Endocrine Abstracts

Background: Pancreatic Neuroendocrine Neoplasms (pNENs) are malignancies arising from the endocrine pancreas. Past in vitro studies have led to a better comprehension and characterization of this malignancy under several points of view. However, effective medical therapies are still not available, therefore, it is essential to continue studying this malignancy in order to identify a successful approach. Due to tumour complexity, techniques such as 3D cultures, able to generate a more realistic model, have been important to study the topic more accurately. On the other hand, several approaches are available to generate 3D cultures and it is still difficult to understand which one should be used to obtain the best results. This study aims to compare three different 3D culture systems in order to possibly identify the most suitable option in studying pNEN.

Methods: The BON1 cell line has been used as a pNEN model and spheroids were treated with Sunitinib 2,5 uM, 5 uM, 7 uM and 10 uM. The first method employed a 48-well plate with a cell-repellent surface and, in order to obtain spheroids formation, the plates were shaken overnight. Subsequently, spheroids were treated and theirsize was measured using Image J software.The second technique employed a 96-well hanging drop plate and spheroids were generated by self-assembly of tumour colonies.Spheroids were moved into another 96-well plate for treatment and MTT analysis was performed to assess metabolic activity. The third method involved the use of ultra-low attachment 96-well plates with clear round bottom: cells were centrifuged in order to generate spheroids and treated by addition of fresh medium and compound to each well. Also in this case MTT analysis was performed.

Results: Spheroids size measurements didn’t highlighted a decrease in spheroids diameter after treatments with Sunitinib at different concentrations while MTT analysis on spheroids cultured with the second method indicated a decrease in metabolic activity after treatment with Sunitinib 2,5 uM, 5 uM, 7 uM of a 10% (treated cells vs. vehicle cells treated with DMSO). MTT results for spheroids cultured with the third approach have shown a decrease of metabolic activity of a 20% (treated cells vs. vehicle cells treated with DMSO) after treatment with Sunitinib 2,5 uM, 5 uM, 7 uM. Results were more homogeneous for cells seeded with the third method in comparison to the second one.

Conclusions: According to our results the third method appears to represent the easiest and most reliable technique to culture spheroids and obtain reproducible results.