samira

AU - Ambrosino, Valeria

AU - Costantini, Daniele

AU - Overi, Diletta

AU - Giancotti, Antonella

AU - Monti, Marco

AU - Bosco, Daniela

AU - De Peppo, Valerio

AU - Oddi, Andrea

AU - De Rose, Agostino Maria

AU - Melandro, Fabio

AU - Bragazzi, Maria Consiglia

AU - Faccioli, Jessica

AU - Massironi, Sara

AU - Grazi, Gian Luca

AU - Panici, Pierluigi Benedetti

AU - Berloco, Paquale Bartomeo

AU - Giuliante, Felice

AU - Cardinale, Vincenzo

AU - Invernizzi, Pietro

AU - Caretti, Giuseppina

AU - Gaudio, Eugenio

AU - Alvaro, Domenico

PY - 2020/9/25

Y1 - 2020/9/25

N2 - 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.

AB - 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.

U2 - 10.1002/hep.31571

DO - 10.1002/hep.31571

M3 - Article

C2 - 32978808

JO - Hepatology

JF - Hepatology

SN - 0270-9139

ER -