Establishment and Characterization of a New Intrahepatic Cholangiocarcinoma Cell Line Resistant to Gemcitabine

Abstract

Intrahepatic cholangiocarcinoma (ICC) is one of the most lethal liver cancers. Late diagnosis and chemotherapy resistance contribute to the scarce outfit and poor survival. Resistance mechanisms are still poorly understood. Here, we established a Gemcitabine (GEM) resistant model, the MT-CHC01R1.5 cell line, obtained by a GEM gradual exposure (up to 1.5 µM) of the sensitive counterpart, MT-CHC01. GEM resistance was irreversible, even at high doses. The in vitro and in vivo growth was slower than MT-CHC01, and no differences were highlighted in terms of migration and invasion. Drug prediction analysis suggested that Paclitaxel and Doxycycline might overcome GEM resistance. Indeed, in vitro MT-CHC01R1.5 growth was reduced by Paclitaxel and Doxycycline. Importantly, Doxycycline pretreatment at very low doses restored GEM sensitivity. To assess molecular mechanisms underlying the acquisition of GEM resistance, a detailed analysis of the transcriptome in MT-CHC01R1.5 cells versus the corresponding parental counterpart was performed. Transcriptomic analysis showed that most up-regulated genes were involved in cell cycle regulation and in the DNA related process, while most down-regulated genes were involved in the response to stimuli, xenobiotic metabolism, and angiogenesis. Furthermore, additional panels of drug resistance and epithelial to mesenchymal transition genes (n = 168) were tested by qRT-PCR and the expression of 20 genes was affected. Next, based on a comparison between qRT-PCR and microarray data, a list of up-regulated genes in MT-CHC01R1.5 was selected and further confirmed in a primary cell culture obtained from an ICC patient resistant to GEM. In conclusion, we characterized a new GEM resistance ICC model that could be exploited either to study alternative mechanisms of resistance or to explore new therapies.

Keywords: cholangiocarcinoma; drug resistance; gemcitabine; gene expression profiling; preclinical models.

Figure 1

Gemcitabine (GEM) effects on survival: Treatment was performed at 72 h using different GEM doses. MT-CHC01: parental cells; MT-CHC01R1.5: GEM resistant cells; MT-CHC01R1.5 no Gem: MT-CHC01R1.5 resistant clone deprived from GEM for 10 days and re-exposed to escalating doses of the drug for 72 h. All the experiments were conducted in three independent experiments in quadruplicate. Error bars represent mean and SD. **** p = 0.00001.

Figure 2

Differences in cell growth and colony formation. (A) Growth curves of MT-CHC01 and MT-CHC01R1.5 cells: 1.5 × 10⁵ cells were plated in 6-well plates in triplicate in three different experiments in optimal medium. Viable cells were counted at 24, 48, and 72 h after seeding. (B) Colony formation assay on MT-CHC01 and MT-CHC01R1.5 cells: Representative images of colony formation after 10 days of seeding. (C) Quantification of colony formation. Colonies formed by more than 10 cells were counted in triplicate in three different experiments. There is a statistically significant difference in terms of the number of colonies between MT-CHC01 and MT-CHC01R1.5 (p = 0.0001). Error bars represent the mean and SD. * p = 0.01, *** p = 0.0001.

Figure 3

Cell cycle analysis: (A) Representative flow cytometry histograms of cell populations in MT-CHC01 and MT-CHC01R1.5 cells assigned to different cell cycle phases (G0/G1, S, and G2) based on the intensity of PI staining (reflecting DNA content), gated on a single cell population. Cells were plated in optimal culture conditions (MT-CHC01R1.5 also in the presence of GEM) for 24 and 48 h and flow cytometry was performed, and data analyzed using FlowJo software. (B) Statistical analysis of the cell cycle distribution was conducted for three independent experiments. A significant increase of phase S was found in MT-CHC01R1.5 (p = 0.0001), while a significant decrease of phases G0/G1 and G2/M was revealed (p = 0.001) after 24 h. No significant differences were found after 48 h. ** p = 0.001 and **** p = 0.00001.

Figure 4

In vivo tumor growth of MT-CHC01 and MT-CHC01R1.5 in NOD/SCID mice. The graph indicates the mean tumor volume (mm³) measured weekly (error bars: SD). Six mice for each cell lines were used and three independent experiments were conducted. **** p = 0.00001.

Figure 5

Effects of several chemotherapeutic agents on MT-CHC01, MT-CHC01R1.5, and 82.3 cells. The different drugs ((A) Carboplatin; (B) 5-FU; (C) Oxaliplatin; (D) Trabectedin) at the indicated concentrations (for Carboplatin, from 40 to 0.156 μg/mL; for 5-FU, from 384 to 0.75 μM; for Oxaliplatin, from 20 to 0.078 μM; and for Trabectedin, from 10 to 0.039 nM) were added to each cell line. The effect was evaluated by the Cell Titer-Glo assay after 72 h. The values obtained are the mean with SD (bars) of three different experiments. Statistical analysis was performed with Student t test. Asterisks indicate a significant p value (* p < 0.01, ** p < 0.001, *** p < 0.0001, **** p < 0.00001). Statistical analysis was performed comparing resistant cells to MT-CHC01 cells.

Figure 6

Effects of Paclitaxel (A) and Doxycycline (B) on MT-CHC01R1.5 cells: MT-CHC01R1.5 cells were sensible at low doses both to Paclitaxel and Doxycycline. The viability was evaluated by Cell Titer Glo assay. All experiments were conducted three times in quadruplicate. ** p = 0.001, *** p = 0.0001, **** p = 0.00001.

Figure 7

Doxycycline restores the sensitivity of MT-CHC01R1.5 cells to Gemcitabine. Viability of Gemcitabine-resistant MT-CHC01 cells was tested in different culture conditions. GEM 1.5: cells routinely growing in the presence of 1.5 µM Gemcitabine for 72 h; DOXY 30-10-5: cells treated with 30 to 10 or 5 µg/mL of Doxycycline, respectively, for 72 h; DOXY 30-10-5 GEM 1.5-0.75-0.373: cells pre-treated with 30, 10, or 5 µg/mL of Doxycycline for 24 h, followed by 1.5, 0.75, or 0.375 µM Gemcitabine for 72 h; NT: not treated cells. The experiment was repeated three times with four replicates for each dose. Error bars represented means and SD. * p = 0.01, ** p = 0.001, **** p = 0.00001.

Figure 8

Differentially expressed genes obtained using panels of cancer drug resistance (A) and EMT (B) related genes. Control: MT-CHC01 parental cells, which represent the reference.

Figure 9

MGMT promoter methylation level in (A) sensitive and (B) resistant cells. The highlighted regions represent the analyzed CpG site of the MGMT promoter with the percentage of C showing the level of methylation each site. The experiment was conducted in duplicate.