The Influence of Pancreatic Cancer Spheroid Three-Dimensional Cultures Modeling Method on Efficacy of Doxorubicin Activity

Introduction Pancreatic cancer is a malignant tumor that develops in the pancreas and spreads to the lymph and lymph nodes, through the blood to the liver and lungs [1]. Pancreatic cancer is treated with surgical removal of the tumor [2] and with radiotherapy, immunotherapy and chemotherapy with paclitaxel in combination with doxorubicin (DOX), gemcitabine, erlotinib, sunitinib [3]. There is a lack of scientific research on DOX effectiveness on PANC-1 and MIA PaCa-2 adenocarcinoma cells spheroids (SPH) growth dynamics and characteristics. Therefore, the effect of SPH modeling on agarose and hanging drop methods on anticancer compound doxorubicin activity in vitro was evaluated. Aim To evaluate the influence of human pancreatic cancer three-dimensional cultures modeling method on the DOX activity. Methods Modeling of PANC-1, MIA PaCa-2 SPH using on agarose and hanging drop methods. Analysis of DOX activity, transport to SPH were performed. A cell suspension was divided into 4 test tubes with 2000, 1000, 500, 250 cells per 2 ml using on agarose and hanging drop method. In both methods, cells were incubated. SPH formation was monitored every 48 h for 8 days and evaluated by phase contrast microscopy (PCM). The effect of DOX on SPH was assessed by the change in SPH diameter (DM) and viability using the MTT reduction method. SPH transferred on agarose and SPH formed on agarose were exposed to 4 µM, 20 µM, 100 µM DOX solutions. A nutrient medium with DMSO was used as a negative control. The SPH were incubated for 8 days, imaged every 48 h under a PCM. In order to assess DOX transport 250 cells SPH grown to approx. 200 µm in DM and 2000 cells SPH grown to approx. 300 µm in DM were incubated with 4 µM, 20 µM, 100 µM DOX, and every 48 h for 8 days imaged with a fluorescence microscope. Statistical analysis achieved with Microsoft Excel. Results Growth dynamics and SPH DM depend on the initial cell concentration and incubation time of the SPH. The DM of MIA PaCa-2 SPH on agarose increased from 147.9±8.9 µm to 426.7±38.6 µm, and MIA PaCa-2 SPH in a hanging drop increased from 151.1±6.9 µm to 432.5±19.3 µm. The DM of PANC-1 SPH on agarose increased from 157.5±6.9 µm to 431.9±8.5 µm, and PANC-1 SPH in a hanging drop increased from 156.6±4.3 µm to 435.1±13.3 µm. There were no difference in both lines cells SPH growth dynamics between used SPH modeling methods (p>0.05). There was no difference in the DM and viability of in both lines cells SPH formed on agarose and in a hanging drop after exposure to DOX (p>0.05). DOX at concentration of 100 µM on the 8th day had a significant effect on the growth of PANC-1 and MIA PaCa-2 SPH of both lines, formed by both methods. Conclusions Growth dynamics of PANC-1 and MIA PaCa-2 SPH were independent of the modeling method. The efficacy of DOX was not depending on the pancreatic cancer SPH modeling method. The incubation time and DOX concentration had the influence on DOX transport to SPH.