Abstract
Pancreatic Neuroendocrine Neoplasms (pNEN) are rare tumors which treatment still represent an important clinical problem, due to the paucity of medical treatments. Due to tumor complexity, techniques as 3D cultures are important to study drug activity in a more realistic model. This study aims to compare three different 3D culture methods in order to understand which one can be considered the best option in terms of experimental easiness and reproducibility in studying the efficacy of a target drug on pNEN. The BON1 cell line was used as a pNEN model and the well-known Receptor Tyrosine Kinase inhibitor Sunitinib was used in order to better investigate the different features of each method. The investigated methods are: (1) 96-well hanging drop plates (HD plates), (2) 24-well plates with a cell-repellent surface, and (3) ultra-low attachment 96-well plates with clear round bottom (ULA plates). The evaluated parameters during the study were: cell seeding, easiness in spheroids formation, morphology, culture maintenance, medium change, spheroids monitoring, picture quality, spheroid perimeter measurement reproducibility error, possibility to perform assays into the seeding plate, overall time of the experiment. Moreover, we investigated how culture methods can influence experimental outcomes evaluating perimeter changes, cell viability and immunohistochemistry of spheroids treated with different Sunitinib concentrations. Results showed that each method has weak and strong points but, considering the easiness of spheroids maintenance and reproducibility results, ULA plates method appears to be the best approach to culture BON1 spheroids and, therefore, to study pNEN.
Highlights
Pancreatic Neuroendocrine Neoplasms are malignancies arising from the Islets of Langerhans and, are different from the more frequently occurring exocrine pancreas cancer [1]. pNEN represent 7% of all NEN and ∼2% of all pancreatic neoplasms with an incidence of 1–2 per 100,000 persons per year [2]. pNEN diagnosis is mostly influenced by hormonalBON1 Spheroids in pNEN Study hypersecretion that usually leads to early diagnosis due to the manifestation of clinical symptoms [3]
BON1 cells were cultured using a 96-well hanging drop plate and treated with Sunitinib at different concentrations chosen on the basis of previous experiments [21]
This evaluation showed that there is no significant difference between mean perimeter of vehicle treated spheroids and mean perimeter of spheroids treated with different Sunitinib concentrations
Summary
Pancreatic Neuroendocrine Neoplasms (pNEN) are malignancies arising from the Islets of Langerhans and, are different from the more frequently occurring exocrine pancreas cancer [1]. pNEN represent 7% of all NEN and ∼2% of all pancreatic neoplasms with an incidence of 1–2 per 100,000 persons per year [2]. pNEN diagnosis is mostly influenced by hormonalBON1 Spheroids in pNEN Study hypersecretion that usually leads to early diagnosis due to the manifestation of clinical symptoms [3]. Sunitinib inhibits several receptor tyrosine kinases (RTKs) such as Vascular Endotherlial Growth Factor Receptor (VEGFR), Platelet-Derived Growth Factor Receptor and KIT [12] This drug is frequently employed in the treatment of pNEN, since this tumor type is strictly dependent on VEGFR activation [12, 13]. Enhanced cell interaction and crosstalk are additional very important characteristics of 3D cultures that contribute to the generation of a complex microenvironment, that, again, cannot be reproduced in 2D cultures All these features together may be useful for specific research aspects, that cannot be provided by 2D systems, representing an essential asset for drug discovery research [16, 18]. We evaluated the results of each 3D method according to their different specific features and tried to identify which method is the best to study drug activity in BON1 cells
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