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.

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Broncho-pulmonary neuroendocrine neoplasms (BP-NENs) are neoplasms orphan of an efficient therapy. Available medical treatments derived from clinical trials are not specific for the management of this malignancy. Sunitinib is a multi-receptor tyrosine-kinases (RTKs) inhibitor that has already shown its efficacy in NENs, but there are no available data about its action in BP-NENs. Therefore, our aim was to understand the effects of RTKs inhibition promoted by sunitinib in order to evaluate new putative targets useful in malignancy treatment. Since our results underlined a role for EGFR and IGF1R in modulating sunitinib antiproliferative action, we investigated the effects of erlotinib, an EGFR inhibitor, and linsitinib, an IGF1R inhibitor, in order to understand their function in regulating cells behaviour. Cell viability and caspase activation were evaluated on two immortalised human BP-NEN cell lines and primary cultures. Our results showed that after treatment with sunitinib and/or IGF1, EGF and VEGF, the antiproliferative effect of sunitinib was counteracted by EGF and IGF1 but not by VEGF. Therefore, we evaluated with AlphaScreen technology the phosphorylated EGFR and IGF1R levels in primary cultures treated with sunitinib and/or EGF and IGF1. Results showed a decrease of p-IGF1R after treatment with sunitinib and an increase after co-treatment with IGF1. Then, we assessed cell viability and caspase activation on BP-NEN cell lines after treatment with linsitinib and/or erlotinib. Results demonstrate that these two agents have a stronger antiproliferative effect compared to sunitinib. In conclusion, our results suggest that IGF1R and EGF1R could represent putative molecular targets in BP-NENs treatment.

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