Characterization of tumor cell biology and development of potential treatment in human cholangiocarcinoma
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Date
2019
Authors
Yang, Jiaqi
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Abstract
Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that can be classified anatomically into intrahepatic and extrahepatic CCA (I-CCA and E-CCA subtypes). Human I-CCA and E-CCA have different growth features, responses to treatment and propensity to metastasize. In this research we hypothesized that these differences could reflect intrinsic differences in tumor cell biology; the prevalence of cancer stem cell (CSC) within the tumor; and/or expression profiles of multidrug resistance associated proteins (MRPs) and chemokine receptors (CRs) within the two cell types.
Tumor cell growth features, CSC prevalence, and stem cell surface markers (SCSMs) were documented in six CCA and one non-malignant cholangiocyte cell line in vitro. Cell proliferation was determined by the WST-1 assay, colony formation by soft-agar colony formation, spheroid formation by 3D sphere-forming, cell migration by wound healing and invasion by Transwell invasion chambers. CSC prevalences and SCSM expression were examined by flow cytometry. MRP inhibition was achieved by lentivirus-based shRNA knockdown and CR activity influenced by a specific pharmaceutical CR agonist and antagonist.
The results revealed that I-CCA cells had significantly increased proliferative activity, shortened doubling times, and were more invasive than E-CCA cells, but colony/spheroid formation and migration rates were similar. There were no clear differences in the prevalence of CSCs or SCSM expression within the I-CCA and E-CCA cell population. Regarding chemoresistance, following gemcitabine exposure, MRP5 and/or MRP6 expression were significantly upregulated in CCA cells. MRP6 knockdown in I-CCA and MRP5 in E-CCA significantly increased gemcitabine-induced cytotoxicity. Finally, the CCR5 antagonist, Maraviroc significantly inhibited cell proliferation, migration and invasion in I-CCA cells, and spheroid formation and invasion in E-CCA cells. The CCR5 agonist RANTES had no effect on I-CCA cells but increased proliferation, migration and invasion of E-CCA cells.
In conclusion, certain features of I-CCA cells are intrinsically more aggressive than E-CCA cells, but the differences cannot be explained by differences in the prevalence of CSCs or SCSM expression profiles. Inhibition of MRP5 and/or MRP6 expression and inactivation of CCR5 should be explored as potential treatments for CCA.
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Keywords
Cholangiocarcinoma, Tumor biology, Chemoresistance, Chemokine receptor, Cancer stem cell, ShRNA, CCR5
Citation
APA 6th edition