In vitro modelling of cholangiocarcinoma to evaluate Curcumin antitumoral potential

Cholangiocarcinoma (CCA) is a rare and highly aggressive malignancy which arises from the epithelial cells of the liver (intrahepatic) or the biliary duct (extrahepatic). Late diagnosis and pathophysiological heterogeneity are linked to poor prognosis, caused by limited effective targeted therapies, and resistance to conventional treatments. This highlights the urgent need for novel therapeutic strategies and more predictive in vitro models able to accelerate preclinical research. In this context, natural compounds such as Curcumin, a polyphenol derived from Curcuma longa, has drown attentions due to its reported antiproliferative, anti-inflammatory, and pro-apoptotic effects properties in different cancer models. This thesis aimed to study the antitumor efficacy of curcumin on extrahepatic CCA cell line, (i.e., TFK1) using both traditional 2D monolayer and 3D matrix-embedded culture systems. Since Curcumin hydrophobic nature, its solubility in cell culture medium was assessed by preliminary spectrophotometric analysis. Importantly, filtration sequestered part of the molecules, changing Curcumin concentration, therefore not-filtered solutions were used in subsequent experiments to preserve its cytotoxic potential. In the 2D model, TFK1 cells were treated with increasing concentrations of Curcumin (from 5 to 80 μM), and cell viability was assessed at different timepoints using the MTT assay, showing a dose- and time-dependent cytotoxic effect. Specifically, 40 μM and 80 μM seem to be the most damaging conditions. In the meanwhile, a combinational treatment was also tested by employing lower curcumin concentration (i.e., 5 μM and 20 μM) with a widely used chemotherapeutic agent (i.e., Gemcitabine) to identify any synergistic effects. Surprisingly, at tested concentration, Gemcitabine alone did not exhibit a clear dose-response trend, with comparable effects observed across all tested concentrations. However, the addition of curcumin both at 5 μM and 20μM enhanced gemcitabine-induced cytotoxicity, suggesting its potential chemosensitizing role. To better mimic the tumor microenvironment, a 3D cell culture system was established to provide more physiologically relevant conditions. Specifically, TFK1 cells were incapsulated in Gelatin MethAcryloyl (GelMA) hydrogels with high degree of functionalization (i.e., around 90%) at two different concentrations, 7.5%w/v and 10%w/v. Viability and cell morphology was evaluated in both hydrogels by Live/Dead assay and nuclear/cytoskeletal staining. After model characterization, Curcumin cytotoxicity was evaluated at higher concentrations (from 20 μM, to 250 μM). In both GelMA concentrations, Curcumin show antiproliferative effect. However, the response appeared less pronounced than 2D coltures, indicating a partial attenuation of curcumin’s cytotoxic effect in the 3D environment. Notably, Gelma 10% had a time-dependent reduction in cell viabiliy showing strong reduction at 72h. GelMA 7.5%, on the other hand, showed a more evident dose-dependent trend throughout timepoints. Overall, the results support the potential of anticancer compound as curcumin to enhance the efficacy of standard chemotherapy. The higher concentrations required in 3D cultures highlight the value of biomimetic 3D models serving as reliable in vitro models for studying drug–tumor interactions, encouraging further investigations of natural compounds in standard combination therapy.